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ANNUAL  REPORT 
OF 
PROGRAM  ACTIVITIES 
NATIONAL  INSTITUTE  OF  ALLERGY  AND  INFECTIOUS  DISEASES 
Fiscal  Year  1979 


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TABLE  OF  CONTENTS 
1979  ANNUAL  REPORT 
NATIONAL  INSTITUTE  OF  ALLERGY  AND  INFECTIOUS  DISEASES 

Director I-I 

Immunology,  Allergic  and  Immunologic  Diseases  Program 2-1 

Microbiology  and  Infectious  Diseases  Program 6-1 

Extramural  Activities  Program I2-I 

Intramural  Research  Program I8-I 

SCIENTIFIC  DIRECTOR I8-I 


Laboratory  of  Biology  of  Viruses I9-I 

Laboratory  of  Clinical    Investigations 20-1 

Laboratory  of  Immunogenetics 2I-I 

Laboratory  of  Immunology 22-1 

Laboratory  of  Infectious   Diseases 23-1 

Laboratory  of  Microbial    Immunity 24-1 

Laboratory  of  Parasitic  Diseases 25-1 

Laboratory  of  Streptococcal    Diseases 26-1 

Laboratory  of  Viral    Diseases 27-1 

Pocky  Mountain  Laboratory 

Laboratory  of  Microbial  Structure  and  Function 28-1 

Laboratory  of  Persistent  Viral  Diseases 29-1 

Epidemiology  Branch 30-1 

Rocky  Mountain  Operations  Branch 3I-I 


ANNUAL  REPORT   OF  PROGRAM  ACTIVITIES 

NATIONAL   INSTITUTE  OF  ALLERGY  AND   INFECTIOUS   DISEASES 

October  1,    1978  Through  September  30,    1979 

Nineteen  Seventy-Nine  has  been  a  busy  year  for  the  NIAID.     This  is  re- 
flected in  the  reports  of  the  program  directors  and  branches  and  labora- 
tories which  follow.     We  have  cause  for  pride  in  the  accomplishments  of 
our  scientific  constituency  and  the  momentum  that  is  being  built  toward 
solution  to  many  of  the  problems  that  have  beset  us  and  been  insolvable. 

The  Director  has  devoted  much  of  his  time  to  interaction  with  constituent 
groups.     These  efforts  to  inform  them  of  the  Institute's  research  programs, 
learn  their  views  and  enlist  their  interest  and  support  have  opened  new 
pathways  to  information  about  the  problems  of  the  practicing  physician  and 
his  views  on  priorities.      In  January   1979,   the  Institute  held  an  open  house 
which  was  attended  by  representatives  of  24  professional  societies  and  volun- 
tary health  organizations.     This  all-day  meeting  was  devoted  to  exchange  of 
information  and  discussions.     The  Director  also  gave  the  following  invited 
lectures  during  the  year. 

Diabetes,   Virology  and  DNA  or  It's  a  Long  Way  from  Amphioxus.     Octo- 
ber 1,    1978,   Annual  ICAAC  Lecture,   Atlanta,   Georgia. 

Introductory  Remarks.     NIAID   30th  Anniversary,   October  27,    1978, 
Masur  Auditorium,   NIH,   Bethesda,   Maryland. 

The  Influence  of  Infection  of  the  Geography  of  Heart  Disease.   Novem- 
ber 13-15,    1978,   T.   Duckett  Jones  Memorial  Lecture,    51st  Annual  Meeting 
of  the  American  Heart  Association,   Dallas,   Texas. 

The  Second  Century  of  Medical  Bacteriology:     Rediscovered  Opportuni- 
ties.    November  20,    1978,   American  Society  for  Microbiology  Archives 
Collection,   Catonsville,   Maryland. 

From  VD  to  STD   (Sexually  Transmitted  Diseases).   'November  21,    1978, 
Medicine  for  the  Layman,   Masur  Auditorium,   NIH,   Bethesda,   Maryland. 

A  Tribute  to  Miss  Helen  Hayes.     November  28,    1978,  Asthma  and 
Allergy  Foundation  of  America,   New  York,   New  York. 

Conference  on  Pharmaceuticals  for  Developing  Countries.     January  21- 
31,    1979,   National  Academy  of  Science,   Washington,   D.C. 

A  Return  to  Nature:     A  Challenge  for  Outdoor  Medicine.     February  2, 
1979,   Columbia  Medical  Society,   Columbia,   South  Carolina. 

International  Symposium  on  Potentiation  of  Immune  Response  to  Vaccines. 
February  20-21,    1979,   Wilson  Hall,   NIH,   Bethesda,   Maryland. 


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From  Glendalough  to  the  Great  Famine:     The  Unexpected  in  Human  His- 
tory.    March  30,    1979,   Annual  Meeting  of  the  American  Epidemiology 
Society,   Atlanta  Hilton  Hotel,   Atlanta,   Georgia. 

Can  Curiosity  Flourish  in  an  Age  of  Diminishing  Expectations?     April  5, 
1979,   Rosenstiel  Basic  Medical  Sciences  Research  Center,   Waltham,   Massa- 
chusetts. 

The  New  York  Academy  of  Medicine  Medal  for   1979  to  Dr.   Maclyn 
McCarty.     April  19,    1979,   The  New  York  Academy  of  Medicine,   New  York, 
New  York. 

INTRODUCTORY  REMARKS:     The  Kinyoun  Lecture.      "The  Role  of  Com- 
plement in  Natural  Resistance  to  Infections."     April  24,    1979,   Wilson  Hall, 
NIH,   Bethesda,   Maryland. 

A  Claim  for  Curiosity  on  a  Vanishing  Frontier.     May  4,    1979,   Center  for 
Interdisciplinary  Research  in  Immunologic  Diseases  (CIRID),   University  of 
California,   Los  Angeles,   California. 

INTRODUCTORY  REMARKS:  The  Kinyoun  Lecture.  "Cell  Mediated  Im- 
munity to  Intracellular  Parasites  and  Polymorphic  Nature  Transplantation 
Antigens."     May  8,    1979,   Wilson  Hall,    NIH,   Bethesda,   Maryland. 

Cystic  Fibrosis  and  the  Historical  Precedent  for  Optimism.     August  9, 
1979,   The  Cystic  Fibrosis  Foundation  President's  Conference,   Dulles 
Airport,   Virginia. 

"Role  of  the  National  Institute  of  Allergy  and  Infectious  Diseases  in  the 
Conquest  of  Disease  —  A  Tenacious  Strategy  to  Match  a  Restless  Tide." 
September  21,    1979,   Wilson  Awards  Symposium:     Prevention  of  Major 
Infectious  Diseases:      Current  Concerns  and  Future  Promise,   Rochester, 
New  York. 

Dr.   Jack  S.   Whitescarver  has  attended  a  number  of  professional  society  and 
voluntary  health  organization  meetings  to  meet  with  officers  and  others  re- 
garding relationships  between  them  and  the  Institute.      He  is  assuming  a 
major  role  in  the  development  of  fiscal  and  program  information  desired  by 
these  organizations,    working  closely  with  the  Director  and  Program  Directors 
in  doing  so. 

International  Medical  Research 

The  absence  of  a  full-time  professional  with  responsibility  for  international 
medical  research  activities  threw  quite  a  burden  on  the  Director  and  Deputy 
Director.     A  new  initiative  in  the  government  to  develop  research  and  techno- 
logical exchanges  and  activities  with  the  People's  Republic  of  China  (PRO 
required  development  of  suggested  program  activities.     With  the  signing  of  a 
formal  agreement  between  the  Ministry  of  Health,   PRC  ,   and  the  DHEW,   the 
NIAID   drew  responsibilities  for  collaboration  in  infectious  diseases,   immunology 


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and  recombinant  DNA  research.     The  activities  now  involving  program  staff 
have  required  representation  at  a  number  of  meetings.     As  the  year  ends, 
definitive  activities  are  evolving. 

The  Director  has  also  been  heavily  involved  with  the  Office  of  International 
Health  in  reviewing  possibilities  for  expanded  relationships  with  medical  re- 
search in  India.  This  stems  from  requests  by  the  Indian  Medical  Research 
Council  and  involved  a  trip  to  India  for  discussions. 

The  Deputy  Director  was  designated  as  Chairman  of  the  Subcommittees  on 
Biomedical  Research  and  Infectious  and  Parasitic  Diseases  of  the  U.S. -Egypt 
Committee  on  Medical  Research.     In  this  role  he  attended  the  joint  meetings 
of  the  subcommittees  and  committees  in  July  where  the  entire  collaboration  in 
research  was  reviewed ,  pending  applications  reviewed  and  approved ,   and 
plans  made  for  the  allocation  of  the  rapidly  dwindling  residual  of  PL  480 
funds. 

Also  as  the  year  ended,   new  initiatives  in  cooperation  with  the  Japanese  were 
developing.     These  stemmed  from  interests  of  Frank  Press,   Director  of  the 
Office  of  Science  and  Technology  Policy,   The  White  House,  in  increasing  the 
investment  being  made  by  the  Japanese  in  research  and  technology  which 
potentially  benefits  the  U.S.     A  July,    1979  trip  in  which  the  Director,   NIH, 
participated  led  to  agreement  in  several  areas  involving  the  NIAID.     These 
were  vaccine  development,  immunology,   and  recombinant  DNA  research.     The 
Director  is  working  with  the  Director,   NIH,   and  staff  toward  specific  propo- 
sals in  these  areas.     Immunology  will  eventually  wind  up  within  the  framework 
of  the  U.S. -Japan  Cooperative  Medical  Sciences  Program  administered  by  the 
NIAID.     Vaccine  development  may  also  go  this  route,   but  recombinant  DNA 
will  require  new  mechanisms  for  cooperation. 

Influenza 

The  Deputy  Director,   along  with  the  Director,   MIDP,   serves  on  an  Inter- 
agency Working  Group   (IWG)   for  DHEW  activities  in  the  control  of  influenza. 

The  IWG  has  coordinated  responses  to  a  number  of  questions  regarding  in- 
fluenza stemming  from  the  Secretary's  Conference  in  1978,   and  the  Congress 
and  the  OSTP.     It  has  also  maintained  an  exchange  of  information  between 
the  CDC,   the  NIAID,   and  the  BoB/FDA  on  occurrence  of  the  disease,   virus 
strain  characteristics  and  research  activities.     Other  activities,  including 
the  Consensus  Conference  on  Amantadine,   are  described  in  the  MIDP  report. 

Recombinant  DNA  Activities 

The  Office  of  Specialized  Research  and  Facilities  continued  to  maintain  high 
containment  facilities  in  an  operational  state  throughout  FY  79.     The  labora- 
tory at  the  Frederick  Cancer  Research  Center  provided  the  main  facility 
support  for  risk  assessment  experiments  being  conducted  by  Drs.   Rowe  and 
Martin  of  the  Intramural  Program.     Those  experiments  have  now  provided 
the  first  direct  evidence  that  some  potential  biohazards  considered  earlier 
are  unlikely  to  occur.     It  is  anticipated  that  those  results,   and  others  yet 
to  come  from  continuing  studies ,    will  have  a  major  impact  on  revisions  of  the 

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NIH  Guidelines  for  Recombinant  DNA  Research.     The   1978  Guidelines  do  not 
explicitly  require  P-4  containment  for  any  permissable  experiment.     Conse- 
quently the  laboratory  in  Frederick  is  being  operated  at  lower  levels  but  it 
can  convert  to  P-4  operations  on  very  short  notice  should  a  need  arise.     The 
Mobile  Containment  Laboratory  has  been  downgraded  to  a  P-3  facility  and 
will  not  be  operated  again  in  the  P-4  mode. 

During  the  year,   the  Architectural  /Engineering  studies  for  the  National  Bio- 
medical Containment  Laboratory  were  completed  in  compliance  with  the  original 
workscope.     The  A/E  construction  cost  estimates  were  significantly  higher 
than  those  developed  by  both  staff  and  the  prime  contractor,   Litton  Bio- 
netics,   Inc.,   at  the  beginning  of  the  project.     Review  of  the  A/E  estimates 
revealed  them  to  be  accurate  and  reasonable  but  significantly  higher  than 
estimating  data  references  for  general  research  laboratories;   this  reflects 
the  extremely  complex  nature  of  the  NBCL.     Accordingly,   the  A/E  has  been 
asked  to  restructure  the  drawings  to  permit  a  basic  bid  package  with  "add- 
ons" to  accomplish  the  construction  of  the  entire  building  in  stages.      Receipt 
of  the  revised  drawings  is  anticipated  near  the  end  of  FY  79  and  a  decision 
as  to  whether  to  solicit  construction  bids  is  expected  at  that  time. 

With  the  issuance  in  December  1978  of  revised  guidelines,   the  Secretary, 
DHEW,   requested  that  the  NIH  prepare  a  Risk  Assessment  Plan.      Since  the 
hypothetical  risks  and  technical  basis  of  recombinant  DNA  research  are  pri- 
marily microbiological  in  nature,   the  responsibility  for  coordination  and 
implementation  of  the  plan  was  assigned  to  the  Director,   NIAID.     In  compli- 
ance with  the  Secretary's  request,   the  NIAID  published  a  proposed  plan  for 
public  comment  and  had  that  document  reviewed  by  the  Recombinant  DNA 
Advisory  Committee.     This  first  Annual  Plan  has  now  been  published  in  the 
Federal  Register.      In  implementing  the  Plan  the  NIAID  will:      (1)   recruit  and 
appoint  an  eminent  scientist  as  a  Special  Assistant  to  the  Director  for  Risk 
Assessment  to  provide  leadership  and  coordination  of  all  activities  concerned 
with  the  evaluation  of  risks;    (2)   develop  and  issue  such  requests  for  appli- 
cations or  proposals  as  are  necessary  to  ensure  the  conduct  of  risk  assess- 
ment research  required  to  answer  specific  questions  or  to  fill  gaps  in  data 
being  accumulated  from  other  research;    (3)   prepare  and  send  periodic  re- 
ports to  the  RAC  identifying  questions,   problems,   and  evaluations  of  scien- 
tific information  pertinent  to  their  various  advisory  functions ;   and  ( 4) 
respond  to  inquiries  from  scientists,   the  public,   DHEW,  or  other  government 
agencies  regarding  available  data  on  risk  assessment  and  evaluation  of  those 
data.     In  carrying  out  these  responsibilities,   the  NIAID  will  enlist  the 
services  of  existing  NIH  offices,   committees,   and  people  to  provide  informa- 
tion,  to  advise  and  evaluate,   and  to  review,   as  appropriate,   reports  for 
completeness  and  accuracy. 

The  consolidation  by  NIH  of  recombinant  DNA  administrative  activities  into 
one  Institute  was  continued  by  the  decision  to  transfer  the  Office  of  Re- 
combinant DNA  Activities  from  NIGMS   to  NIAID.      As  FY   79  draws  to  a  close, 
the  operational  aspects  of  the  transfer  have  all  been  effected  and  it  is 
anticipated  that  at  the  start  of  FY   80  final  administrative  approval  will  be 
received . 


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Office  of  Research  Reporting  and  Public  Response 

The  loss  of  experienced  professional  and  secretarial  staff  during  this  year 
weakened  the  efforts  of  ORRPR.     Assistant  Chief  Margaret  McElwain  had  been 
a  member  of  our  staff  for  more  than   10  years;  Ms.   Bobbie  Plocinik,   our 
"resident  immunologist,"  had  been  with  us  for  more  than  five  years,   and 
Ms.    Sylvia  Glukenhaus  had  been  ORRPR  secretary  for  more  than  eight  years. 
These  personnel  losses  hitting  ORRPR  in  one  year  resulted  in  a  reduction  in 
overall  productivity,  particularly  in  the  area  of  research  reports. 

We  were  successful  in  recruiting  Mr.   Thomas  Coleman,   former  executive  di- 
rector of  the  National  Association  of  Hearing  and  Speech  Agencies,   to  serve 
as  an  expert  in  medical  communications.     He  will  be  developing  the  Institute's 
capability  in  the  audiovisual  and  electronic  media.     He  also  will  be  available 
to  work  with  the  Director  and  other  Institute  personnel  on  communications 
projects,  including  "outreach"  programs  and  minority  efforts.     Also,   Ms. 
Lydia  Woods  Schindler  has  been  employed  under  contract  to  work  on  a  varie- 
ty of  writing  assignments  with  Dr.   Krause. 

Publications  —  Much  of  ORRPR's  staff  effort  this  year  went  into  producing 
publications  which  were  a  part  of  or  an  outgrowth  of  two  NIAID  task  forces. 
As  a  result,   NIAID  now  has  the  beginnings  of  a  Scientific  Monograph  Series 
with  a  six- volume  report  of  the  Virology  Task  Force  and  a  single  volume 
reporting  the  Task  Force  on  Asthma  and  the  Other  Allergic  Diseases.     Our 
staff  devoted  a  great  deal  of  time  in  editing,   proffing  and  distributing  these 
monographs  to  appropriate  scientists  and  physicians.     In  addition,   we  helped 
to  prepare  and  distribute  summary  reports  of  the  Virology  Task  Force  chair- 
man and  the  Asthma  and  Allergic  Disease  Task  Force.     The  latter  summary 
report  contained  NIAID  Council  Recommendations. 

One  aspect  of  both  Task  Force  charges  was  to  produce  a  document  aimed  at 
the  general  public.     The  publication,   Allergies  and  Asthma — An  Optimistic 
Future,   was  written  under  contract  by  Patrick  Young,   freelance  science 
writer.     Approximately  70  illustrations  were  selected  and  developed  by  ORRPR 
staff  to  be  included  in  this  lay  version,   which  is  scheduled  for  distribution 
early  next  year.     The  manuscript  for  the  lay  publication  in  virology,   written 
under  contract  by  Elaine  Blume  Wilson,   a  former  ORRPR  staffer,   has  the 
working  title  of  Intimate  Enemies — An  Introduction  to  Viruses.      It  also  is  in 
final  stages  of  preparation  and  is  being  considered  as  an  "occasional  paper" 
from  the  Institute. 

Our  staff  assisted  in  the  preparation  of  the  Institute's  chapter  on  "Tobacco 
— Its  Role  in  Allergy  and  Immunity,"  for  the  DHEW  publication,   Smoking  and 
Health:     A  Report  of  the  Surgeon  General.      Copies  of  this  chapter  have 
been  reprinted  and  are  being  distributed  to  the  nation's  allergists. 

ORRPR  has  collaborated  with  NIH's  Medical  Arts  Department  in  the  design  of 
a  new  format  for  our  fact  sheet  series.     The  new  format  is  half  the  former 
page  size,   easier  to  display  and  store,   more  attractive  and  economical.     Six 
fact  sheets  have  been  printed  in  the  new  format. 


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Other  publication  efforts  include  a  new  fact  sheet  on  hereditary  angioedema, 
a  backgrounder  on  staph  infections,   and  a  Q  and  A  on  genital  herpes;   major 
revisions  in  three  pamphlets  —  "Asthma,"   "Sexually  Transmitted  Diseases," 
and  "Drug  Allergy"  —  and  revisions  of  four  fact  sheets  —   "Malaria,"  "Toxo- 
plasmosis,"  "Viral  Hepatitis,"  and  "Rocky  Mountain  Spotted  Fever." 

We  continue  to  prepare  columns  for  the  "Search  for  Health"  series  distributed 
by  the  NIH  to  local  newspapers  around  the  country.     Over  the  years,   the 
response  to  these  health  columns  has  been  excellent  with  thousands  of 
follow-up  requests  for  NIAID  publications.     This  year's  columns  resulted  in 
requests  for  our  publications  on  pollen  allergy,  insect  allergy,   poison  ivy 
allergy,   and  Rocky  Mountain  spotted  fever. 

We  also  have  made  a  special  effort  to  supply  allergy  publications  on  a  con- 
tinuing basis  to  the  local  chapter  of  the  Asthma  and  Allergy  Foundation  of 
America  for  distribution  at  its  public  meetings,   which  are  often  attended  by 
an  ORRPR  staff  member. 

In  our  continuing  cooperation  with  the  Consumer  Service  (CIS)  ,  ORRPR  pro- 
vided copies  of  the  backgrounders  on  "Acne"  and  "Q&A  About  Allergies," 
both  of  which  were  promoted  in  the  spring  and  summer  CIS  catalogues.     A 
CIS  spokesperson  has  advised  ORRPR  that  in  response  to  requests  approxi- 
mately 90,000  copies  of  "Acne"  and  75,000  copies  of  "Q&A  About  Allergies" 
have  been  distributed  this  year. 

In  total,   more  than  a  quarter  of  a  million  NIAID  publications  were  distri- 
buted by  ORRPR  during  the  year. 

Public  Inquiries  —  The  number  of  public  inquiry  requests  continues  to  in- 
crease. Approximately  700  individualized  letters  were  prepared  to  respond 
to  a  variety  of  requests,  including  nearly   40  responses  to  the  Congress. 

Scientific  Meetings  and  Information  —  During  the  past  year,   ORRPR  staff 
managed  press  briefings  tied  to  the  International  Symposium  on  Pertussis, 
the  International  Symposium  on  Potentiation  of  Immune  Response  to  Vaccines, 
and  the  results  from  the  initial  polyoma  recombinant  DNA  risk  assessment 
experiments  conducted  by  NIAID  scientists   (Martin  and  Rowe)   at  Ft.   Detrick. 

Working  closely  with  Institute  scientists,   representatives  of  NIH's  Office  of 
Medical  Applications  for  Research  (OMAR)   and  a  private  contractor,   ORRPR 
staff  developed  promotional  materials  and  advance  publicity  for  the  NIH  Con- 
sensus Development  Conference  on  the  Use  of  Amantadine.     This  conference, 
to  be  held  on  October  15,    1979,   will  be  followed  by  a  press  briefing  and  the 
recommendations  of  the  conference  panel  will  be  disseminated  by  ORRPR 
staff. 

Exhibits  were  produced  and  manned  at  the  annual  meetings  of  the  following 
organizations:      American  Public  Health  Association   (APHA):    the  Association 
of  Military  Surgeons  of  the  U.S.;   the  American  Academy  of  Allergy ;   the 
American  Society  for  Microbiology  (AMS)  ;  and  the  American  Lung  Association/ 
American  Thoracic  Society.     This  represents  an  increased  effort  in  reaching 


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members  of  NIAID   "constituent"  organizations.     ORRPR  staff  also  assisted 
NIAID's  EEO  Coordinator  in  developing  an  exhibit  for  the  Minority  Biomedical 
Science  Symposium  in  Atlanta.     We  furnished  pamphlets  and  information  on 
infectious  diseases  for  two  panels  of  an  NIH  Year-of-the-Child  exhibit,   which 
is  being  sent  to  many  meetings,   including  next  year's  APHA  meeting. 

At  the  ASM  meeting,   ORRPR  employed  a  videotape  presentation  for  the  first 
time.     This  included  two  short  segments:      (1)   a  description  of  NIAID's  high- 
risk  containment  facility  at  Ft.   Detrick;   and  (2)   scientific  footage,  provided 
by  Dr.   Adel  Mahmoud  of  Case-Western  Reserve  University,   visualizing  an 
antibody  potentiated  attack  on  trichina  larvae  by  eosinophils. 

Last  year,  ORRPR  staff  developed  our  first  patient-education  slide /tape  show 
on  "What  You  Should  Know  About  Asthma."     Because  of  its  success,   we  were 
developing  a  second  show,    "Coping  with  Your  Allergy  —  At  Home,   At  School 
and  On  the  Job."     This  was  previewed  at  our  exhibit  at  the  American  Acade- 
my of  Allergy  meeting.     It  will  be  pretested  in  selected  patient  populations 
next  year  for  suitability  of  material  and  its  comprehension  level  prior  to  be- 
ing offered  to  the  General  Services  Administration  (GSA)   for  possible  sale. 
GSA  has  reported  that  nearly  300  copies  of  "What  You  Should  Know  About 
Asthma"  have  already  been  sold  to  physicians,   hospitals,   and  nonprofit  or- 
ganizations.    In  addition,  ORRPR  has  lent  copies  of  the  show  to  several 
groups,  including  the  PTA,   Bronco  Junction  (a  camp  for  asthmatics)   and 
local  chapters  of  the  Asthma  and  Allergy  Foundations  of  America  and  the 
American  Lung  Association. 

Special  Events  —  ORRPR  handled  publicity  in  connection  with  the  Institute's 
30th  anniversary  celebration.     Members  of  the  Institute  staff  attending  the 
formal  ceremony  in  Masur  Auditorium  on  October  27  received  a  special  four- 
page  feature  of  NIAID,  prepared  by  ORRPR  staff,   which  later  appeared  in 
the  NIH  Record's  special  anniversary  issue. 

ORRPR  worked  closely  with  Dr.   Krause  and  Dr.    Gordon  Wallace  on  the  pro- 
gram, poster  and  plaque  design  for  the  NIAID  Kinyoun  Lecture  Series. 
Established  by  Dr.   Krause  (with  Dr.   Fredrickson's  verbal  approval),   this 
new  series  of  invited  lecturers  will,   for  the  most  part,   emphasize  the  inter- 
dependence of  infection  and  immunity.     The  first  two  lecturers  were  Dr. 
Hans  Muller-Eberhard  and  Dr.   Rolf  Zinkernagel,  both  from  Scripps  Clinic, 
La  Jolla,   California. 

EEO  Activities  and  Functions 

Report  from  the  Chairperson,   EEO  Advisory  Committee  —  The  EEO  Advisory 
Committee  consists  of  16  voting  members.     Twelve  were  elected  by  the  NIAID 
staff,   two  were  appointed  by  Dr.   Krause,   and  two  are  the  NIAID  EEO  Coun- 
selors for  the  Bethesda  campus.     There  is  an  additional  EEO  Counselor  at  the 
Rocky  Mountain  Laboratory  and  she  participates  in  a  newly  organized  EEO 
Advisory  Subcommittee  for  RML.     In  addition  to  active  participation  by  the 
RML  Subcommittee,   a  Federal  Women's  Program  Subcommittee  has  some  mem- 
bers on  the  Advisory  Committee  and  some  other  Institute  members.     The 
Federal  Women's  Program  Coordinator  (FWPC)   for  NIAID  heads  the  FWP 


1-7 


Subcommittee  and  is  an  ex  officio  member  of  the  Advisory  Committee.     Also 
participating  as  ex  officio  members  are  the  Training  Specialist  and  Coordina- 
tor for  Employment  of  Handicapped  and  the  Hispanic  Employment  Program 
Coordinator,   along  with  the  EEO   Coordinator  for  NIAID.     Members  of  the 
Office  of  the  Director,   Personnel  Office,   Information  Office,   Scientific  Direc- 
tor's Office  and  others  in  various  divisions  of  management  participate  in 
advisory  capacities.     The  EEO  Advisory  Committee  is  directly  advisory  to  the 
Director  on  all  matters  relating  to  EEO  concerns,   programs  and  objectives. 
Much  of  the  committee's  activities  involve  monitoring  of  the  existing  Affirma- 
tive Action  Plan,   encouraging  completion  of  EEO  program  objectives  and 
development  of  new  EEO  and  affirmative  action  objectives.     A  summary  of 
recent  work  is  presented  in  this  report. 

Reports  from  Subcommittees  of  the  EEO  Advisory  Committee  —  Chairpersons 
for  various  subcommittees  of  the  EEO  Advisory  Committee  prepared  final  re- 
ports on  activities  conducted  by  their  subcommittees  during  the  past  fiscal 
year.     These  detailed  reports,   which  were  submitted  to  the  Chairperson  of 
the  EEO  Advisory  Committee,   are  summarized  below: 

1.  Subcommittee  on  Program  Evaluation  —  The  subcommittee  recognized  the 
need  for  a  system  to  monitor  the  implementation  of  the  Affirmative  Action 
Plan  (AAP)   for  the  NIAID.      It  recommended  that  the  Major  Initiative 
Tracking  System  (MITS),   or  a  similar  system,   be  used  by  the  EEO  Advi- 
sory Committee  to  facilitate  program  evaluation  in  this  regard.     This 
tracking  system  entails  (a)   the  selection  of  the  most  impacting  objectives 
from  the  AAP  for  tracking,    (b)   the  collection  of  monthly  progress  re- 
ports from  responsible  officials,    (c)   the  charting  of  progress  made  with 
respect  to  the  attainment  of  these  objectives  on  a  quarterly  and  annual 
basis,   and  (d)   the  dissemination  of  all  charted  information  to  NIAID  em- 
ployees at  the  end  of  each  fiscal  year.     By  means  of  this  tracking  sys- 
tem,  the  EEO  Advisory  Committee  should  be  made  aware  of  problems 

and /or  obstacles  that  may  impede  progress  in  implementing  the  AAP. 
The  subcommittee  acknowledged  that  the  EEO  Coordinator  is  involved  in 
monitoring  the  implementation  of  the  AAP.     However,   the  EEO  Advisory 
Committee,   as  an  elected  body,   has  a  special  obligation  to  carefully  and 
systematically  monitor  affirmative  action  programs.     This  may  be  done  in 
conjunction  with  the  EEO  Coordinator's  office,   but  it  should  also  be  done 
by  the  EEO  Advisory  Committee  separately. 

2.  Subcommittee  on  Statistics  —  This  subcommittee  is  responsible  for  col- 
lecting, interpreting,   and  reporting  statistical  information  to  the  EEO 
Advisory  Committee  for  further  evaluation.      In  order  to  perform  these 
functions ,   access  to  a  vast  source  of  statistical  information  must  be  ob- 
tained.     Since  this  type  of  information  is  not  presently  available  to  the 
subcommittee ,   it  is  exploring  the  possibility  of  establishing  a  computer- 
ized personnel  master  file  for  NIAID.     This  file  would  be  accessible  to 
both  the  EEO  Advisory  Committee  and  the  EEO   Coordinator.     The  pur- 
pose of  such  a  file  would  be  to  provide  up-to-date  information  on  current 
trends  in  hiring,   promotions  and  transfers;   it  would  serve  as  a  means 
for  detecting  flagrant  deficiencies  in  these  areas. 


3.  Subcommittee  on  Training  —  On  the  basis  of  the  response  to  a  question- 
naire submitted  to  employees  of  the  NIAID,   the  subcommittee  prepared  an 
extensive  report  on  training  opportunities  within  the  NIAID .      Several 
barriers  to  training  were  identified  and  suggestions  were  made  as  to  how 
these  barriers  might  be  overcome.     The  subcommittee's  general  recom- 
mendations included  (a)   the  proper  distribution  of  NIAID  policy  state- 
ments on  training  opportunities  to  all  interested  employees,    (b)  increased 
efforts  to  eliminate  advancement  barriers  for  research  employees,    (c)   the 
requirement  that  all  new  employees  be  informed  of  NIAID-sponsored  train- 
ing programs,    (d)   the  encouragement  of  supervisors  to  discuss  training 
possibilities  with  employees,    (e)   communication  on  possible  continued 
employment  to  stay-in- school  employees  now  on  board,   and  (f)   a  request 
for  training  slots  for  employees  in  the  animal  care  and  secretarial  job 
series . 

4.  Subcommittee  on  Communication  —  The  major  function  of  this  subcommit- 
tee is  to  disseminate  information  on  EEO  activities  to  employees  within 
the  NIAID  and  to  seek  ways  by  which  this  communications  process  and 
awareness  of  the  objectives  of  EEO  programs  can  be  improved.     The 
subcommittee  is  currently  seeking  input  from  employees  within  the  NIAID 
on  the  format,   agenda,   and  organization  of  the  coming  EEO  Workshop  at 
which  a  new  five-year  AAP  will  be  formulated. 

5.  Subcommittee  on  Awards  —  This  ad  hoc  subcommittee  reviewed  several 
nominations  for  annual  EEO  awards.      In  accordance  with  NIH   guidelines 
for  service  and  dedication  to  the  principles  of  EEO ,  three  nominees  were 
selected  for  awards  and  special  recognition:     Ms.   Thelma  Gaither,   LCI; 
Dr.   Richard  Asofsky,   LMI;   and  Ms.   Edna  Miller,   OD .     These  individuals 
were  presented  with  certificates  and  cash  awards,  in  recognition  of  their 
outstanding  contributions  to  the  EEO  program,   at  the  NIAID  All  Hands 
Staff  Meeting  on  June  21,    1979. 

6.  Report  from  the  Coordinator  of  the  Federal  Women's  Program  —  Late  in 
1977,   nominees  or  volunteers  for  a  Federal  Women's  Program  Coordina- 
tor (FWPC)   were  requested  of  the  NIAID   staff.      Selection  was  made  by 
the  EEO  Coordinator  (Vincent  Thomas)   with  Advisory  Committee  concur- 
rence.    Weltha  Logan   (Bio  Lab  Tech,   LMI)   was  Dr.   Krause's  appointment 
for  a  two-year  collateral  duty,   from  September  1977  to  1979. 

A  brief  survey  was  conducted  by  Ms.   Logan,  in  the  course  of  a  month's 
detail  in  January  1978,   to  assess  the  concerns  of  the  women  of  the  Insti- 
tute and  to  determine  support  for  an  NIAID  Federal  Women's  Program. 
Major  among  the  concerns  listed  by  the  responders  were  those  of  posi- 
tion management,   career  advancement,   and  training. 

A  subcommittee  of  the  Advisory  Committee  was  the  preferred  organiza- 
tional choice  of  management ,   based  on  the  desire  to  maintain  all  EEO 
concerns  as  an  entity.     Ten  members,   selected  from  among  responders  to 
the  survey  and  representing  various  Institute  programs  and  EEO  pur- 
views,  were  appointed  in  September  1978.     They  received  training  in 
November  1978  to  assist  them  in  understanding  their  functions  and  to  set 


1-9 


specific  goals  for  the  coming  year.     Working  groups  were  established  for 
(a)   survey,    (b)   program,    (c)   communications,   and  (d)   network.     Also, 
an  ad  hoc  group  was  established  to  formulate  an  amendment  to  the  Advi- 
sory Committee  charter  describing  the  subcommittee. 

Prior  to  the  establishment  of  the  subcommittee,    group  discussion  lunches 
had  been  scheduled  periodically.     Early  in   1978,   their  purpose  was  to 
discuss  possible  goals  for  the  program.      Later,   Ms.   Rachelle  Mandlebaum 
of  the  Employee  Assistance  Branch  spoke  on  "Coping  with  Stress  on  the 
Job."     Dr.   Elizabeth  Hearne  of  NIGMS  discussed  statistics  on  women  in 
the  sciences;   and  Alice  Sargent,   an  organizational  consultant,   spoke  on 
women  in  management. 

Presently  the  subcommittee  is  working  mainly  in  two  areas:     program  and 
survey.     Programs  approved  and  scheduled  include:      (a)   two  presenta- 
tions for  Secretaries'  Week  on  effective  communications  by  Dr.   Gloria 
Harris,   offered  to  all  of  NIAID's  office  support  staff;  and  (b)   a  two-hour 
seminar  open  to  all  of  NIH,   entitled  "Women  in  the  Workforce."     Dr.   Ann 
Briscoe,   biochemist  at  Harlem  Hospital  Center  in  New  York  City,   spoke 
on  the  problems  faced  by  professional  scientific  women.     To  speak  on 
issues  which  affect  all  women  who  work  was  Dr.   Fierst,   economist  and 
member  of  the  Interdepartmental  Task  Force  on  Women  established  by  the 
White  House. 

Survey  questions  have  been  formulated  and  sent  around  for  review; 
these  suggestions  were  revised  for  final  approval  by  the  EEO  Advisory 
Committee  and  EEO  Team.      It  is  hoped  that  information  received  from  the 
survey  will  initiate  action  items  for  the  five-year  AAP  to  be  devised  this 
summer. 

Meetings  of  the  Federal  Women's  Subcommittee  are  scheduled  for  the  third 
Wednesday  of  each  month  from   12:00  to  1:30,   held  alternately  in  the 
Westwood  Building  and  Building  31.     The  meetings  are  open  to  visitors. 
Minutes  of  meetings  are  distributed  to  the  Institute  with  the  minutes  of 
the  NIAID  EEO  Advisory  Committee  meetings  and  posted  for  staff  perusal. 

Special  Projects  Related  to  the  Affirmative  Action  Plan. 

1.     Recruitment  Initiatives 

A.     Outreach  to  Local  Minority  High  Schools  —  A  program  was  developed 
during  the  past  fiscal  year  to  stimulate  minority  and  women's  interest 
in  biomedical  careers.     Students  from  four  high  schools  in  the  Dis- 
trict of  Columbia  (Wilson,   McKinley,   Coolidge,   and  Spingarn)   were 
invited  to  visit  the  NIAID;   students  from  more  high  schools  could 
have  been  invited,   but  the  four- week  D.C.   teachers'  strike  created 
serious  communication  problems  in  this  regard.     Each  visiting  group 
was  comprised  of  from   12-17  students  plus  at  least  one  of  their 
teachers.     The  program  for  the  students  consisted  of  a  welcome  and 
orientation  session  at  which  the  Scientific  Director  presented  back- 
ground information  on  research  programs  being  conducted  at  NIH 


1-10 


and  NIAID;   also,   there  was  a  discussion  of  volunteer  and  salaried 
positions  available  for  high  school  students  within  the  NIAID.     Then 
students  were  escorted  to  several  laboratories  where  members  of  the 
scientific  staff  explained  the  type  of  research  being  conducted. 
Here,  the  students  had  an  opportunity  to  observe  experiments  ac- 
tually in  progress  and  the  operation  of  instruments  and /or  techniques 
used  in  biomedical  research.     Although  Dr.   Lois  A.   Salzman,   LBV, 
was  largely  responsible  for  planning  and  coordinating  all  aspects 
connected  with  this  program,   the  individual  efforts  of  many  senior 
staff  scientists  contributed  greatly  to  its  success. 

B .     Introduction  to  Biomedical  Research  —  The  NIAID  recently  hosted 
39  outstanding  science  students  who  participated  in  a  seminar, 
Introduction  to  Biomedical  Research.     The  students  who  came  from 
20  states,   the  District  of  Columbia,   and  Puerto  Rico  were  chosen  by 
their  colleges  to  participate  in  this  special  program  held  February  27 
to  March   1.     The  seminar  was  part  of  the  Minority  Biomedical  Sci- 
ences Program  that  assists  financially  and  educationally  disadvan- 
taged students  to  enter  biomedical  research  and  health-related  pro- 
fessions . 

Dr.    Zora  J.   Griffo,  OD,   and  Dr.   Richard  M.   Krause,   Director  of 
NIAID ,   welcomed  the  students  and  discussed  career  opportunities 
at  NIH.     Dr.   Ciriaco  Q.    Gonzalez,   Chief,   MBS  Program,   DRR ,   empha- 
sized the  need  for  minority  participation  in  the  biomedical  research 
sciences.     The  students  also  heard  from  NIAID  intramural  scientists 
and  Dr.   Kenneth  Sell,   the  Institute's  Scientific  Director. 

The  students  visited  NIAID  laboratories  where  they  spoke  with  in- 
vestigators and  observed  various  experiments  in  progress.     They 
were  also  interviewed  by  researchers  for  their  interest  in  NIH's  sum- 
mer employment  program ;    10  of  these  students  were  invited  to  return 
to  NIAID  in  the  summer  to  work  in  various  laboratories. 

The  program  was  organized  by  Dr.   Kathleen  Jaouni  Cook,   assistant 
to  Dr.    Sell,   and  Mr.   Frank  Fountain,   NIAID 's  EEO  Coordinator. 

2.  Community  Affairs 

The  Community  Outreach  Program  —  As  a  follow-up  to  the  Personnel  Of- 
fice's participation  in  a  Career  Fair  held  at  St.  Paul's  Christian  Communi- 
ty Church,    414  Tennessee  Avenue,   N.E.,  in  June  of  1978,   the  following 
activities  were  initiated  and  are  now  in  progress:     a  tutorial  reading 
service;   an  alcohol  and  drug  abuse  program;   and  safety  and  security 
workshops. 

3.  Employee  Development  Programs  —  The  Employee  Development  Specialist 
(Mrs.   Edna  Miller)  instructed  and  coordinated  a  course  in  Laboratory 
Animal  Care  for  16  employees,   as  well  as  coordinated  three  seminars  on 
the  Factor  Evaluation  System.     The  latter  were  designed  for  administra- 
tive and  supervisory  personnel.     Mrs.   Miller  has  established  a  Mini 


1-11 


Information  Center  for  all  employees  in  her  office  (Room  Bl-01,   Building 
5) .     Catalogs  and  a  variety  of  information  on  educational  opportunities 
for  various  career  specialties  are  available.      In  addition  to  these  activi- 
ties,  tours  of  the  Institute  were  conducted  for  summer  employees,   new 
employees,   and  administrative  personnel;    such  tours  are  scheduled  to  be 
held  at  least  four  times  each  year. 

4.     Employment  of  the  Handicapped  —  The  Coordinator  for  the  Handicapped 
Persons  Program  (Mrs.   Edna  Miller)   reported  that  three  handicapped  em- 
ployees successfully  completed  a  course  in  Laboratory  Animal  Care  in 
April  1979.     The  Coordinator  has  attended  two  courses  so  as  to  keep 
abreast  of  policies  and  laws  concerning  (a)   the  selective  placement  of 
the  handicapped,   and  (b)   cooperative  training  and  employment  programs 
for  the  handicapped. 

Report  from  the  Office  of  the  EEO  Coordinator  —  The  National  Institute  of 
Allergy  and  Infectious  Diseases  is  undertaking  a  program  of  affirmative  ac- 
tion to  which  good  faith  efforts  will  be  directed  for  achievement  of  its 
Major  Civil  Rights  and  Affirmative  Action  Initiatives.     Our  goals  and  initia- 
tives are  consistent  with  those  of  DHEW,   PHS ,   and  NIH.      In  addition,    goals 
and  initiatives  have  been  established  which  are  unique  to  NIAID. 

The  EEO   Coordinator  has  direct  access  to  the  Institute  Director.     Addition- 
ally,  the  EEO  staff  has  direct  access  to  all  NIAID  personnel,  including  the 
Executive  Officer,   Personnel  Officer,   and  Associate  Directors.     There  are 
Federal  Women's  Program  and  Hispanic  Employment  Program  Coordinators  as- 
signed on  a  part-time  basis,   each  spending  at  least  20  percent  of  their  time 
toward  accomplishing  program  objectives.      It  has  been  difficult  for  both  the 
FWPC  and  HEPC  to  actively  pursue  program  objectives  based  on  a  20  per- 
cent time  allocation.     All  other  EEO  officials  who  have  other  principal  assign- 
ments devote  at  least  20  percent  of  official  time  to  EEO  program  matters  and 
serve  as  technical  advisors  to  the  Personnel  Officer.     The  Institute  has  an 
EEO  Advisory  Committee  and  three  EEO  Counselors.     Adequate  management 
and  fiscal  controls  are  established  to  track  all  resources  devoted  to  the  EEO 
program  and  expenses  have  been  charged  to,   and  tracked  by,   the  EEO 
Coordinator  and  Budget  Office. 

Our  present  recruitment  sources  do  yield  qualified  minority  and  female  ap- 
plicants who  meet  organization  needs;   however,   there  has  not  been  an  ade- 
quate number  of  minorities  to  select  from  in  the  M.D.   and  Ph.D.   community. 
Our  recruitment  literature  reflects  the  desire  to  reach  all  segments  of  the 
potential  for  employment,   and  this  effort  is  also  across  internal  NIH  organi- 
zational lines  to  obtain  maximum  effectiveness  and  efficiency .     All  paid 
advertisement  used  for  recruitment  includes  minority  media  coverage.     These 
recruitment  efforts  are  monitored  to  ensure  equal  treatment  regardless  of 
race,   color,   religion,   sex,   national  origin,   handicap,    and  age. 

Plans  are  being  developed  to  increase  the  representation  of  Hispanics  and 
Native  Americans  through  our  Outreach  Programs. 


1-12 


NIAID's  Extramural  Activities  Program,  through  training  grants  and  con- 
tracts,  participates  in  the  Minority  Access  to  Research  Careers  Program 
(MARC)   which  is  designed  to  help  minority  institutions  train  greater  num- 
bers of  scientists  and  teachers  in  the  biomedical  discipline.     Presently,  two 
postdoctoral  fellows  are  being  supported  through  this  program.      No  new 
hires  resulted  from  this  program  during  FY   1979. 

Minority  Biomedical  Support  Program  (MBS)  is  designed  to  increase  minority 
participation  in  biomedical  research,   to  develop  a  pool  of  minority  scientists, 
to  strengthen  biomedical  research  capabilities  of  minority  institutions ,   and  to 
utilize  the  talents  of  minority  biomedical  investigators  in  the  mission  of  the 
NIH.     NIAID  contributed  $125,858  during  FY   1979  as  part  of  its  support 
agreement. 

NIAID  is  supportive  of  the  Cooperative  Education  Program  and  has  two  Black 
female  biomedical  student  participants. 

Expert  appointments  are  those  appointments  of  individual  scientists  or  pro- 
fessionals qualified  in  fields  related  to  the  program  needs  of  NIAID.     Two 
white  females  have  received  appointments. 

Intergovernment  Personnel  Agreement  (IPA)   —  One  Black  female  is  presently 
in  our  Laboratory  of  Clinical  Investigation. 

The  Clinical  Associates  Program,   class  of  1979,   provided  two  white  females. 

NIAID  is  committed  to  increasing  the  participation  of  minorities  and  women  in 
the  scientific  community  and  in  those  grades  and  occupational  series  where 
there  is  underrepresentation.     No  hiring  goals  were  set  for  any  of  our  re- 
cruitment programs  during  FY   1979. 

Sixty  employees  responded  to  a  qualifications  analysis  questionnaire.     The 
survey  was  forwarded  to  all  nonprofessional  employees.     A  consultant  was 
hired  for  this  project.     During  the  follow-up,   approximately  45  NIAID  em- 
ployees were  interested  in  a  qualifications  analysis.     Workshops  were  con- 
ducted which  focused  on  specifically  identified  career  fields,  i.e.,   personnel, 
administration,   program  analysis,   etc.     Employees  were  counseled  as  to  their 
specific  qualifications,   what  was  needed  for  entry  into  primary  career  fields 
of  their  interest,   and  exposed  to  the  prospective  opportunities  at  NIH  in 
those  fields.     Two  such  surveys  were  conducted  during  FY   1979,  one  by 
NIH.     The  skills  file  is  referred  to  when  considering  applicants  for  all  vacan- 
cies. 

Three  workshops  were  conducted  on  the  Factor  Evaluation  System.     The  con- 
tent of  these  workshops  was  designed  and  geared  to  the  needs /interests  of 
the  audience.     A  film  was  shown  and  a  discussion  was  held  on  how  this  sys- 
tem would  affect  those  individuals  in  the  auditing  and  classification  of  their 
positions . 

Training  opportunities  are  available  for  all  employees  to  facilitate  their 
career  goals.     All  denials  of  training  must  be  supported  in  writing.     An 


1-13 


analysis  of  training  sponsored  by  the  Institute  is  ongoing  to  determine  if 
there  is  any  maldistribution  by  sex,   race,    grade,   etc. 

The  majority  of  this  Institute's  supervisors  and  managers  have  received 
supervisory  and  EEO  training.     An  assessment  is  being  conducted  to  examine 
our  supervisory  and  management  practices  and  to  identify  our  needs  for  im- 
plementing training  in  specific  areas  of  concern.     The  Institute  is  committed 
to  the  assurance  that  all  managers  and  supervisors  will  receive  training  in 
accordance  with  established  principles. 

This  Institute  supports  the  NIH  Upward  Mobility  Programs  including  the  Merit 
Promotion  plan.     At  present  we  have  no  internal  training  programs  or  posi- 
tions.     However,   NIAID  provides  training  for  other  Institutes'  Management 
Interns.     Career  counseling  is  available  to  all  employees  through  our  Em- 
ployment Development  Specialist. 

The  EEO  Plan  is  updated  annually.     The  various  program  evaluations  are  in- 
corporated into  the  plan  as  changes  or  additions  at  that  time.     The  evaluation 
is  conducted  by  the  EEO  Coordinator,   the  Director,   the  EEO  Advisory  Com- 
mittee,  and  the  EEO  Team.     Other  management  officials  participate  when  the 
topics  to  be  discussed  require  their  input.     An  AAP  Update  Workshop  is 
planned  for  the  transition  year.     One  of  the  objectives  is  long-  and  short- 
range  AAP  objectives.      The  EEO   Advisory  Committee  will  receive  EEO  Train- 
ing for  Advisory  Committee  Members.     This  will  also  facilitate  monitoring  and 
evaluation  of  our  EEO  Program. 

Currently,   there  is  no  program  in  effect  to  cross-train  the  EEO  Specialists 
or  the  Personnel  Management  Specialists.     The  Personnel  Management  Spe- 
cialists are  scheduled  to  attend  the  EEO  courses  in  accordance  with  one  of 
our  AAP  objectives.     One  Equal  Opportunity  Specialist  progressed  to  the 
Management  Analysis  field. 


1-14 


Annual  Report 

Immunology,  Allergic  and  Immunologic  Diseases  Program 

Table  of  Contents 


Director1  s  Report 2 

A.  Administrative  Summary 2-1 

B.  Scientific  Summary 2-5 

Allergy  and  Clinical  Immunology  Branch 3 

A.  Scope 3-1 

B.  Awards  and  Support  Levels 3-2 

C .  Program  Areas  and  Highlights 3-2 

Genetics  and  Transplantation  Biology  Branch 4 

A.  Scope 4-1 

B .  Awards  and  Support  Levels 4-1 

C.  Programs  Areas  and  Highlights 4-2 

Immunology  and  Immunochemistry  Branch 5 

A.  Scope 5-1 

B.  Awards  and  Support  Levels 5-1 

C .  Program  Areas  and  Highlights 5-2 


REPORT  OF  THE  DIRECTOR 

IMMUNOLOGY,  ALLERGIC  AND  IMMUNOLGIC  DISEASES  PROGRAM 

Fiscal  Year  1979 

A.  Administrative  Summary 

1.  Organization  and  Function 

The  Immunology,  Allergic  and  Immunologic  Diseases  Program  (IAIDP) 
was  initiated  on  October  1,  1976,  coincident  with  the  reorganization  of 
NIAID  into  major  programs  characterized  by  professional  function.  With 
this  change  the  IAIDP  assumed  the  responsibility  for  research,  training, 
and  conference  activities  that  formerly  were  assigned  to  the  Allergy  and 
Immunology  Branch  of  the  Extramural  Programs  and  contract  activities  in 
transplantation  immunology  previously  administered  within  the  Collaborative 
Research  Program. 

During  FY  1979,  the  following  organization  served  the  IAID  Program: 

Office  of  the  Director 

Director-Sheldon  G.  Cohen,  M.D. 

Special  Assistant  to  the  Director-Dorothy  D.  Sogn,  M.D. 

Assistant  for  Program  Management-George  R.  Jenkins,  B.S. 

Collaborative  Projects  Section 

Head-Daniel  I.  Mullally,  M.D.,  M.P.H. 
Program  Officer- John  G.  Ray,  Jr.,  Ph.D. 

Allergy  and  Clinical  Immunology  Branch 

Chief-Robert  A.  Goldstein,  M.D.,  Ph.D. 
Immunology  and  Immunochemistry  Branch 

Chief-Bernard  W.  Janicki,  Ph.D. 

Genetics  and  Transplantation  Biology  Branch 

Chief-Henry  Krakauer,  M.D.,  Ph.D. 

Serum  Bank  Manager-Katherine  A.  Hopkins,  R.N. 

Each  of  the  three  IAIDP  Branches  assumes  responsibility  for  adminis- 
tering investigator  initiated  research  grants  and  collaborative  research 
contract  awards,  developing  programmatic  initiatives  and  new  projects, 
serving  on  trans-NIH  coordinating  committees  and  taking  active  roles  in  the 
development  and  conduct  of  workshops  and  conferences  in  areas  of  the  bio- 
logical disciplines  and  medical  specialties  with  which  they  are  concerned. 


2-1 


All  three  Branches  are  also  involved  with  programs  related  to  Research 
Career  Development  Awards  and  training,  i.e.,  individual  and  institutional 
fellowships.   In  addition,  the  Allergy  and  Clinical  Immunology  Branch 
administers  Allergic  Disease  Academic  Awards  which  are  designed  to  further 
the  careers  of  mid-level  investigators  preparing  for  careers  in  Academic 
Allergy  Research  and  the  Young  Investigator  Research  Grant  Awards  which 
are  designed  to  assist  and  encourage  investigators  in  early  stages  of 
their  careers  to  develop  research  interests  and  capabilities  in  clinical 
aspects  of  immunology. 

The  LAID  Program  also  assists  investigators  in  their  research  by 
providing  certain  reagents  that  are  not  otherwise  available  from  commer 
cial  sources.   Such  reagents  are  intended  either  for  reference  standard 
materials  such  as  human  and  mouse  histocompatibility  typing  sera  and 
various  purified  allergens.   Program  staff  has  the  responsibility  for 
maintaining  adequate  inventories  of  reagents  as  well  as  for  developing  new 
sources  and  identifying  new  materials  for  acquisition. 

A  new  program  activity,  initiated  in  FT  1979  is  designed  to  evaluate 
the  efficacy  of  skin  testing  with  major  and  minor  determinants  of  penicil- 
lin prior  to  prophylactic  administration  of  this  drug  or  its  analogues. 
Using  multi-institution  contracts  for  clinical  testing  data  will  be  pro- 
vided on  the  predictive  value  of  newly  developed  diagnostic  reagent 
material  through  the  collaborative  efforts  of  many  physicians  working  in 
diverse  clinical  situations  with  patients  manifesting  a  variety  of 
infectious  illnesses,  some  rare.   It  is  anticipated  that  the  use  of  common 
protocol  with  standardized  material  prepared  and  furnished  to  participa- 
ting investigators  will  help  to  establish  the  criteria  necessary  for 
developing  penicillin  pre- tests  as  an  important  practical  diagnostic  tool 
and  accelerate  its  clinical  application.   Of  especial  concern  are  the 
problems  faced  by  patients  with  positive  histories  of  penicillin  reactions 
presenting  indications  for  therapy  with  benzylpenicillin,  ampicillin, 
carbenicillin,  phenoxyethylpenicillin,  phenoxymethylpenicillin  and  amoxi- 
cillin.  Contracts  for  participation  in  this  field  trial  were  awarded  to 
six  geographically  dispersed  university  sections  having  interdisciplinary 
programs  in  allergy  and  collaborative  efforts  with  infectious  diseases 
sections  (Mayo  Foundation;  University  of  Rochester;  Northwestern  Univer- 
sity;  University  of  Colorado,-   University  of  Washington,  and  University 
of  California  at  Los  Angeles)   and  one  interagency  agreement  was 
established  with  the  Allergy  Section  of  Walter  Reed  Army  Medical  Center. 
The  Clinical  Centers  Program  consisting  of  Centers  for  Interdisciplinary 
Research  on  Immunologic  Diseases  (CIRID)  and  Asthma  and  Allergic  Disease 
Centers  (AADC)  continues  to  focus  upon  multidisciplinary  collaboration, 
interactions  between  basic  and  clinical  investigators  and  the  application 
of  research  leads  in  the  biomedical  sciences  to  clinical  problems  in  diag- 
nosis, treatment  and  prevention.   Project  Directors  have  been  exploring 
possibilities  in  their  regional  areas  to  increase  the  involvement  of  their 
staff  in  outreach  areas  of  education,  referral  services  and  the  socioeco- 
nomic aspects  of  disease  and  disability.   The  AADCs  now  total  16  in  number 
with  three  new  Centers  added  to  this  network,  State  University  of  New  York 
at  Stony  Brook;   for  the  study  of  urticaris,  angioedema  and  vaculitis; 


2-2 


State  University  of  New  York  at  Buffalo;  for  the  study  of  asthma,  and 
University  of  Kansas;  for  the  study  of  respiratory  disease  and  systemic 
lupus  erythematosus. 

The  rapid  developments  and  progress  in  research  on  physiologic 
function,  chemistry  and  genetics  of  immunocompetent  cells  nave  not  only 
been  important  for  the  fundamental  knowledge  revealed  but  additionally 
have  pointed  to  areas  where  clinical  relevance  may  be.  Accordingly,  this 
year  we  have  instituted  efforts  to  facilitate  interactions  and  exchanges 
between  the  Directors  of  NIAID  supported  clinical  centers  (CIRID  and  AADC) 
and  Directors  of  Program  Projects  in  Lymphocyte  Biology.  The  effective- 
ness of  this  program  has  been  sufficiently  productive  to  move  in  the 
direction  of  expansion  through  the  addition  of  one  new  "Lymphocyte  Biology 
Center"  at  Jewish  Hospital  of  St.  Louis/Washington  University,  bringing 
the  total  of  participating  institutions  to  five. 

Our  mission  as  the  designated  lead  Institute  in  the  study  of  immune 
mechanisms  has  alerted  us  to  the  work  of  other  NIH  B/I/D  concerned  with 
organ  and  disease  oriented  interests  involving  dysfunctions  of  the  immune 
system.  Accordingly,  we  have  developed  joint  program  efforts  especially 
related  to  conference  activities  and  definition  of  research  initiatives 
with  NIAMDD  (dermatology,  diabetes),  NEI  (occular  immunopathology) ,  NCI 
(tumor  immunology)  and  NHLBI  (hypersensitivity  lung  diseases). 

During  FY  1979,  the  work  of  the  Task  Force  on  Asthma  and  Other 
Allergic  Diseases  was  completed.   One  hundred  and  fifty  contributors  and 
consultants  participated  in  the  work  of  twelve  panels.  The  final  report 
of  their  Task  Force  dealt  with  the  state-of-the-art  and  put  forth  specific 
recommendations  for  extended  study  in  twelve  specific  areas  concerned  with 
social  economic  aspects,  basic  mechanisms,  asthma,  rhinitis,  special 
problems  of  allergic  children,  farmer's  lung  and  related  disorders,  the 
expanding  problem  of  occupational/environmental  asthma  and  related  respira- 
tory diseases;   proposed  development  of  integrated  consultative  and 
research  facilities,  dermatologic  allergy,  allergic  emergencies,  drug 
allergy,  allergic  and  immunologic  aspects  of  other  diseases,  and  educa- 
tional needs. 

The  Scientific  Report  has  been  published  and  distributed  to  the 
community  of  health  professionals  concerned  withclinical  and  investigative 
endeavors  in  asthma  and  allergic  diseases.   An  interpretative  summary 
version  is  in  the  process  of  completion  for  distribution  to  the  concerned 
lay  community  of  health  action  groups. 

Staff  members  of  the  IAIDP  cooperated  in  the  preparation  of  the 
updated  Report  of  the  Surgeon  General  on  Smoking  and  Health.   For  this 
DHEW  publication  we  assumed  the  responsibility  of  developing  a  state-of- 
the-art  review  concerned  with  the  effects  of  tobacco  and  its  products  on 
the  immune  system  in  a  chapter  entitled  "Tobacco  -  Allergy  and  Immunity" . 

The  phase  of  data  collection  and  correction  of  Kidney  Transplant 
Histocompatibility  Study  (KTHS)  has  been  completed  and  the  first  stage  of 
evaluation  and  analysis  is  currently  being  carried  out  by  the  Naval  Medical 


2-3 


Research  Institute  for  the  IAIDP.   This  work  consists  of  characterization 
of  donor  and  recipient  populations,  of  selection,  operative  and  post- 
operative procedures,  of  outcomes,  and  of  certain  elementary  correlations 
among  them. 

The  analysis  of  the  data  will  be  extended  under  contract  with  the 
Peter  Bent  Brigham  Hospital,  Doctor  C.B.  Carpenter,  Principal  Investigator, 
through  a  series  of  in-depth  studies  of  specific  issue  in  renal  transplan- 
tation. These  studies  will  be  directed  by  groups  of  scientists  representing 
the  KTHS  contractors  and  program  staff. 

2.  Budget 

The  following  shows  the  distribution  of  support  by  award 
mechanism  for  the  activities  of  the  Program  during  FY  1979. 

Immunology,  Allergic  and  Immunologic  Diseases  Program 
FY  1979  Awards 


Award  Mechanism 


Number 


Amount 


Research  Grants  450 

Career  and  Career  Development  Awards    58 

Subtotal    508 


$  46,195,862 

1,744,640 

$  47,940,502 


Fellowship  Awards 
Training  Programs 


Subtotal 


42 

34   _ 

76   $ 


539,221 
2,311,604 
2,850,825 


Contracts 


Total 


54 
638 


2,373,061 
$  55,118,775 


Total  costs,  estimated  using  a  40%  overhead  for  indirect  costs. 

Conference  Support 

During  FY  1979,  the  following  meetings,  conferences,  and  workshops 
relevant  to  the  activities  and  functions  of  the  Immunology,  Allergic 
and  Immunologic  Diseases  Programs  were  supported. 

October  1979    Mediation  of  Effector  Function  by  Antibodies. 
Bethesda,  Maryland 

Immune  Mechanisms  in  Renal  Diseases, 
Bethesda,  Maryland 

Conference  on  B  Lymphocytes  in  Immune  Responses, 
Scottsdale,  Arizona 


2-4 


November  1978   Asthma,  Allergic  and  Immunologic  Centers  Workshop, 
Bethesda,  Maryland 

January  1979    The  Role  of  Non-HLA  Tissue  Antigens  in  Human 
Transplantation , 
Bethesda,  Maryland 

February  1979   Review  of  Kidney  Transplant  Histocompatibility  Study, 
Bethesda,  Maryland 

1979   Conference  on  Biological  Recognition  and  Function, 
Keystone,  Colorado 

Conference  on  Cell  Surface  and  Malignancy, 
Keystone,  Colorado 

The  Regulatory  Role  of  Macrophage  Products  in  Immunity, 
Brook  Lodge,  Michigan 


March 


May 


1979   Second  International  Lymphokine  Workshop, 
Ermatingen,  Switzerland 


September  1979  Third  International  Workshop  on  Nude  Mice, 
Bozeman,  Montana 


Workshop  on  Immune  Mechanisms  in  Cutaneous  Disorders, 
Brook  Lodge ,  Michigan 

B.   Scientific  Summary 

Central  to  the  activities  and  interests  of  the  Immunology  Allergic, 
and  Immunologic  Diseases  Program  (IAIDP)  is  the  immune  system,  its  func- 
tions in  the  maintenance  of  health,  its  involvement  in  immunologic  and 
allergic  diseases  and  its  genetic  relationships  in  transplantation 
phenomena  and  organ  tranplantation  surgery.   Within  the  programmatic 
activities  of  allergy,  diseases  of  the  immune  system  and  tissue  transplan- 
tation is  original  work  focusing  on  the  structure  and  function  and  the 
basic  biology  of  the  immune  system  and  its  application  to  pathophysiologic 
changes.   Responsibility  for  involvement  in  these  biomedical  areas  is 
accomplished  through  investigator  initiated  research  projects,  collabora- 
tive research  and  development  contracts,  clinical  centers  concerned  with 
asthma  and  allergic  disease  (AADC)  and  Interdisciplinary  Research  on 
Immunologic  Diseases  (CIRID),  Lymphocytes  Biology  Program  Projects 
("Lymphocytes  Biology  Centers"),  procurement  and  distribution  of  research 
resource  and  standard  research  reagent  materials,  and  the  maintenance  of  a 
serum  bank  for  histocompatibility  testing. 

Designed  to  accomplish  these  objectives,  the  IAIDP  is  organized  into 
three  biomedical  and  scientific  Branches:  Immunobiology  and  Immuno- 
chemistry,  Allergy  and  Clinical  Immunology  and  Genetics  and  Transplantation 


2-5 


Biology.   It  is  from  the  work  of  these  three  programmatic  functional 
entities  that  the  IAIDP  has  gained  an  insight  into  important  developments 
and  aspects  of  the  state-of-the-art  of  both  basic  and  clinical  endeavors  in 
immunology  and  allergy.   A  review  of  studies  in  immunobiology ,  immuno- 
chemistry  and  immunogenetics  reveals  not  only  progress  in  the  generation  of 
fundamental  knowledge  but  also  important  potential  applications  of  basic 
research  data  to  problems  concerned  with  health  and  disease  through  the 
function  of  the  immune  system.   Investigations  in  asthma,  allergic 
mechanisms,  immunologic  diseases,  biology  and  inflammation,  immunopathology 
and  transplantation  biology  present  promising  leads  for  the  prompt  appli- 
cation of  technologic  advances  to  developing  improved  methods  of  diagnosis, 
treatment  and  prevention  of  relevant  disorders. 

An  understanding  of  immunocompetency  will  depend  upon  the  ultimate 
definition  of  the  origin,  differentiation  pathways  and  functional  roles  of 
the  various  subsets  of  lymphocyte  cell  populations.   It  is  therefore 
important  to  focus  studies  upon  both  the  biology  of  progenitor  cells  and 
the  identification  of  mechanisms  of  functional  maturation.   The  most 
promising  leads  in  this  area  are  emerging  from  cell  surface  molecular 
markers  on  lymphocytes  of  both  bone  marrow  and  thymus  derivation.  Critical 
to  the  functional  adequacy  of  differentiation  and  function  of  immunocompe- 
tent cells  may  be  their  specific  enzyme  contents.  Keeping  alert  to 
expressions  of  defects  in  critical  points  of  both  T  and  B  lymphocyte 
development  may  offer  leads  to  deal  with  immunodeficiency  diseases  by 
appropriate  reconstitution.   The  study  and  identification  of  cell  surface 
antigenic  markers,  especially  in  embryos  undergoing  differentiation,  can 
offer  clues  to  immunologic  association  and  involvement  in  the  regression  of 
cell  maturation  processes  leading  to  neoplasia.   Accordingly,  it  will  be 
important  to  direct  such  studies  to  the  investigation  of  both  cell  surface 
markers  and  cytochemical  processes. 

One  of  the  most  important  benefits  of  the  ascendency  of  research  in 
cellular  immunology  is  the  increasing  insight  gained  on  cell  to  cell  inter- 
actions.  This  is  especially  important  in  the  potential  application  of 
incoming  knowledge  on  both  suppressor  and  helper  influences  of  T  lymphocyte 
cell  subsets  on  antibody  producing  B  lymphocytes  in  immune  defense  to 
microbial  agents,  hypersensitivity  phenomena  and  transplantation  reactions. 
Of  especial  pertinence  is  the  regulatory  role  of  products  of  the  major 
histocompatibility  complex  on  the  functional  role  of  other  antigens 
expressed  on  the  cell  surfaces  in  the  immune  response.   In  searching  for 
key  factors  for  reconstitution  in  immune  aberrations,  a  pertinent  research 
approach  which  must  be  extended  concerns  new  knowledge  pointing  to  the 
necessity  to  match  for  functional  interactions  based  on  cells  having  same 
or  self  markers . 

It  becomes  increasingly  clear  that  immunocompetence  depends  upon  a 
programed  series  of  events  among  cells  of  different  types  to  complete  the 
process  of  recognition,  uptake  and  processing  of  antigen  and  regulation  of 
the  critical  signal  to  those  cells  involved  in  either  humoral  immunity 
through  specific  antibody  production  or  cell  mediated  immune  processes. 
However,  it  is  not  sufficient  to  recognize  only  physical  contact  between 


2-6 


macrophages  and  lymphocytes,  and  lymphocyte  to  lymphocyte  of  different 
subsets.  It  will  be  important  to  search  out  and  identify  cell-derived 
soluble  factors  that  can  be  utilized  in  manipulation  of  the  immune  system. 
Central  to  this  is  the  need  to  understand  regulatory  mechanisms  whether 
through  feedback  or  suppressor  dysfunction  in  hopeful  anticipation  of  the 
ability  to  design  mechanisms  and  specific  approaches  to  the  control  of  both 
allergic  and  autoimmune  diseases.   Thus,  such  technologic  developments  that 
can  lead  to  the  separation  and  isolation  of  reactive  cells  with  their 
subsequent  cloning  in  culture  are  encouraging  developments. 

One  of  the  early  developments  in  the  study  of  cell-mediated  immunity 
was  the  identification  of  soluble  products  of  lymphocytes  termed  lympho- 
kines.   Demonstrated  only  by  their  biologic  activity  in  in  vitro  systems, 
this  finding  continues  to  give  an  implication  of  T  cell  reactivity  apart 
from  systems  giving  evidence  of  immunoregulatory  functions.  Though  lympho- 
kines  have  been  purported  to  be  responsible  in  a  variety  of  immunologic 
reactions  including  inflammation,  homograft  rejection,  delayed  hypersen- 
sitivity, tumor  and  other  cell  killing,  progress  in  this  research  area  will 
require  chemical  identification.  Another  critical  point  is  that  lymphokine 
activity  has  thus  far  only  been  appreciated  in  in  vitro  systems.  We  will 
therefore  be  carefully  looking  at  work  that  suggests  the  possibility  of 
suppression  of  in  vivo  manifestations  of  cell-mediated  immunity. 

Though  the  direction  of  research  endeavor  in  recent  years  has  progres- 
sively moved  to  studies  at  the  cellular  level,  important  work  continues  on 
immunochemistry  attempting  to  define  the  molecular  components  of  the  immune 
system  utilizing  chemical  approaches.   Supplementing  earlier  studies  on  the 
chemical  composition  and  structure  of  humoral  and  secretory  immunoglobulins, 
structure  of  antigens  and  the  kinetics  of  immunocomplex  formation  are 
sophisticated  investigations  at  the  molecular  level  of  cell  surface 
components.   Additionally,  we  see  the  emergence  of  a  newly  developing 
field,  immunopharmacology,  centering  on  the  identification,  characteriza- 
tion, isolation  and  attempted  synthesis  of  chemical  regulators  of  immune 
function.  Elucidation  of  these  factors  can  contribute  to  our  understanding 
of  immune  dysfunction  and  measures  to  correct  altered  mechanisms  in  immuno- 
logic and  allergic  diseases.   To  understand  the  role  of  antibody  synthesis 
in  physiologic  and  pathologic  situations,  it  will  be  important  to  have  a 
clear  definition  of  basis  of  antibody  diversity.   Accordingly,  immuno- 
chemical studies  offer  the  possibility  for  ascertaining  whether  antibody 
diversification  depends  upon  a  germ-line  or  a  somatic  process.   An 
important  indication  is  the  likelihood  that  antibody  diversity  can  arise  by 
somatic  cell  mutation. 

The  past  year  has  seen  an  escalation  in  technologic  advances  in  an 
area  bridging  immunobiology  and  immunochemistry,  especially  concerned  with 
the  development  of  hybridomas.   Cell  fusion  techniques  and  selection  are 
being  employed  for  the  insertion  of  antibody  producing  characteristics  into 
cultured  malignant  plasma  cells  so  that  by  in  vitro  methods  large 
quantities  of  pure  immunoglobulins  may  be  recovered.   Another  intriguing 
approach  has  been  the  development  of  methods  for  inserting  functional 
messenger  RNA  into  cultured  cells.   By  these  techniques,  investigators  now 


2-7 


may  be  able  to  obtain  an  unlimited  source  of  homogenous  antibodies  and 
other  immunologically- important  molecules  for  study.  The  application  of 
recombinant  DNA  technology  to  the  problem  of  suppling  large  amounts  of 
immunologically  active  reagents  also  is  feasible  and  is  being  watched  with 
considerable  interest. 

Another  technologic  advance  has  been  the  development  of  a  fluores- 
cence-activated cell  sorter  which  can  be  utilized  to  prepare  pure 
populations  of  human  mononuclear  cells  from  peripheral  blood  for  functional 
studies.   In  addition  to  the  contribution  that  this  methodology  has  made  to 
immunobiologic  studies  it  may  be  utilized  in  the  detection  of  neoplastic 
lymphocytes  types  and,  in  turn,  be  utilized  to  monitor  therapy  in  patients 
with  lymphomas . 

The  major  objective  of  research  in  immunogenetics  is  the  acguisition 
of  sufficient  understanding  of  the  immune  processes  that  lead  to  acceptance 
or  rejection  of  a  graft  to  render  human  organ  transplantation  a  successful 
therapeutic  modality.  Indeed  much  of  the  knowledge  of  immunogenetics  has 
resulted  from  attempts  to  define  the  genetic  and  antigenic  relations  that 
determine  histocompatibility,  i.e.  from  "tissue  typing." 

In  all  the  mammalian  species  studied,  histocompatibility  is  governed 
principally  by  an  array  of  genes  in  a  well-delimited  region  of  a  single 
chromosome,  the  so-called  major  histocompatibility  complex  (MHC).   It  is 
designated  H-2  in  mouse,  the  most  exhaustively  studied  animal,  and  HLA  in 
man.  The  genes  of  the  MHC  code  for  a  series  of  cell  surface  antigens,  most 
of  which  have  been  identified  by  serologic  techniques  as  a  result  of  their 
ability  to  stimulate  antibody  production  and  some  by  their  ability  to 
induce  the  proliferation  of  nonsyngeneic  lymphocytes. 

The  genes  of  the  MHC  are  very  highly  polymorphic.   In  man,  the  two 
most  extensively  studied  loci  are  designated  A.  and  B.  An  ever  growing  list 
of  serological  specificities,  presumably  reflecting  antibodies  against 
specific  antigens,  currently  about  70,  have  been  identified.   Until 
recently,  matching  to  attempt  to  obtain  histocompatibility  in  organ  trans- 
plantation was  restricted  to  these  loci.   Such  matching  has  very 
substantial  predictive  power  value  for  renal  graft  acceptance  when  donor 
and  recipient  are  related.   Whether  matching  at  the  A  and  B  loci  improves 
graft  survival  when  donor  and  recipient  of  the  kidney  are  not  related  is 
not  at  all  clear,  especially  in  the  highly  heterogeneous  U.S.  population. 
In  the  case  of  transplantation  between  related  persons,  matching  at  the  A 
and  B  loci  implies  very  strongly  matching  over  the  entire  MHC.   In  man,  at 
least  three  other  loci  have  been  identified.  The  C  locus  codes  for  sero- 
logically determined  antigens,  but  has  relatively  little  effect  on  graft 
survival.   The  D  locus  codes  for  antigens  determined  by  cellular  methods: 
lymphocytes  disparate  at  the  D  locus  stimulate  each  other  to  proliferate. 
Matching  at  the  D  locus  is  particularly  critical  in  bone  marrow  transplan- 
tation. Indeed,  the  mixed  lymphocyte  reaction  involving  cells  of  donor  and 
recipient  may  be  characterized  as  a  transplant  in  the  test  tube.   It  is 
usable  in  renal  allografting  from  a  living  donor,  but,  because  of  the  time 
it  currently  requires,  it  can  not  yet  be  used  when  the  donor  is  a  cadaver. 


2-8 


Under  intense  investigation  at  the  present  time  through  an  International 
Workshop  sponsored  by  NIAID  (IAIDP)  is  the  role  of  the  DR  locus  which  is 
very  closely  linked  to  the  D  locus  in  renal  transplantation.  The  initial 
reports  from  Europe  are  very  encouraging,  but  the  data  beginning  to  become 
available  on  the  experience  in  the  U.S.  are  much  more  ambiguous. 

Overall,  it  is  clear  from  work  in  human  organ  transplantation  as  well 
as  in  animal  models  that  matching  at  the  MHC  does  not  guarantee  graft 
survival  even  when  donor  and  recipient  are  related,  unless,  of  course,  they 
are  identical  twins.   There  are  many  other  loci  on  various  chromosomes 
which  code  for  cell-surface  antigens  which,  should  they  be  polymorphic, 
could  function  as  histocompatibility  antigens.   In  addition,  evidence  for 
tissue-specific  alloantigens  is  accumulating.   Current  histocompatibility 
testing  focuses  on  lymphocytes  as  the  antigen-bearing  cells.   The  expres- 
sion of  antigens  on  various  tissues  is  not  uniform.   The  existence, 
therefore,  of  tissue-specific  histocompatibility  antigens  is  to  be  expected. 
It  does,  however,  increase  the  difficulties  of  matching  donors  and 
recipient  very  substantially. 

Alternative  maneuvers  are  being  attempted  to  improve  graft  survival. 
Among  selection  procedures  there  are  tests  of  general  reactivity  of  the 
recipient  to  antigenic  stimulation.   For  example,  the  vigor  of  the  response 
to  challenge  with  dinitrochlorbenzene  prior  to  transplantation  correlates 
significantly  with  the  vigor  of  graft  rejection.   Pretreatment  of  the 
cadaveric  kidney  to  reduce  its  immunogenic ity  is  under  active  investigation. 
Preculture  of  thymic  cells  and  of  pancreatic  islet  cells  has  been  found  to 
sufficiently  enhance  their  survival  after  implatation.   There  are  now 
numerous  reports  of  the  beneficial  effects  of  blood  transfusions  on  the 
survival  of  renal  allografts,  whether  from  cadaveric  or  living  related 
donors.   Our  own  Kidney  Transplant  Histocompatibility  Study  has  shown  that 
the  much- feared  consequence  of  blood  transfusions,  the  sensitization  of  the 
recipient  to  a  variety  of  donors,  with  the  consequent  increased  difficulty 
of  finding  an  appropriate  donor,  does  not  in  fact  occur  with  significant 
frequency. 

Given  the  practical  near  impossibility  of  obtaining  true  histocom- 
patibility, manipulation  of  the  immune  system  of  the  recipient,  and  of  the 
engrafted  donor  cells  in  bone  marrow  transplantation,  is  the  sole  remaining 
approach  to  maintenance  of  the  graft  survival.   The  mainstays  of  immuno- 
suppressive therapy  are  adrenal  cortical  steroids  and  cytotoxic  agents, 
principally  azathioprine.   Because  of  their  toxicity,  the  transplanter 
treads  a  narrow  path  between  graft  rejection  and  patient  death.  A  clear 
goal  is  the  minimization  of  immunosuppression.   There  is  thus  a  constant 
search  for  alternative  strategies  and  more  specific  suppressants.   One 
approach  under  investigation,  with  a  clinical  trial  just  now  begun, 
involves  massive  irradiation  of  the  recipient's  lymphoid  tissue  with  either 
sparing  of  the  bone  marrow  or  followed  by  transfusion  with  previously 
collected  autologous  marrow.   The  post-irradiated  state  in  which  the 
lymphoid  organs  are  reseeded  with  lymphoid  precursors  is  apparently  one  of 
great  susceptibility  to  induction  of  tolerance,  for  example  of  a  graft.  A 
more  selective  immunosuppressant,  under  study  for  a  number  of  years,  is 


2-9 


anti- lymphocyte  or  anti- thymocyte  globulin.   The  weight  of  the  evidence  is 
for  its  efficacy.   Yet  its  effect  is  to  improve  graft  survival  only  some- 
what, and  it  is  not  certain  that,  like  steroids  and  cytotoxic  agents,  it  is 
not  so  generally  immunosuppressive  as  to  endanger  the  life  of  the  trans- 
plant recipient  without  adequately  enhancing  the  probability  of  graft 
survival.   Thus,  in  one  study,  its  abandonment  led  to  improved  patient 
survival  without  appreciable  increases  in  graft  losses. 

A  still  more  specific  immunosuppressant,  the  cyclosporin  family  of 
drugs,  whose  target  is  a  lymphocyte  subpopulation,  is  now  coming  under 
careful  study.   There  is  very  clearly  great  need  for  such  highly  specific 
agents ,  given  the  great  complexity  and  delicate  balance  in  the  regulatory 
mechanisms  that  govern  the  immune  response.  It  is  of  no  use  to  so  disturb 
these  mechanisms  with  immunosuppressants  as  to  produce  an  uncontrolled 
active  response.  Yet  this  does  occur  on  occasion.   It  is,  further,  clearly 
necessary  when  intervening  massively  in  the  function  of  the  immune  system 
to  carefully  monitor  the  activity  of  its  various  regulatory  components. 
The  availability  of  reagents  which  identify  various  lymphocyte  subpopu- 
lations  that  function  as  helpers,  suppressors  and  effectors  made  this 
possible  in  the  mouse  recently.   Similar  reagents  are  now  becoming 
available  for  man.   Before  long,  the  careful  tailoring  of  immunosuppressive 
treatment  of  the  transplant  recipient  should  become  possible.   Together 
with  careful  selection  to  avoid,  for  example  transmission  of  potentially 
lethal  viral  infections  as  in  the  case  of  cytomegalovirus  with  the 
transplanted  organ  and  pretreatment  of  the  recipient,  solid  organ  trans- 
plantation should  significantly  improve  its  status  as  an  effective  form  of 
therapy.   Bone-marrow  transplantation  is  considerably  more  difficult 
because,  in  addition  to  the  possible  rejection  of  the  marrow  by  the 
recipient,  there  frequently  occurs  an  immune  reaction  of  the  marrow  against 
the  recipient.   Here  also  success  is  predicated  on  careful  and  selective 
immunosuppression.   Significant  advances  are  being  made. 

In  recent  years,  numerous  statistical  associations  between  disease 
syndromes  and  HLA  types  have  been  established.   These  associations  have 
practical  diagnostic  utility.   However,  much  more  interesting  are  the 
theoretical  implications.   In  the  mouse,  genes  that  control  response  to 
various  defined  antigens  have  been  mapped  to  the  MHC.   In  addition,  the 
serologically  identified  antigens  have,  in  certain  cases,  been  involved  in 
the  presentation  of  foreign  antigen  in  the  recognition  phase  of  the  immune 
response.   Thus,  there  is  promise  that  the  association  of  the  disease  with 
a  cell  surface  structure  will  lead  to  an  understanding  of  factors  that 
determine  susceptibility  to  the  disease. 

While  studies  in  basic  immunology  and  immunogenetics  have  been  respon- 
sible for  rapidly  expanding  knowledge  of  fundamental  biologic  processes,  at 
the  same  time  they  have  been  increasingly  generating  data  to  provide 
insights  into  mechanisms  of  allergic  and  immunologic  diseases.   During 
recent  years  the  escalation  of  rewarding  productive  investigation  into 
allergy,  clinical  immunology  and  immunopathology  has  been  a  direct  product 
of  a  maintained  awareness  of  the  potential  of  basic  experimental  leads  and 
our  ability  to  seek  their  application  to  important  clinical  problems.   An 


2-10 


understanding  of  etiologic  and  pathophysiologic  factors  of  disorders  of  the 
immune  system  in  turn  has  changed  the  direction  of  our  approaches  to  work 
directed  to  the  development  of  improved  methods  of  diagnosis,  prevention 
and  treatment  of  allergic  and  immunologic  diseases.  Research  focused  on 
pharmacologic,  biochemical,  and  molecular  aspects  of  immunology  by  eluci- 
dating of  functions  and  abberations  of  the  immune  system  is  providing  the 
necessary  base  of  information  for  promising  approaches  to  deal  with  hyper- 
sensitivity, inflammatory  and  immune  deficiency  disorders  by  immune 
manipulation  and  immunopharmacologic  techniques . 

A  number  of  the  multifactorial  influences  contributing  to  the 
expression  of  asthma  as  a  clinical  disorder  are  being  more  clearly  defined. 
Along  with  the  established  role  of  inhalant  and  food  allergens  there  is 
increasing  evidence  of  the  sensitizing  role  being  played  by  chemical  and 
viral  agents.   The  multiplicity  of  allergenic,  inflammatory  and  infectious 
etiologic  entities  appear  to  have  a  common  denominator  of  pathophysiologic 
action  effected  through  the  release  of  chemical  mediators  such  as 
histamine,  slow  reactive  substance  of  anaphylaxis,  eosinophil  chemotactic 
factor  and  platelet  activating  factor.   It  is  expected  that  ongoing  immuno- 
genetic  studies  will  help  to  explain  the  mechanism  of  genetic  predisposi- 
tion to  allergic  involvement  of  the  respiratory  tract  and  other  target 
organs  of  allergic  processes. 

Because  asthma  continues  to  be  an  incompletely  understood  disease  com- 
plex, it  is  necessary  to  direct  attention  to  investigative  approaches  from 
several  different  standpoints.   Among  pathologic  factors  to  be  delineated 
are  the  physiochemical  character  and  control  of  mucus  secretion,  the  pro- 
tective role  and  possible  alterations  in  bronchial  mucosal  barriers  and 
bronchial  epithelial  permeability,  and  structure  and  function  studies 
involving  mast  cell  distribution  and  smooth  muscle  of  the  bronchi.  Since 
neurogenic  influences  play  an  important  role  in  the  control  of  bronchial 
smooth  muscle  tone,  it  will  be  important  to  more  clearly  establish  contri- 
butions by  improperly  functioning  autonomic  nervous  control  and  excessive 
cholinergic  activity,  nonadrenergic  inhibitory  nervous  system  influences  in 
the  tracheobronchial  tree  and  whatever  specific  afferent  fiber  receptors 
may  be  involved  in  the  physiology  of  reflex  mechanisms.   Though  smooth 
muscle  spasm  has  been  indicated  as  an  important  cause  of  airway  obstruction 
in  asthma,  the  relative  roles  of  pathophysiologic  involvement  within  the 
larger  central  airways  or  in  the  smaller  periphreal  airways  are  still  to  be 
determined.  Developing  improved  methods  for  the  treatment  of  asthma  will 
depend  upon  information  that  can  be  obtained  from  extended  approaches  on 
the  study  of  the  pathophysiology  of  airway  obstruction,  the  neurogenic 
control  of  bronchial  smooth  muscle  and  the  pharmacodynamics  of  asthma.   In 
the  area  of  diagnosis  we  are  looking  to  the  answers  that  will  be  provided 
by  standardization  of  provocation  testing  based  upon  the  challenges  of 
exercise,  tartrazine  and  aspirin  inhalation,  occupational  exposures  and 
pharmacologic  agents.  Studies  under  way  which  hopefully  may  contribute  to 
preventive  aspects  include  pathogenic  factors  of  bronchial  hyper-reactivity, 
food  and  environmental  restrictions  in  infancy,  the  natural  history  of 
asthma,  and  the  effect  of  immunotherapy  on  established  asthmatic  patterns. 


2-11 


Along  with  asthma  there  is  evidence  for  immune  mechanisms  in  a  variety 
of  other  bronchopulmonary  conditions  associated  with  occupational  and 
environmental  factors,  e.g.,  chronic  bronchitis,  fibrosis,  granulomatous 
disease  and  hypersensitivity  pneumonitis.  Allergic  reactions  appear  to  be 
central  to  the  pathogenesis  of  many  types  of  occupational  asthma  and  hyper- 
sensitivity pneumonitides.  Among  the  isocyanates,  (e.g.,  TDI),  cotton 
linters  and  wood  dust,  experimental  studies  are  focusing  on  a  delineation 
of  the  respective  irritant,  allergic  and  pharmacologic  pathogenic  proper- 
ties.  In  addition  to  occupational  and  environmental  asthma,  immune 
mechanisms  are  being  shown  to  play  important  roles  in  the  production  of 
certain  fibrotic  and  granulomatous  lung  processes  associated  with  inorganic 
dusts,  e.g.,  beryllium,  silica  and  asbestos  fibers.  Important  relevant 
investigations  include  those  concerned  with  the  study  of  basic  immune 
mechanisms,  clinical  expressions,  epidemiologic  studies  especially  in  the 
plastics,  pharmaceutical  and  wood  industries.   It  is  anticipated  that 
specially  designed  materials  and  methods  for  bronchial  provocation, 
respiratory  function  tests,  immunologic  skin  tests  and  serologic  studies 
correlated  with  environmental  monitoring  will  help  us  assess  important 
causative  factors  and  measures  for  their  control.  While  the  largest  number 
of  instances  of  hypersensitivity  pneumonitis  occur  following  occupational 
exposure,  (e.g.,  moldy  fodder  in  farmer's  lung,  moldy  sugar  in  bagassosis, 
bird  droppings  in  pigeon  breeders  disease,  mold  spores  in  maple  bark 
stripper's  disease)  there  is  increasing  evidence  that  sensitizing  organisms 
contaminating  forced  air  heating,  humidification  and  air  conditioning 
systems  produce  comparable  pulmonary  disease  risks  in  homes  and  offices. 

Important  areas  of  ongoing  research  on  hypersensitivity  pneumonitis 
require  the  isolation  of  well  defined  antigens,  controlled  inhalation 
exposure,  pathogenic  mechanisms  involving  disposition  of  antigen,  various 
parameters  of  the  immune  response,  and  immunopathologic  and  toxicity 
studies.  Pertinent  points  for  extended  investigation  will  be  focused  on  the 
immunochemical  characteristics  of  suspected  offending  organic  dusts  and  the 
definition  of  environmental  factors  involved  in  the  generation  and 
dispersion  of  such  causative  agents. 

An  understanding  of  immediate  hypersensitivity  will  require  a  precise 
definition  of  structural  molecular  characteristics  of  basophil  and  mast 
cell  receptors  for  IgE  and  the  regulation  of  IgE  synthesis  and  secretion. 
In  turn,  an  important  goal  of  research  is  the  development  of  methodology  to 
block  IgE  fixation  and  release  mechanisms.   In  this  connection  studies  of 
eosinophil  biology  and  eosinophil-mast  cell  relationships  can  be  expected 
to  provide  insights  into  the  potential  residing  within  such  cellular 
mechanisms  for  modulation  of  hypersensitivity  reactions  and/or  possible 
secondary  cytotoxic  tissue  damage.  Since  mast  cell  hyperproliferation  and 
eosinophil  migration  are  prominent  accompaniments  of  helminth  tissue 
invasion,  studies  dealing  with  the  immunologic  and  immunopathologic  aspects 
of  parasitic  infection  assume  increasing  importance. 

Important  areas  of  definition  of  basic  immediate  hypersensitivity 
mechanisms  relate  both  to  chemical  mediators  of  inflammation  and  inter- 
actions between  immunocompetent  cells.   Approaches  to  developing 


2-12 


pharmacologic  agents  capable  of  preventing  or  modulating  allergic  inflam- 
mation have  been  aided  by  emerging  information  on  the  two  types  of 
histamine  cell  receptors  (HI  and  H2)  and  their  specific  antagonists  capable 
of  blocking  this  amine 's  bronchial  vascular  effects.  Additionally,  infor- 
mation on  the  surface  characteristics  of  subclasses  of  lymphocytes  may  make 
it  possible  to  selectively  impair  the  function  of  suppressor,  helper  or 
cytotoxic  lymphocytes  and  possibly  to  approach  the  treatment  of  both 
allergic  and  immunodeficiency  disorders  by  immune  modulation  or  reconsti- 
tution. 

In  the  area  of  drug  reactions  we  are  looking  to  the  possibility  of 
providing  tests  of  predictive  value  in  allergic  reactivity.  Additionally, 
the  morbidity  due  to  allergic  drug  reactions  could  be  considerably  reduced 
by  attention  to  such  factors  as  isolation,  purification  and  possible 
elimination  of  the  defined  antigenic  moiety.  Identification  of  the  actual 
chemical  determinants  for  haptens  responsible  for  drug  allergy  will  assume 
increasing  importance  in  our  search  for  the  development  of  specific 
diagnostic  reagents  in  drug  allergy  situations.   In  addition  to  concen- 
trating on  immunological  detection  systems  potentially  capable  of  detecting 
cellular  or  humoral  immunity  applicable  to  drug  sensitization  an  equally 
important  approach  is  being  directed  to  developing  sensitive  in  vitro 
systems.   Another  approach  which  may  eventually  be  helpful  in  reducing  the 
incidence  of  serious  drug  reactions  to  specific  drugs  awaits  ongoing 
studies  on  the  identification  of  HLA  or  other  genetic  markers  that  may  be 
associated  with  allergic  reactivity  based  on  altered  drug  metabolism, 
alterations  in  reactive  metabolite  information  or  innate  immune  responsive- 
ness. Additionally  important  in  the  area  of  drug  allergy  are  approaches  to 
understanding  the  mechanisms  of  drug  modulation  of  the  immune  response 
associated  with  exacerbation  of  autoimmune  disease  during  the  drug  therapy. 
We  also  are  looking  to  those  studies  on  immunotherapy  in  allergic  disease 
based  upon  the  induction  of  tolerance  as  models  for  the  design  of 
tolerogenic  analogues  of  therapeutic  agents  having  allergic  potential. 

The  importance  of  allergic  mechanisms  and  immune  system  function  has 
been  demonstrated  in  several  dermatologic  disorders,  especially  atopic 
dermatitis,  allergic  contact  dermatitis,  urticaria  and  angioedema,  the 
vesiculobullous  diseases  (pemphigus,  dermatitis  herpitiformis,  bullous 
pemphagoid)  necrotizing  vasculitis  and  lupus  erythematosus.   Conversely, 
the  study  of  these  skin  diseases  has  added  considerably  to  our  under- 
standing of  basic  immune  mechanisms.   An  appreciation  of  the  ready  access 
of  skin  for  study  of  disease  expressions  in  systemic  immunopathologic 
processes  merits  our  continued  attention.   We  have  been  especially 
concerned  with  studies  directed  to  identifying  disorders  and  their 
mechanisms  where  skin  may  be  primarily  involved  as  target  tissue  by  immuno- 
humoral  and/or  cellular  reactants,  or  where  cells  of  the  integument  may 
serve  as  natural  sources  of  sepcific  antigens  in  immune  processes. 
Important  pathogenic  mechanisms  have  been  shown  to  include  IgE  involvement, 
activation  of  the  complement  cascade  and  formation  and  deposition  of  immune 
complexes.   Atopic  dermatitis  once  believed  to  be  solely  an  immediate 
hypersensitivity  disorder  has  now  been  shown  to  involve  complex  mechanisms 
including  T  cell  abnormalities  and  possible  immunodeficiency  factors.  As  a 


2-13 


multifactorial  disorder,  work  on  this  disorder  is  being  concentrated  on 
genetic  predisposition  and  cyclic  AMP  cell  biologic  functions  interacting 
with  IgE  mechanisms.   In  urticaria  and  angioedema  where  the  role  of 
chemical  mediators  of  inflammation  and  vascular  permeability  can  be 
demonstrated,  the  importance  of  complement  activation  and  associated 
vasculitis  are  providing  helpful  approaches  to  the  design  of  specific 
pharmacologic  agents. 

The  importance  of  immune  mechanisms  has  been  demonstrated  in  a  variety 
of  connective  tissue,  renal,  gastrointestinal,  endocrine,  hemotologic  and 
neoplastic  conditions.   Among  those  of  established  definition  meriting 
immunopathologic  experimental  approaches  are  rheumatoid  arthritis, 
hepatitis,  inflammatory  bowel  diseases  (ulcerative  colitis  and  Crohn's 
disease),   intestinal  malabsorption  syndromes,  neurologic  disorders 
(Guillain-Barre  syndrome,  myasthenia  gravis,  multiple  sclerosis),  erythro- 
blastosis fetalis,  glomerulonephritis  and  interstitial  nephritis,  systemic 
lupus  erythematosus,  agranulocytosis,  juvenile  diabetes  and  T  and  B  cell 
lymphomas.   Over  the  past  several  years  significant  advances  in  basic 
immunology  have  exerted  a  major  impact  on  our  study  and  understanding  of 
this  broad  range  of  diseases.  Accordingly,  many  can  now  be  classified  into 
those  related  to  specific  immune  mechanisms,  e.g.,  immunodeficiency,  immune 
complex  deposition  and  autoimmunity.  Accordingly,  we  are  utilizing  highly 
sensitive  methodologies  for  the  identification  of  etiologically  important 
antigens,  antibodies  and  immune  complexes  involved  directly  or  indirectly 
in  their  suspected  pathogenesis  and  techniques  for  identifying  and 
fractionating  various  subpopulations  of  lymphocyte  cells  involved  in 
immunologic  reactivity  and  immunoregulation.   Fruitful  approaches  include 
the  identificaion  of  antigens  and  immune  complexes  in  vasculitis  related 
disorders  and  the  identification  of  antibodies  against  cell  surface 
receptors.  The  identification  and  functional  delineation  of  subpopulations 
of  lymphoid  cells  is  expected  to  allow  for  a  greater  understanding  of  the 
primary  and  secondary  immunodeficiency  disorders  and  in  turn  an  explanation 
of  some  immunoregulatory  abnormalities  in  autoimmune  diseases.  We,  there- 
fore, continue  to  direct  our  programmatic  concerns  to  technological 
advances  emerging  from  basic  research  to  provide  useful  tools  for  probing 
disease  states  and  applying  basic  immunologic  investigative  data  to 
clinically  relevant  disorders. 


2-14 


ALLERGY  AND  CLINICAL  IMMUNOLOGY  BRANCH 

A.    Scope 

This  Branch  is  concerned  with  the  etiology,  pathogenesis,  diagnosis, 
prevention  and  treatment  of  both  naturally  occurring  and  acguired  allergic 
and  immunologic  diseases . 

Relevant  studies  of  allergic  diseases  supported  by  the  Branch  include: 
(1)  factors,  both  primary  and  predisposing,  which  contribute  to  the  produc- 
tion of  asthma,  such  as  extrinsic  allergy,  infection,  abnormalities  of  the 
sympathetic  nervous  system,  cellular  and  chemical  mediators  of  inflammation, 
and  pharmacologic  agents,-  (2)  immediate  type  hypersensitivity  and  its 
disorders  (allergic  rhinitis,  atopic  dermatitis,  urticaria  and  angioedema)  ,- 
(3)  allergic  phenomena  affecting  respiratory,  gastrointestinal  and 
cutaneous  tissues;  (4)  allergic  reactions  and  disorders  caused  by  insect 
bites  and  stings,  foods,  airborne  allergens,  and  infectious  agents,-  (5) 
humoral  antibody,  particularly  IgE,  and  the  chemical  mediators  released  by 
the  interaction  of  antigen  and  antibody  on  target  cells,-  (6)  therapy  and 
prevention  of  allergic  disorders  and  hypersensitivity  reactions  by  immuno- 
therapy with  specific  antigens  or  drugs,-  (7)  mechanisms  of  delayed  hypersen- 
sitivity and  contact  dermatitis,-  (8)  mechanisms  of  drug  reactions  and 
chemical  sensitization,-  and  (9)  isolation  and  chemical  characterization  of 
the  active  fractions  of  known  allergenic  agents;  and  (10)  epidemiologic  and 
environmental  studies  designed  to  ascertain  those  agents  or  substances 
which  may  be  of  clinical  relevance  to  allergic  individuals — either  as 
causal  or  contributory  to  their  disease  process. 

In  the  area  of  immunologic  diseases,  the  Branch  activities  are  focused 
on  studies  of  the  underlying  mechanisms  of  disease  and  the  application  of 
basic  knowledge  to  the  etiology,  prevention  and  management  of  immunologic 
disorders.   Either  or  both  of  two  disciplinary  approaches.  Clinical  Immun- 
ology and  Immunopathology ,  are  involved  in  this  effort.  Studies  in  Clinical 
Immunology  are  directed  toward  acquired  and  inherited  diseases  associated 
with  dysfunctions  of  the  immune  system.   Immunopathology  studies  include 
genetics,  cytology,  biochemistry,  physiology  and  pharmacology  of  the  immune 
system.   Relevant  areas  supported  by  the  Branch  include  studies  of  (1) 
immune  deficiency  diseases  arising  from  primary  defects  in  development  or 
maturation  of  the  immune  response  or  secondarily  resulting  from  disorders 
affecting  immune  responses,  (2)  clinical  manifestations  mediated  by 
products  of  lymphocytes,  (3)  diseases  associated  with  immune  complexes  and 
autoimmune  phenomena,   (4)  immunodermatology ,  i.e.,  immune  disorders 
involving  the  cutaneous  system,  and  (5)  immunotherapy  of  disease  processes. 

The  ultimate  goal  of  the  Allergy  and  Clinical  Immunology  Program  is  to 
promote  the  acquisition,  translation,  and  application  of  research  findings 
to  the  diagnosis,  prevention,  and  treatment  of  allergic  and  immunologic 
diseases.   To  achieve  this  goal,  the  Branch  is  responsible  for  and  manages 
Programs  of  Institute  Emphasis  (PIE)  in  Asthma  and  Allergic  Diseases, 
Immunologic  Diseases,  and  Immunology  Centers  and  Program  Projects.  Colla- 
borative clinical  as  well  as  basic  research  studies  are  supported  among 
Centers  and  Program  Projects  in  an  effort  to  take  advantage  of  the 

3-1 


nationwide  character  of  these  programs  and  to  facilitate  achieving  our 
goals.  To  support  these  efforts,  the  Branch  funds  targeted  contract  efforts 
and  also  obtains  and  distributes  selected  research  reagents  and  materials 
to  investigative  allergists  and  clinical  immunologists. 

B.   Awards  and  Support  Levels 

Allergy  and  Clinical  Immunology 

FY  '79  Awards 

Award  Mechanisms  Number  Amount 


Research  Grants 
Career  Awards  and 
Career  Development  Awards 
Subtotal 

Fellowship  Awards 
Training  Awards 

Subtotal 


Contracts 


Total 


151 

21 
172 

16 
24 
40 

10 
222 


$  15,930,164 

783,711 
$  16,713,875 

216,015 

1,611,585 

$   1,827,600 

452,156 
$  18,993,631 


Total  Costs,  estimated  using  a  40%  overhead  for  indirect  costs. 

The  distribution  of  these  awards  by  area  of  study  is  approximately  30% 
for  asthma  and  allergic  diseases  and  70%  for  immunologic  diseases.  Approxi- 
mately 34%  of  these  awards  were  for  competing  new  or  renewal  applications; 
the  remainder  represents  commitments  to  support  awards  made  in  prior  years. 

Support  for  the  PIE  activities  is  included  in  the  research  grant 
category.  During  FY  '79,  the  Branch  supported  these  activities  with  awards 
for  15  Asthma  and  Allergic  Disease  Centers  at  a  total  cost  of  $1,787,467,  6 
Clinical  Immunology  and  Immunopathology  Program  Projects  at  a  total  cost  of 
$3,093,499,  and  4  Centers  for  Interdisciplinary  Research  in  Immunologic 
Diseases  at  a  total  cost  of  $1,184,446. 

C.   Program  Areas  and  Highlights 

1.   Asthma  and  Allergic  Disease  Centers  Program  (AADC) 

A  network  of  15  Asthma  and  Allergic  Disease  Centers,  located  through- 
out the  United  States,  is  actively  engaged  in  a  collaborative  fashion  to 
gain  new  knowledge  and  insights  into  the  field  of  allergic  disorders 
including  those  manifestated  in  the  upper  and  lower  respiratory  tract, 
skin,  and  other  organs  such  as  kidney,  gastrointestinal  tract,  etc. 
Research,  both  fundamental  and  applied,  is  conducted;  and  through  this 
multi- faceted  approach,  methods  have  already  been  developed  to  improve  the 
diagnosis  and  treatment  of  asthma  and  other  allergic  diseases.   Although 


3-2 


the  emphasis  of  the  Centers  Program  is  directed  toward  a  better  under- 
standing of  the  basic  mechanisms  involved  in  the  various  allergic  disorders, 
they  also  have  provided  the  field  of  allergy  with  unique  academic  resources 
in  the  discipline  of  allergy. 

By  serving  as  referral  centers  for  patients  in  their  areas,  by 
engaging  in  collaborative  clinical  trials  to  assess  diagnostic  and  thera- 
peutic discoveries,  and  finally  by  acting  as  an  important  resource  for 
training  academic  allergists  for  the  future ,  they  have  had  a  profound 
impact  on  the  delivery  of  health  care  to  allergic  individuals.   Separately 
each  of  these  Centers  have  also  been  successful  in  competing  for  additional 
support  through  either  the  regular  grant  program,  training  grants,  or 
research  career  awards. 

At  the  Eighth  Annual  AADC  Workshop  in  Bethesda  on  November  30  to 
December  1,  1978,  directors  and  professional  staffs  from  the  15  Asthma  and 
Allergic  Disease  Centers  (over  125  participants)  met  at  NIH  to  review 
progress  by  grantees  in  this  Centers  Program.   While  the  presentation  of 
scientific  papers  (more  than  50)  provided  the  focal  point  of  interest,  the 
ultimate  success  of  this  workshop  can  be  measured  better  by  the  broad  and 
open  discussion  and  the  significant  opportunities  for  investigators  to 
exchange  ideas  for  future  study.   A  measure  of  the  importance  of  this 
meeting  came  from  the  overwhelming  requests  by  people  outside  the  Centers 
Program  to  participate. 

Brief  highlights  of  some  of  the  activities  conducted  by  our  Asthma  and 
Allergic  Disease  Centers  follows. 

John  Salvaggio,  AI  13401,  Tulane  University  and  his  colleagues  have  focused 
on  Environmental/Occupational  Hypersensitivity  lung  diseases.   Purification 
of  coffee  and  castor  bean  allergens  enabled  them  to  perform  studies  showing 
that  workers  in  the  coffee  industry  were  sensitized  by  exposure  to  sacks 
that  had  previously  been  used  to  transport  castor  beans.   Persons  with 
asbestosis  and  silicosis  have  been  found  to  have  a  high  frequency  of  anti- 
nuclear  antibody,  a  finding  which  may  lead  to  better  understanding  of  the 
pathogenesis  of  fibrotic  lung  disease.   Finally,  studies  of  mold  sensitive 
asthma  have  progressed  with  clarification  of  antigenic  relationship  among  3 
species  of  aspergillus — an  important  effort  in  attempting  to  clarify  and 
control  environmental  sources  of  these  allergens. 

Roy  Patterson,  AI  11403,  Northwestern  University  has  continued  efforts  to 
reduce  the  allergenicity  of  ragweed  antigen  E  while  retaining  antigenicity 
by  use  of  polymerized  fractions,-  preliminary  clinical  trials  with  this 
material  has  demonstrated  efficacy  when  compared  with  monomer  preparations 
but  with  greater  safety.   Expanded  trials  in  collaboration  with  other 
centers  are  underway. 

Richard  Hong,  AI  10404,  University  of  Wisconsin  and  colleagues  have 
continued  studies  investigating  the  role  of  viral  infections  in  predis- 
posing or  causing  asthma. 


3-3 


Oscar  L.  Frick,  AI  11010,  University  of  California  at  San  Francisco 
similarly  has  been  in  following  children  prospectively  in  order  to  clarify 
the  meaning  of  a  coincidental  association  between  certain  virusinduced 
colds  and  flu- like  symptoms  and  the  onset  of  allergy  during  the  first  year 
of  life.   They  found  in  a  small  group  that  antibodies  to  influenza- like 
viruses  and  certain  environmental  or  food  materials  (dog,  cat,  house  dust, 
grass  or  cow  and  soy  milk)  increased  at  time  of  onset  of  allergic  symptoms. 
Studies  were  conducted  to  learn  whether  certain  viruses  can  act  to  alter 
the  immune  system  so  as  to  trigger  the  onset  of  allergy. 

Kenneth  P.  Mathews,  AI  10391,  University  of  Michigan  has  continued  studies 
of  "vasomotor  rhinitis"  in  an  effort  to  define  the  varied  mechanisms  by 
which  people  may  be  affected.   Clinical  trials  are  also  being  conducted 
assessing  efficacy  of  intranasal  treatment  with  gluteraldehydeaggregated 
ragweed  extract  in  treatment  of  ragweed  hay  fever--which  if  successful 
could  greatly  reduce  morbidity  and  cost  to  patients. 

Philip  S.  Norman,  AI  10304,  Johns  Hopkins  University  and  colleagues  have 
continued  studies  of  modified  allergens  or  allergoid  (formaldehyde  modified 
whole  extract  and  allergen).   Also  they  provided  fundamental  data  which 
permitted  commercial  release  of  venom  extracts  in  prevention  of  anaphylaxis 
from  bee  stings.  A  double  blind  controlled  trial  is  in  progress  to  study 
the  effects  of  Rinkel  immunotherapy — no  difference  has  been  found  to  date 
between  this  and  standard  immunotherapy. 

Gerald  J.  Gleich,  AI  11483,  Mayo  Foundation  and  colleagues,  taking  advan- 
tage of  ongoing  studies  to  refine  allergens  and  develop  sensitize 
diagnostic  tests,  have  shown  that  efficacy  of  ragweed  pollen  immunotherapy 
is  associated  with  reduction  of  activity  of  IgE  antibodies  in  the  radio- 
allergo-sorbent  test,  thus  giving  a  means  of  precise  monitoring  of  efficacy. 

2.    Centers  for  Interdisciplinary  Research  in  Immunologic  Diseases  (CIRID) 

In  September  1978,  4  Centers  for  Interdisciplinary  Research  in  Immuno- 
logic Diseases  were  funded  by  NIAID.   This  program  is  designed  to  foster 
integration  and  coordination  of  research  projects  in  clinical  immunology 
being  pursued  in  other  clinical  specialties  (e.g.  dermatology,  pulmonary 
medicine,  hematology,  nephrology,  rheumatology,  infectious  diseases  and 
otorhinolaryngology)  with  those  in  basic  research  (e.g.,  immunochemistry, 
microbiology,  virology  genetics,  biochemistry,  pharmacology,  general  physi- 
ology, and  pathology).  An  important  additional  component  of  these  Centers 
is  the  funding  of  specific  projects'  in  demonstration/outreach  activities, 
which  are  designed  to  impact  on  the  health  care  of  the  community  in  a 
tangible  and  immediate  way  by  bringing  research  advances  directly  to  the 
patient.  While  it  is  obvious  that  during  the  start  up  year  efforts  are  of 
necessity  preliminary  in  nature,  the  following  is  intended  to  provide  an 
idea  of  how  progress  is  anticipated  in  this  area. 

At  UCLA  (John  Fahey,  AI  15332)  an  evaluation  of  a  supplementary  program  of 
nonmedical  intervention  on  the  course  of  childhood  asthma  is  being  con- 
ducted by  (E.  Lewis,  M.A.  Lewis  &  G.  Rachelfsky) .  A  curriculum  has  been 
designed  to  teach  children  and  their  parents  what  asthma  is,  to  recognize 

3-4 


initial  symptoms,  and  then  to  utilize  self -management  techniques  such  as 
relaxation  and  breathing  exercise  to  terminate  or  minimize  each  episode;  to 
recognize  indications  for  use  of  certain  pharmacologic  agents;  and  finally 
to  conduct  field  trials.  Early  evidence  from  an  initial  group  of  10 
children  and  their  families  suggest  that  the  desired  level  of  information 
can  be  acquired  and  more  importantly  that  families  are  enthusiastic  in 
their  participation. 

John  Leddy,  AI  15312,  University  of  Rochester  has  designed  and  implemented 
a  12session  concentrated  course  in  Clinical  Immunology  for  physicians  and 
other  medical  personnel  (e.g.,  nurse  practitioners)  affiliated  with  out- 
lying community  hospitals  in  the  large  semi- rural  region  served  by  the 
University  of  Rochester.  It  is  hoped  that  this  will  lead  to  better  compre- 
hension and  therefore  diagnosis  and  treatment  of  immunologic  diseases  in 
rural  upstate  New  York.   To  prove  this  point  a  means  to  assess  the  benefit 
to  the  patient  will  be  undertaken  not  only  by  sequentially  testing  partici- 
pants, but  also  by  tracking  referrals  and  as  well  open  lines  of 
communication  fully  between  the  CIRID  and  the  regional  community. 

Joseph  Bellanti,  AI  15321,  Georgetown  University  and  his  collaborators  have 
sponsored  a  symposium  on  public  concerns  of  immunization  (October  1978)  as 
part  of  a  broader  effort  to  facilitate  the  transfer  of  basic  science  know- 
ledge to  the  general  public.  They  have  also  initiated  an  Asthma  Education 
Intervention  Project  emphasizing  self -management  techniques  for  pediatric 
asthmatics  and  their  parents.  They  have  further  initiated  study  of 
prevalence  of  asthma  and  other  allergic/ immunologic  disorder  and  cost  and 
utilization  of  health  services  in  the  metropolitan  Washington,  D.  C.  area. 

Charles  W.  Parker,  AI  15322,  Washington  University   St.  Louis   has 
initiated  a  program  whose  intent  is  similar  to  that  of  the  Rochester  CIRID, 
in  that  it  is  designed  to  enhance  knowledge  of  immunologic  disorders  in  the 
urban  community  served  by  the  CIRID. 

It  should  be  mentioned  in  closing  that  while  this  report  has  emphasized  the 
outreach/demonstration  activities  of  the  4  CIRIDs ,  the  largest  proportion — 
more  than  90  percent--of  their  efforts  continue  to  relate  to  basic  and 
clinical  research. 

3.   Program  Projects  in  Mechanisms  of  Immunologic  Diseases 

Program  Projects  were  established  to  encourage  the  development  of 
collaborative  basic  science  and  clinical  investigation  in  immunologic 
diseases.   As  such  this  program  is  designed  to  further  investigate  under- 
lying mechanisms  of  disease  and  to  enhance  basic  knowledge  relevant  to 
eiology,  prevention  and  management  of  immunologic  disorders.   Studies  are 
effected  from  either  one  of  two  disciplinary  approaches:   clinical  immun- 
ology or  immunopathology.   Clinical  immunology  studies  are  directed  toward 
acquired  and  inherited  diseases  associated  with  dysfunctions  of  the  immune 
system.   Immunopathology  studies  include  specific  areas  of  genetics,  cyt- 
ology, biochemistry,  physiology,  and  pharmacology  of  the  immune  system  and 
its  disorders . 


3-5 


In  FY  1979  there  were  7  active  grants  in  this  program.  The  following 
will  be  an  attempt  to  present  sufficient  detail  from  each  to  show  the  depth 
and  breadth  of  this  program. 

The  project  headed  by  Frank  J.  Dixon  (AI  07007  -  Scripps  Clinic  and 
Research  Foundation)  is  designed  to  gain  a  better  understanding  of  the 
molecular  basis  of  diseases  caused  by  aberrant  immune  responses.   Its  focus 
is  upon  1)  normal  controls  of  immune  responses,  2)  the  means  by  which 
unusual  presentation  of  an  antigen,  particularly  associated  with  chronic 
viral  infections,  may  initiate  pathologic  responses,  3)  the  chemistry  and 
function  of  the  several  mediator  systems  which  are  activated  by  immunologic 
reactions  and  cause  injury,  and  4)  the  multiple  complex  immunopatho logic 
events  found  in  spontaneous  or  experimental  immunologic  disease.   The 
diseases  most  immediately  related  to  this  research  include  glommerlar 
nephritis,  systemic  Lupus  erythematosus,  rheumatoid  arthritis,  other  auto- 
immune diseases,  acute  and  chronic  viral  infections,  allergic  and  anaphy- 
lactic reactions,  allograft  rejection,  and  disorders  of  complement  and 
Hagemann  factor  occurring  in  mediator  systems. 

Measurements  of  immune  complexes  and  biologic  fluids  appear  to  be 
providing  useful  information  helpful  in  monitoring  the  clinical  courses  of 
patients  and  guiding  therapy.   It  is  hoped  that  isolation  of  immune  com- 
plexes may  contribute  to  the  identification  of  pathogens  and  diseases  of 
unknown  etiology,  and  to  the  development  of  practical  diagnostic  and  thera- 
peutic measures.   An  example  of  the  value  of  such  studies  relates  to  the 
belief  that  the  immune  complexinduced  degenerative  vascular  lesions  seen  in 
murine  Lupus  may  have  relevance  to  degenerative  arterial  disease  in  man; 
supported  by  the  knowledge  of  increasing  occurrence  of  coronary  artery 
disease  and  myocardial  infarction  in  humans  with  Lupus,  confirmed  by  pre- 
liminary studies  in  autopsies,  as  well  as  examination  of  the  deposition  of 
immune  complexes  in  arterial  walls.   It  is  thus  thought  that  repeated 
vascular  deposition  of  immune  complexes  associated  with  incidental  immune 
reactions  might  provide  a  significant  component  in  the  genesis  of  athero- 
sclerotic disease. 

Studies  by  Joseph  Feldman  as  part  of  this  program  project  have  deter- 
mined that  survival  times  of  skin  grafts,  repeatedly  applied  to  BN  and 
Lewis  rats,  were  unaffected  by  the  levels  of  circulating  immune  complexes, 
and  in  rats  grafted  with  allogeneic  kidneys  (vascular  graft)  and  in  dams 
repeatedly  impregnated,  no  circulating  immune  complexes  were  detected.   The 
signifance  of  these  studies  is  related  to  the  desire  to  examine  the  hypo- 
thesis of  the  aging  process  and  neoplasic  (which  occur  with  increased 
freguency  in  aged  hosts)  may  well  be  associated  with  some  membrane  changes 
(microviscosity,  cholesterol,  phospholipid)  and  with  cytoskeletal  membrane 
interactions  that  revealed  deficiencies  in  immune  surveillance  and  response. 

In  vivo  studies  by  Charles  G.  Cochrane  have  examined  the  participation 
of  the  Hagemann  factor  system  and  complement  component  in  the  development 
of  immunologic  injury,  which  were  made  possible  by  biochemical  analyses  of 
the  individual  components  and  their  mechanisms  of  action.   With  the  avail- 
ability of  components  of  the  Hagemann  factor  system  in  both  rabbit  and 
human,  together  with  methods  for  their  detection,  definitive  experiments 

3-6 


were  made  possible  to  detect  the  active  participation  of  such  components, 
thus  indicating  that  any  deficiency  in  this  pathway  will  abrogate  humoral 
mediated  destruction  of  virus-infected  cells. 

Studies  by  Curtis  B.  Wilson  have  concentrated  on  examining  the  role  of 
monocytes  and  macrophages  in  immune-complex  induced  glommerular  nephritis 
in  rabbits.   An  extension  of  previous  studies  regarding  the  contribution  of 
monocytes  and  macrophages  have  shown  that  monocytes  and  macrophages  can  be 
recovered  in  large  numbers  by  tissue  culture  of  isolated  glommeruli  taken 
from  rabbits  with  the  most  proliferative  forms  of  experimental  serum  sick- 
ness glommerular  injury.   It  is  hoped  that  results  of  these  studies  will 
enable  characterization  of  differing  patterns  of  immune  complex  localiza- 
tion in  kidneys  and  efforts  have  been  made  to  relate  these  to  host  genetic 
factors  which  could  influence  the  susceptibility  of  individuals  to  immune 
complexinduced  glommerular  injury. 

William  0.  Weigle  has  continued  studies  examining  the  activation  of  T 
and  B  cells  and  the  interaction  of  those  cells  with  macrophages  in  the 
induction  of  immune  tolerance  and  autoimmunity.  These  studies  led  to  the 
description  of  a  model  of  immunologic  tolerance  to  human  gammaglobulin 
which  is  maintained  in  the  absence  of  demonstrable  suppressor  cells.   Other 
studies  showed  that  the  tolerance  state  induced  in  B  cells  in  adult  mice  to 
human  gammaglobulin  is  the  result  of  central  unresponsiveness  rather  than 
of  antigen  blockade.   A  thymus  replacing  factor  (TRF)  has  been  produced  by 
the  activation  of  spleen  cells  with  concanavalin  A,  the  cell  type  in  the 
spleen  responsible  for  the  generation  of  thymus  replacing  factor  is  a  T 
cell,  that  is,  LYT  1+  and  LYT  2-.  Preliminary  studies  indicate  that  TRF 
obtained  from  mice  sensitized  with  sheep  red  blood  cells  support  a  primary 
response  in  nude  mice.   Further  studies  by  Hans  Muller-Eberhard  into  the 
mechanism  and  functional  role  of  complement  have  shown  that  the  isolated 
component  mixture  containing  the  proteins  of  the  alternative  and  of  the 
membrane  attack  pathway  is  capable  of  generating  bactericidal  activity, 
thus  showing  that  the  system  is  complete  and  antibody  is  not  required  for 
alternative  pathway  initiation.   These  observations  relate  the  cytolytic 
alternative  pathway  to  natural  resistance  to  infection.   Thus,  in  vivo, 
although  bacteria  are  usually  phagocytized  and  killed  intercellular ly, 
complement  is  required  for  opsonization  (C3B)  and  to  some  extent  for  intra- 
cellular killing  (C5  through  C8) .   Individuals  with  homozygous  deficiency 
of  one  of  the  precursor  proteins  of  the  membrane  attack  complex  have ,  as 
expected,  an  increased  susceptibility  to  infections,  particularly  of  the 
Neisseria  type.   Thus,  these  reported  structural  and  functional  analyses  of 
the  membrane  attack  complex  have  provided  a  basic  understanding  of  the 
manner  in  which  complement  lysis  envelope  the  viruses  and  kills  bacteria  in 
animal  cells. 

Hugh  0.  McDevitt,  AI  11313,  Stanford  University  has  been  engaged  in  an 
overall  effort  to  examine  Humoral,  Cellular  and  Genetic  Mechanisms  in  Auto- 
immunity.  Studies  concerned  with  the  relationship  between  the  major  histo- 
compatibility complex  (MHC)  have  demonstrated  the  presence  of  immune 
complexes  in  patients  with  two  related  forms  of  arthritis,  ankylosing 
spondylitis  and  Reiter's  syndrome.   The  suggestion  is  made  that  these 
patients  may  have  an  abnormal  immune  response  to  certain  bacterias,  which 

3-7 


leads  to  formation  of  immune  complexes  which  may  be  the  cause  of  the 
arthritis.   Other  diseases  being  studied  for  possible  genetic  suscepti- 
bility include  rheumatoid  arthritis,  multiple  sclerosis,  diabetis  mellitus, 
and  systemic  Lupus  erythematosus.   Further  studies  aimed  at  improving  MHC 
typing  are  being  conducted  by  F.  Carl  Grumet  in  this  program. 

A.  Cacin  has  been  following  seroepidemiologically  persons  with  enteric 
infection  and  Reiter's  syndrome,  finding  that  Shigella  flexneri  2a  and 
flexneri  1  cause  Reiter's  disease. 

Samuel  Strober  has  been  conducting  studies  assessing  the  use  of  total 
lymphoid  irradiation  in  treatment  of  auto-immune  diseases  and  in  the  pre- 
vention of  organ  graft  rejection.   Four  of  6  patients  whose  rheumatoid 
arthritis  failed  to  respond  to  all  conventional  therapies  had  significant 
improvement  in  their  disease  activity  which  lasted  from  6 — 18  months. 

Roy  Patterson,  AI  11759,  Northwestern  University  and  his  colleagues 
have  concentrated  their  efforts  on  immune  aspects  of  lung  disease.   Main- 
tenance of  a  colony  of  rhesus  monkeys  giving  consistent  asthmatic  responses 
to  ascaris  as  well  as  control  animals  has  permitted  investigation  of 
response  to  prostaglandins  and  histamines  as  well  as  other  allergic 
mediator  substances.   Studies  of  airways  physiology  as  well  as  peripheral 
immune  responses  have  been  made  possible.   Dog  models  of  allergy  have  also 
been  utilized.   Studies  of  human  asthma  have  investigated  physiologic 
responses  to  inhaled  antigens  and  methacholine.  Hay  fever  patients  were 
found  to  respond  normally  in  contrast  to  asthmatic  subjects  when  challenged 
with  methacholine;  similarly  however,  they  responded  to  asthmatics  when 
challenged  with  antigen-this  response  was  not  mediated  by  cholinergic 
reflexes  (atropine  pretreatment  made  no  difference).   This  group  also  has 
shed  further  light  on  the  role  of  trimellitic  anhydride  (TMA)  as  an  occupa- 
tional hazard  which  may  produce  not  only  asthma  but  pulmonary  infiltrative 
disease.   Their  development  of  accurate  diagnostic  tests  will  permit  exten- 
sion of  these  findings  to  a  large  number  of  potentially  exposed  workers. 

Other  investigations  of  environmental  hazards  have  included  the  obser- 
vation that  di-2-ethyl-hexyl-phthalate  was  found  to  migrate  from  polyvinyl 
chloride  tubing  into  human  blood  in  dialysis  patients.  Studies  are  under- 
way to  learn  whether  this  finding  can  be  related  to  the  clinical  obser- 
vation that  such  foreign  substances  could  produce  the  accelerated  form  of 
atherosclerosis  in  dialysis  patients  or  be  responsible  for  other  vascular 
damage . 

Fred  S.  Rosen,  AI  05877,  Children's  Hospital  Medical  Center,  Boston 
and  his  coworkers  have  been  examining  the  reactions  and  metabolism  of 
immune  proteins  and  cells.   Continuing  studies  of  genetic  deficiency  of 
complement  components  led  to  the  description  of  the  first  homozygous  (C3 
deficient  individuals  and  that  C3  nephritic  factor  was  an  IgG  molecule  (an 
autoantibody  to  C3bBb) .   Further  they  documented  a  polypeptide  produced  in 
plasma  of  patients  with  hereditary  angiodema,  affects  the  postcapillary 
venule  of  man  and  is  generated  from  the  second  component  of  complement  by 
action  of  plasmin.   Patients  with  acquired  or  common  varied  agammaglobu- 
linemia were  found  to  have  no  B  cells  or  to  have  B  cells  that  were 


3-8 


unreactive  in  the  presence  of  lymphocyte  mitogenic  factor.   Restoration  of 
function  in  some  of  these  individuals  has  been  accomplished  with  use  of 
steroids.   Bone  marrow  transplants  were  found  to  restore  function  in 
Wiskott-Aldrich  Syndrome  and  adenosine  deaminase  deficiency.   Finally,  the 
dermatomyositis-like  syndrome  of  agammaglobulinemia  was  found  to  result 
from  infection  of  the  CNS  with  ECHO  virus.   It  should  be  mentioned  that  all 
of  these  studies  are  being  accomplished  in  the  setting  of  a  large  referral 
center  for  study  of  immunologic  disorders,  especially  in  children. 

David  G.  Marsh,  AI  13370,  Johns  Hopkins  University   has   continued 
studies  of  the  Genetics  of  Immune  Response  in  Man.   Studying  a  group  of  400 
allergic  patients  and  their  families  since  1971  has  resulted  in  the  obser- 
vation of  significant  associations  between  specific  HLA  types  and  specific 
IgE  mediated  allergic  responses  to  4  different  highly  purified  pollen 
allergens.   In  addition  an  IgE  regulator  gene,  not  linked  to  HLA,  has  been 
found  to  exert  an  additional  control  over  specific  IgE  responses.  Continued 
studies  are  planned  to  test  the  hypothesis  that  under  immunogenically 
limiting  dosage  exposure,  response  to  a  specific  antigen  is  regulated  by 
relatively  few  immune  responses  (IR)  and  immune  suppressor  (IS)  genes  and 
that  all  IgE  antibody  responses  may  be  controlled  by  a  gene  which  regulates 
the  overall  synthesis  of  IgE  to  any  specificity. 

4.   Mechanisms  of  Allergic  Diseases 

Studies  within  this  program  have  been  concerned  with  investigations  of 
the  etiologic  factors,  pathogenetic  mechanisms,  diagnostic  measures,  and 
therapeutic  approaches  related  to  the  effective  management  of  allergic 
disorders. 

The  release  of  inflammatory  molecules  from  tissue  mast  cells  is  a 
necessary  step  in  the  evolution  of  allergic  reaction  of  the  immediate  type. 
It  is  interesting  to  note  that  such  reactions  may  also  be  relevant  in  other 
immune  responses,  such  as  those  implicated  in  graft  rejection,  poison  ivy 
and  immune  kidney  diseases. 

Timothy  J.  Sullivan,  AI  10090,  Washington  University,  in  studies 
investigating  the  mechanism  by  which  mediators  are  released  from  mast 
cells,  has  detected  reactions  which  appear  to  control  the  release  of  his- 
tamine.  The  formation  of  1,2  diacylglycerol  (DAG)  appears  to  be  a  key 
step,  and  drugs  of  the  me thy Ixan thine  class  have  been  shown  to  inhibit  the 
formation  of  DAG  and  mediators  in  parallel.   From  these  studies  it  is  hoped 
that  drugs  can  be  developed  to  control  mast  cell  mediated  inflammation. 

Kimishige  Ishizaka,  AI  10060,  Johns  Hopkins  University,  also  looking 
at  the  mechanism  of  mast  cell  secretion,  found  that  bridging  of  receptor 
molecules  rather  than  polymerization  of  cell-bound  IgE  molecules  was 
responsible  for  mast  cell  triggering. 

Lawrence  Lichtenstein,  AI  07290,  Johns  Hopkins  University,  has  shown 
that  human  basophils  have  an  adenosine  receptor  which  is  linked  to 
adenylate  cyclase  and  increases  cyclic  AMP.   When  this  receptor  is  occupied 
by  adenosine,  histamine  release  is  stopped.   Interestingly,  this  receptor 


3-9 


is  antagonized  by  theophyllin,  a  drug  commonly  used  for  the  treatment  of 
allergic  disorders. 

Marshall  Plaut,  AI  12810,  Johns  Hopkins  University,  has  studied  the 
role  of  histamine  type  2  receptors  in  vivo  in  man  by  use  of  the  drug, 
cimetidine  (an  H2  blocker),  and  found  no  significant  changes  in  either 
immediate  type  or  delayed  type  skin  reactions.   Continued  studies  such  as 
these  should  help  clarify  the  role  of  mediators  in  acute  allergic  responses 
and  further  permit  development  of  pharmacologic  manipulation  of  these 
immune  responses. 

Stephen  Wasserman,  AI  00254,  Harvard  University,  has  shown  that 
heparin  is  present  in  human  mast  cells  and  that  in  a  rat  model  of  allergic 
disease,  mast  cell  enzymes  have  been  found  and  released  after  immunologic 
challenge.   These  enzymes,  beta  glucuronidase  and  hexosaminidase,  could 
destroy  connective  tissue  and  explain  prolongation  of  problems  in  human 
allergic  diseases.   Finally,  he  showed  that  disodium  cromoglycate,  which 
prevents  mast  cell  mediator  release,  proved  effective  in  the  treatment  of 
systemic  mastocytosis.   Andrew  Grant,  AI  12621,  University  of  Texas  Medical 
Branch,  Galveston,  examining  histamine  release  from  basophils,  has 
continued  studies  assessing  the  role  of  C5A,  a  small  fragment  from  the 
fifth  component  of  complement,  which  has  been  found  to  bind  directly  to  the 
blood  basophil,  inducing  release  of  histamine. 

The  role  of  reaginic  antibody,  also  known  as  IgE,  which  circulates  in 
the  blood,  is  well  established  in  the  allergic  response.   Many  investi- 
gators have  been  examining  ways  to  determine  how  this  immune  response  is 
regulated,  in  order  to  learn  whether  or  not  this  response  could  be 
abrogated  in  some  general  way  in  order  to  benefit  allergic  individuals. 
For  example,  Kimishige  Ishizaka,  AI  11202,  Johns  Hopkins,  showed  that  the 
IgE  antibody  response  in  the  mouse  is  dependent  upon  the  dose  and  nature  of 
antigen--that  is,  induction  of  antigen-specific  suppressor  T  cells  is 
involved  in  the  transient  IgE  antibody  response  found  in  the  genetically 
high  IgE  responder  animal. 

Andrew  Saxon,  AI  15251,  UCLA,  found  that  control  of  B  lymphycyte 
production  was  dependent  upon  the  presence  of  T  lymphocytes;  that  radiated 
T  lymphocytes  provided  helper  function  without  suppression  of  IgE  at  high 
T-to-B  cell  ratios. 

Albert  Kalisker,  AI  13592,  has  shown  among  mouse  strains  normally 
insensitive  to  ragweed  antigen,  who  have  been  treated  with  moderate  doses 
of  cyclophosphamide,  results  in  an  enhancement  of  their  ability  to  produce 
high  anti-ragweed  IgE  titers.   By  way  of  explanation,  it  is  thought  that  the 
drug  eliminated  from  those  mice  cells  which  were  involved  in  the  actual 
suppression  of  IgE  production.   Other  investigators  such  as  Rebecca  Buckley, 
AI  12026,  Duke  University,  has  also  been  examining  the  role  of  suppressor 
cells  in  the  IgE  response.   Samuel  B.  Lehrer,  AI  14891,  additionally  has 
been  assessing  and  detecting  IgE  antibody  producing  cells  in  different 
lymphoid  organs,  and  selectively  suppressing  IgE  production  in  the  mouse 
with  anti-IgE.  Alec  Sehon,  AI  14526,  has  carried  the  studies  of  IgE  to 
penicillin  allergy,  showing  that  suppression  of  IgE  antibodies  to  a 

3-10 


constituent  of  penicillin,  that  is,  the  benzyl  penicilloyl  (BPO)  group  was 
induced  by  the  administration  of  non- immunogenic  conjugates  of  BPO  with 
normal  gammaglobulins  of  the  corresponding  species .   David  Katz  at  Scripps 
Clinic  has  shown  that  IgE  levels  can  be  altered  by  a  humoral  factor  which 
may  either  suppress  or  enhance  the  igE  response  in  genetically  different 
animals.   It  is  hoped  that  this  may  lead  to  an  ability  to  alter  man's 
response  to  external  allergens  and  enable  investigators  to  modulate  the 
immune  response  in  such  a  way  as  to  abrogate  any  high  Ige  responders.   If 
this  material  can  be  deemed  safe  for  clinical  trials,  it  could  be  an  alter- 
nate and  universal  "therapy"  for  allergy. 

Other  studies  related  to  IgE  antibody  include  those  of  Frederick  J. 
Grundbacher,  AI  12360,  Peoria  School  of  Medicine,  who  has  shown  that  low 
levels  of  IgA  may  constitute  a  predisposing  factor  to  the  development  of 
allergies  in  children.   Floyd  J.  Malveaux,  AI  05385,  Johns  Hopkins  has 
shown  that  the  total  number  of  IgE  receptors  per  basophil  is  closely 
correlated  with  the  level  of  circulating  IgE.  This  relationship  implies  a 
genetic  association  between  serum  IgE  and  the  number  of  basophil  IgE 
receptors  with  a  modulation  of  the  number  of  receptors  by  circulating  IgE. 

Charles  W.  Parker,  AI  10405,  Washington  University,  has  been  studying 
the  IgE  receptor  on  the  rat  basophilic  leukemia  cell  line,  which  has  main- 
tained in  tissue  culture.  This  receptor  has  been  solubilized  and  purified 
and  may  permit  possible  design  of  analogs  to  this  receptor  which  would 
compete  with  the  receptor  for  IgE,  thus  inhibiting  its  ability  to  respond 
when  confronted  with  an  external  allergen. 

The  interaction  of  IgE  with  mast  cells  and  basophils  has  been  amply 
demonstrated.   Hans  L.  Speigelberg,  AI  10734,  Scripps  Clinic  and  Research 
Foundation,  has  shown  that  IgE  binds  to  a  subpopulation  of  bone  marrow 
derived  lymphocytes.   Peripheral  blood  contains  approximately  4  percent  IgE 
binding  cells  in  contrast  to  tonsils  and  spleens  which  contain  four  to  five 
times  as  many.   While  it  is  unclear  as  to  the  biologic  role  of  the  lympho- 
cytes binding  IgE,  studies  are  underway  to  determine  the  number  of  IgE 
binding  sites  binding  B  cells  in  patients  with  allergies  and  other 
disorders. 

5.   Asthma  and  the  Other  Allergic  Diseases 

Studies  within  this  program  have  been  concerned  with  investigations  of 
the  etiologic  factors,  pathogenetic  mechanisms,  diagnostic  measures,  and 
therapeutic  approaches  to  the  effective  management  of  various  allergic 
disorders.   Among  some  of  the  diseases  covered  are  asthma,  allergic 
rhinitis,  hay  fever,  hymenoptera  sensitivity,  drug  allergy,  as  well  as 
basic  studies  in  allergic  dermatitis. 

Approximately  9  million  Americans  have  asthma,  and  although  they  may 
present  with  a  variety  of  clinical  presentations,  in  essence,  it  consti- 
tutes a  disorder  of  reversible  airways  obstruction.   This  obstruction  has 
three  major  elements:   1)  contraction  of  bronchial  smooth  muscles,  2) 
increased  or  retained  secretions  of  mucus,  and  3)  inflammation  of  the 
respiratory  tract  mucosa.   In  individual  asthmatics  attacks  can  be  provoked 


3-11 


by  exposure  to  specific  allergens,  and  some  of  these  individuals  have  other 
allergies  such  as  allergic  rhinitis.   On  the  other  hand,  many  asthmatics  do 
not  have  a  demonstrable  allergy,  and  in  these  individuals  a  variety  of 
infectious,  nervous  (adrenergic  and  cholinergic),  genetic  or  inflammatory 
factors  may  play  a  primary  predisposing  or  contributory  role.   The  program 
on  asthma  is  structured  to  investigate  all  of  the  major  parameters  contri- 
buting to  describing  the  mechanisms  of  causation  in  this  disease.   Areas 
under  investigation  include:   the  natural  history,  allergens,  broncho- 
provocation  challenge,  animal  models,  pulmonary  function  testing,  chemical 
meditors,  cell  mediators,  agernergic  agonists  and  antagonists,  parasym- 
pathomemedic  agents,  drug  allergy,  diagnostic  methodology,  and  therapeutic 
agents . 

Efforts  to  elucidate  what  causes  asthmatic  reactions  in  individuals 
have  included  studies  like  those  of  Stanley  P.  Gallant,  AI  00304,  University 
of  Utah,  who  has  examined  the  neutrophils  of  asthmatic  individuals,  showing 
that  the  number  and  quality  of  beta  agernergic  receptors  to  a  variety  of 
pharmacologic  agents  are  essentially  normal.   Further  dissection  of  the 
pathways  of  how  while  blood  cells  from  asthmatic  individuals  respond  should 
be  helpful  in  elucidating  means  of  pharmacologic  action.   The  role  of 
respiratory  viral  infections  which  induce  asthma  has  continued  under  the 
leadership  of  Richard  Hong,  AI  10404,  University  of  Wisconsin,  whose  colla- 
borators have  shown  that  respiratory  virus  infection,  particularly  with 
rhino  virus,  may  produce  transient  airway  obstruction  similar  to  asthma, 
and  that  the  white  blood  cells  from  these  individuals  with  rhino  virus 
illness  do  have  an  impaired  inhibition  of  lysosomal  enzyme  release  from 
neutrophils.   These  observations  have  been  extended  to  influenza  virus, 
which  has  been  shown  to  induce  changes  in  the  intracellular  levels  of 
cyclic  AMP.   Hopefully,  these  findings  may  provide  leads  for  using  either 
measures  of  peripheral  blood  leukocyte  responses  as  indicators  of  the 
asthmatic  condition,  or  to  monitor  therapeutic  modalities.   Other  investi- 
gators are  similarly  engaged  in  these  kinds  of  studies. 

During  the  past  several  years,  broncho-provocation  and  inhalation 
challenge  techniques  have  been  refined  to  study  how  to  induce  asthmatic 
attacks,  as  well  as  being  employed  to  corroborate  the  diagnosis  of  asthma 
and  to  ascertain  the  constituents  of  the  chemical,  cellular,  and  physio- 
logic events  in  the  ontogeny  of  the  asthmatic  response,  as  well  as  to 
evaluate  pharmacologic  agents . 

Againdra  Bewtra,  AI  14233,  University  of  Iowa  has  established  that  the 
sensitivity  of  airways  to  methacholine  and  histamine  are  comparable  in 
asthmatics.  Further  studies  have  demonstrated  that  the  chemical  compound 
SCH-1000  protects  against  methacholine  challenge  on  both  the  large  and  small 
airways;  however,  it  protects  against  histamine  only  on  the  larger  airways. 
Studies  of  responses  to  allergens  are  underway.   Similar  broncho-provocation 
studies  have  been  conducted  in  asthmatic  patients  by  Roy  Patterson,  AI 
117  59,  Northwestern  University  with  inhaled  antigen,  such  as  ragweed 
antigen  E,  as  well  as  with  methacholine.   He  has  found  evidence  that 
asthmatic  subjects  appear  to  be  more  sensitive  to  inhaled  antigen  when 
tested  after  a  period  of  natural  environmental  exposure  than  before.  It  has 
been  impossible  to  date  to  correlate  pulmonary  function  findings  with 

3-12 


clinical  results  of  immunotherapy.  Since  the  inhalation  of  allergen  per  se 
can  be  a  highly  sensitizing  event,  as  well  as  a  provoking  event,  studies  of 
broncho-provocation  challenge  with  use  of  allergens  have  been  impeded  some- 
what because  of  these  undesirable  side  effects. 

As  an  alternative,  Dr.  Patterson  has  been  following  a  colony  of  asth- 
matic monkeys  which  appear  to  be  to  date  the  best  animal  model  of  asthma 
which  may  simulate  the  human  disease  condition,  especially  insofar  as 
airways  are  concerned.   For  example,  this  antigen- induced  asthma  occurs  only 
in  selected  monkeys,  which  like  human  subjects,  have  hyper-reactive  airways. 
Dr.  Patterson  has  shown  that  living  antigen  reactive  mast  cells  reside  free 
in  the  bronchial  lumens  of  these  monkeys,  as  well  as  in  dogs  and  man,  and 
that  these  cells  can  transfer  the  asthmatic  type  airway  responsiveness  to 
non-reactive  animals. 

A  variety  of  chemical  mediators,  such  as  prostaglandins,  histamines, 
and  other  agents,  which  have  been  postulated  as  being  important  in 
describing  human  airways  responses,  have  been  tested  in  this  animal  model 
and  shown  to  simulate  the  human  asthmatic  condition.   Such  a  model  will 
provide  for  evaluating  therapeutic  agents  prior  to  tests  in  humans  and 
offers  the  unique  opportunity  to  perform  repetitive  studies  under 
controlled  conditions  that  otherwise  could  not  be  undertaken  in  humans. 

Additional  basic  studies  which  may  prove  relevant  to  the  asthmatic 
condition  include  those  of  H.  Benfer  Kaltreider,  AI  12296,  University  of 
California,  San  Francisco,  who  has  studied  immune  functions  of  the  lung, 
showing  that  sensitized  lymphocytes  capable  of  killing  foreign  cells  could 
be  induced  to  appear  in  lung  tissue  by  either  local  or  intraperitoneal 
immunization  in  animals.   These  killer  lymphocytes  have  been  found  to  be 
critical  for  the  control  of  virally- infected  or  neoplastically  induced 
cells.   Additionally,  alveolar  macrophages  may  exert  a  suppressive  effect 
on  the  responses  of  lymphocytes  to  several  immune  stimuli,  and  this  appears 
to  occur  through  substances  such  as  prostaglandins,  which  may  be  released 
by  the  macrophages  themselves.   His  efforts  are  at  understanding  the 
mechanisms  of  non-cellular  and  cellular  suppression  of  local  lung  lymphocyte 
function,  and  thereby  develop  strategies  for  combating  hypersentitivity 
reactions  in  the  lung.   Direct  work  studying  alveolar  macrophage  function 
in  asthmatic  individuals  is  inhibited  by  the  difficulties  of  performing 
bronchial  lavage  in  individuals  with  hyperactive  airways.   However,  studies 
are  underway  in  several  laboratories  at  this  time,  which  should  shed 
further  light  on  these  areas. 

Similar  studies  of  basic  immune  functions  of  the  lung  have  been  per- 
formed by  Barbara  A.  Nichols,  AI  00294,  who  has  used  the  parasite  model  of 
Toxoplasma  gondii  to  study  the  interactions  between  this  parasite  and  lung 
macrophages . 

Ragweed  allergy  is  a  disorder  affecting  approximately  15  million 
Americans.   For  most  individuals  it  is  a  mild  seasonal  disorder  commonly 
called  hay  fever,  which  is  characterized  by  eye  irritation  and  nasal  dis- 
charge; symptoms  are  usually  relieved  by  antihistamines;  however,  in  some 
individuals,  although  not  life  threatening,  are  a  source  of  considerable 


3-13 


morbidity.   In  others  ragweed  has  been  indicated  as  being  causal  in 
producing  an  asthmatic-like  syndrome. 

Most  of  the  advances  in  understanding  allergic  rhinitis  have  occurred 
in  this  model,  particularly  in  the  areas  of  etiology,  diagnosis,  patho- 
genesis, treatment  and  prevention.   Further  progress  can  be  expected  to 
lead  to  greater  understanding  of  factors  involved  in  all  allergic  diseases. 

Pertinent  in  this  regard  is  the  difficulty  in  obtaining  purified 
allergen  preparations.   Despite  the  fact  that  there  are  at  least  100 
different  proteins  in  ragweed,  only  five  have  been  demonstrated  to  produce 
allergy  in  man.   J.  Donald  Capra,  AI  12796,  University  of  Texas  at  Dallas 
has  determined  the  chemical  structure  of  the  smallest  of  these  five 
proteins,  termed  RA  5,  and  is  completing  studies  on  defining  the  structure 
of  further  components.   More  recent  studies  are  making  an  effort  to 
determine  the  amino  acid  sequence  of  cedar  and  sage  allergens.  T.  P.  King, 
AI  14422 ,  has  prepared  conjugates  of  purified  Antigen  E  with  non-immuno- 
genetic  polymers.   These  conjugates  of  Antigen  E  show  reduced  allergenic 
activity,  yet  retain  the  immunosuppressive  property  of  the  native  antigen, 
and  would  not  have  been  possible  without  the  clarification  of  antigen 
structure.   He  is  continuing  further  studies  in  the  immunochemical  charac- 
terization of  vespid  venoms  with  similar  goals. 

The  clarification  of  the  structure  of  individual  allergens  is  impor- 
tant both  chemically  and  biologically.   Commercial  suppliers  of  pollen  for 
use  in  human  desensitization  work  may  vary  in  their  source,  so  if  an 
individual  is  receiving  treatments  in  various  parts  of  the  country,  there 
may  be  increasing  or  decreasing  response  to  treatment.   In  addition  and 
more  importantly,  if  an  allergist  clinician  should  change  the  supplier  of 
his  pollen  extracts  or  if  the  supplier  should  obtain  pollen  from  a 
different  region  of  the  country,  the  potency  of  the  extract  might  be 
markedly  different.   Efforts  are  underway  in  this  program  to  provide 
standardized  antigen  materials. 

Gerald  Gleich,  AI  07047,  The  Mayo  Clinic,  has  successfully  separated 
the  two  chains  of  Antigen  E,  the  major  allergen  of  ragweed  pollen.  Biologic 
activity  of  the  individual  chains  was  preserved,  and  studies  are  underway 
to  determine  the  amino  acid  sequence  of  each  chain,  including  especially 
those  portions  of  the  chains  which  are  the  determinants  of  the  aller- 
genicity  of  the  molecule.   Presumably,  if  the  ability  to  confer  allergy 
resides  in  only  a  small  portion  of  the  molecule,  then  these  studies  could 
lead  to  a  new  and  improved  type  of  immunotherapy  for  ragweed  pollen 
sensitivity.   On  the  other  hand,  David  Marsh,  AI  09565,  Johns  Hopkins 
University,  has  focused  his  research  on  the  highly  basic  proteins  which  are 
rapidly  released  from  ragweed  pollen  grains,  as  contrasted  with  the  acidic 
proteins,  such  as  Antigen  E,  which  are  slowly  released.   Since  allergenic 
reagents  currently  employed  in  immunotherapy  contain  a  large  propotion  of 
acidic  proteins,  such  as  Antigen  E,  the  addition  of  these  rapidly  released 
substances  may  lead  to  an  improvement  in  immunotherapy. 

Several  investigators,  including  Roy  Patterson,  AI  11403,  Northwestern 
University,  and  Philip  Norman,  AI  04866,  Johns  Hopkins  University,  have 

3-14 


pursued  the  development  of  chemically  modified  ragweed  allergens  termed 
allergoids.   A  variety  of  chemical  agents  (formaldehyde,  urea,  gluteralde- 
hyde,  ethylene,  glycol,  etc.)  have  been  employed,  but  the  main  allergen 
studied  in  immunotherapy  is  the  more  rapid  induction  of  blocking  IgG  anti- 
bodies with  greater  safety  than  conventional  materials  now  in  use.   Larger 
amounts  of  immunogenic  material  can  be  administered  in  a  shorter  period  of 
time  without  the  risk  of  generalized  allergic  reaction.   Clinical  trials 
are  underway  to  assess  the  value  of  these  materials  and  they  should  be 
licensed  shortly  for  more  widespread  use. 

Other  investigators  such  as  Rebecca  Buckley,  AI  12016,  AI  14314,  and 
AI  70830,  Duke  University,  have  utilized  the  purified  ragweed  antigens, 
such  as  Antigen  E,  which  are  supplied  by  our  institute's  allergenic 
reagents  program.   With  these  purified  materials,  both  Drs.  Buckley  as  well 
as  Dr.  Patterson  have  been  able  to  demonstrate  the  synthesis  of  ragweed 
specific  IgE  from  lymphocytes  cultured  in  vitro,  thus  establishing  a 
laboratory-based  system  for  further  in-depth  studies  of  human  allergic 
diseases.   Because  of  the  need  for  more  widespread  use  of  purified  ragweed 
antigens,  combined  with  the  depletion  of  existing  stock,  it  became 
necessary  for  the  Allergy  and  Clinical  Immunology  Branch  to  acguire  new 
materials.   T.  P.  King,  AI  82567  Rockefeller  University,  has  produced  RA  3, 
RA  5,  Antigen  K  and  Antigen  E.  These  materials  will  be  available  to  investi- 
gators and  insure  maintenance  of  a  supply  of  highly  purified  and  well- 
characterized  allergens  for  further  studies.   Exciting  recent  advances  have 
been  further  carried  forward  in  the  field  of  the  sting  of  insects  of  the 
bee  family  called  Hymenoptera,  which  induce  an  overwhelming  and  sometimes 
fatal  reaction  in  sensitive  individuals.   While  firm  data  is  lacking,  it  is 
believed  that  about  2  million  Americans  may  be  sensitive  to  these  stinging 
insects,  and  although  the  number  of  fatalities  each  year  is  not  precisely 
known,  it  is  believed  to  be  less  than  500.   For  individuals  who  develop  an 
overwhelming  reaction  to  a  Hymenoptera  sting,  an  injection  of  epinephrine 
may  be  lifesaving.   Since  exposure  to  these  insects  may  occur  in  areas 
where  prompt  medical  assistance  is  not  available,  it  was  important  to 
develop  an  effective  preventive  measure.   The  Hymenoptera  include  honey 
bees,  yellow  jackets,  hornets  and  wasps.   Their  venoms  contain  several 
vaso-active  amines  and  other  noxious  simple  chemicals  which  are  responsible 
for  the  immediate  irritation  experienced  by  anyone  stung  by  these  insects. 
In  addition,  their  venoms  contain  several  proteins,  and  it  is  these 
proteins  which  are  responsible  for  the  induction  of  the  hypersensitivity 
state  and  the  subsequent  development  of  anaphylaxis  in  very  sensitive 
individuals . 

Until  recently  the  only  materials  available  for  testing  and  treating 
allergic  patients  to  insect  stings  were  so-called  whole  body  extracts  which 
were  prepared  by  grinding  up  insects.   Through  the  collaborative  efforts  of 
Lawrence  M.  Lichtenstein,  AI  02870,  Johns  Hopkins  University  and  Philip 
Norman,  AI  10304,  Johns  Hopkins  University,  it  was  established  that  whole 
body  extracts  available  to  practicing  allergists  contain  little  if  any 
active  venom  protein,  and  that  their  studies  showed  that  injection 
desentization  therapy  with  these  extracts  have  virtually  no  utility  in 
diagnosis  or  prevention  of  insect  sting  allergy.   Subsequently,  these 
investigators  obtained  venom  itself,  prepared  solutions  for  clinical 

3-15 


evaluation.   They  found  an  excellent  corrolation  between  results  of  venom 
skin  testing  with  clinical  history  of  Hymenoptera  sensitivity.   When  these 
same  venom  materials  were  administered  in  a  series  of  immunotherapy  injec- 
tions to  sensitive  individuals,  protection  against  serious  reactions  ensued 
in  well  over  95  percent  of  individuals.   Other  investigators  have  confirmed 
the  efficacy  of  these  materials,  and  in  the  spring  of  1979,  owing  to  their 
work,  venom  for  desensitization  was  in  fact  made  commercially  available. 
Since  this  material  has  only  been  in  the  hands  of  practitioners  for  a  short 
period  of  time,  more  data  will  be  required  to  determine  which  individuals 
will  require  this  form  of  treatment,  further  clarification  of  therapeutic 
regimens  will  have  to  occur,  etc.   What  is  clear,  however,  is  that  through 
clarification  of  the  immunochemical  properties  of  the  active  venom  protein, 
efficacious  immune  therapy  with  this  material  has  been  possible,  and  the 
development  of  specific  diagnostic  procedures  combined  with  skin  testing, 
followed  by  immunotherapy  in  appropriate  individuals,  has  resulted  in 
protection  in  the  vast  majority  of  persons. 

6.    Immunodeficiency 

The  program  on  immunodeficiency  is  concerned  with  the  etiology, 
ontogony,  prevention,  and  treatment  of  structural  and  functional 
deficiencies  of  the  immune  system.   Both  naturally  occurring  and  acquired 
disease  states  are  included.   Ongoing  studies  have  focused  on  the 
congenital  absence,  failure  of  development,  or  other  disorders  affecting 
thymus  or  bone  marrow  cellular  elements;  abnormalities  in  production, 
inhibition  or  catabolism  of  immunoglobulin;  deficiencies  of  specific 
complement  components;  and  defective  host  defenses  due  to  abnormalities  of 
leukocytes.   These  naturally  occurring  or  acquired  defects  of  immunity 
provide  a  unique  opportunity  to  expand  the  understanding  of  normal  immune 
function.   Only  by  its  absence  do  we  see  a  role  for  its  presence  in  the 
sequence  of  steps  which  make  up  the  normal  immune  response.  Although  these 
diseases  are  rare,  the  information  gained  is  relevant  to  the  general  field 
of  immunology  and  to  many  health  problems  including  infectious  diseases, 
allergy  and  arthritis. 

Certain  immune  deficient  subjects  have  been  shown  to  demonstrate  high 
serum  IgE  levels.   Rebecca  Buckley,  AI  12016,  Duke  University,  has  examined 
the  function  of  accessory  cells  such  as  macrophages  and  polymorphonuclear 
white  blood  cells  in  these  patients ,  and  compared  them  with  allergic 
individuals.  Abnormalities  have  in  fact  been  shown  in  both  groups  of 
persons.   Richard  Hong,  AI  14354,  University  of  Wisconsin  at  Madison,  has 
suggested  that  the  exaggerated  response  in  these  allergic  individuals  may 
be  related  to  a  defect  in  T  lymphocyte  regulation.  Dr.  Hong  has  also 
studied  the  role  of  thymus  epithelial  cells  in  reconstituting  certain 
immuno-deficient  patients.   He  is  examining  means  to  improve  methods  of 
culturing  thymus  tissue  prior  to  transplantation,  and  learn  by  in  vitro 
methods  whether  or  not  this  is  of  benefit  to  such  individuals.   He  has 
found  that  thymus  glands  may  be  preserved  if  kept  properly  in  an  incubator, 
and  shown  that  thymic  transplantation  can  provide  restoration  not  only  for 
immune  function  in  classically  immune  deficient  individuals,  but  has 
extended  studies  into  work  in  cancer  patients  who  have  secondary  immune 
deficiencies.   Bone  marrow  transplantation  has  accomplished  cures  in 

3-16 


individuals  with  aplastic  anemia,  as  demonstrated  by  Robert  A.  Good,  AI 
11843,  Sloan  Kettering  Institute  for  Cancer  Research.    In  addition  to 
generalized  immune  deficiencies,  other  important  observations  have  been 
made  which  have  permitted  further  extension  in  this  exciting  area  of  thera- 
peutics.  For  example,  Ralph  Wedgewood,  AI  07073,  has  shown  that  in  the 
Wiscott-Aldrich  Syndrome  a  combination  of  immune  deficiencies  may  be 
observed,  and  further  that  a  hereditary  enzyme  deficiency  of  adenosine 
deaminase  could  be  reconstituted  with  use  of  bone  marrow  transplantation. 
Ronald  C.  Scott,  AI  12617,  University  of  Washington,  by  screening  indivi- 
duals who  are  immunodeficient  for  adenosine  deaminase  and  nucleoside 
phosphorylase,  has  provided  further  insight  into  biochemical  causes  of 
childhood  immune  deficiency  states. 

Edward  J.  Moticka,  AI  12191,  University  of  Texas  Health  Science  Center, 
Dallas,  has  studied  the  acquired  immunologic  incompetence  which  accompanies 
antigen- induced  hypergammaglobulinemia.   This  interference  with  reactivity, 
an  important  regulatory  mechanism  for  the  immune  system,  may  be  responsible 
for  the  diminished  immune  capacity  observed  in  patients  with  multiple 
myeloma. 

Sandra  S.  Kaplan,  AI  14218,  University  of  Pittsburgh,  studying  the 
defect  in  polymorphonuclear  leukocytes  which  occurs  in  the  Chediak-Higashi 
Syndrome,  which  is  almost  uniformly  fatal,  has  demonstrated  that  a 
defective  blood  platelet  may  be  responsible  for  this  susceptibility  to 
infection  because  it  fails  to  activate  the  white  blood  cell.   Studies  of 
drugs  are  underway  to  overcome  this  deficiency  by  activating  leukocytes 
through  biochemical  changes  similar  to  those  mediated  by  normal  platelets. 

Harvey  J.  Cohen,  AI  00311,  Childrens  Hospital  Medical  Center,  Boston, 
studying  children  with  chronic  grammulomatous  disease,  who  are  known  to  be 
missing  an  enzyme  in  their  white  cells,  has  preliminary  evidence  permitting 
the  prenatal  diagnosis  of  this  disease  by  study  of  fetal  granular  sites 
obtained  by  amniocentesis.   This  work  emphasizes  how  the  study  of  basic 
mechanisms  may  have  a  broad  range  of  potential  diagnostic  and  therapeutic 
applications. 

Hugh  Fudenberg,  AI  13484,  Medical  University  of  South  Carolina,  has 
also  been  studying  immune  deficiency  diseases  and  has  described  genetic 
markers  on  certain  lymphocytes  of  patients  with  common  variable  agamma- 
globulinemia who  develop  a  fatal  kidney  disease;  and  that  IgA  deficiency 
and  X-linked  hyper-IgM  syndrome  are  heterogeneous  diseases.  Work  on  cystic 
fibrosis  has  shown  a  basic  defect  which  may  be  related  to  a  defect  in 
alpha-2  macroglobulin  in  the  serum. 

7 .    Immune  Complex  Disease 

Immune  complexes  result  from  the  binding  of  antigens  by  antibodies . 
Subsequently,  components  of  the  complement  system  also  become  involved. 
Such  complexes,  therefore,  arise  during  the  course  of  the  development  of 
the  normal  immune  response.   Under  some  circumstances,  however,  these 
entities  appear  within  lesions  and  indeed  are  necessary  for  the  development. 
The  pathogenetic  mechanisms  by  which  immune  complexes  cause  tissue  damage, 


3-17 


particularly  in  the  auto- mmune  diseases  with  which  they  are  increasingly 
associated  are  still  poorly  understood.   The  lack  of  sensitive,  quantita- 
tive assays  for  all  types  of  immune  complexes  has  impeded  work  in  this  area 
to  some  extent.   Recent  studies  by  Vincent  Agnelo,  AI  70968,  New  England 
Medical  Center  Hospital,  who  has  refined  two  such  assays,  one  for  rheuma- 
toid factor  and  anti-IgG  antibody,  and  one  for  the  ClQ  component  of 
complement,  are  helping  to  clarify  these  measures.   An  unexpected  finding 
is  that  some  rheumatoid  factors  crossreact  with  nuclear  protein.  A  further 
advance  by  Douglas  B.  Cines,  AI  05477,  University  of  Pennsylvania  was  made 
by  his  utilization  of  a  radio- immunoassay  to  quantitate  IgG  and  the  C3 
component  of  complement.  This  technique  can  be  used  also  to  estimate  the 
affinity  of  an  antibody  for  an  antigen  (R.  M.  Rothberg,  AI  07854,  Univer- 
sity of  Chicago) . 

William  P.  Arend,  AI  10975,  University  of  Washington,  has  made  an 
effort  to  determine  what  it  is  that  renders  an  immune  complex  damaging.  He 
reports  that  in  rheumatoid  arthritis,  anti-IgG  antibodies  (rheumatoid  factor) 
specific  for  the  FC  portion  reacted  with  IgG  to  produce  innocuous  complexes, 
whereas  anti-IgG  antibodies  specific  for  the  FAB  portion  produced  injurious 
complexes . 

Systemic  Lupus  Erythematosus  is  an  auto- immune  disease  in  which  anti-DNA 
antibodies  play  a  permanent  pathogenetic  role.   The  cause  for  the 
appearance  of  the  auto-antibodies  is  being  studied  in  a  genetically  defined 
mouse  strain  which  develops  an  SLE-like  syndrome  spontaneously.  Defective 
suppression  of  the  immune  response  in  these  animals  can  break  the  self- 
tolerance  usually  seen  (M.  Eric  Gershwin,  AI  00193,  University  of 
California,  Davis) .  Many  people  are  studying  the  strain  and  have  shown  that 
the  induction  of  tolerance  to  DNA  can  be  accomplished  by  treatment  with 
appropriately  constructed  antigen.   Bernard  Stollar,  AI  14534,  Tufts 
University  has  undertaken  studies  to  define  the  chemical  requirements  for 
developing  reagents  that  can  specifically  prevent  the  formation  of  anti- 
bodies to  nucleic  acid.   In  detailed  studies  of  the  specificity  of 
tolerance,  in  studies  done  in  collaboration  with  Yves  Borel,  AI  13867, 
Boston  Children's  Hospital  Medical  Center,  he  has  shown  that  tolerance  to 
one  nucleoside  does  not  prevent  the  formation  of  antibodies  to  other 
nucleosides  of  DNA.   This  finding  provides  an  approach  to  the  study  of  the 
regulation  of  responsiveness  and  specific  nonresponsiveness  at  the  cellular 
level.   The  obvious  hope  in  this  regard  is  to  develop  information 
sufficient  to  permit  appropriate  manipulation  of  the  immune  response  in 
order  to  effect  therapeutic  benefits.   Thus,  the  development  of  specific 
immune  tolerance  would  be  a  significant  improvement  over  the  use  of  non- 
specific immunosuppressive  or  anti- inflammatory  therapy  in  the  treatment  of 
auto-immune  disorders. 

Immune  complexes  which  may  be  also  associated  with  chronic  infectious 
diseases  and  cause  inflammation  and  tissue  damage  are  also  found  to  be 
involved  in  graft  rejection.   That  they  may  have  a  beneficial  effect  is 
suggested  by  the  evidence  that  such  immune  complexes  can  modulate  the 
response  to  the  antigen  contained  within  them,  and  in  fact  prolong  graft 
survival .   Thus ,  Joseph  P.  Feldman,  AI  07104,  Scripps  Clinic  and  Research 
Foundation,  and  associates  have  been  studying  circulating  immune  complexes 

3-18 


which  appear  after  graft  implantation  and  their  effect  on  the  host  immune 
response. 

Many  studies  are  now  nationwide  measuring  serum  immune  complexes  and 
trying  to  define  their  role  in  clinical  disorders,  whether  they  may  be 
valuable  in  assessing  diagnostic  or  other  modalities.   Continued  emphasis 
by  this  program  in  developing  expeditious  ways  whereby  these  measurements 
can  be  made  standard  and  by  which  other  investigators  can  compare  data 
among  several  groups  of  individuals  is  of  particular  importance . 

8.    Inflammation 

The  process  of  inflammation  is  mediated  by  cells  and  by  humoral 
factors.   This  process  is  essential  for  host  defense  against  foreign  sub- 
stances.  However,  in  certain  disease  states,  inflammation  plays  a  major 
role  in  tissue  damage.   An  understanding  of  what  initiates  and  what 
modulates  inflammation  is,  therefore,  crucial  to  explaining  and  controlling 
these  disease  states.   The  noncellular  mediators  of  inflammation  can  be 
divided  into  two  general  categories ,  those  of  small  molecular  weight  and 
those  of  high  molecular  weight.   The  group  of  serum  proteins  derived  from 
the  complement  system  and  the  associated  enzyme  systems  of  coagulation, 
fibrinolysis,  and  kinin  fall  into  the  latter  category. 

The  initiation  of  the  complement  casade  by  either  the  classic  or 
alternate  pathway  makes  a  major  contribution  to  the  development  of  the 
inflammatory  response.   Once  activated,  complement  components  have 
functions  including  opsonization,  immune  adherence,  chemotaxis,  cell  lysis, 
and  anaphylatoxin  generation.   Derangements  of  complement  have  been 
implicated  in  causing  or  contributing  to  a  wide  range  of  disorders 
including  cancer,  kidney  diseases,  collagen  vasular  diseases,  infectious 
diseases,  and  inherited  diseases. 

Understanding  of  the  complement  system  depends  on  isolation  and 
identification  of  its  components.   Through  work  done  by  our  grantees,  it  is 
known  that  there  are  approximately  20  complement  components.  Paul  A. 
Liberti  (AI  12365,  Thomas  Jefferson  University)  has  devised  a  new  isolation 
procedure  for  C12  which  results  in  stable  preparations  retaining  all  native 
biological  activities.   He  has  used  this  procedure  to  measure  the  physical 
properties  of  Clq  in  order  to  determine  its  most  likely  molecular 
structure,  thereby  enabling  them  to  relate  its  structure  to  function.   Irma 
Gigli  (AI  13809,  New  York  University)  has  continued  studies  examining  the 
structural  and  functional  proteins  of  the  complement  system,  and  has  shown 
that  Ch  serves  an  important  function  in  the  initial  steps  of  complement 
activation  and  that  C4bp  (previously  reported)  functions  in  the  control  of 
the  formation  of  C42  convertase,  confirming  the  presence  of  a  control 
mechanisms  in  serum  which  regulates  the  classical  pathway  of  convertase. 
Shaun  Ruddy,  AI  13049,  Virginia  Commonwealth  University,  has  shown  that  a 
newly  discovered  control  protein,  beta-1  H  globulin,  actually  combine  with 
one  of  the  active  complement  proteins  and  blocks  its  function  in  producing 
inflammation.   Understanding  the  mechanism  of  action  should  allow  develop- 
ment of  new  methods  to  control  inflammation  induced  by  complement. 


3-19 


Particular  interest  has  been  placed  on  studies  of  the  properdin  or 
alternate  complement  pathway.   The  alternate  pathway  was,  until  recently, 
thought  to  have  specific  recognition  proteins  which  bound  to  and  thereby 
identified  foreign  particles  for  destruction.  Hans  Muller-Eberhard  (AI 
07007,  Scripps  Clinic  and  Research  Foundation)   reports  that  the  complement 
protein,  C3 ,  has  recognition  function  in  the  so-called  alternate  pathway. 
Antibody  is  not  required  for  this  reaction.   A  cell  (fungal,  bacterial, 
etc.)  that  is  an  activator  of  this  pathway  allows  C3,  from  the  plasma,  to 
be  deposited  on  its  surface.   The  deposited  C3  interacts  with  surface 
structures  of  activators  such  that  a  regulatory  protein  which  ordinarily 
initiates  inactivation  of  deposited  C3 ,  cannot  reach  it.   As  a  consequence, 
the  orderly  assembly  of  complement  enzymes  procedes  without  interference  by 
regulators  and  the  cell  is  killed  by  the  membrane  attack  complex. 

Other  studies  of  basic  mechanisms  have  included  those  of  Celso  Bianco, 
AI  15221,  S.U.N.Y.  Downstate  Medical  College,  who  has  examined  the  role  of 
certain  complement  components  in  studies  of  macrophage  motility.  Prelim- 
inary experiments  have  shown  that  peritoneal  macrophages  respond  to  Bb,  a 
cleavage  product  of  factor  B  of  the  properdin  pathway  by  spreading  an 
inhibition  of  migration.  Hans  Muller-Eberhard,  AI  13010,  Scripps  Clinic  and 
Research  Foundation,  has  also  studied  the  responsiveness  of  human  mono- 
nuclear phagocytes  to  activated  factor  B  (Bb  of  the  properdin  system) 
showing  that  mononuclear  phagocytes  synthesize  and  secrete  complement 
proteins,  and  that  monocytes  synthesize  C5  which  is  intimately  involved  in 
processes  which  lead  to  activation  of  monocytes  to  spread  in  response  to 
both  Bb  and  anti-C5F  (AB  prime)2.   Thus  the  Bb  complement  activation  frag- 
ment influences  monocytes,  therefore  creating  a  possible  role  for 
complement  protein  as  mediators  of  cellular  immunity.   To  whatever  extent 
that  it  correct,  it  is  of  interest  that  this  could  have  relevance  in  cancer 
study,  and  in  fact,  Alfred  Esser,  AI  14099,  Scripps  Clinic  and  Research 
Foundation,  has  shown  that  certain  RNA  tumor  viruses  can  be  lysed  by  human 
complement,  supporting  the  proposition  that  complement  can  provide  a  major 
defense  against  infection  with  reto  viruses  (oncornaviruses,  C-type  viruses, 
mouse  leukemia  viruses  which  may  be  associated  with  the  production  of 
tumors  in  animals). 

These  basic  studies  have  been  very  rewarding  because  they  are  useful 
in  explaining  mechanisms  for  several  clinical  problems.   Understanding  of 
these  mechanisms  is  important  for  possible  prevention  and  treatment  of 
these  diseases.   For  example,  K.  Frank  Austen  (AI  07722,  Robert  B.  Brigham 
Hospital)  reports  that  he  has  been  able  to  work  out  the  molecular  structure 
and  mechanisms  of  action  of  C3  nephritic  factor,  the  abnormal  protein 
present  in  patients  with  membranoproliferative  glomerulonephritis.  This 
protein  causes  the  complement  system  to  be  fully  activated  in  the  serum  of 
patients  with  various  types  of  nephritis.   This  protein  is  a  host  antibody 
directed  against  protein  of  the  alternate  complement  pathway  and  serves  to 
stablize  the  interaction  of  the  complement  protein  thereby  circumventing 
all  host  regulatory  mechanisms.   This  leads  to  virtually  complete  depletion 
of  the  complement  system  with  resultant  inflammatory  reaction.   He  is 
currently  working  out  a  role  of  the  alternate  complement  pathway  in  bullous 
pemphigoid,  a  disease  in  which  the  mast  cell  effector  system  also  appears 
to  be  abnormal. 


3-20 


Other  tantalizing  clinical  leads  have  come  from  basic  research  in 
complement.   For  example,  Dr.  Gigli  states  that  recent  studies  have  shown 
that  the  complement  system  may  play  an  important  role  in  the  localization 
of  myocardial  infarction.   Depletion  of  one  of  the  components,  C3,  is 
accompanied  by  better  outcome  of  animals  in  which  experimental  infarcts 
have  been  induced.   Kenneth  Mathews,  AI  14950,  University  of  Michigan,  Ann 
Arbor,  has  described  a  patient  with  a  deficiency  of  serum  carboxy-peptidase 
B,  which  might  predispose  to  radiographic  contrast  media  reactions  or 
chronic  idiopathic  urticaria  or  angiodema  (worked  on  in  collaboration  with 
Tony  Hugli,  AI  15782,  Scripps  Clinic) .   The  potential  significance  of  this 
observation  is  that  there  is  an  implication  that  there  is  a  subpopulation 
of  patients  who  not  only  react  adversely  to  radio  contrast  dyes,  but  who 
also  may  have  the  syndrome  of  chronic  urticaria  or  angiodema,  where  serum 
carboxy-peptidase  B  deficiency  may  contribute  to  their  clinical 
difficulties. 

John  P.  Leddy  (AI  12568,  University  of  Rochester)  has  been  able  to 
correlate  the  inordinate  white  cell  depression  described  after  donation  of 
white  cells  to  the  activation  and  consumption  of  the  complement  system. 
This  white  cell  activation  is  coincident  with  the  formation  of  some  white 
cell  depressing  factor  in  blood  passing  through  the  nylon  filter  used 
during  this  donation  procedure  (leukapheresis) .   By  determining  the  precise 
mechanisms  for  this  problem  it  may  in  the  future  be  eliminated. 

The  genetics  of  the  complement  system  have  been  studied  by  various 
investigators.   Harvey  R.  Colten  (AI  12791,  Boston  Children's  Hospital 
Medical  Center)  has  been  looking  at  congenital  absences  of  complement 
protein  in  regard  to  increased  susceptibility  to  infection,  hereditary 
attacks  of  edema,  and  increased  susceptibility  to  rheumatoid- like  diseases. 
Chester  A.  Alper  (AI  14157,  Center  for  Blood  Research)  has  found  that  the 
second  complement  component  and  factor  B  but  not  the  fourth  component  are 
controlled  by  genes  in  the  region  near  the  HLA  genes.   In  a  more  basic  way, 
John  Goldman,  AI  12792,  Michael  Reese,  has  continued  studies  investigating 
the  relationship  of  early  components  of  complement  to  the  H2  complex  in 
mice,  finding  that  functional  levels  of  CI,  C4,  and  C2  are  under  the 
control  of  genes  within,  or  closely  linked  to  the  S  region  of  the  H2 
complex. 

A  second  category,  that  of  low  molecular  weight  mediators  of  inflam- 
mation, was  previously  mentioned.  A  discussion  of  basophils  (or  mast 
cells)  must  accompany  any  discussion  of  these  chemicals,  because  these  are 
the  cells  which  release  them.  The  sequence  of  events  leading  to  mediator 
secretion  is  as  follows.-  sensitization  takes  place;  IgE  attaches  to  the 
surface  of  mast  cells  and  basophils,-  on  re-exposure,  the  cell-bound  anti- 
bodies are  bridged  by  the  allergen  signaling  release  of  the  mediators  from 
the  cells. 

Research  in  this  area  has  been  facilitated  by  several  new  advances: 
Herve  Bazin  (AI  12830,  Catholic  University  of  Louvain)  through  the  develop- 
ment of  a  colony  of  rats  with  IgE  secreting  plasmacytomas,  has  made  this 
immunoglobulin  readily  available;  and  both  Dr.  Parker  and  Dr.  Ishizaka  have 
established  rat  basophilic  leukemia  cells  in  culture. 

3-21 


Both  of  the  latter  investigators  are  also  studying  basophil  receptors. 
Dr.  Ishizaka  has  been  able  to  demonstrate  histamine  release  from  these 
cells  via  divalent  anti-receptor  antibody  without  participation  of  IgE.  Also 
studying  these  receptors  in  Floyd  J.  Malveaux  (AI  05383,  Johns  Hopkins 
University)  who  has  noted  the  relationship  between  serum  IgE  and  the  number 
of  basophil  IgE  receptors. 

While  Dr.  Ishizaka  has  shown  that  mast  cells  can  discharge  allergic 
mediators  without  involvement  of  IgE,  Lawrence  M.  Lichtenstein  (AI  11334, 
Johns  Hopkins  University)  has  demonstrated  release  of  mediators  from  other 
than  mast  cells.   Namely  polymorphonuclear  (PMN)  leukocytes  contain  and 
release  slow  reacting  substance  of  anaphylaxis  (SRS-A)  and  eosinophilic 
chemotactic  factor  (ECF) . 

Philip  S.  Askenase  (AI  12211,  Yale  University)  is  studying  so-called 
cutaneous  basophil  hypersensitivity  (CBH) .   Traditional  views  of  delayed 
hypersensitivity  and  immediate  hypersensitivity  as  being  separate  patterns 
of  immune  reactivity  may  no  longer  hold.   For  example,  otherwise  typical 
delayed  reactions  contain  significant  numbers  of  basophils.   CBH  can  be 
transferred  in  animal  models  by  either  immune  cells  or  serum.  Similarly, 
the  late  IgE  mediated  reactions  in  man  may  be  analogous  to  serum  transfer 
of  CBH. 

Despite  this  clouding  of  hypersensitivity  reactions,  some  discussion 
of  what  is  known  classically  as  the  type  IV  hypersensitivity  reaction- 
cellular  hypersensitivity,  mediated  by  T  cells  is  needed.   These  T  cells 
also  function  as  cytotoxic  effector  cells,  have  a  role  in  secreting 
mediators  of  delayed  hypersensitivity,  regulate  immune  responses  as  helper 
or  suppressor  cells  and  act  as  memory  cells. 

The  first  part  of  this  discussion  will  be  confined  to  studies  of 
hypersensitivity  relevant  to  clinical  diseases.   Contact  dermatitis  is  a 
prototype  for  cell  mediated  immunity.   Henry  Claman,  AI  12685,  University 
of  Colorado  Medical  Center)  has  used  a  mouse  model  to  study  the  development 
of  delayed  hypersensitivity  to  dinitrofluorobenzene .   This  experimental 
model  is  important  because  the  complete  antigen  made  after  contact 
sensitization  is  DNP-coupled-to-self ,  and  thought  of  in  this  way,  contact 
allergy  can  be  considered  a  form  of  autoimmunity  where  at  least  part  of  the 
antigen  is  self.   Vera  Byers  (AI  12947,  University  of  California,  San 
Francisco,  is  studying  poison  oak  and  poison  ivy,  two  of  the  main  causes  of 
workman's  compensation  in  California.   She  is  developing  systems  to  study 
the  effects  of  these  chemicals  on  human  lymphocytes  in  vitro,  in  an  effort 
to  produce  a  state  of  unresponsiveness.   Henry  C.  Maguire,  AI  13337,  Hahne- 
mann Medical  College  and  Hospital  of  Philadelphia,  is  also  studying  contact 
dermatitis.   He  is  trying  to  determine  the  mechanisms  of  an  interesting 
clinical  observation   the  use  of  cyclophosphamide  under  particular 
conditions  will  increase  the  intensity  of  allergic  contact  dermatitis 
reactions.  Fred  S.  Kantor  (AI  060706,  Yale  University)  is  studying  the 
opposite  phenomenon--anergy .   He  has  induced  anergy  in  an  animal  model  and 
found  the  effect  to  be  mediated  by  a  subset  of  T  lymphocytes  which  adhere 
to  nylon-wool  columns . 


3-22 


When  sensitized  T  lymphocytes  are  cultured  in  the  presence  of  specific 
antigen,  they  release  a  variety  of  nonspecific  mediators,  or  lymphokines, 
that  act  on  other  lymphocytes  and  macrophages  among  other  cells.   Manfred 
Mayer  (AI  12371,  Johns  Hopkins  University,  has  been  doing  basic  research  to 
determine  what  lymphokines  are.   In  particular  he  has  looked  at  lymphotoxin 
which  plays  a  role  in  certain  cytotoxic  reactions  mediated  by  lymphocytes, 
mitogenic  factor,  lymphocyte  activating  factor  which  is  an  amplifier  of 
immune  and  allergic  processes,  and  hydrophobic  peptide.   Louis  W.  Heck  (AI 
05207,  Robert  Beck  Brigham  Hospital,  has  been  studying  migration  inhibitor 
factor.   He  has  shown  that  macrophages,  pretreated  with  proteinase 
inhibitors  demonstrated  an  augmented  response  to  subthreshold  doses  of  MIF. 

Eosinophils  are  known  to  be  involved  in  inflammatory  processes, 
particularly  those  allergic  in  nature.   For  this  reason,  some  mention 
should  be  made  of  research  in  this  area.   Gerald  Gleich  (AI  09728,  Mayo 
Clinic,  has  localized  the  basic  protein  of  the  eosinophil  to  the  core  of 
the  granule.   The  clinical  relevance  of  this  finding  is,  as  yet,  unclear. 
Thomas  T.  Hubscher  (AI  15001,  Georgetown  University)  has  discovered  that 
eosinophils  may  serve  to  dampen  the  allergic  response. 

Macrophages  which  are  differentiated  blood  monocytes  differ  from  the 
other  phagocytes  in  that  they  are  long  lived,  are  capable  of  continuing 
lysosomal  enzyme  synthesis,  and  may  further  differentiate  into  epithelioid 
cells  which,  in  turn,  may  fuse  to  form  multinucleated  giant  cells.   The 
technology  for  these  studies  has  been  advanced  by  the  work  of  Steven 
Douglas  (AI  12478,  University  of  Minnesota,  who  has  developed  a  technigue 
of  freeze-fracture  in  conjunction  with  electron  microscopy  making  it 
possible  to  study  macrophage  membranes  at  high  resolution.   In  particular, 
he  is  interested  in  developing  technigues  for  the  study  of  the  genetic 
regulation  of  their  plasma  membrane  receptors  for  immunoglobulin.   Victor 
Najjar,  AI  09116,  Tufts  University,  is  studying  a  peptide  called  tuftsin 
which  stimulates  phagocytosis.  He  is  looking  at  the  possible  curative 
ability  of  this  compound  in  animals,  especially  those  that  have  had  their 
spleens  removed. 


3-23 


GENETICS  AND  TRANSPLANTATION  BIOLOGY  BRANCH 

A.  Scope 

This  Branch  supports  and  manages  research  on  the  immunologic  factors 
that  determine  the  acceptance  or  rejection  of  grafts  and  on  the  genetic 
relationships  that  underlie  them. 

The  increasing  prevalence  of  degenerative  disease  and  of  trauma  that 
may  impair  the  function  of  particular  organs  sufficiently  to  be  incapac- 
itating or  life-threatening  identifies  the  perfection  of  a  successful 
transplantation  methodology  as  one  of  the  objectives  of  highest  priority 
in  current  medical  science.  The  technical  surgical  aspects  have  proved 
quite  manageable  but  immunological  rejection  of  the  graft  is  the  obstacle 
that  has  yet  to  be  surmounted. 

Research  efforts  supported  by  this  Branch  include: 

1.  Studies  of  strategies  designed  to  minimize  the  likelihood  of 
rejection  by  assuring  the  best  possible  antigenic  match  between 
donor  and  recipient. 

2.  Explorations  of  immunosuppressive  regimens  whose  objective  is  to 
reduce  the  capacity  of  the  host  to  reject  the  graft. 

3.  Studies  of  treatments  of  the  graft  that  will  reduce  its  ability 
to  provoke  a  response. 

Studies  in  animal  models  are  supported  that  explore  the  genetic 
controls  of  the  immune  response,  and,  on  a  more  practical  level,  test 
methodologies  whose  ultimate  application  is  to  man. 

Genetically  conditioned  associations  between  antigenic  characteristics 
and  susceptibility  to  various  diseases  have  recently  been  brought  to  light 
and  also  fall  within  the  purview  of  this  Branch. 

Two  important  activities  of  the  Branch  are  the  management  of  the 
Kidney  Transplant  Histocompatibility  Study  which  is  now  entering  the 
analysis  stage  and  the  management  of  the  HLA  Serum  Bank,  a  national  cell 
typing  resource  for  transplantation  as  well  as  basic  research  studies. 
Stocks  of  human  B  lymphocytes  are  being  accumulated  and  eventually  will  be 
made  available  to  the  scientific  community  for  use  in  immunological  studies 
and  histocompatibility  typing.  A  comparable  bank  of  mouse  typing  alloanti- 
sera  is  also  maintained  by  the  Branch. 

B.  Awards  and  Support  Levels 

The  Genetics  and  Transplantation  Biology  Branch  supports  research 
activities  through  individual  grants,  research  career  development  awards, 
fellowships,  training  grants,  and  very  importantly,  in  conncection  with  its 
kidney  transplant  program  and  serum  banks ,  through  contracts . 


4-1 


Research  Grants 

64 

Career  and  Career 

Development  Awards 

9 

Subtotal 

73 

Fellowship  Awards 

7 

Training  Awards 

1 

Subtotal 

8 

Contracts 

39 

Total 

120 

The  following  shows  the  distribution  of  support  for  the  activities  of 
the  Branch  during  FY  1979. 

Genetics  and  Transplantation  Biology  Branch 

FY '79  Awards 

Award  Mechanism  Number  Amount 

$  6,531,867 

300,783 
$  6,832,650 

78,760 

12,298 

$    91,058 

1,895,292 
$  8,819,004 

Total  costs,  estimated  using  a  40%  overhead  for  indirect  costs. 

The  majority  of  these  awards,  approximately  80%,  are  concerned  directly 
with  transplantation  biology  and  the  remainder  support  research  on  the 
genetics  of  the  immune  system  and  of  disease  associations.  Approximately 
30%  of  these  awards  were  for  competing  new  or  renewal  applications;  the 
remainder  represents  commitments  to  support  awards  made  in  prior  years. 

C.   Program  Areas  and  Highlights 

1 .   Immunogenetics 

An  immune  response  is  the  result  of  a  complex  set  of  events  involving 
recognition  of  the  antigen  and  of  interacting  cells  of  the  lymphoid  system 
and  of  macrophages.  Such  recognition  is  in  all  instances  determined  by  the 
genetic  constitution  of  the  responder.  On  it  depend  the  ability  to  resist 
infection,  the  ability  to  accept  or  reject  grafted  tissue,  and  suscepti- 
bility to  diseases  of  currently  uncertain  etiology,  many  of  which  appear  to 
have  an  immune  component. 

The  pattern  of  recognition  is  predominantly,  but  not  exclusively, 
determined  by  genes  organized  into  a  so-called  Major  Histocompatibility 
Complex  (MHC)  located,  in  all  mammals  studied  to  date,  on  a  specific  seg- 
ment of  a  specific  chromosome.   The  organization  of  the  MHC  is  best 
understood  in  the  mouse  and  in  man.   It  contains  genes  that  code  for  cell 
surface  proteins  that  act  as  strong  transplantation  antigens  because,  if 
the  compositions  of  these  proteins  are  disparate  in  the  donor  and  recipient 
of  a  transplanted  organ  or  tissue,  the  graft  is  usually  rapidly  rejected. 
On  the  other  hand,  certain  foreign  antigens  such  as  viruses  must  become 
associated  with  these  structures  to  elicit  an  immune  response  in  the  host. 


4-2 


These  major  transplantation  antigens  have  been  recognized  to  be  highly 
polymorphic  both  in  studies  on  rejection  of  grafts  and  by  their  ability  to 
stimulate  the  production  of  specific  antibodies.  Approximately  70  speci- 
ficities have  been  identified  at  3  loci  in  man  on  the  basis  of  serologic 
reactivities,  and  the  list  continues  to  grow. 

There  are  other  polymorphic  loci  within  the  MHC,  also  definable,  by 
the  ability  of  their  gene  products  to  stimulate  antibody  production  on 
alloimmunization.   These  antigens  have  been  designated  la  antigens  in  the 
mouse  and  DR  antigens  in  man.   The  loci  coding  for  them  are  clearly 
identical  with  or  very  closely  linked  to  genes  which  control  several  of  the 
components  of  the  immune  response  in  the  mouse  and  probably  also  in  man. 
Thus,  the  I-A,  I-B  and  I-C  control  helper  functions.   The  I-C  subregion 
also  controls  suppressor  functions  related  to  the  mixed  lymphocyte  reaction 
(see  below),  while  the  IE  subregion  codes  for  antigens  on  T  cells  capable 
of  generating  soluble  suppressor  factors.  Helper  and  suppressor  functions 
of  T  cells  are  also  identifiable  by  means  of  the  Ly  cell  surface  proteins, 
which,  however,  appear  to  represent  a  separate  system  of  differentiation 
antigens . 

Other  regions  within  the  MHC  control  the  ability  of  lymphocytes  to 
proliferate  in  response  to  exposure  to  other  lymphocytes  from  a  non- 
syngeneic  donor,  ultimately  producing  progeny  capable  of  lysing  the 
stimulator  cell.  The  proliferative  response  is  called  the  mixed  lymphocyte 
reaction  (MLR)  and  is  associated  with  the  leukocyte  activating  determinants 
(LAD),  the  principal  one  of  which  in  man  is  designated  the  D  locus. 

Because  of  their  role  as  transplantation  antigens,  much  effort  has 
gone  into  attempts  to  assure  their  identity  in  the  donor  and  recipient  of  a 
graft,  be  it  a  solid  organ  such  as  the  kidney  or  tissue  such  as  the  bone 
marrow.  Determination  of  the  characters  of  these  antigens  is  called  tissue 
typing.  The  success  of  such  efforts  both  in  attaining  good  matches  and  in 
the  prediction  of  the  acceptance  of  the  graft  is  limited  by,  on  the  one 
hand,  the  very  extensive  polymorphism  of  the  genes  and  antigens,  and,  on 
the  other  hand,  by  the  existence  of  very  numerous  other  loci,  as  many  as 
perhaps  500  in  the  mouse,  coding  for  so  called  "minor"  histocompatibility 
antigens,  whose  role  in  graft  rejection  becomes  prominent  when  mismatch  at 
the  MHC  is  absent. 

Research  on  the  MHC  is  currently  focused  on  three  areas.   Genetic 
studies  are  being  carried  out  on  recombinant  strains  of  mice  (Schreffler) 
to  characterize  the  topology  of  the  MHC  and  the  interdependence  among  the 
various  gene  products  in  the  expression  of  their  function.   For  example,  it 
has  become  evident  that  the  expression  of  the  structural  gene  which  codes 
for  the  I-A  region  antigen  is  controlled  by  a  gene  near  the  I-J  region 
(McDevitt) .   The  appearance  of  the  I-A  incoded  molecule  on  the  cell  surface, 
not  its  synthesis,  requires  the  presence  of  the  second  gene  either  on  the 
same  (cis-complementation)  or  on  the  other  (trans-complementation) 
chromosome  6 . 


4-3 


Such  studies  on  recombinants  make  use  of  highly  inbred  colonies  of 
mice.   Such  colonies  are  also  necessary  for  the  production  of  highly 
defined  mutants  at  the  MHC  and  other  histocompatibility  loci  (Melvold) . 
Such  mutants  become  an  important  resource  for  studies  of  structure- function 
and-antigenicity  relations  in  the  gene  products  when  the  mutations  involve 
single-amino  acid  changes  (Forman)  or  for  the  identification  of  loci  when 
the  mutations  are  deletions.   It  is  with  such  a  mutant  line  that  a  new 
mouse  MHC  locus  H-2  L  was  identified  very  recently  (Kohn  and  Melvold) . 

A  third  approach  to  the  study  of  genetic  regulation  of  the  MHC  employs 
wild  mice  (Klein).   This  permits  estimation  of  the  true  degree  of  poly- 
morphism of  the  genes  as  well  as  of  heterozygosity.  Thus,  the  finding  of 
very  high  levels  of  hetezygosity,  about  100%  at  the  K  and  D  loci  and  80%  at 
the  I-A  locus,  even  in  inbreeding  colonies  in  the  wild  strongly  implies  the 
existence  of  selective  pressures  that  may  well  be  associated  with  the 
observed  correlations  of  MHC  types  with  disease  (see  below).  Thus,  homo- 
zygosity might  accentuate  the  susceptibility  to  a  disease  associated  with  a 
particular  antigen  which  would  be  present  in  a  double  dose. 

The  second  area  in  which  much  activity  is  evident  is  that  of  the 
functional  roles  of  the  histocompatibility  antigens.   The  control 
mechanisms  ascribed  to  the  various  loci  or  sub- regions  of  the  I  region  of 
the  MHC  have  already  been  discussed  (David).   Such  a  thorough  dissection, 
analysis  and  correlation  with  function  of  a  chromosomal  region  represents  a 
most  significant  advance  in  mammalian  genetics  and  begins  to  approach  the 
level  insight  achieved  in  single  celled  organisms. 

The  genetic  control  of  the  immune  response  has  also  been  investigated 
through  immunizations  with  well-defined  antigens  in  animals,  where  control 
of  the  response  to  many  antigens  has  been  found  to  reside  in  the  I  region, 
or  through  associations  with  diseases  which  have  an  immunological  component 
with  MHC  antigens  in  man  (Amos,  Duquesnoy,  Fudenberg,  Hsia,  Marsh,  Stastny, 
and  Walford) .  Sufficient  progress  has  been  made  in  determining  the 
molecular  basis  for  the  involvement  of  MHC  gene  products  in  the  recognition 
and  control  of  the  response  to  antigens  in  the  mouse  to  permit  the  con- 
struction of  well  based  hypothetical  models.   It  appears  that  I-A  and  I-E 
region  products  must  associate  with  the  foreign  antigen  for  it  to  be 
effectively  recognized  by  the  afferent  limb  of  the  immune  system.   This 
also  seems  to  be  the  case  in  the  recognition  of  certain  viral  antigens 
which  must  interact  in  some  manner  with  the  host's  major  transplantation 
antigens  to  elicit  an  immune  response. 

The  products  of  other  I  region  genes  are  evidently  involved  in 
recognitions  among  the  regulatory  subsets  of  the  lymphocyte  population. 

The  required  associations  of  foreign  antigens  with  MHC  gene  products 
have  clear  implications  as  to  the  mechanisms  that  underlie  the  observed 
statistical  correlations  between  human  MHC  (HLA)  types  and  various 
degenerative  or  infectious  diseases.   Direct  investigations  of  these 
possible  mechanisms  are  now  much  needed. 


4-4 


The  third  area  of  intense  investigation  of  the  MHC  consists  of 
physical  and  chemical  studies  of  the  surface  antigens.   Such  studies  employ 
sensitive  separations  of  the  cell  surface  proteins  to  identify  the  extent 
of  the  similarities  and  differences  among  the  various  gene  products  and 
among  their  various  antigenic  forms  (Jones  and  McDevitt) ,  and  of  determina- 
tions of  exact  amino  acid  sequence  to  establish  evolutionary  patterns 
(Hood),  relations  to  other  products  of  the  immune  system  such  as  antibodies, 
or  the  chemical  basis  of  alloantigenicity.   In  this  connection,  it  might  be 
pointed  out  that  the  carbohydrate  moiety  of  the  HLA  antigens  is  not 
recognized  in  alloimmunizations  (Strominger) . 

2.   Transplantation 

The  major  application  of  studies  on  the  immunogenetics  of  man  remains 
transplantation.   The  principal  objective  is  to  so  reduce  the  antigenic 
disparity  between  donor  and  recipient  as  to  minimize  the  tendency  to  reject 
the  graft.  Until  recently  efforts  to  match  focused  on  two  serologically 
identified  loci  of  HLA,  A  and  B  (C  is  of  minor  importance),  and  at  the 
locus  identified  by  the  mixed  lymphocyte  reaction  (MLR),  D.  Matching  is 
very  clearly  highly  beneficial  in  transplants  among  related  donors  and 
recipients ,  as  is  matching  at  the  D  locus  in  renal  and  probably  bone  marrow 
transplantation.  The  inference  that  may  be  drawn  is  that  matching  a  living- 
related  donor  and  recipient  at  the  two  most  accessible  loci  is  effective 
because  it  also  implies  matching  along  the  entire  MHC  and  beyond.   Its 
utility  in  the  transplantation  of  organs  and  tissues  from  unrelated  donors 
is  much  less  clear  and  is  the  subject  of  continuing  debate.  As  a  result, 
the  search  continues  for  "the"  transplantation  antigen.   A  new  candidate 
has  appeared  in  the  field  of  kidney  transplantation:   the  product  of  the  DR 
locus .   This  region  is  very  closely  linked  to  the  D  locus  and  appears  to 
dominate  the  responses  observed  in  the  primed  lymphocyte  test  (PLT)  in 
which  the  proliferation  of  lymphocytes  stimulated  once  by  exposure  to 
allogeneic  cells  is  measured  following  a  second  exposure  (Dausset).  Matching 
at  the  DR  locus  has  been  found  very  promising  in  Europe,  where  matching  at 
the  A  and  B  loci  also  prolongs  graft  survival,  and  is  now  being  attempted 
in  the  U.S.   The  technical  aspects  of  the  procedure  are  difficult  and  a 
satisfactory  level  of  consistency  in  DR  typing  is  only  now  being  approached 
(Third  American  Workshop,  Fran  Ward,  Ed.).   An  extensive  international 
study  of  the  efficacy  of  DR  matching  in  renal  transplantation  is  being 
conducted  under  the  auspices  of  the  Eighth  International  Workshop  on  HLA 
Testing  (Terasaki) . 

The  limitations  of  the  approach  to  matching  at  the  MHC  by  typing 
lymphocytes  must  be  kept  clearly  in  mind,  however.   Thus,  there  are 
numerous  other  loci  on  various  chromosomes  that  code  for  polymorphic  cell 
surface  structures  which  can,  as  a  result,  serve  as  transplantation 
antigens.   The  red  blood  cell  antigens  are  an  obvious  example,  but  many 
other  examples  exist.   The  skin  displays  the  effects  of  mismatching  of 
these  antigens  most  clearly:  it  is  the  most  difficult  tissue  to  transplant 
because  it  is  at  highest  risk  of  rejection.   It  is  in  connection  with  these 
non-MHC  antigens  that  the  phenomenon  of  cyclic  variation  in  the  intensity  of 
the  response  became  apparent:   alternate  periods  of  stimulation  and 


4-5 


suppression  (Graff) .   This  variation  is  undoubtedly  not  restricted  to 
responses  to  non-MHC  alloantigens.  A  further  complication  is  introduced  by 
the  existence  of  developmental  or  tissue-specific  alloantigens,  for  example 
endothelial  and  skin  antigens  (Bailey  and  Sakai).   These  will  be  missed 
when  only  lymphocytes  are  typed  and  yet  will  cause  rejection  on  transplan- 
tation.  Clearly,  additional  knowledge  of  these  alloantigens  must  be  sought. 

Complete  matching  is  possible  only  in  identical  twins.   In  all  other 
cases,  survival  of  the  graft  is  totally  dependent  on  immunosuppression  of 
the  recipient.   The  current  mainstays  of  immunosuppression  are  adrenal 
cortical  steroids  and  the  cytotoxic  agent  azathioprine,  used  in  rather 
standardized  regimens.   The  philosophy  of  such  treatment  in  renal  trans- 
plantation is  shifting  towards  minimization  of  drug  doses  with  acceptance 
of  increased  risk  of  graft  loss  (Tilney) .   Concurrently,  there  is  a  search 
for  more  specific  immunosuppressants.   One  such  agent,  anti- lymphocyte  or 
thymocyte  globulin,  has  been  under  investigation  for  a  number  of  years, 
with  variable  results.  The  reason  may  well  be  the  highly  variable  potency 
of  this  biological  material.  This  difficulty  is  being  overcome  by  assessing 
the  potency  of  the  preparations  in  monkeys  and  by  carefully  monitoring  the 
lymphocyte  and  T  cell  (E-rosetting  cells)  levels  in  the  recipient  (F. 
Thomas  and  J.  Thomas).   A  further  advance  in  immunological  monitoring  is 
impending  as  a  result  of  the  development  of  reagents  (sera)  capable  of 
identifying  the  helper,  suppressor  and  effector  T  cell  subpopulations  in 
man  (Schlossman) .   In  addition  to  helping  tailor  drug  doses,  the  sera 
should  facilitate  the  elucidation  of  the  regulatory  mechanisms  that 
determine  the  fate  of  the  graft. 

An  apparently  still  more  highly  selective  immunosuppressant  coming 
under  investigation  at  this  time  is  the  drug  cyclosporin  A.   Its  mechanism 
of  action  is  not  yet  clear  but  appears  to  involve  suppression  of  the 
recipient's  cells  stimulated  specifically  by  the  graft. 

An  alternative  approach  to  immunosuppression  depends  on  irradiation  of 
the  recipient.   Studies  in  dogs  employing  supralethal  irradiation  followed 
by  infusion  of  autologous  bone  marrow  drawn  earlier  suggest  that  a  state  of 
high  susceptibility  to  tolerance  induction  results,  facilitating  the 
acceptance  of  the  graft  (Rapaport).   A  similar  state  of  susceptibility  to 
tolerance  induction  follows  the  less  drastic  total  lymphoid  irradiation  in 
which  the  marrow  of  the  skull  and  long  bones  and  the  lungs  are  spared 
(Strober).   This  state  applies  not  only  to  the  recipient  but  also  to  the 
donor  tissue  in  bone  marrow  transplantation  and  so  avoids  the  development 
of  graft-versus-host  disease  (GVHD).  This  approach  clearly  merits  further 
development,  and  TLI  is  currently  being  attempted  in  renal  transplantation 
at  the  University  of  Minnesota  (Simmons). 

At  present,  the  preparation  of  patients  for  bone  marrow  transplanta- 
tion with  radiation  and  cytotoxic  agents  would  be  lethal  were  not  donor 
marrow  given.  The  recipient  is  thus  at  risk  both  from  rejection  of  the 
marrow  and  from  the  development  of  GVHD.   The  two  principal  diseases 
treated  with  bone  marrow  transplantation  are  aplastic  anemia  and  acute 
leukemia.   In  the  former,  survival  rates  of  85%  are  attained  if  prior  blood 


4-6 


transfusions  are  avoided.  The  major  danger  is  of  rejection  of  donor  cells. 
In  acute  leukemia,  two  year  survivals  of  55-60%  have  been  achieved  if  the 
transplantation  is  performed  prior  to  the  end  stages  of  the  disease  (E.  D. 
Thomas).   The  major  advances  in  bone  marrow  transplantation,  other  than 
those  mentioned,  have  resulted  from  refinements  in  supportive  care. 

3.   Kidney  Transplantation  Histocompatibility  Study 

The  Kidney  Transplantation  Histocompatibility  Study  (KTHS)  is  a  pro- 
spective study  of  the  natural  history  renal  transplantation  carried  out 
under  contract  by  a  consortium  of  over  40  U.S.  and  Canadian  centers.  At 
its  inception,  its  principal  objective  was  to  determine  the  utility  of 
matching  at  the  A  and  B  loci.  However,  comprehensive  data  were  collected 
on  all  phases  of  renal  transplantation,  including  characterizations  of 
donors  and  recipients,  selection  procedures,  intraoperative  events,  post 
operative  management  and  outcome.  A  total  of  2434  transplants  were  entered 
into  the  study  in  1974-1976.   Complete  followup  continued  until  the  end  of 
1978,  with  some  additional  information  still  to  be  acquired  in  1979.   The 
data  to  the  end  of  1978  have  been  reviewed  and  corrected  and  are  currently 
being  analyzed  by  the  Naval  Medical  Research  Institute's  Data  Analysis 
Office  and  GTBB.   The  salient  conclusions  evident  at  this  point,  some 
previously  known,  include  the  following:   (a)  Graft  survival  is  substan- 
tially higher  when  the  donor  is  related  to  the  recipient.  This  advantage 
depends,  however,  on  the  adequacy  of  matching  at  the  A  and  B  loci.  In 
transplants  involving  related  donors  in  which  a  two  haplotype  match  is 
achieved,  two-year  graft  survival  is  about  85%,  as  against  about  50%  when 
the  donor  is  unrelated.   If  only  one  haplotype  match  is  attained,  results 
are  much  inferior  though  still  better  (about  65%)  than  in  the  latter  case. 
Matching  has  no  significant  effect  on  graft  survival  when  the  donor  is  an 
unrelated  cadaver,   (b)  The  age  of  the  recipient  has  an  important  effect  on 
survival  of  kidneys  from  cadaveric  donors,  significant  deterioration 
setting  in  beyond  age  30,  but  not  when  the  donor  is  living  and  related,  (c) 
The  effect  of  blood  transfusions  prior  to  transplantation  is  very  signifi- 
cantly beneficial.   The  improvement  in  graft  survival,  evident  even  after 
very  few  (less  than  5)  transfusions  is  not  counterbalanced  by  rapid  broad 
sensitization  of  the  recipient  with  consequent  increased  difficulty  in 
finding  a  cross-match  negative  donor.   (d)  The  results  of  transplantation 
even  of  kidneys  of  unrelated  cadaveric  donors  are  very  satisfactory  from  a 
personal  and  a  societal  point  of  view.   Patient  survival,  in  contrast  to 
graft  survival  of  cadaveric  kidneys,  is  over  80%  at  one  year  after  implan- 
tation. Moreover,  of  those  who  retain  a  functioning  kidney,  approximately 
70%  are  rehabilitated  and  return  to  fully  normal  activity.   (e)  Prior 
transplants  do  not  reduce  graft  survival  of  unrelated  cadaveric  grafts. 

The  second  phase  of  analysis  of  KTHS  data  which  will  involve  in-depth 
evaluation  of  specific  issues  in  renal  transplantation  will  be  carried  out 
under  contract  by  the  Harvard  School  of  Public  Health. 

It  is  also  hoped  that  the  formulation  of  additional  multi-center 
trials  and  studies  will  be  stimulated  as  a  result  of  information  generated 
by  KTHS.  One  such  trial,  of  immunological  pre  treatment  of  cadaveric  kidneys 


4-7 


with  cyclophosphamide,  has  been  proposed,  under  the  auspices  of  the 
American  Society  of  Transplant  Surgeons  and  will  shortly  undergo  review  for 
scientific  merit. 

4.   HLA  Serum  Bank 

The  HLA  Serum  Bank  is  an  international  resource  that  supports  all 
aspects  of  histocompatibility  testing.   Through  a  series  of  contracts  it 
acguires,  processes,  stores  and  distributes  human  sera  in  bulk  form  and  on 
trays.  Sera  are  also  acquired  by  direct  purchase  and  by  donation,  the  last 
accounting  for  approximately  one- third  of  the  current  inventory.   Bulk  sera 
are  made  available  predominantly  for  research  while  the  principal  use  of 
the  trays  is  in  clinical  applications,  chiefly  renal  and  bone  marrow  trans- 
plantation.  Sera  accepted  by  the  bank  are  subjected  to  careful  screening 
to  characterize  their  behavior  and  validate  their  utility.  They  thus  serve 
as  standards  for  the  research  community. 

During  the  past  year,  the  bank  distributed  13  liters  of  human  sera  in 
1  ml  freeze-dried  samples  to  150  U.S.  and  94  foreign  investigators.   These 
sera  were  used  in  studies  in  genetics,  immunology  and  anthropology,  among 
others.   A  continuing  application  of  great  interest  is  study  of  the 
association  of  various  diseases  with  HLA  types. 

The  Bank  has  also  recently  acquired  homozygous  typing  cells  which  will 
also  serve  as  reference  standards. 

Approximately  35  thousand  typing  trays  were  distributed  during  the 
past  year.  Previously  restricted  to  use  in  human  transplantation,  they  have 
now  also  been  made  available  for  the  characterization  of  panels  of  cells 
used  to  identify  sera  with  useful  specificities,  as  well  as  for  selected 
research  purposes.  As  a  result  of  recent  policy  decisions,  of  the  increas- 
ingly routine  clinical  use  of  sera  and  trays ,  and  of  our  increasing 
understanding  of  where  these  materials  can  be  used  to  greatest  advantage, 
investigational  applications  will  receive  a  higher  priority  and  efforts 
will  be  made  to  have  the  routine  clinical  aspects  provided  for  by  more 
appropriate  mechanisms.   In  this  connection,  the  typing  tray  is  being 
redesigned.   A  basic  tray  most  directly  useful  in  typing  related  graft 
donors  and  recipients  and  adequate  in  the  majority  of  other  applications 
will  be  produced  and  will  be  supported  by  a  more  limitedly  available  trays 
encompassing  the  rarer  specificities  and  useful  in  studies  of  special 
problems  and  populations. 


Grants  and  Contracts  Cited 

1.  Amos,  D.  Bernard,  Duke  University;  5  K06  AI-18399-17 ;  5  ROl  AI-08897-11 

2.  Bailey,  Donald  W.,  Jackson  Laboratory,-  5  ROl  AI-13130-04. 


4-8 


3.  Dausset,  Jean,  University  of  Paris;  2  R01  AI-09293-18. 

4.  David,  Chella  S.,  Mayo  Foundation;  5  R01  AI-14764-02. 

5.  Duquesnoy,  Rene  J.,  Milwaukee  Blood  Center;  5  R01  AI-12507-05. 

6.  Eighth  International  Histocompatibility  Task  Force  and  Workshop, 
Terasaki,  Paul,  Conference  Chairman,-  Contract  Pending. 

7.  Forman,  James  M. ,  University  of  Texas  Health  Science  Center;  5  R01  AI- 
13111-03. 

8.  Fudenberg,  H.  Hugh,  Medical  University  of  South  Carolina;  1  T32  AI- 
07063-01A2. 

9.  Graff,  Ralph  J.,  Jewish  Hospital  of  St.  Louis;  3  R01  AI-07437-llsl . 

10.  Hood,  Leroy  E.,  California  Institute  of  Technology,-  5  R01  AI-10781-08. 

11.  Hsia,  Shyuan,  Medical  College  of  Georgia;  1  ROl  AI-16180-01. 

12.  Jones,  Patricia  P.,  Stanford  University;  1  ROl  AI-15732-01. 

13.  Klein,  Jan,  Max  Planck  Institute  for  Biology;  5  ROl  AI-14736-02. 

14.  Kohn,  Henry,  Harvard  University,-  5  ROl  AI-14039-02. 

15.  Marsh,  David  G.,  Johns  Hopkins  University;  5  P01  AI-13370-03. 

16.  McDevitt,  Hugh  0.,  Stanford  University,-  5  ROl  AI-07757-13;  5  P01  AI- 
11313-07. 

17.  Melvold,  Roger  W.,  New  England  Deaconess  Hospital;  1  ROl  AI/CA-15969- 
01. 

18.  Naval  Medical  Research  Institute,  Kidney  Transplant  Histocompatibility 
Study;  2-Y01-AI-50001-06. 

19.  Rapaport,  Felix  T.,  State  University  of  New  York  Stony  Brook;  5  ROl 
AI-14453-03. 

20.  Sakai,  Akiyoshi,  Downstate  Medical  Center,-  5  ROl  AI-12824-03. 

21.  Schlossman,  Stuart  F.,  Sidney  Farber  Cancer  Institute;  2  ROl  AI-12069- 
06. 

22.  Schreffler,  Donald  C,  Washington  University,-  5  ROl  AI-12734-04;  1  T32 
AI-07163-01. 

23.  Simmons,  Richard  L.  ,  University  of  Minnesota  at  Minneapolis,-  5  ROl  AI- 
12754-05;  5  ROl  AI-14032-02. 


4-9 


24.  Stastny,  Peter,  University  of  Texas  Health  Science  Center  Dallas;  2 
R01  AI-12563-04. 

25.  Strober,  Samuel,  Stanford  University;  2  R01  AI-10293-08  Al . 

26.  Strominger,  Jack  L.,  Sidney  Farber  Cancer  Institute;  1  R01  AI-15669-01; 
5  R01  AI-10736-05. 

27.  Third  American  Workshop,  Frances  Ward,  Ed.;  NO1-TW-8-2110. 

28.  Tilney,  Nicholas  L. ,  Harvard  University;  5  R01  AI-12250-03. 

29.  Thomas,  Edward  D.,  University  of  Washington;  4  K06  AI-02425-16. 

30.  Thomas,  Francis,  Medical  College  of  Virginia;  2  R01  AI-12822-04. 

31.  Thomas,  Judith,  Medical  College  of  Virginia;  2  R01  AI/CA- 12586-04. 

32.  Walford,  Roy  L.,  University  of  California  at  Los  Angeles;  5  R01  AI- 
10089-09. 


4-10 


IMMUNOBIOLOGY  AND  IMMUNOCHEMISTRY  BRANCH 

A.  Scope 

This  Branch  is  concerned  with  the  biology  and  chemistry  of  the  immune 
system  and  its  products.   The  fundamental  studies  which  it  supports  on  the 
structure  and  function  of  the  immune  system  are  directed  toward  acquiring  a 
complete  understanding  of  immune  response  mechanisms  at  their  basic 
cellular  and  molecular  levels  as  they  function  in  health  and  disease. 

Activities  in  this  broadly-based  program  area  cross  traditional  disci- 
plinary lines  of  biology  and  chemistry  and  encompass  anatomic,  physiologic, 
pharmacologic,  and  microbial  biology  and  organic,  physical,  and  biological 
chemistry.   Its  scope  includes  studies  of  the  origin,  maturation,  localiza- 
tion, and  function  of  immunologically  active  cell  populations,  and  the 
mechanisms  involved  in  the  induction,  modulation,  regulation,  and  expres- 
sion of  immune  reactivity,  as  well  as  physicochemical  studies  of  antigens 
and  their  homologous  antibodies  and  the  mechanisms  and  kinetics  of  antigen- 
antibody  reactions. 

Research  activities  supported  within  this  program  area  are  grouped  in 
the  general  programs  for  Immunobiology  and  Immunochemistry  and  in  a  Program 
of  Institute  Emphasis  (PIE)  for  Lymphocyte  Biology. 

B.  Awards  and  Support  Levels 

Relevant  activities  in  immunobiology  and  immunochemistry  are  supported 
through  various  mechanisms  including  contracts,  individual  post-doctoral 
fellowships,  institutional  pre-  and  post-doctoral  training  grants,  career 
and  career  development  awards,  as  well  as  investigator-initiated  research 
grants . 

The  following  shows  the  distribution  of  support  by  award  mechanism  for 
the  activities  of  the  Branch  during  FY  1979. 

Immunobiology  and  Immunochemistry  Branch 
FY  1979  Awards 


Award  Mechanism 

Number 

Amount 

Research  Grants 
Career  and  Career 

Development  Awards 
Subtotal 

235 

28 

263 

$23,733,831 

660,146 

24,393,977 

Fellowship  Awards 
Training  Awards 

Subtotal 

19 

9 

28 

244,446 
687,721 
932,167 

Contracts 

Total 

5 
296 

25,609 
$25,351,753 

Total  costs,  estimated  using  a  40%  overhead  for  indirect  costs. 

5-1 


The  distribution  of  these  awards  by  discipline  is  approximately  3/4 
for  immunobiology  and  1/4  for  immunochemistry .  Approximately  35%  of  these 
awards  were  for  competing  new  or  renewal  applications;  the  remainder  repre- 
sents commitments  to  support  awards  made  in  prior  years. 

Support  for  the  Lymphocyte  Biology  PIE  is  included  in  the  research 
grant  category.  During  FY  1979,  five  program  project  awards,  at  a  total 
cost  of  $4,280,855,  were  made  to  support  this  activity. 

C.   Program  Areas  and  Highlights 

1.   Immunobiology 

This  program  is  concerned  with  the  processes  leading  to  immunocyte 
differentiation,  proliferation,  and  production  of  biologically-active  sub- 
stances which  mediate  immune  reactions. 

Research  supported  within  this  program  includes  studies  on  the  origin, 
maturation,  and  localization  of  immunologically  active  cell  populations, 
the  interactions  of  lymphocyte  subpopulations  and  their  interrelationships 
with  macrophages  and  other  leukocytes,  the  cellular  phenomena  of  antigen 
processing,  immunologic  tolerance  and  enhancement,  and  the  mechanisms 
involved  in  the  induction,  modulation,  and  regulation  of  immune  responses. 
The  cellular  mechanisms  involved  in  the  induction,  maintenance,  expression 
and  pathophysiology  of  delayed-type  hypersensitive  immune  reactivity  are 
also  included  in  this  category.   Also  relevant  are  studies  of  the  lympho- 
kines  and  other  lymphocyte  substances  which  activate  macrophages  and  have 
pharmacologic  effects  on  lymphocyte  and  other  leukocytic  cell  functions 
such  as  chemotaxis,  phagocytosis,  blastogenesis ,  cytotoxicity,  and 
microbial  resistance. 

Definition  of  the  origin,  differentiative  pathways,  and  functional 
roles  of  the  various  subpopulations  of  lymphocytes  continues  to  be  a  focus 
of  considerable  investigative  effort.   Although  it  is  recognized  that  one 
population  of  mature  lymphocytes  (T)  is  thymus  dependent  and  another  popu- 
lation (B)  is  thymus  independent,  their  progenitors  and  their  mechanisms  of 
functional  maturation  are  still  not  precisely  identified.   Studies  on  the 
ontogeny  of  lymphocytes  being  conducted  by  Irving  Goldschneider  (AI  09649 
and  AI  14743,  University  of  Connecticut  Health  Center)  are  centered  on  the 
identification  and  isolation  of  lymphohemopoietic  stem  and  progenitor  cells 
from  rat  bone  marrow.   Using  specific  antilymphocyte  sera  and  the  fluores- 
cence-activated cell  sorter,  he  has  enriched  preparations  of  pluripotent 
hemopoietic  stem  cells  and  thymocyte  progenitors  and  has  identified  two 
subsets  of  B  lymphocytes  which  represent  sequential  stages  in  B  cell 
development.   He  has  demonstrated  that  the  Thy-1  antigen,  and  T  cell  surface 
molecule,  is  present  on  the  least  mature  members  of  the  B  cell  series  but 
is  absent  on  mature  B  cells;  for  this  reason,  the  Thy-1  antigen  can  serve  as 
a  useful  marker  of  the  early  stages  of  B  cell  development.  He  also  has 
shown  that  a  subset  of  bone  marrow  cells  which  contain  the  enzyme  terminal 
deoxynucleotidyl  transferase  (TdT)  is  composed  almost  exclusively  of  thymo- 
cyte progenitors.  Other  data  indicate  that  nucleotide  metabolizing  enzymes 


5-2 


such  as  adenosine  deaminase  (ADA)  and  purine  nucleoside  phosphorylase  (PNP) 
play  important  roles  at  different  stages  of  T  lymphocyte  development.  Of 
interest  are  the  results  of  related  studies  in  man  by  Rochelle  Hirschhorn 
(AI  10343,  New  York  University)  who  has  found  that  approximately  one-half  of 
the  patients  with  severe  combined  immunodeficiency  disease  are  deficient  in 
ADA  and  PNP.   She  believes  that  these  enzymes  are  crucial  for  normal 
differentiation  and  function  of  immunocompetent  cells  and  suggests  that  the 
pathophysiology  of  this  disease  involves  a  primary  inhibition  of  T  cell 
maturation  and  a  toxic  effect  on  cells  differentiating  after  antigenic 
stimulation.   She  has  found  that  some  of  the  immunodeficient  and  enzyme 
deficient  patients  can  be  immunologically  reconstituted  by  infusion  of 
irradiated  erythrocytes  which  contain  ADA  and  has  proposed  that  deoxyadeno- 
sine  triphosphate  is  the  mediator  of  the  toxic  effect  in  ADA  deficiency. 
Defects  in  human  B  lymphocyte  development  also  have  clinical  expressions. 
Alexander  R.  Lawton,  III  (AI  11502,  University  of  Alabama  in  Birmingham) 
has  found  that  a  defect  in  differentiation  of  B  lymphocytes  from  their  pre-B 
cell  progenitors  occurs  in  one  form  of  congenital  agammaglobulinemia,  while 
failure  of  pre-B  cell  development  occurs  in  a  type  of  hypogammaglobulinemia 
associated  with  a  tumor  of  the  thymus  gland. 

The  results  of  other  ontogenic  studies  similarly  have  led  to  recogni- 
tion of  new  developmental  markers.   On  immunization  of  mice  with 
teratocarcinoma  cells,  Barbara  Knowles,  recipient  of  a  Career  Development 
Award  (AI  00053,  Wistar  Institute),  has  obtained  an  antibody  which  reacts 
with  embryonic  carcinoma  cells  of  both  mouse  and  human  origin  and  with  some 
preimplantation  stage  mouse  embryos.   She  believes  that  this  stage-specific 
antigen  on  the  cell  surface  is  a  hallmark  at  the  undifferentiated  state;  it 
is  first  detected  on  cells  of  the  8-cell  stage  embryos  and  is  lost  by 
embryonic  carcinoma  cells  when  they  differentiate.  Using  a  histochemical 
stain  for  viable  DNA,  Michael  R.  Loken  (AI  14872,  University  of  Chicago) 
has  developed  a  method  to  quantitatively  discriminate  between  viable  B  and 
T  lymphocytes  in  both  mouse  and  man.  He  believes  that  this  histochemical 
stain  will  serve  as  an  important  marker  to  differentiate  lymphocyte  subpop- 
ulations  based  on  their  cytochemical  composition  rather  than  on  their  cell 
surface  molecules.  The  results  of  preliminary  studies  of  mouse  bone  marrow 
cells  suggest  that  differentiating  lymphocytes  can  be  discriminated  on  this 
basis. 

Although  the  functional  distinction  of  lymphocyte  populations  is 
convincingly  documented,  the  purpose  and  mechanisms  of  their  interactions 
with  each  other  and  with  macrophages  is  still  being  examined.   It  is  clear 
that  B  lymphocytes  differentiate  to  become  antibody- secreting  plasma  cells 
and  that  T  lymphocyte  subsets  can  exert  helper  or  suppressor  effects  on  B 
lymphocytes  as  well  as  effector  functions  in  transplantation  reactions  and 
in  cell-mediated  immune  reactions  to  various  microbial  agents.  The  coopera- 
tive and  effector  functions  of  T  lymphocytes  have  been  found  to  correlate 
well  with  the  presence  of  a  cell  surface  molecule,  the  Ly  antigen.   It  also 
is  known  that  these  functions  are  controlled  and  regulated  by  other  cell 
surface  antigens  which  are  products  of  the  genes  in  the  major  histocompati- 
bility complex  (MHC).   The  regulatory  role  of  MHC  products  and  the 
functional  role  of  other  cell  surface  antigens  in  the  immune  response  have 
been  defined  most  extensively  in  the  mouse  although  systems  analogous  to 

5-3 


the  H-2  component  of  the  murine  MHC  have  been  identified  in  other  animals 
and  in  man. 

On  recognition  of  the  regulatory  role  which  the  MHC  exerts  on  the 
immune  system,  efforts  to  clarify  the  responsible  mechanisms  have  intensi- 
fied. To  facilitate  research  in  this  area,  the  program  has  contractually 
supported  the  acquisition  and  distribution  of  antisera  with  specificities 
against  various  mouse  cell  surface  antigens.  A  supply  of  antiserum  against 
many  of  the  H-2  gene  products  has  been  available  and  antisera  specific  for 
antigens  of  the  I  region  of  the  H-2  complex  have  recently  been  prepared  by 
Donald  C.  Shreffler  (AI  6-2502,  Washington  University)  for  distribution  to 
investigators. 

Evidence  obtained  in  several  experimental  systems  has  convincingly 
demonstrated  that  efficient  physiological  interactions  among  macrophages,  T 
cells,  and  B  cells  require  that  these  cells  share  membrane  molecules 
encoded  for  by  the  major  histocompatibility  complex  of  the  species.   David 
H.  Katz  (AI  13874,  Scripps  Clinic  and  Research  Foundation)  has  provided 
substantial  evidence  that  the  genes  controlling  interactions  between  T  and 
B  lymphocytes  are  located  in  the  I  region  of  the  mouse  H-2  complex.  He  has 
found  that  T  lymphocytes  will  not  exert  effective  helper  functions  for  B 
cells  when  these  cells  differ  at  the  relevant  I  region  locus.  He  believes 
that  self-recognition  is  the  critical  mechanism  by  which  cell-cell  communi- 
cation takes  place  in  the  immune  system.   In  a  related  project  (AI  13781), 
he  has  obtained  evidence  indicating  that  the  process  of  differentiation  of 
stem  cells  and  their  progeny  also  may  be  critically  regulated  by  MHC  gene 
products.   Using  bone  marrow  chimeras,  he  has  demonstrated  that  lymphocyte 
differentiation  may  be  "adaptive"  to  the  environment  in  which  it  takes 
place.   He  believes  such  an  "adaptive  differentiation"  results  in 
preferential  interactions  among  cells  that  have  undergone  their  differen- 
tiative  processes  in  the  same  genotypic  environment;  "adaptive 
differentiation"  thus  may  represent  a  manipulative  approach  to  define 
genetic  and  molecular  mechanisms  responsible  for  cell-cell  interactions. 

Genetic  restrictions  imposed  by  products  of  the  I  region  of  the  H-2 
complex  on  interactions  between  macrophages  and  immune  T  cells  are  being 
investigated  by  Carl  W.  Pierce  (AI  13915,  Jewish  Hospital  of  St.  Louis). 
Using  antigen  pulsed  macrophages,  he  has  found  that  naive  lymphocytes  will 
respond  to  antigen  even  if  it  is  presented  on  an  histoincompatibile  macro- 
phage.  However,  the  secondary  immune  response  is  genetically  restricted; 
the  immune  T  cell  must  be  stimulated  with  antigen-bearing  macrophages  which 
are  genetically  identical  to  the  macrophage  that  presented  the  antigen 
during  the  primary  immunization.   He  has  demonstrated  that  such  genetic 
restrictions  are  controlled  by  products  of  the  I-A  subregion  of  the  H-2 
complex  and  believes  that  these  restrictions  involve  active  suppressive 
phenomena  when  antigen  is  presented  on  an  inappropriate  macrophage.   In  a 
related  study,  Judith  A.  Kapp-Pierce  (AI  13987,  Jewish  Hospital  of  St. 
Louis)  has  found  that  an  antigenspecific  suppressor  T  cell  factor  is 
activated  when  nonresponder  lymphocytes  are  stimulated  by  antigen;  suppres- 
sion by  this  factor  is  mediated  by  a  molecule  encoded  by  the  I-J  subregion 
of  the  H-2  complex.   She  also  has  shown  that  this  suppressor  factor  does  not 
require  participation  of  macrophages  for  effective  suppression;  she 

5-4 


suggests  that  the  suppressor  factor  acts  directly  on  primed  B  cells.  The 
results  of  similar  studies  by  Donal  B.  Murphy  (AI  14349,  Yale  University) 
suggest  that  the  different  functional  subpopulations  of  immunocompetent 
lymphocytes  express  different  products  of  the  I  region  on  their  surface. 
He  has  demonstrated  that  suppressor  T  cells  bear  an  I  region  antigen  not 
found  on  suppressor  T  lymphocytes.   Of  interest  is  his  observation  that, 
although  different  T  cell  subpopulations  bear  distinct  I  region  products, 
all  or  many  of  these  products  appear  to  be  expressed  on  the  surface  of  the 
macrophage  so  that  its  interaction  with  these  lymphocytes  would  not  be 
encumbered  by  this  type  of  genetic  restriction. 

The  mechanisms  by  which  antigens  are  presented  to  T  cells  by  macro- 
phages to  initiate  the  immune  response  are  being  examined  by  David  W. 
Thomas  (AI  14226,  Jewish  Hospital  of  St.  Louis).  He  is  using  macrophages 
modified  with  trinitrophenyl  (TNP)  as  a  model  to  study  the  "immunologically 
relevant"  processing  of  antigen  by  macrophages  which  is  necessary  for  its 
subsequent  recognition  by  T  cells  and  which  is  different  from  the  degrada- 
tive  and  metabolic  breakdown  of  antigen  by  macrophages.   His  results 
indicate  that  T  cell  recognition  of  TNP  modified  macrophages  does  not 
simply  involve  a  surface  display  of  TNP  but  that  macrophages  somehow 
process  TNP  to  create  an  efficient  immunogen.   He  has  concluded  that  T 
cells  recognize  and  respond  to  macrophage-bound  antigen  only  when  it  is 
associated  with  surface  products  of  the  I  region  of  the  MHC.  His  physioco- 
chemical  studies,  however,  have  provided  evidence  that  TNP  is  not  directly 
conjugated  to  macrophage  I  region  products.   On  this  basis,  he  believes 
that  antigen  processing  by  macrophages  involves  a  physical  association  of 
antigen  with  I  region  products  rather  than  an  alteration  of  "self"  by 
chemical  linkage  of  antigen  with  I  region  products. 

Although  it  is  recognized  that  physical  contact  between  macrophages 
and  lymphocytes  is  necessary  in  the  immune  response,  there  is  considerable 
evidence  that  each  of  these  cell  types  produce  factors  which  also  are 
involved  in  the  initiation  of  the  immune  response.   Byron  H.  Waksman  (AI 
06455,  Yale  University),  for  example,  has  demonstrated  that  the  lymphocyte 
activating  factor  which  stimulates  helper  T  cells  is  a  macrophage  enzyme. 
He  has  provided  evidence  to  indicate  that  this  enzyme  alters  lymphocyte 
surfaces  so  that  calcium  incorporation  is  stimulated  and  cyclic  nucleotide 
activity  is  consequently  enhanced.   In  a  reciprocal  fashion,  Michael  P. 
Rabinovitch  (AI  10969,  New  York  University),  has  demonstrated  that 
interferon,  an  antiviral  agent  produced  by  lymphocytes,  controls  the 
phagocytic  functions  of  macrophages.  He  has  demonstrated  that  macrophages 
obtained  from  animals  treated  with  interferon  inducers  exhibit  enhanced 
phagocytosis  while  macrophages  obtained  from  mice  treated  with  an  antibody 
which  neutralized  interferon  had  diminished  phagocytic  activity.  Interferon 
also  has  been  shown  to  have  immunoregulatory  effects  on  lymphocytes;  Barry 
R.  Bloom  (AI  09807,  Albert  Einstein  College  of  Medicine)  has  found  that 
interferon  was  produced  and  generated  suppressor  T  cells  when  lymphocytes 
of  normal  individuals  were  cultured  with  measles  virus.  Of  interest  is  his 
observation  that  lymphocytes  from  patients  with  multiple  sclerosis  failed 
to  produce  interferon  when  similarly  exposed  to  measles  virus.  He  believes 
this  selective  defect  in  response  to  measles  virus  in  patients  with 


5-5 


multiple  sclerosis  may  play  a  significant  role  in  the  persistence  of  this 
disease. 

Cell-cell  interactions  are  required  not  only  to  initiate  the  immune 
response  but  also  to  regulate  it.   It  is  now  recognized  that  the  help  and 
suppression  provided  by  T  cell  subsets  is  carefully  balanced  within  the 
responding  host.  As  demonstrated  by  Richard  K.  Gershon  (AI  10497,  Yale 
University),  helper  T  cells,  on  stimulating  B  cells  to  produce  antibody, 
also  become  susceptible  to  regulation  through  a  feedback  inhibition 
mechanism  by  inducing  suppressor  T  cells.   In  collaboration  with  Harvey 
Cantor  (AI  13600,  Sidney  Farber  Cancer  Institute),  the  functional  subsets 
have  been  identified  on  the  basis  of  differentiation  molecules,  termed  Ly 
antigens,  present  on  their  surfaces.  Thus,  helper  cells  have  been  shown  to 
be  Ly  1+  while  suppressor  cells  are  Ly  2 ,  3+.   Cantor  recently  has  provided 
convincing  evidence  that  the  Ly  1  helper  cell  can  induce  an  uncommitted  Ly 
1,  2,  3+  subset  to  participate  in  specific  suppressor  activity.   In  this 
context,  Gershon  believes  from  his  current  studies  on  the  cellular  feedback 
inhibition  mechanism  that  suppressor  dysfunction  is  a  cause  of  at  least 
some  autoimmune  diseases.   He  acknowledges,  however,  that,  in  some  auto- 
immune diseases,  the  aberrant  activity  of  suppressor  T  cell  is  not  due  to 
inherent  dysfunction  of  that  cell  but  due  to  defects  in  other  cells  that 
regulate  it  or  are  regulated  by  it. 

Although  research  on  functional  subsets  of  T  lymphocytes  is  proceeding 
at  an  accelerated  pace,  it  is  recognized  that  this  work  is  technically 
limited  by  the  need  to  separate  the  subsets  in  adequate  numbers  for  study. 
This  difficulty  may  be  obviated  by  the  recent  work  of  Frank  W.  Fitch  (AI 
04197,  University  of  Chicago)  who  has  developed  a  method  to  isolate  and 
culture  cloned  lines  of  reactive  T  cells ,  including  separate  lines  with 
distinct  helper  and  suppressor  activities.   In  a  similar  fashion,  research 
in  this  area  should  be  facilitated  with  the  Program's  acquisition  of  a 
supply  of  antisera  against  Ly  antigens  which  now  are  available  for  distri- 
bution to  qualified  investigators.   These  sera  were  prepared  under  contract 
through  the  efforts  of  Jeffrey  A.  Frelinger  (AI  7-2528,  University  of 
Southern  California).   Development  of  methods  and  materials  for  preparing 
antisera  against  newly- recognized  Ly  antigens  and  other  immunologically- 
relevant  cell  surface  molecules  also  is  being  supported  through  a  contract 
with  Edward  A.  Boyse  (AI  8-2541,  Sloan  Kettering  Institute  for  Cancer 
Research) . 

Approaches  to  obtain  helper  and  suppressor  factors  from  T  cell  subsets 
also  are  being  explored.   Sirkka  K.  Kontiainen  (AI  13145,  University  of 
Helsinki)  has  developed  methods  to  obtain  active  suppressor  factor  from 
cultured  mouse  suppressor  T  cells.   Using  anti-Ly  antiserum,  she  has  demon- 
strated that  the  target  for  suppression  is  the  Ly  1+  helper  T  cell  and  has 
characterized  the  suppressor  factor  as  having  both  an  antigen  combining 
site  and  a  determinant  encoded  by  the  I  region  of  the  H-2  complex.  Using  a 
similar  approach.  Marc  Feldmann  (AI  15653,  University  College,  London)  has 
obtained  a  helper  factor  from  cultured  human  peripheral  blood  lymphocytes 
and  has  developed  an  assay  for  its  activity  using  mouse  spleen  cells.   This 
technology  will  permit  analysis  of  human  lymphocyte  immunoregulatory 


5-6 


function  in  disease  states  and  should  facilitate  attempts  to  selectively 
manipulate  the  immune  response  in  immunologically  impaired  individuals. 

In  addition  to  the  factors  produced  by  T  cells  which  have  immuno- 
regulatory  functions,  another  group  of  lymphocytes  products,  termed 
lymphokines,  is  receiving  considerable  investigative  attention.   Lympho- 
kines  have  been  recognized  as  being  the  mediators  of  a  variety  of 
immunologic  reactions  including  inflammation,  graft  rejection,  tumor  and 
other  cell  killing,  and  delayed  hypersensitive  reactions.  The  cellular  and 
molecular  mechanisms  of  action  of  lymphotoxin,  a  cell  lytic  effector,  are 
being  investigated  by  Gale  A.  Granger  (AI  09460,  University  of  California 
at  Irvine).  He  recently  has  found  that  human  lymphotoxin  molecules  form  an 
interrelated  system  of  cell  toxins  which  is  composed  of  noncovalently 
associated  subunits.   He  has  identified  three  separate  types  of  molecules 
in  this  system;  it  contains  lytic  subunits,  condensing  molecules  which 
facilitate  association  of  the  subunits,  and  antigen  binding  receptors.  He 
has  found  all  three  types  of  molecules  are  released  predominantly  from  T 
lymphocytes  and  that  the  lytic  action  of  the  molecular  complex  is  directed 
by  the  component  containing  the  antigen  binding  receptors.   In  similar 
studies  of  another  human  lymphokine,  macrophage  migration  inhibition  factor, 
Stanley  Cohen  (AI  13258,  University  of  Connecticut  Health  Center)  has 
obtained  evidence  that  it  also  may  consist  of  subunits,  noncovalently 
associated  into  an  active  complex.   In  a  related  study  (AI  12477),  he  is 
exploring  ways  to  therapeutically  alter  lymphokine-mediated  reactions  in 
the  intact  host.   He  has  found  that  various  monosaccharides  which  are 
capable  of  inhibiting  lymphokine  activity  in  vitro  also  are  effective  in 
suppressing  in  vivo  manifestations  of  cell-mediated  immunity.   For  example, 
L-fucose  was  demonstrated  to  inhibit  the  delayed  hypersensitive  skin 
reaction  induced  by  antigen  in  actively  immunized  guinea  pigs.   Furthermore, 
he  and  Takeshi  Yoshida,  recipient  of  a  Career  Development  Award  (AI  00082, 
University  of  Connecticut  Health  Center),  have  been  able  to  induce  mediator 
production  in  vivo  and  abolish  delayed  hypersensitive  skin  reactivity  in 
actively  immunized  guinea  pigs.   They  found  that  desensitization  could  be 
passivley  transferred  using  mediator-containing  serum  from  desensitized 
animals;  the  results  of  physicochemical  studies  of  such  sera  excluded  the 
participation  of  antibodies  or  immune  complexes  and  suggested  that  the 
desensitizing  substance  may  represent  another  type  of  endogenous 
lymphokine . 

2.   Immunochemistry 

Research  grouped  in  this  program  category  centers  on  the  defined 
molecular  components  of  the  immune  system  and  utilizes  chemical  and  physico- 
chemical  approaches  for  study  of  immunologic  reactants  and  reactions. 
Studies  on  the  chemical  composition  and  structure  of  humoral  and  secretory 
immunoglobulins  and  of  natural  and  synthetic  antigens,  as  well  as  of  the 
mechanisms  and  kinetics  of  antigen-antibody  reactions  and  of  the  mechanisms 
of  immunoglobulin  biosynthesis  and  its  regulation,  are  included  within  this 
program  area.   Also  relevant  are  studies  at  the  molecular  level  of  cell 
surface  components  related  to  immunologic  activity  and  of  subsurface  cyto- 
skeletal  and  contractile  assemblies  believed  to  play  a  role  in  cellular 
activation.   In  addition,  the  newly-developing  field  of  immunopharmacology , 


5-7 


which  is  concerned  with  the  identification,  characterization,  isolation,  or 
synthesis  of  chemical  regulators  of  immune  function  and  the  elucidation  of 
their  mechanisms  of  action  is  supported  in  this  program  area. 

A  major  aim  of  studies  supported  by  this  program  is  to  elucidate  the 
primary  structure  of  antibodies  and  to  correlate  their  structure  with  their 
antigen  binding,  specificity,  and  other  biological  functions.   The  results 
of  structural  studies  have  established  that  an  immunoglobulin  basically 
consists  of  two  identical  light  (L)  and  two  identical  heavy  (H)  chains, 
linked  by  disulfide  bridges  to  form  a  two-fold  symmetrical  molecule,  each 
side  of  which  consists  of  an  L  and  an  H  chain.   Two  clases  of  L  chains 
(kappa  and  lambda)  and  five  H  chains  (gamma,  alpha,  mu,  episolon  and  delta), 
which  determine  the  immunoglobulin  class  (IgG,  IgA,  IgM,  IgE,  and  IgD, 
respectively)  have  been  recognized.   Limited  proteolysis  of  a  typical  IgG 
molecule  results  in  three  fragments  of  nearly  egual  size,  2  Fab  fragments 
and  an  Fc  piece.   It  is  now  clear  that  the  Fab  components  contain  the 
antigen  binding  (combining)  site  and  that  the  Fc  fragment  plays  an  impor- 
tant role  in  complement  fixation.  Amino  acid  sequence  studies  of 
immunoglobulins  have  demonstrated  the  existence  of  three  domains  with 
constant  sequences  on  the  heavy  chain  (C  )  and  one  domain  of  variable 
sequence  (V  ) ,-  each  light  chain  consists  or  two  domains,  a  constant  region 
(C  )  and  a  variable  domain  (V.).   The  close  association  of  the  V  and  V 
domains  in  the  Fab  fragment,  containing  the  combining  site,  produces  a 
continuous  hypervariable  surface  which  can  be  readily  altered  by  amino  acid 
substitution,  insertions  or  deletions,  and  provides  an  extremely  large 
number  of  structural  combinations  which  are  believed  to  confer  immunologic 
specificity. 

Considerable  effort  now  is  being  expended  to  gain  an  understanding  of 
the  mechanisms  controlling  and  regulating  immunoglobulin  biosynthesis  and 
of  the  basis  for  antibody  diversity.   Attempts  to  explain  the  diversity  of 
variable  region  sequences  have  centered  on  a  multiple  germ- line  gene  theory 
which  presupposes  that  all  information  necessary  for  antibody  production  is 
encoded  in  the  germ  line  and  is  inherited  or  on  a  theory  of  somatic 
diversification  which  assumes  that  a  limited  number  of  germ-line  genes  are 
diversified  during  an  individual's  lifetime  by  somatic  processes,  such  as 
point  mutations,  recombinations  or  introduction  of  errors  into  the  gene 
sequence,  followed  by  repair  to  yield  a  new  set  of  encoding  genes. 

Amino  acid  sequence  analysis  and  structural  studies  provide  one 
approach  to  understanding  immunoglobulin  diversity.   The  results  of  studies 
by  Fred  Karush  (AI  09492,  University  of  Pennsylvania)  support  the  view  that 
the  V  genes  expressed  in  murine  IgG  antibody  are  encoded  in  the  germ  line. 
A  contract  project  under  the  direction  of  Elvin  A.  Kabat  (AI  8-2158, 
Columbia  University)  in  collaboration  with  Tai  T.  Wu,  recipient  of  a  Career 
Development  Award  (AI  70497,  Northwestern  University),  supports  the  tabu- 
lation and  analysis  of  immunoglobulin  sequence  data  reported  in  the  world's 
scientific  literature.   Using  the  PROPHET  Computer  System,  this  project 
provides  the  concerned  scientific  community  with  a  unique  and  invaluable 
data  resource  and  serves  as  a  source  of  data  for  molecular  model  building 
and  statistical  predictions  of  immunoglobulin  molecular  structures.   The 
accumulated  data  base  on  immunoglobulin  variable  region  sequences  has  been 

5-8 


published  and  a  series  of  computer-assisted  analyses  of  the  data  base  has 
been  completed.   Evidence  has  been  obtained  to  suggest  that  the  amino  acid 
sequence  characteristic  of  segments  in  the  variable  region  of  an  immuno- 
globulin is  uniquely  associated  with  antibody  specificity.   Computer- 
generated  inferrences  concerning  amino  acids  at  key  positions  in  light  and 
heavy  chains  agreed  well  with  structural  conclusions  obtained  by  X-ray 
crystallography.  Additional  predictions  of  the  three  dimensional  structure 
of  the  combining  site  have  led  to  the  suggestion  that  the  structural  gene 
for  the  variable  region  of  an  antibody  may  be  assembled  from  "mini  genes" 
which  have  been  conserved  in  the  germ-line.  Support  for  the  germline  theory 
also  has  been  provided  from  studies  conducted  by  Joseph  M.  Davie  (AI  11635, 
Washington  University)  who  interprets  the  uniformity  of  isoelectric 
focusing  patterns  of  rat  antibodies  as  being  consistent  with  the  view  that 
these  antibodies  are  products  of  germ-line  genes. 

In  contrast,  using  antibodies  prepared  against  the  combining  site,  or 
idiotype,  of  an  immunoglobulin,  Alfred  Nisonoff  (AI  12895,  Brandeis 
University)  believes  that  it  is  not  necessary  for  a  mouse  to  inherit  V 
structural  genes.  He  feels  that  antibody  diversity  can  be  generated  through 
a  random  process  of  somatic  mutation,  rather  than  through  a  programmed, 
genetically-controlled  series  of  mutations.   The  results  of  his  related 
studies  (AI  12907  and  AI  12908)  are  providing  strong  evidence  for  the 
importance  of  an  idiotype-antiidiotype  network  as  a  mechanism  regulating  the 
immune  response.   He  believes  that  the  idiotypespecific  regulation  by 
antiidiotype  antibody  and  by  suppressor  T  cells  is  an  important  physio- 
logical mechanism  determining  the  magnitude  of  a  given  immune  response. 
The  results  of  research  by  Matthew  D.  Scharff  (AI  05231,  Albert  Einstein 
College  of  Medicine)  and  Malcolm  L.  Gefter  (AI  13357,  Massachusetts 
Institute  of  Technology)  support  the  view  that  immunoglobulin  diversity  is 
generated  by  somatic  diversification.  Studies  of  immunoglobulin  synthesis 
at  the  level  of  the  nucleus  of  a  cell  also  has  served  as  an  approach  to 
evaluate  theories  of  immunoglobulin  diversity.  The  results  of  sequence 
studies  of  DNA  and  messenger  RNA  and  of  their  function  in  immunoglobulin 
synthesis  conducted  by  several  investigators  including  Ursula  B.  Storb  (AI 
10685,  University  of  Washington),  Randolph  Wall  (AI  13410,  University  of 
California  at  Los  Angeles),  and  Janet  M.  Stavnezer  (14617,  Sloan-Kettering 
Institute  for  Cancer  Research)  are  difficult  to  reconcile  with  a  strict 
germ-line  theory  and  argue  for  a  somatic  process  of  immunoglobulin 
diversification. 

Efforts  are  now  being  made  to  extend  such  studies  to  man  although  they 
are  still  in  their  preliminary  stages.  Moyra  Smith-Wright  (AI  14287,  Mt. 
Sinai  School  of  Medicine),  using  a  somatic  cell  hybridization  technique, 
has  demonstrated  that  the  structural  genes  encoding  IgG  heavy  chains  are 
located  on  human  chromosome  6.   Using  a  serologic  approach  to  examine  the 
diversity  and  inheritance  of  immunoglobulin  genes,  An  Chuan  Wang  (AI  13388, 
Medical  University  of  South  Carolina)  has  discovered  a  new  genetic  marker 
which  is  located  in  the  V   region  of  human  immunoglobulins  G,  M,  and  A. 
This  observation  represents  the  first  description  of  a  human  immunoglobulin 
variable-region  genetic  marker;  the  results  of  pedigree  and  population 
analyses  suggests  that  it  has  a  dominant  mode  of  inheritance. 


5-9 


Although  sequence  and  serologic  studies  of  serum  immunoglobulins  are 
not  generally  restricted  by  the  availability  of  material  for  study,  similar 
studies  of  cell-bound  immunoglobulins  and  other  cell  surface  receptor 
molecules  have  indeed  been  limited  by  the  small  amounts  of  material 
available  for  study.   Recent  technical  advances  may  obviate  this  problem, 
however.  Gerald  M.  Edelman  (AI  09273,  Rockefeller  University)  had  developed 
a  system  to  synthesize  authentic  mouse  S --microglobulin  and  its  precursor 
in  rabbit  reticulocytes  using  messenger  RNA  from  murine  tumor  cells;  its 
authenticity  has  been  verified  by  radiochemical-sequence  analysis.   The 
same  system  is  now  being  used  to  synthesize  H-2  antigens,  TL  antigens,  and 
other  cell  surface  proteins  found  on  lymphoid  cells.   Similarly,  Sheldon 
Dray  (AI  04073,  University  of  Illinois  Medical  Center)  has  been  employing 
liposomes,  synthetic  micellar  structures  which  mimic  cell  surface  lipid 
structures,  to  selectively  insert  functional  messenger  RNA  into  cultured 
cells.   He  recently  has  obtained  evidence  which  indicates  that  cultured 
human  epithelial  carcinoma  cells  treated  with  liposomally-encapsulated 
rabbit  globin  messenger  RNA  are  stimulated  to  produce  a  globlin-like 
protein.  Matthew  D  Scharff  (AI  05231,  Albert  Einstein  College  of  Medicine) 
is  employing  somatic  cell  genetic  technology  to  refine  the  Kohler-Milstein 
technique  for  production  of  hybridomas,  the  product  of  fusing  malignant 
plasma  cells  and  normal  antibody- forming  cells.  He  has  developed  an  exten- 
sive library  of  drug-marked  myeloma  cells  for  use  in  fusion  experiments  and 
has  adapted  the  fusion  technology  to  significantly  improve  the  frequency  of 
functional  hybridomas.   Further  application  of  the  hybridoma  technology,  as 
well  as  the  insertion  and  translation  of  messenger  RNA  in  cultured  cells, 
has  the  potential  to  provide  an  unlimited  source  of  homogeneous  antibody 
and  other  immunologically- important  molecules  for  study.  Application  of 
recombinant  DNA  technology  to  this  problem  can  similarly  be  expected  to 
expand  the  supply  of  study  material.   Randolph  Wall  (AI  13410,  University 
of  California  at  Los  Angeles)  already  has  successfully  inserted  mouse 
myeloma  immunoglobulin  DNA  into  a  bacterial  plasmid  and  has  isolated 
recombinant  clones  which  contained  all  of  the  light  chain  constant  and 
variable  region  coding  sequences. 

Efforts  to  define  and  understand  the  molecular  basis  of  antigenicity 
and  of  the  antigenic  or  determinant  site  similarly  has  progressed  by  appli- 
cation of  amino  acid  sequence  analysis.   For  example,  M.  Zouhair  Atassi  (AI 
13181,  Mayo  Foundation)  and  Ahmed  F.  Habeeb  (AI  14791,  University  of  Puerto 
Rico)  have  employed  hen  egg  white  lysozyme  as  a  model  antigen  and  have 
systematically  sequenced  it.   They  have  identified  three  peptide  fragments 
which  contain  the  antigenic  sites  of  this  protein.   Using  a  method  of 
surface  simulation,  they  have  synthesized  peptides  containing  the  component 
amino  acid  residues  in  appropriate  structural  order  and  have  been  able  to 
accurately  define  the  voundary,  residues,  and  conformational  structure  of 
the  three  determinants  of  the  native  protein.  Their  results  clearly  demon- 
strated that  these  three  sites  quantitatively  accounted  for  the  total 
antigenic  reactivity  of  the  native  protein.   Thus,  the  entire  antigenic 
structure  of  lysozyme  has  been  precisely  defined.   In  a  related  study,  Eli 
E.  Sercarz  (AI  11183,  University  of  California  at  Los  Angeles  has  demon- 
strated that  peptide  fragments  containing  these  determinants  can  be 
distinguished  by  their  different  immunologic  functions.   The  L,  peptide, 
containing  amino  acid  residues  1-12,  induced  a  suppressor  cell  response ,- 

5-10 


the  L„  peptide  containing  amino  acids  13-105  induced  helper  cells  while  the 
L_  peptide  had  no  effect.   This  demonstration  of  helper  and  suppressor 
sites  on  the  same  molecule  provides  a  powerful  tool  to  investigate  the 
cellular  mechanisms  of  immune  recognition. 

A  somewhat  different  approach  to  the  problem  of  antigenicity  is  being 
employed  by  Conrad  Scheurch  (AI  12509,  State  University  of  New  York  at 
Syracuse)  who  has  prepared  a  variety  of  synthetic  polysaccharides,  modeled 
after  the  naturally  occurring  bacterial  polysaccharide  dextran  as  well  as 
yeast  and  fungal  mannans.  In  addition  to  providing  synthetic  antigens  with 
well  defined  chemical  structure  for  immunologic  study,  this  work  is 
purposely  designed  to  identify  clinically  useful  plasma  expanders,  like 
dextran,  which  would  be  safer  for  use  because  they  do  not  possess  dextran' s 
allergenic  properties.   These  synthetic  polysaccharides  are  now  being 
examined  for  allergenic  potential. 

3.   Program  of  Institute  Emphasis  for  Lymphocyte  Biology 

This  Program  currently  supports  five  program  projects,  each  of  which 
is  a  multidisciplinary  research  effort  integrating  the  technical  expertise 
of  cell  biologists,  geneticists,  cellular  immunologists  and  immunochemists 
and  is  directed  by  an  acknowledged  leader  in  the  field.   Their  combined 
studies  are  designed  to  expand  knowledge  of  the  morphologic  and  functional 
heterogeneity  of  lymphocyte  populations  and  to  develop  the  capability  for 
identification  and  selection  of  lymphocyte  subpopulations  with  specific 
immune  reactivity  or  antigenic  composition,  for  hybridization  of  such 
populations  and  for  selective  production  of  specific  lymphocyte  products. 
The  objective  of  this  Program  is  to  acquire  as  complete  a  knowledge  as 
possible  of  the  life  history  of  immunocompetent  cells  and  of  the  physio- 
logic and  external  factors  that  determine  their  fate  and  function  in  vivo 
and  in  vitro. 

This  program  was  initiated  approximately  seven  years  ago  with  the 
award  of  four  program  project  grants,  each  directed  toward  the  goal  of 
understanding  the  biology  of  the  lymphocyte  and  having  different  but  com- 
plementary approaches.  Three  of  the  original  projects  have  successfully 
undergone  competitive  peer  review  and  have  received  renewal  of  support  for 
a  five-year  period.   In  addition,  support  for  two  new  program  projects  has 
recently  been  provided,  one  each  in  FY  1978  and  FY  1979. 

The  program  project  directed  by  Matthew  D.  Scharff  (AI  10702,  Albert 
Einstein  College  of  Medicine)  has  focused  its  approaches  on  the  molecular 
biology  and  somatic  cell  genetics  of  the  immune  system  to  define  the 
genetic  and  molecular  control  mechanisms  of  immunoglobulin  synthesis,  the 
structure  and  synthesis  of  the  antigens  of  the  major  histocompatability 
complex  (MHC),  and  the  functions  and  interactions  of  T  and  B  lymphocyte 
populations  and  macrophages.  In  many  of  these  studies,  continuous  cloned 
cell  lines  and  variants  derived  from  these  cell  lines  are  being  used  to 
study  the  role  of  the  cells  and  their  products  in  the  immune  response.   For 
example,  Dr.  Scharff  and  his  associates  have  devoted  considerable  time  and 
effort  to  generate  immunologically  functional  hybrids  between  mouse  myeloma 
cells  and  spleen  cells  from  immunized  mice.  They  have  developed  a  technology 

5-11 


for  fusing  meyloma  and  spleen  cells  at  a  high  freguency  and  some  of  the 
drug  marked  cells  which  are  now  widely  used  to  prepare  hybridomas 
origniated  in  their  laboratory.   They  have  provided  cell  lines  and 
assistance  to  well  over  a  hundred  investigators  around  the  world  to 
establish  this  technology  in  their  own  laboratories.   Studies  within  the 
program  project  have  been  productive  and  important  contributions  have  been 
made.   Cultured  mouse  myeloma  cell  lines  have  been  used  to  examine  the 
genetic  control  of  the  expression  of  immunoglobulin  genes.   Analysis  of 
antigen-binding  variants  of  these  cell  lines  indicate  that  the  variants  have 
small  structural  changes  in  the  V  region,  arise  spontaneously  at  a  very 
high  freguency,  and  may  be  relatea  to  the  normal  process  which  regulates 
the  generation  of  antibody  diversity.   Primary  seguence  analysis  of 
constant  region  variants  has  established  that  they  arise  as  a  result  of 
crossing  over  between  two  closely  related  constant  region  genes.   Studies 
of  the  biochemical  properties  and  primary  structure  of  the  H-2  MHC  prodcuts 
in  an  attempt  to  relate  functional  and  genetic  properties  with  molecular 
structure  have  suggested  that  discrete  amino  acid  changes  in  the  H-2  glyco- 
proteins are  directly  related  to  their  immunologic  specificity.   In  order 
to  pursue  the  mechanisms  underlying  the  complex  and  coordinated  functions 
of  macrophages  and  to  identify  the  molecular  basis  for  modulation  of  these 
functions  by  products  of  activated  lymphocytes,  physiologic  and  functional 
studies  of  cloned  macrophage- like  cell  lines  have  been  initiated.   An 
unusual  macrophage-like  cell  line  has  been  established  which  can  migrate, 
suppress  antibody  production  by  hybridoma  cell  lines  and  suppress  mitogenic 
responses  of  T  and  B  cells.   These  differentiated  functions  have  not  been 
previously  observed  in  continuous  cell  lines.   Insulin  was  found  to  depress 
phagocytosis  in  these  cells  but  this  effect  could  be  overcome  by  cyclic 
nucleotides,  establishing  for  the  first  time  that  cyclic  nucleotides  play 
an  important  regulatory  role  on  the  phagocytic  process.   The  functional 
status  of  immunoregulatory  mechanisms  in  chronic  infections  also  is  being 
investigated.   When  stimulated  with  specific  antigen,  T  lymphocytes  from 
patients  with  lepromatous  leprosy  have  been  found  to  suppress  mitogenic 
responses;  this  observation  may  explain  the  anergic  state  freguently  seen 
in  these  patients.   In  contrast,  patients  with  multiple  sclerosis  exhibit 
defective  T  suppressor  and  cytotoxic  functions  which  may  contribute  to  the 
persistence  of  the  disease. 

The  approach  directed  by  Gerald  M.  Edelman  (AI  11378,  Rockefeller 
University)  centers  on  the  analysis  of  molecules  and  molecular  assemblies 
involved  in  the  control  and  recognition  of  antigenic  stimuli  at  the  surface 
and  within  the  cells  of  the  immune  system.   The  results  of  his  studies  have 
provided  several  insights  into  the  nature  of  recognition  and  control  events 
in  lymphocyte  function.   Of  special  interest  is  his  evidence  for  the 
existence  of  a  surface  modulating  assembly  (SMA)  which  regulates  cell 
surface  receptors  and  mediates  transmembrane  signalling  for  functional 
cellular  responses.   Dr.  Edelman  and  his  associates  picture  the  SMA  as  a 
three-component  system  consisting  of  cell  surface  receptors  that  penetrate 
the  cell  membrane,  submembranous  microfilaments  which  coordinate  receptor 
movement,  and  microtubules  which  provide  anchorage  for  the  receptors  and 
propogate  signals  to  and  from  the  cell  surface.   To  study  the  mobility  of 
the  cell  surface  receptors  and  the  molecular  basis  for  transmembrane 
control  of  receptor  mobility,  the  fluorescence  photobleaching  recovery 

5-12 


method  has  been  adapted  to  directly  measure  surface  receptor  mobility  on 
lymphocytes.   Using  this  technique,  different  classes  of  receptors  have 
been  found  to  be  under  separate  control  mechanisms.  A  viral  gene  protein 
has  been  established  as  a  valid  probe  to  dissect  the  stimulatory  sequence 
leading  from  the  cell  membrane  to  the  cytoplasm  and  the  nucleus  and  to 
study  the  enzymes  involved  in  cellular  DNA  replication.   Evidence  has  been 
obtained  which  indicates  that  the  microtubules  and  microfilaments  which 
form  the  cytoskeleton  act  as  signal  regulators  in  controlling  cell  growth 
and  division.   Lectins  also  are  being  employed  to  probe  the  mechanisms  of 
mitogenesis;  favin,  like  concanavalin  A,  is  mitogenic  but  has  been  found  to 
have  a  subunit  structure  very  different  from  that  of  concanavalin  A  and, 
consequently,  a  different  capacity  to  bind  cell  surface  glycoprotein 
receptors.   Favin  thus  offers  an  alternative  probe  for  examining  cell 
surface  events.   The  results  of  structural  studies  of  cell  surface  glyco- 
proteins provide  evidence  that  these  surface  receptors  from  aggregates  with 
each  other  and  with  antigenic  components  through  formation  of  a  transient 
disulfide  bond.  To  further  understand  the  nature  of  molecular  interactions 
at  the  cell  surface,  X-ray  crystallography  is  being  applied  to  study  the 
structural  organization  of  mitogenic  stimuli,  particularly  concanavalin  A. 

Evidence  has  been  obtained  to  suggest  that  the  normal  geometry  of 
concanavalin  A  is  dramatically  altered  in  the  presence  of  calcium  and 
manganese  ions.   These  changes  in  structural  geometry  are  associated  with 
mitogenic  activity  and  are  believed  to  be  involved  in  the  binding  of  con- 
cavalin  A  to  the  carbohydrate  components  of  surface  glycoproteins. 
Although  Dr.  Edelman's  research  is  centered  on  the  role  of  the  SMA  in  the 
immune  response,  his  results  suggest  that  the  SMA  has  a  key  role  in 
controlling  cell  movement,  growth,  and  differentiation  in  general. 

The  program  project  directed  by  Jonathan  W.  Uhr  (AI  11851,  University 
of  Texas  Health  Science  Center  at  Dallas)  is  centered  on  the  study  of  the 
biochemistry  and  biology  of  lymphocyte  surface  molecules.   The  structure  of 
immunoglobulins  on  the  membrane  of  B  cells  is  being  studied  in  order  to 
define  its  attachment  to  the  plasma  membrane  and  to  clarify  the  molecular 
interactions  that  occur  after  surface  immunoglobulins  react  with  specific 
antigens.   The  broader  purpose  of  such  studies  is  to  obtain  an  under- 
standing of  the  molecular  mechanisms  which  underlie  signalling  of  B 
lymphocytes  and  control  their  differentiation.   Studies  of  T  cells 
emphasize  the  process  of  immune  recognition;  efforts  are  being  made   to 
biochemically  characterize  the  T  cell  receptor  and  to  define  the  role  of  la 
antigens  in  the  interaction  of  antigen-pulsed  macrophages  with  T  cells. 
Relevant  studies  include  the  role  of  major  histocompatibility  complex 
products  in  recognition  of  viral  infected  cells  and  structural  analysis  of 
these  products  to  develop  insights  into  their  evolution  genetic  control  and 
function.   The  major  concern  and  principal  contributions  of  Dr.  Uhr's 
project  have  been  in  the  area  of  the  ontogeny,  regulation,  and  function  of 
B  cell  surface  immunoglobulins.  An  immunoglobulin  D  (IgD)-like  molecule  was 
found  on  the  surface  of  circulating  murine  lymphocytes ;  ontogenic  studies 
showed  that  it  appeared  after  immunoglobulin  M  (IgM)  when  mice  were 
approximately  one  week  of  age.   The  results  of  additional  studies  have  led 
to  the  acceptance  of  a  model  which  assigns  an  important  role  for  IgD  in  the 
development  and  function  of  B  cells.   In  this  model,  immature  B  cells  first 

5-13 


develop  surface  IgM,  then  acquire  surface  IgD  to  become  "double  bearers"; 
some  cells  subsequently  lose  surface  IgM  to  become  cells  that  bear  only 
surface  IgD.   Dr.  Uhr  and  his  associates  have  demonstrated  that  cells  which 
bear  only  surface  IgM  become  tolerized  after  encounter  with  antigens.  They 
also  have  shown  that  surface  IgD  acts  as  a  triggering  receptor  so  that 
cells  that  bear  both  surface  IgM  and  IgD  give  a  primary  IgM  response  after 
encounter  with  antigen;  cells  that  bear  only  surface  IgD  give  an  IgG 
response  after  encounter  with  antigen.   On  stimulation  with  lipopoly- 
saccharide,  cells  with  surface  IgM  enlarge  and  produce  a  polyclonal  IgM 
response;  cells  with  surface  IgD  do  not  exhibit  an  IgM  polyclonal  response 
but  instead  undergo  blastogenesis  and  proliferation.   Similar  results  were 
obtained  using  antigenic  stimulation.   It  was  concluded  from  such  studies 
that  cells  bearing  surface  IgD  are  the  major  progenitors  of  IgG  antibody 
forming  cells  in  the  secondary  antibody  response.   Long-term  memory  cells 
bear  small  amounts  of  both  surface  IgM  and  IgG.   Studies  of  surface 
receptors  on  T  cells  have  similarly  led  to  important  conceptual  advances. 
Evidence  has  been  obtained  to  indicate  that  specific  binding  of  antigens 
coupled  to  autologous  macrophages  by  T  cells  represents  the  initial  event 
in  immune  stimulation.   The  macrophage  plays  a  key  role  in  this  stage 
because  T  cell  receptors  do  not  specifically  bind  to  native  exogenous 
antigens.   Structural  analyses  of  T  cell  surface  receptors  have 
demonstrated  a  marked  homology  between  the  products  of  the  major  histocom- 
patibility complex  of  humans,  mice,  and  guinea  pigs,  implying  analogies  in 
function. 

The  program  project  directed  by  Baruj  Benacerraf  (AI  14732,  Harvard 
Medical  School)  is  designed  to  gain  understanding  of  the  genetic  controls 
of  the  immune  system  and  of  the  regulation  of  functional  expressions  of 
immunocompetent  cells.   Component  projects  are  concerned  with  the  identifi- 
cation and  function  of  immune  response  genes,  the  induction,  regulation, 
and  function  of  helper  and  suppressor  T  cells ,  and  the  identification  and 
function  of  surface  receptors  on  T  and  B  cells.   Using  genetically- 
determined  responder  and  nonresponder  strains  of  mice,  Dr.  Benacerraf  and 
his  associates  have  found  that  antigen-specific  suppressor  factor  derived 
from  T  cells  nonresponder  mice.   They  also  have  found  that  specific 
suppression  can  be  eliminated  by  administration  of  antisera  specific  for  I-J 
gene-controlled  determinants;  immunopotentiation  of  nonresponder  mice  with 
this  antiserum  was  demonstrated  to  reflect  a  reduction  of  specific 
suppressor  responses.   In  other  studies,  macrophages  of  responder  mice  were 
shown  to  be  able  to  present  antigen  in  an  immunogenic  form  and  to  play  a 
central  role  in  regulating  the  balance  of  activated  helper  and  suppressor  T 
cells,-  these  findings  suggested  that  suggested  that  an  Ir  gene  defect  at 
the  macrophage  level  any  account  for  the  predominant  suppressor  T  cell 
responses  in  nonresponder  mice.   The  studies  of  murine  B  cells  have  led  to 
the  detection  of  monovalent  and  divalent  IgD  on  the  cell  surface;  evidence 
has  been  obtained  to  suggest  that  both  forms  of  IgD  are  native  to  the  cell 
surface  and  may  represent  different  forms  of  attachment  of  these  molecules 
to  the  membrane  of  the  cell.   A  detailed  analysis  of  the  aggregation  or 
"capping"  of  cell  surface  molecules  has  led  to  the  demonstration  of  two 
functionally  different  mechanisms.   The  first  type  which  occurs 
spontaneously  appears  to  be  an  active  process  involving  a  direct  link 
between  the  surface  molecules  and  the  cytoplasmic  contractile  apparatus. 

5-14 


This  type  appears  to  be  related  to  the  process  of  locomotion;  myosin  was 
found  to  be  concentrated  in  the  cytoplasm  directly  beneath  the  capped  area. 
The  other  type  occurred  when  B  cells  were  treated  with  antigens,  antibodies 
against  cell  surface  receptors,  or  mitogens  and  appeared  to  result  simply 
from  aggregation  of  crosslinked  molecules  in  the  plane  of  the  membrane; 
this  type  of  capping  did  not  result  in  a  redistribution  of  cytoplasmic 
myosin  or  stimulation  of  cell  locomotion.   It  is  implied  that  the  inter- 
action of  surface  immunoglobulins,  at  the  molecular  level,  with  other 
components  of  the  cell  membrane  and  cytoplasm  represents  the  earliest  step 
of  specific  antigen  recognition  and  subsequent  triggering  of  B  cells.  In 
other  studies,  the  IgG-bearing  B  cell  subpopulation  from  human  spleen  has 
been  isolated  and  characterized;  these  cells  have  IgM  or  IgD  on  their 
surface  and  give  rise  to  nearly  all  of  the  IgG-  and  a  significant  franction 
of  the  IgM-secreting  cells  after  stimulation  by  mitogen.  The  recognition  of 
an  antigenic  determinant  found  both  on  murine  macrophages  and  on  human 
monocytes  using  hybridoma  antibody  has  made  it  possible  to  use  the  fluores- 
cence-activated cell  sorter  to  prepare  pure  populations  of  human  monocytes 
from  peripheral  blood  for  subsequent  functional  studies.  Also,  by  using  a 
cytofluorometric  technique,  an  approach  has  been  developed  to  recognize 
small  numbers  of  malignant  B  lymphocytes  in  a  population  of  normal  lympho- 
cytes.  This  method  may  facilitate  the  planning  and  monitoring  of  therapy 
in  patients  with  lymphoid  malignancies. 

The  program  project  directed  by  Dr.  Carl  W.  Pierce  (AI  15353,  Jewish 
Hospital  of  St.  Louis)  was  awarded  in  FY  1979  and  has  the  immunobiology  of 
the  major  histocompatibility  gene  complex  as  its  central  theme.  Dr.  Pierce 
is  an  outstanding  cellular  immunologist  who  has  made  important  contribu- 
tions to  this  area;  in  recognition  of  his  accomplishments,  he  was  the  1979 
recipient  of  the  Parke-Davis  award  presented  by  the  American  Society  for 
Experimental  Pathology.  His  co-principal  investigators  at  Washington 
University,  Dr.  Donald  Shreffler  and  Dr.  Joseph  Davie,  are  equally  out- 
standing in  their  fields.   Their  combined  efforts  represent  a  multidis- 
ciplinary  approach  to  several  problems  of  fundamental  importance  to  an 
understanding  of  the  mechanisms  and  functions  of  the  major  histocompat- 
ability  complex.   Areas  to  be  examined  include  the  nature  of  the 
interactions  between  histocompatability  gene  products  and  conventional 
antigens  in  the  generation  of  effector  lymphocytes,  the  structure  of 
genetically  restricted  suppressor  factors,  the  structure  and  genetic 
relationships  of  components  of  the  complement  system  that  are  produced  by 
genes  of  the  major  histocompatability  complex,  the  identification  and 
structure  of  gene  products  on  T  cells  and  macrophages,  and  the 
identification  and  characterization  of  receptor  molecules  on  the  surface  of 
T  lymphocytes . 

This  Program  of  Institute  Emphasis  represents  a  major  investment  of 
the  IAIDP  and  NIAID  in  the  concept  that  advances  in  the  understanding  of 
the  behavior  of  lymphocytes  can  be  effectively  achieved  through  carefully 
organized  efforts  by  large,  highly  interactive  groups  led  by  well- 
established  and  highly  productive  senior  scientists.   To  date,  there  is 
every  indication  that  this  program  has  been  successful  and  productive  and 
that  it  will  continue  so  in  the  future. 


5-15 


MICROBIOLOGY  AND  INFECTIOUS  DISEASES  PROGRAM 

N  I  A  I   D 
TABLE  OF  CONTENTS 

REPORT  OF  THE  DIRECTOR , 6-1 

Bacteriology 6-1 

Mycology 6-2 

Parasitology 6-3 

Virology „ 6-3 

International  Heal  th 6-8 

I.  BACTERIOLOGY  AND  VIROLOGY  BRANCH 7-1 

Program  Summary 7-1 

Bacteriology 7-2 

Sexual ly  Transmitted  Diseases 7-2 

Hospital  Associated  Infections 7-3 

Streptococcal  Diseases  and  Sequelae 7-5 

Other  Disease  Problems 7-7 

Leprosy 7-8 

Tuberculosis 7-9 

Mycology 7-10 

Virology 7-12 

II.  CLINICAL  STUDIES  BRANCH 8-1 

Closed  Vol  unteer  Facil  ities 8-1 

Collaborative  Clinical  Trials  with  Antibiotic  Therapy 8-2 

Rapid  Viral  Diagnosis 8-3 

Nutrition,  Infection  and  Immunity 8-4 

Other  Activities 8-5 

III.  DEVELOPMENT  AND  APPLICATIONS  BRANCH 9-1 

Introduction 9-1 

Program  Summary 9-2 

Research  Highl  ights 9-4 

Influenza 9-4 

Respiratory  Diseases 9-5 

Vi  ral    Hepati  ti  s 9-6 

Anti  vi  ral    Substances 9-8 

Bacterial    Vaccines 9-10 

Enteric   Diseases 9-13 

Viral    Vaccine   Program 9-15 


MICROBIOLOGY  AND  INFECTIOUS  DISEASES  PROGRAM 
N  I  A  I  D 
TABLE  OF  CONTENTS 

Continued 

IV.      EPIDEMIOLOGY  AND  BIOMETRY   BRANCH 10-1 

HLA  and   Infectious   Disease  Epidemiology 10-1 

Influenza 10-1 

CI  inical    Trials 10-2 

Impact  of  Infections 10-3 

Epidemiology  of  Nosocomial    Infections 10-3 

Trans-NIH 10-3 

V.      MOLECULAR  MICROBIOLOGY  AND  PARASITOLOGY  BRANCH 11-1 

Introduction 11-1 

Molecular  Microbiology 11-2 

Program  Summary 11-3 

Research  Highl  ights 11-3 

Recombinant   DNA  Molecular  Research 11-5 

Research  Highl  i  ghts 11-6 

Parasitology 11-7 

Program  Summary 11-8 

Research  Highl  ights 11-8 

Contract  Activity 11-11 


MICROBIOLOGY  AND  INFECTIOUS  DISEASES  PROGRAM 

Annual  Report,  1978-1979 

Director's  Report 


The  accomplishments  of  the  five  branches  of  the  Microbiology  and  Infectious 
Diseases  Program  (MIDP)  are  summarized  in  the  following  sections;  activities 
in  international  health  are   reviewed  at  the  end  of  this  section.  This  report 
addresses  the  implications  of  these  accomplishments  and  activities  for  the 
future,  with  particular  emphasis  on  virology. 

Humans  are  infected  by  an  astounding  number  and  variety  of  organisms,  ranging 
in  size  and  complexity  from  tiny  viruses  which  are  little  more  than  nucleic 
acids  to  large  multi-cellular  parasites.  These  infectious  agents  and  their 
progeny  have  been  living  together  with  man  and  his  progeny  for  millenia,  and 
both  have  changed  as  a  result.  Concurrently,  the  physical  and  socio-economic 
environments  shared  by  man  and  his  microbial  companions  have  changed  markedly, 
at  least  in  some  parts  of  the  world,  and  chemicals  have  been  developed  which 
may  be  introduced  into  man  or  his  environment  to  inhibit  the  growth  of  microbes 
and  parasites  or  to  limit  their  transmission.  And,  in  the  last  two  hundred 
years,  man  has  tamed  certain  of  these  agents,  or  their  toxins,  so  as  to  pro- 
duce attenuated  vaccines  (smallpox,  yellow  fever,  poliomyelitis,  measles, 
mumps,  rubella)  or  toxoids  (diphtheria,  tetanus)  which,  if  properly  used, 
provide  virtually  complete  protection  against  "wild"  or  natural  strains.  The 
net  result  of  all  this  has  been  that,  in  the  developed  world,  many  serious 
life-threatening  infectious  diseases,  particularly  those  of  childhood,  have 
been  prevented  with  a  consequent  increase  in  life  expectancy.  A  major  victory 
for  man! 

But  this  is  no  time  to  be  complacent.  Infectious  diseases  are  still  respon- 
sible for  nearly  25%  of  all  visits  to  physicians  in  the  U.S.  each  year,  and 
they  account  for  65%  of  all  such  visits  for  children  under  16  years.  In  some 
developing  countries,  up  to  40%  of  children  still  die  before  the  age  of  five 
years  because  of  infectious  diseases.  Microbes  and  their  vectors  have  fought 
back  around  the  world.  Malarial  parasites  have  become  resistant  to  once 
curative  drugs;  mosquitoes  which  transmit  encephalitis  viruses  and  malarial 
parasites  have  become  resistant  to  insecticides;  bacteria  have  developed 
resistance  to  antibiotics  and  the  ability  to  transfer  this  resistance  to  other 
bacteria.  A  potential  victory  for  microbes! 

Bacteriology 

It  is  appropriate,  then,  that  NIAID  encourage  additional  studies  on  mechanisms 
of  antibiotic  resistance.  In  doing  so  it  will  not  only  improve  the  care  of 
patients  with  bacterial  infections,  but  it  will  also  contribute  basic  infor- 
mation regarding  man  and  microbes,  for  current  knowledge  of  molecular  genetics 
derives  largely  from  studies  of  bacteria  and  viruses. 


6-1 


One  of  the  major  recent  discoveries  in  this  field  is  that  a  number  of  specific 
DNA  sequences  existing  at  multiple  locations  on  prokaryotic  genomes  can 
transpose  and  translocate  spontaneously  to  new  sites  by  a  mechanism  that  is 
independent  of  the  host's  normal  recombination  system.  Progress  in  the 
identification,  mapping  and  characterization  of  these  insertion  sequences  has 
been  made  possible  by  the  development  of  techniques  for  the  analysis  of  DNA 
structure.  These  techniques  have  shown  that  multiple  copies  of  insertion 
sequences  are  normal  constituents  of  the  bacterial  chromosome  and  also  of 
extra-chromosomal  genomes;  their  location  on  bacterial  plasmids  has  suggested 
that  they  play  a  key  role  in  chromosome  evolution.  Insertion  sequences  have 
been  identified  among  the  repititious  DNA  sequences  which  flank  drug-resistance 
genes  on  bacterial  plasmids,  strengthening  earlier  evidence  that  the  spread 
of  multiple  drug  resistance  among  bacterial  pathogens  is  a  manifestation  of 
genetic  plasticity  made  possible  by  DNA  insertions. 

There  are  encouraging  signs  that  studies  of  other  properties  of  bacteria  will 
benefit  from  the  application  of  new  technologies.  Already,  there  is  new  in- 
formation about  mechanisms  of  attachment  to  mucosal  surfaces,  and  the  role  of 
pili  in  such  attachment.  Studies,  of  chemotaxis,  of  enzymes,  of  membrane 
biogenesis,  and  of  the  structural  dynamics  and  functions  of  outer  membranes, 
are  providing  insights  regarding  bacterial  virulence  and  the  prospects  for 
new  bacterial  vaccines.  Work  continued  during  the  year  on  such  vaccines.  A 
polysaccharide  vaccine  has  been  developed  and  licensed  for  Groups  A  and  C 
meningococci,  but  attempts  to  produce  an  immunogenic  and  protective  Group  B 
polysaccharide  vaccine  have  failed.  Groups  B,  C,  and  Y  meningococci  share 
an  outer  membrane  protein  antigen,  type  2,  and  a  type  2  protein  vaccine  is 
being  developed  for  the  prophylaxis  of  Group  B  meningococcal  infections.  Two 
proteins  for  the  gonococcus  --  the  principal  outer  membrance  protein  and  pili 
protein  --  have  been  shown  to  be  single  proteins  with  only  a  small  amount  of 
1 ipopolysaccharide  (endotoxin),  and  are  ready  for  Phase  I  trials.  Of  several 
protein  antigens  from  toxigenic  E.  coli,  one  --  described  as  "colonizing 
factor  antigen"  --  when  used  as  a  sub-cutaneous  vaccine  protected  against 
illness  and  reduced  intestinal  colonization  in  initial  volunteer  challenge 
studies.  Another  antigen  related  to  attachment  --  pili  --  will  soon  be  tested 
in  volunteers.  Work  is  progressing  on  characterization  of  the  antigens  of 
Groups  B  streptococci  as  potential  vaccine  components. 

Mycology 

Systemic  fungal  infections  have  long  been  recognized  as  important  causes  of 
severe,  sometimes  life-threatening  disease.  Primary  infections  with  two 
fungi  --  Coccidioides  immites  and  Histoplasma  capsulatum  --  are  particularly 
common  in  the  southwestern  and  central  U.S.,  respectively.  Other  fungi  are 
opportunistic  pathogens,  and  infections  with  such  organisms  are  being  recog- 
nized with  increasing  frequency  as  the  use  of  immunosuppressive  therapies 
increases  in  the  management  of  patients  with  malignant  diseases  or  organ 
transplants.  There  are  few  effective  drugs  for  the  treatment  of  mycotic 
infections  in  patients  with  impaired  immune  defenses. 

Consideration  of  these  problems  by  an  MIDP  sponsored  workshop  led  to  the 
recommendation  that  several  national  mycology  centers  be  established  to  assemble 


6-2 


a  critical  concentration  of  scientists  knowledgeable  in  biochemistry,  molecular 
biology  and  genetics,  as  well  as  in  the  clinical  aspects  of  medical  mycology, 
to  provide  a  focus  for  contemporary  research.  Two  institutions,  the  University 
of  Washington,  St.  Louis,  Missouri,  and  the  University  of  California  at  Los 
Angeles,  successfully  competed  for  program  project  grants  for  the  support  of 
such  mycology  research  units.  It  is  believed  that  augmented  research  at  these 
two  strategically  located  units  will  revitalize  the  field  of  mycology  and  lead 
to  more  effective  prevention  and  treatment  of  fungal  infections. 

Concurrently,  multi-center  clinical  trials  of  antifungal  agents  in  selected 
infections  such  as  cryptococcal  meningitis  have  gotten  underway.  In  prospect 
is  a  controlled  trial  of  a  new  drug,  ketoconazole,  a  synthetic  oral  antifungal 
compound  which  inhibits  the  biosynthesis  of  ergosterol  ,  the  major  sterol  in 
most  yeast  and  fungi.  The  drug  apparently  can  be  used  without  impairing 
cholesterol  synthesis  in  man.  It  should  be  possible  to  design  other  such 
targeted  approaches  as  more  is  learned  about  the  molecular  structure  and 
dimorphism  of  the  pathogenic  fungi. 

Parasitology 

The  new  and  expanded  programs  in  international  health  and  tropical  medicine 
implemented  this  year  through  the  support  of  collaborative  research  overseas 
(International  Collaboration  in  Infectious  Diseases  Research  -  ICIDRs)  and  of 
multidisciplinary  programs  in  the  U.S.  (Tropical  Disease  Research  Units  - 
TDRUs)  will  augment  research  on  parasitic  diseases,  particularly  those  target- 
ed by  the  World  Health  Organization:  malaria,  schistosomiasis,  trypanosomiasis, 
leishmaniasis,  and  filariasis.  This  effort  was  facilitated  by  the  many  ties 
that  University  scientists  have  maintained  with  foreign  investigators,  and  by 
the  already  existing  studies  of  the  Parasitic  Diseases  Panel  of  the  U.S. -Japan 
Cooperative  Medical  Science  Program.  Four  of  six  ICIDRs  will  be  funded  in 
FY  79;  two  ICIDRs  and  at  least  two  TDRUs  are  projected  for  FY  80.  Details 
are  provided  at  the  end  of  the  Director's  Report  and  in  the  report  of  the 
Molecular  Microbiology  and  Parasitology  Branch. 

This  increased  effort  comes  at  an  opportune  time.  In  preparation  for  a 
"Conference  on  Pharmaceuticals  for  Developing  Countries"  held  in  January, 
1979,  by  the  Institute  of  Medicine,  National  Academy  of  Sciences,  a  survey 
was  made  of  current  programs  in  U.S.  government  and  academic  laboratories 
for  the  development  of  preventive,  prophylactic,  diagnostic  and  therapeutic 
agents  for  the  five  parasitic  diseases  listed  above  plus  leprosy  and  enteritis. 
In  1978,  of  the  $36,013,000  spent  by  federal  agencies  for  these  purposes, 
$11,181,000  was  invested  by  NIAID,  $9,135,000  in  behalf  of  extramural  research. 
Considering  the  billions  of  world  citizens  infected  with  these  and  related 
diseases,  an  increasing  number  of  whom  are  being  offered  refuge  in  the  U.S., 
this  is  a  sensible  investment  indeed. 

Virology 

One  of  the  outstanding  accomplishments  of  the  year  was  the  publication  of  the 
six  volume  report  of  the  Virology  Task  Force,  culminating  two  years  of  review 
by  distinguished  scientists  for  all  parts  of  the  U.S.  who  assessed  the  state 
of  the  art  in  virology  and  identified  opportunities  for  fostering  the  under- 

6-3 


standing,  prevention  and  control  of  viral  diseases.  Each  of  the  five  Task 
Force  panels  made  a  number  of  recommendations  which  were  combined  into  a  total 
of  25  in  the  final  volume  of  the  Report.  These,  in  turn,  were  consolidated 
by  the  National  Advisory  Allergy  and  Infectious  Diseases  Council  into  ten 
research  areas  which,  the  Council  recommended,  should  be  emphasized  and 
expanded  by  more  than  doubling  the  Institute's  investment  in  research  on 
virology  in  the  next  five  years.  That  such  an  investment  will  yield  significant 
dividends  is  buttressed  by  the  Task  Force's  accounting  of  recent  accomplishments 
and  its  projection  of  what  lies  ahead.  As  illustrated  by  paraphrased  or  ex- 
cerpted sections  of  the  report,  the  future  is  bright. 

The  history  of  virus  research  mirrors  the  progression  from  descriptive 
approaches  to  the  era  of  molecular  genetics.  The  development  of  improved 
biochemical  and  biophysical  techniques  for  studying  the  structure  and  assembly 
of  viruses  has  led  to  increased  knowledge  of  the  biosynthesis  of  viral  compon- 
ents. This,  coupled  with  the  isolation  of  suitable  mutants,  has  opened  the 
way  for  a  concentrated  attack  on  the  precise  mechanisms  involved  in  assembly 
processes  and  their  regulation.  Further  efforts  should  lead  to  greater  know- 
ledge of  important  pathogens  on  the  one  hand,  and  of  normal  macromolecular  and 
cellular  organization  and  function  on  the  other. 

The  fact  that  viruses,  as  bearers  of  unique  genetic  information  encoded  in 
their  DNA  or  RNA,  bring  into  cells  conveniently  small  probes  of  essential 
life  processes  has  made  viral  research  the  cornerstone  of  molecular  biology. 
Viruses  occur  in  all  living  creatures,  and  progress  toward  an  understanding 
of  those  which  cause  disease  in  man  has  derived  from  concepts  and  techniques 
first  applied  to  viruses  of  plants  and  bacteria.  Viruses  which  infect  bacteria 
are  known  as  bacteriophages.  Historically,  the  ideas  of  messenger  RNA,  of  the 
triplet  code  of  stop  and  start  codons  ,  even  the  very   definition  of  a  gene 
arose  from  their  study.  Future  research  is  likely  to  yield  results  of  impor- 
tance to  the  medical  community  in  many  ways. 

Temperate  phages,  along  with  plasmids,  are  members  of  a  class  of  important 
genetic  elements  known  collectively  as  "episomes."  A  recent  finding  in  the 
field  of  episomal  genetics  has  been  that  the  genes  which  confer  resistance  to 
the  antibiotics  are  carried  on  translocatable  DNA  elements  capable  of  trans- 
position from  one  bacterial  genome  to  another.  Thus,  phage  research  is 
applicable  directly  to  the  mechanisms  of  pathogenesis  in  bacteria  since  many 
bacteria  are  dangerous  only  when  they  contain  particular  plasmids  or  phage 
genomes.  Understanding  the  mechanisms  of  maintenance ,  immunity,  incompatibil- 
ity, gene  expression,  and  the  evolution  of  episomes  will  be  of  direct  benefit 
just  as  it  will  be  in  the  case  of  infectious  drug  resistance.  One  might  even 
imagine  devising  competitor  plasmids  and  phages  which  could  effectively  compete 
with  undesirable  plasmids  or  phages  from  the  bacterial  cell. 

Ideas  about  the  fundamental  mechanisms  of  gene  expression,  recombination, 
replication,  and  evolution,  as  well  as  related  technology  derived  from  phage 
research,  are  often  directly  applicable  to  the  study  of  animal  viruses  or 
other  organisms.  Genetic  analysis  of  viruses  will  seek  to  determine  the 
number  of  viral  genes,  the  function  of  each  gene,  and  the  order  or  position 
of  these  genes  on  the  nucleic  acid  chromosome  of  the  virus.  Studies  involving 
the  process  of  recombination  will  then  position  the  genes  relative  to  each 

6-4 


other,  yielding  a  genetic  map  of  the  virus.  Once  this  has  been  accomplished, 
several  steps  may  be  taken  with  viral  genes  of  known  function  to  elucidate 
the  mechanisms  of  viral  virulence  or  to  produce  candidate  viruses  or  antigens 
for  vaccine  production.  Mutations  in  viral  genes  can  be  sought  which  alter 
pathogenicity,  e.g.,  by  affecting  temperature  of  growth,  to  produce  live, 
attenuated  vaccine  strains.  These  viruses  can  then  be  used  to  induce  immunity 
similar  to  that  following  natural  disease,  but  without  illness.  Alternatively, 
the  gene  for  an  immunizing  antigen,  e.g.,  influenza  virus  hemagglutinin,  may 
be  transferred  to  an  E_.  col  i  host  via  recombinant  DNA  technology,  permitting 
replication  of  the  gene  and  synthesis  of  the  desired  antigen  as  the  bacterium 
grows  in  culture.  Hepatitis  and  influenza  genes  are  now  being  tested  in  such 
systems.  Successful  production  and  purification  of  specific  antigens  would 
have  a  great  impact  on  vaccine  manufacture  and  immunization  practices  for  the 
prevention  of  acute  viral  infections. 

Selected  Acute  Infections:  Infections  are  the  most  common  cause  of  illness; 
viruses  are  the  most  common  cause  of  infections;  respiratory  viruses  and 
enteric  viruses  are  the  most  common  causes  of  viral  infections,  ranking  first 
and  second,  respectively,  as  causes  of  illness.  Although  considerable  progress 
as  been  made  in  identifying  the  viruses  responsible  for  these  illnesses,  they 
persist  as  major  public  health  problems  because  of  the  inadequacy  of  knowledge 
essential  to  the  development  of  effective  preventive  measures  and  therapy. 

Acute  respiratory  infections  are  caused  by  a  multiplicity  of  viruses,  the  most 
dramatic  of  which  is  influenza  A,  the  cause  of  periodic  world-wide-epidemics 
associated  with  high  attack  rates,  significant  morbidity  and  excess  mortality. 
The  most  recent  influenza  A  variant  to  appear  --  in  this  instance,  to  reappear 
--  is  the  Russian  strain  (H-jN-])  which  caused  outbreaks  in  children  and  young 
adults  in  1978  and  1979.  In  1978,  MIDP  coordinated  clinical  trials  of  A/USSR 
influenza  vaccine,  combined  with  A/Texas  and  B/Hong  Kong  antigens,  in  approxi- 
mately 2,100  subjects.  Reaction  and  serologic  data  from  these  trials  provided 
the  basis  for  the  formulation  of  vaccines  and  recommendations  as  to  dosage 
schedules  for  the  1978-79  season.  At  a  series  of  technical  meetings  during 
the  past  year,  culminating  with  the  Surgeon  General's  Meeting  on  February  12, 
and  the  Secretary's  Conference  on  March  6,  1979,  these  data  were  updated  for 
those  developing  immunization  recommendations  for  1979-80.  These  reviews  were 
greatly  facilitated  by  the  availability  of  NIAID's  own  computer  competence  in 
the  Epidemiology  and  Biometry  Branch,  and  by  the  close  working  relationship  of 
the  three  agencies  responsible  for  surveillance  (CDC),  control  of  vaccine 
production  (BoB/FDA),  and  research  (NIAID).  The  Director's  office  emphasized 
this  collaboration  when  it  elected  to  display  DHEW  research  on  influenza 
according  to  Science  Base,  Application,  Transfer,  and  Training,  the  SATT  model. 
The  Director,  NIH,  then  requested  that  this  model  be  used  as  a  basis  for 
drafting  a  DHEW  trans-agency  five  year  research  plan  for  influenza  as  an 
example  of  health  research  planning. 

The  most  important  respiratory  disease  of  children  is  that  caused  by  respiratory 
syncytial  (RS)  virus.   In  infants  and  young  children  RS  produces  severe, 
sometimes  fatal  bronchiolitis.  Older  children  and  adults  experience  less 
serious  illness  during  reinfection  than  do  infants  undergoing  primary  infection. 
Since  serious  RS  virus  disease  occurs  most  often  during  the  first  few  months 
of  life,  when  infants  possess  passively  transferred  serum  antibody,  it  is  clear 

6-5 


that  such  neutralizing  antibody  in  serum  does  not  provide  effective  protection 
against  the  most  serious  effects  of  the  virus.  Nor,  as  noted,  does  it  protect 
adults;  reports  during  this  year  have  documented  the  occurrence  of  moderately 
severe  upper  respiratory  illnesses  in  adults  exposed  to  children  during  the 
epidemics  which  occur  every   year  in  urban  centers.  Clearly,  vaccines  cannot 
be  expected  to  prevent  RS  infection,  so  the  current  approach  is  to  seek 
amelioration  of  the  first  infant  illness  by  inducing  local  antibody  with 
attenuated  virus  prior  to  infection  with  wild  virus.  Volunteer  studies  now 
in  progress  may  permit  this  hypothesis  to  be  tested  in  the  coming  year. 

Acute  viral  gastroenteritis  affects  a  broad  segment  of  the  population  through- 
out the  world.  In  the  developed  countries  it  is  a  major  cause  of  morbidity 
in  infants  and  young  children,  whereas  in  the  developing  countries  it  is  a 
major  cause  of  both  morbidity  and  mortality  in  this  same  age  group.  Consider- 
able progress  has  been  made  in  elucidating  the  etiologic  agents  of  viral 
gastroenteritis.  Of  note  is  the  fact  that  these  advances  have  been  made 
without  the  use  of  classical  cell  culture  systems  which  have  been  the  keystone 
in  the  progress  of  modern  virology  but  which  are  of  no  value  in  detecting 
these  fastidious  gastroenteritis  agents  in  clinical  specimens. 

Two  groups  of  etiologic  agents  of  viral  gastroenteritis  --  the  parvovirus-! ike 
group  of  which  the  27  nm  Norwalk  particle  is  the  prototype,  and  the  70  nm 
rotavirus  group  --  have  been  identified.  Studies  of  rotaviruses  have  depended 
heavily  on  the  use  of  the  electron  microscope  (EM)  and  studies  of  the  Norwalk 
group  of  viruses  have  relied  exclusively  on  the  electron  microscope,  utilizing 
almost  always  the  technique  of  immune  electron  microscopy  (IEM)  --  a  method 
which  might  be  defined  as  the  direct  observation  of  antigen-antibody  interaction 

It  is  evident  now  that  a  70  nm  rotavirus  is  a  major  etiologic  agent  of  sporadic 
infantile  gastroenteritis.  This  virus  has  been  associated  etiologically  with 
up  to  50  percent  of  the  hospitalized  cases  of  diarrheal  illness  in  many 
developed  countries  in  temperate  climates.  Viruses  of  the  Norwalk  group  are 
being  related  to  more  and  more  local  outbreaks.  Currently,  second  generation 
tests  which  are  being  or  have  been  developed  offer  great  promise  for  further 
unravelling  the  natural  history  of  these  agents.  Future  studies  will  have  to 
address  (i)  the  efficient  propagation  of  these  agents  in  cell  culture,  (ii) 
a  comprehensive  definition  of  their  overall  importance  over  a  sustained  period 
in  the  etiology  of  gastroenteritis  in  various  populations,  (iii)  elucidation 
of  the  immune  mechanisms  involved  in  host  defense,  (iv)  the  development  of 
effective  methods  to  prevent  or  treat  illnesses  due  to  these  agents,  and  (v) 
the  continuing  search  for  other  etiologic  agents  of  actue  gastroenteritis. 

Similarly,  the  ability  to  recognize  infections  due  to  hepatitis  A  or  B  viruses 
has  disclosed  the  need  to  research  for  other  etiologic  agents  of  hepatitis. 
The  existence  of  "non-A,  non-B"  hepatitis  viruses  is  inferred  from  the 
identification  of  hepatitis  cases  that  lack  serologic  evidence  of  infection 
by  hepatitis  A  or  B  viruses,  cytomegalovirus  or  Epstein-Barr  virus.  Non-A, 
non-B  hepatitis  cases  have  been  detected  throughout  the  world.  At  present 
approximately  90  percent  of  post-transfusion  hepatitis  in  the  U.S.  is  type 
non-A,  non-B.  Recent  studies  of  patients  with  repeated  cases  of  apparently 
acute  hepatitis  suggested  that  at  least  two  agents  not  related  to  type  A  or 
type  B  viruses  exist,  and  recent  transmission  studies  in  chimpanzees  have 

6-6 


provided  electron  microscopic  evidence  of  two  different  viruses.  Thus,  with 
the  prospects  of  an  effective  vaccine  for  hepatitis  B,  and  the  recent  culti- 
vation of  hepatitis  A  virus,  it  is  important  to  determine  what  proportion  of 
hepatitis  cases  occurring  annually  in  the  U.S.  is  caused  by  as  yet  unidentified 
agents. 

Influenza  and  other  respiratory  viruses  are  being  studied  at  the  Influenza 
Center  and  at  several  Vaccine  Evaluation  Centers;  viral  gastroenteritis  is 
being  studied  at  three  Enteric  Diseases  Centers;  viral  hepatitis  is  being 
studied  in  several  populations.  There  are,  of  course,  many  other  viruses 
which  infect  man.  A  number  of  these  infections  can  be  related  etiologically 
to  specific  agents,  but,  unfortunately,  viruses  have  yet  to  be  isolated  from 
many  presumed  viral  illnesses.  The  time  is  ripe  for  expanded  epidemiologic 
studies  of  appropriate  populations  to  define  the  natural  history  of  these 
infections.  In  the  view  of  the  Virology  Task  Force  this  will  best  be  done 
for  the  many  widely  prevalent  viruses  by  continuing  surveillance  of  family 
units.  Although  costly,  relatively  difficult,  and  requiring  a  long  period 
of  time  to  be  productive,  such  family  studies  have  two  important  advantages: 
(1 )  a  given  study  can  serve  to  describe  the  behavior  of  a  large  number  of 
known  viruses;  and  (2)  it  will  yield  a  valuable  "library"  of  specimens  and 
illness  information  that  can  be  exploited  anew  when  new  methods  become 
available  for  studying  infections  with  important  new  viruses  or  with  currently 
known  viruses  which,  for  lack  of  readily  applicable  methods,  have  not  yet 
been  extensively  studied.  MIDP  wishes  to  initiate  one  or  more  "Family  Studies" 
as  recommended  by  the  Task  Force. 

Chronic  Disease:  It  has  long  been  suggested  that  viruses  may  cause  chronic 
as  well  as  acute  diseases.  It  is  now  known  that  one  or  more  of  the  viruses 
that  cause  actue  hepatitis  may  persist  and  cause  chronic  liver  disease.  There 
are  other  viruses  which  induce  no  acute  symptoms,  but  which  as  a  result  of 
"slow",  progressive  persistent  infections,  are  responsible  for  such  rare  CNS 
syndromes  as  Kuru,  Creutzfel d- Jakob,  and  Familial  Alzheimer  Diseases.  Obser- 
vations in  the  past  year  have  contributed  new  knowledge  regarding  the  role  of 
viral  infections  in  a  major  chronic  disease,  diabetes.  These  studies  support 
the  hypothesis  that  acute  destruction  of  specialized  tissue  cells  may  lead  to 
chronic  loss  of  function  of  those  cells,  specifically  the  beta  cells  of  the 
islets  of  Langerhans  of  the  pancreas. 

The  common  cold  and  influenza,  along  with  measles,  chicken-pox,  and  mumps, 
are  examples  of  acute  viral  diseases  from  which,  barring  complications,  patients 
recover.  In  rare  instances,  measles  virus  may  cause  a  severe  and  fatal  brain 
disease  called  subacute  sclerosing  pan  encephalitis  (SSPE).  In  other  instances, 
not  so  rare,  the  chicken-pox  virus  may  persist  in  ganglion  cells  of  the  nervous 
system  and  be  reactivated  to  produce  the  painful  lesions  of  herpes  zoster. 
Mumps  virus,  which  sometimes  produces  meningitis  and  pancreatitis,  as  well  as 
parotitis,  has  been  questioned  as  a  cause  of  diabetes.  While  the  association 
of  mumps  with  diabetes  has  yet  to  be  established,  at  least  one  virus  now  has 
been  incriminated  as  a  cause  of  juvenile  onset  diabetes  in  humans.  This  is 
a  coxsackie  B4  virus  isolated  by  investigators  at  the  National  Institute  of 
Dental  Research  from  the  pancreas  of  a  previously  healthy  ten-year-old  boy 
who  died  in  diabetic  coma  ten  days  after  the  onset  of  symptoms  of  an  acute 
febrile  illness.  Previous  studies  in  animals  had  shown  that  Venezuelan  equine 

6-7 


encephalomyelitis   virus,  encephalomyocarditis   virus,   reo virus  type  1,  and 
coxsackie  B4  virus   could  induce  diabetes   in  certain  strains  of  mice.     They 
do  so   by  destroying  the  beta  cells.     Since  it  is   these  cells  which  produce 
insulin,   their  malfunction  or  destruction  lead  to   a  lack  of  insulin,  and  thus, 
to   insulin  dependent,  or  juvenile,  diabetes  mellitus.     The  coxsackie  B4  virus 
isolated   from  the  young  boy  produced  diabetes   in  susceptible  mice. 

Many  questions   remain  to  be  answered  in   FY  80  and  beyond.      Is   human  suscepti- 
bility genetically  determined  as   is   the  case  in  mice?     Antibodies  to  coxsackie 
B4  are  present  in  about  half  the  population;  even  more  common  are  antibodies 
to  mumps.      Diabetes  may  occur  in  children  who  lack  antibody  to  either  virus. 
Perhaps   initiation  of  juvenile-onset  diabetes   requires  more  than  a  simple 
virus   infection  in   genetically  predisposed  individuals.     Why  do  damaged  beta 
cells  not  regenerate?     Clearly,  additional   epidemiological    studies  are  needed 
to  examine  these  questions.     Such  prospective,   longitudinal,  mul tidiscipl inary 
studies  will    be  costly  and  demanding,  but  the  answers  they  seek  would   form  the 
basis  of  new  approaches   to  prevention  of  a  particularly  devastating  chronic 
disease. 

International    Health 

International    health  programs   are  administered  by  Dr.    Earl    Beck,  Special 
Assistant  to   the   Director,  MIDP,   in  conjunction  with   individual    program  officers 
and  coordinators. 

The   International    Centers   for  Medical    Research    (ICMR)   that  have  been  operative 
for  nearly  20  years  will    be  phased-out  on  or  about  May  31,   1980.     The   four 
institutions   participating  in  the   ICMR  program  are  the  Johns   Hopkins   University 
with  an  overseas   base  presently  at  the  Gorgas  Memorial    Laboratories,   Balboa 
Heights,   Canal    Zone;  Tulane  University  with  a   research  unit   in   Cali,   Colombia; 
the  University  of  Maryland  with  a  base  in   Lahore,   Pakistan;  and  the  University 
of  California  with  a  unit  in   Kuala   Lumpur,  Malaysia. 

ICMR  Evaluation:     The  plan  to  evaluate  the   ICMR  program  with  1%  set-aside 
funds  was   dropped  because  of  prohibitive  cost  projections  and  timing.     Since 
the   ICMRs  will    be  in  their  phase-out  year,   it  was   decided  to  make  plans   now 
to  evaluate  the   ICIDR  program  in  about  three  years. 

International  Collaboration  in  Infectious  Diseases  Research  (ICIDR)  is  a  new 
initiative  in  the  Microbiology  and  Infectious  Diseases  Program.  It  is  divided 
into  Part  A,  International  Program  Project  Grants,  and  Part  B,  International 
Exploratory/Developmental  Research  Grants.  The  research  emphasis  of  the  new 
ICIDR  program  is  tropical  infectious  diseases  and  the  immunology  of  these 
diseases.  Special  attention  is  given  to  the  six  diseases  of  the  WHO  Special 
Program  for  Research  and  Training  in  Tropical    Diseases. 

Part  A,    International    Program  Project  Grants,  embraces   broadly  based  mul ti- 
discipl inary  research  programs   that  have  a  well-defined  central    focus  or 
objective,  with  major  portions  of  the   research  being  conducted  overseas  with 
an  acceptable   foreign  affiliate.      Fourteen  applications  were   received  and 
reviewed;   nine  were  approved,  and  five  disapproved.      Of  the  nine  approved, 
six  were  within  the   fundable   range.     The   Institute  plans   to   fund  four  of  the 

6-8 


program  project  grants   in  late   FY  79,  and  two  in   FY  80,  depending  on  the 
availability  of  funds.     The  grantees  will    be  collaborating  with  scientists 
in  Brazil,  Thailand,  Sudan,  Colombia,   and  Pakistan. 

Part  B,   International    Exploratory/Developmental    Research  Grants,  encourages 
an  individual    investigator  to  develop  a  biomedical    research  program  with  an 
overseas  affiliate,  with  most  of  the  work  being  done  in  the  host  country. 
Thirty-nine  proposals  were  received  and  reviewed;  sixteen  were  approved,  and 
twenty-three  disapproved.     Of  the  sixteen  approved  grants,   five  were  within 
the  fundable  range.     These  grantees  will    be  collaborating  with  scientists  in 
Mexico,  Brazil,   India,  and  Nigeria.     Based  upon  the  Institute's  Advisory 
Council's  recommendation  that  the  Part  B  portion  of  the   ICIDR  program  be 
readvertized,  consideration  is  now  being  given  to  this   recommendation  so  as 
to  provide  applicants  as  much  time  as  possible  for  the  preparation  of  appli- 
cations.    Funding  of  these  grants   is  projected  for  FY  81. 

Diplomatic  initiatives   by  successive  U.S.  administrations  have  involved  NIH 
in  a  growing  number  of  bilateral   health  agreements,  with  MI  DP  being  assigned 
responsibility  for  coordinating  participation  of  NIAID  investigators   in 
collaboration  in  infectious  diseases  research.     The  oldest  and  most  productive 
of  these  bilateral   programs  is  that  with  Japan,   now  completing  its   fifteenth 
year. 

The  U.S. -Japan  Cooperative  Medical    Science  Program  provides  the  mechansim  for 
scientists  of  the  United  States  and  Japan  to  collaborate  on  the  following 
diseases  or  disease  categories  of  importance  to  the  health  of  the  people  of 
Asia:     cholera,  environmental   mutagenesis  and  carcinogenesis,  leprosy,  mal- 
nutrition, parasitic  diseases,  tuberculosis,  and  viral   diseases. 

The  Joint  Subcommittee  on  Program  Review  and  Planning  met  in  Honolulu  on 
February  8  and  9,   1979,  and  again  on  July  25,   1979,  at  NIH,  just  prior  to  the 
Fifteenth  Joint  Committee  meeting.     Major  topics  discussed  and  acted  upon  at 
the  latter  meeting  included  the  following: 

1.  The  formal    review  of  the  Joint  U.S. -Japan  Panels  on   (Methods   for 
Evaluating)   Environmental   Mutagenesis  and  Carcinogenesis,   initiated  in  1978, 
was  completed.     The  Committee  accepted  the  report  with  revised  guidelines  as 
recommended  by  the  Subcommittee.     The  future  emphasis  of  this   program  will 
change  from  developmental   methodology  to  population  monitoring. 

2.  The  formation  of  a  Hepatitis   Panel   was   formally  approved  along  with 
appropriate  guidelines  and  Panel   members.     The  first  joint  meeting  of  the  new 
panels  will   be  held  in  Japan  in  1980. 

3.  Revised  guidelines   for  the  Tuberculosis   Panels  were  considered  and 
approved.     The  guidelines  were  sharpened  to  reflect  the  heavy  emphasis  on 
research  relating  to  the  pathogenesis  and  immunological   aspects  of  the  disease. 

4.  Preliminary  discussions  were  held  to  explore  the  possibility  of 
introducing  discipline  oriented  panels   into  the  program,  with  the  first  such 
Panel    to  be  one  on   Immunology.     The  matter  is  to  be  considered  further  at  the 
next  meeting  of  the  Subcommittee  on   Program  Review  and  Planning  in   February, 

6-9 


1980.  The  Subcommittee  will  make  these  recommendations  to  the  next  Joint 
Committee  meeting  in  Tokyo  in  1980. 

5.  A  schedule  was  developed  for  publishing  a  Third  Five  Year  Report, 
1975-1980.  A  rough  draft  is  to  be  ready  for  Subcommittee  review  at  its 
February,  1980  meeting. 

U.S.-U.S.S.R.  Health  Cooperation:  The  Institute's  responsibility  under  this 
program  consists  of  its  participation  in  the  U.S.-U.S.S.R.  Collaborative 
Agreement  on  Influenza  and  Acute  Respiratory  Diseases.  For  the  period  October 
8  through  November  5,  1978,  Dr.  Fred  Hayden,  University  of  Virginia,  partici- 
pated in  clinical  trials  of  anti-infl uenzal  drugs  at  the  All  Union  Institute 
for  Influenza,  Leningrad.  As  part  of  a  month-long  visit  to  the  U.S.,  Dr.  Yuri 
Ivannikov  of  that  Institute  spent  a  week  at  NIH  in  March,  1979,  discussing 
epidemiological  studies. 

As  U.S.  Coordinator  of  Problem  Area  IV  -  Chemotherapy  and  Chemoprophylaxis  - 
Dr.  George  Galasso  organized  a  meeting  on  this  subject  at  the  Influenza  Center 
in  Houston,  Texas,  on  February  1-2,  1979.  Five  Soviet  scientists  participated, 
with  three  remaining  for  visits  to  U.S.  laboratories.  It  is  expected  that 
several  Soviet  investigators  will  review  the  Russian  experience  with  amantadine 
at  the  Consensus  Conference  on  the  use  of  this  anti-infl uenzal  drug  scheduled 
for  mid-October,  1979.  Two  days  later,  a  joint  meeting  in  Bethesda  will  address 
Problem  Area  II  -  Immunoprophyl axis  -  and  its  two  topic  areas.  Dr.  W.  S.  Jordan, 
Jr.  is  U.S.  Coordinator  for  Topic  1,  Development  of  killed  and  live  influenza 
vaccines;  Dr.  Frank  Ennis  (BoB/FDA)  is  U.S.  Coordinator  for  Topic  2,  Standardiz- 
ation and  control  of  influenza  vaccines. 

U.S. -China  Cooperation  in  the  Science  and  Technology  of  Medicine  and  Health: 
On  June  22,  1979,  representatives  of  the  Ministry  of  Public  Health  of  the 
People's  Republic  of  China  and  of  the  Department  of  Health,  Education,  and 
Welfare  of  the  U.S.  signed  a  Protocol  of  agreement,  and  later  participated  in 
the  first  meeting  of  the  U.S.-P.R.C.  Joint  Committee  for  Cooperation  in  Medicine 
and  Public  Health.  One  of  the  subjects  designated  for  cooperative  research  is 
to  be  Infectious  and  Parasitic  Diseases,  with  initial  cooperation  in  four  areas: 
1)  viral  hepatitis;  2)  schistosomiasis;  3)  influenza;  4)  malaria.  Plans  are 
now  being  made  to  implement  this  new  bilateral  agreement. 

Cholera  Research  Laboratory:  For  nearly  twenty  years  the  NIH/NIAID  has  operated 
the  Cholera  Research  Laboratory  (CRL)  in  Dacca  for  the  U.S.  Agency  for  Inter- 
national Development  (AID)  as  a  field  station  for  studying  cholera  and  related 
diarrheas.  The  CRL  was  launched  under  the  Southeast  Asia  Treaty  Organization 
in  1960,  and  became  the  International  Center  for  Diarrheal  Disease  Research/ 
Bangladesh  this  year.  This  change  in  name  reflects  a  change  in  administration 
negotiated  by  the  CRL  with  its  various  sponsors.  The  final  steps  in  the  trans- 
formation of  CRL  to  ICDDR/B  occurred  during  the  last  week  of  June,  1979,  when  a 
new  Board  of  Trustees  met  in  Dacca  and  assumed  responsibility  for  operating  the 
Center.  Dr.  Carl  Miller  has  served  the  U.S.  administrative  needs  of  the  CRL  for 
many  years,  and  was  particularly  helpful  during  the  recent  period  of  transition. 
NIH  will  now  relate  to  the  ICDDR/B  in  its  new  status  as  an  autonomous  institu- 
tion. It  is  anticipated  that  the  laboratory  will  continue  to  make  important  con- 
tributions to  the  treatment  and  control  of  cholera  and  other  diarrheal  diseases. 


6-10 


BACTERIOLOGY  AND  VIROLOGY  BRANCH 

The  Bacteriology  and  Virology  Branch  is  responsible  for  the  administration 
of  a  broad-ranging  program  of  biomedical  research;  it  supports  program 
project  grants,  individual  research  grants,  training  grants,  career  develop- 
ment awards,  individual  postdoctoral  fellowships,  and  contract  programs  of 
targeted  research  in  bacteriology,  virology,  and  mycology.  Dr.  Milton  Puziss 
administers  the  Branch  and  the  Bacteriology  Program.  Dr.  William  P.  Allen 
administers  the  Virology  Program,  and  also  serves  as  Executive  Secretary  of 
the  Virology  Panel,  U.S. -Japan  Cooperative  Medical  Science  Program.  One  of 
his  recent  activities,  for  which  he  received  the  NIH  Director's  Award,  was 
bringing  to  a  successful  conclusion  the  work  of  the  Virology  Task  Force. 
Dr.  Darrel  D.  Gwinn  administers  the  Mycology  and  Mycobacterial ogy  Programs, 
and  also  serves  as  Executive  Secretary  of  the  Leprosy  Panel  and  of  the  Tuber- 
culosis Panel,  U.S. -Japan  Cooperative  Medical  Science  Program. 

Approximate  Level  of  Support 

Bacteriology  and  Mycology 

Activity  Number  Amount 

Research  Grants 

Research  Program  Projects 

Career  Awards 

Training  Grants 

Fellowships 

Research  Contracts 

Total 

Virology 

Research  Grants 

Research  Program  Projects 

Career  Awards 

Training  Grants 

Fellowships 

Research  Contracts 

Total 
Branch 

Total 
Program  Summary 

The  program  of  this  Branch  focuses  upon  projects  in  both  fundamental  and  applied 
biomedical  research,  with  the  ultimate  objective  being  the  translation  of  the 
knowledge  gained  into  more  practical  methods  for  the  diagnosis,  prevention 

7-1 


178 

$12,821,120 

7 

2,167,800 

11 

399,169 

19 

1,563,342 

10 

142,200 

12 

1,003,379 

237 

$18,097,010 

194 

$16,016,949 

2 

464,087 

21 

699,908 

13 

1,177,476 

17 

211,150 

1 

22,000 

248 

$18,591,570 

485 

$36,688,580 

and  therapy  of  bacterial,  fungal  and  viral  diseases.  Certain  research  areas 
have  been  considered  as  Programs  of  Institute  Emphasis;  research  in  these 
targeted  endeavors  is  continuing  and  expanding  where  possible.  These  ongoing 
Institute  Emphasis  Programs  are  in  Sexually  Transmitted  Diseases,  Hospital 
Associated  Infections,  Streptococcal  Diseases  and  Sequelae,  and  Mycology, 
plus  several  in  Virology.  Viral  infections  have  a  significant  and  continuing 
impact  on  the  health  of  the  public;  control  of  these  infections  is  a  vital 
part  of  the  Virology  Program  where  studies  in  Persistent  Infections  and 
Chronic  Viral  Diseases,  Clinical  Virology,  and  Biology  of  Viruses  have  been 
highlighted  for  increased  attention.  Targeted  research  on  rabies,  dengue  and 
other  arthropod  transmitted  diseases  under  the  auspices  of  the  U.S. -Japan  CMSP 
is  also  a  part  of  the  thrust  toward  expanding  knowledge  of  viral  diseases 
commonly  found  in  the  developing  countries  of  the  world. 

Bacteriology 

Sexually  Transmitted  Diseases  (STD) 

Sexually  Transmitted  Diseases  continue  as  one  of  the  most  pressing  problems 
in  public  health  today,  with  little  indication  of  any  significant  diminution 
in  their  prevalence.  Gonorrhea  is  still  the  most  important  of  these  diseases, 
although  many  experts  now  consider  that  this  has  been  superseded  by  the  rapid 
increases  in  nongonococcal  urethritis  (NGU)  and  in  herpes  simplex  genital 
infections  (HSV-2).  There  is  also  increasing  concern  regarding  other 
diseases,  such  as  hepatitis,  amebic  dysentery,  and  shigellosis  that  are  now 
being  seen  with  increasing  frequency,  particularly  among  homosexual  groups. 
The  Institute's  overall  research  program  in  support  of  the  STD  problem 
includes  program  projects  (STD  Centers),  grants,  contracts,  and  training. 
An  important  new  thrust  in  FY  1979  was  the  initiation  of  a  contract  to 
develop  candidate  vaccine  materials  for  the  eventual  control  of  gonorrhea. 

Research  Highlights 

P01  AI  12192-04  K.  K.  Holmes  (University  of  Washington):  Dr.  Holmes,  the 
director  of  this  project,  has  reported  that  chlamydial  infections  in  females 
have  a  strong  association  with  neonatal  death,  in  addition  to  fetal  wastage 
and  low  birth  weights.  He  also  found  that  these  infections  may  be  a  cause 
of  peri -hepatitis  (liver  surface  infection),  as  detected  by  a  rise  in  anti- 
body titers.  Dr.  Falkow  of  this  group  has  found  that  Chlamydia  trachomatis 
and  C_.  psittaci  have  a  group  plasmid  of  apparently  the  same  size,  4.5  daltons. 
This  plasmid  was  also  found  in  Lymphogranuloma  venereum  strains  of  Chlamydia; 
there  is,  therefore,  a  very  close  genetic  relationship  between  these  chlamy- 
dial organisms.  The  chlamydial  plasmid  DNA  has  been  cloned  into  an  E_.  coli 
plasmid;  the  role  of  this  plasmid  as  a  virulence  factor  in  disease,  however, 
is  not  yet  clear.  Dr.  Eschenbach  has  undertaken  a  study  of  vaginitis  as 
an  STD  problem.  He  has  reported  that  Hemophilus  vaginalis  infections  can 
be  successfully  treated  with  metronidazole  (Flagyl]"?  In  a  study  of  male 
partners  of  infected  females,  he  isolated  H_.  vaginalis  from  both  the  urethra 
and  semen.  He  further  noted  that  vaginitis  may  be  a  mixed  infection  with 
H_.  vaginalis  together  with  an  anaerobic  flora.  By  DNA  homology  studies 
H_.  vaginalis  was  determined  to  be  unrelated  to  Corynebacteria  species  or 
to  any  other  similar  organism  --  it  may  be  a  new  genus. 

7-2 


AI  13233-03  J.  A.  Yorke  (University  of  Maryland):  This  investigator  is 
studying  the  epidemiology  of  gonorrheal  infections  by  developing  a  mathemati- 
cal model  of  the  existing  and  potential  effectiveness  of  various  control 
programs.  His  study  indicates  that  screening  programs  for  gonorrhea 
discover  only  one-tenth  of  all  infected  women,  usually  in  the  asymptomatic 
stage.  He  also  emphasizes  the  importance  of  relatively  small  "core"  groups 
who  are  repeatedly  reinfected  and  are  efficient  disease  transmitters.  In 
a  model  based  on  use  of  a  hypothetical  gonorrheal  vaccine  administered  to 
this  "core"  group,  he  reported  that  there  would  be  a  substantial  reduction 
in  gonorrhea  prevalence,  even  with  immunity  of  short  duration.  Dr.  Yorke 
emphasizes  that  it  is  roughly  three  to  five  times  as  important  to  identify 
and  treat  the  source  of  the  primary  infection  as  it  is  to  treat  a  secondary 
infection.  This  concept  of  a  "core"  group  of  efficient  disease  transmitters 
can  be  valuable  in  understanding  the  transmission  dynamics  of  other  diseases. 

P01  AI  12618-04  J.  M.  Knox  (Baylor  College  of  Medicine):  Dr.  G.  Dreesman, 
an  investigator  in  this  program  project,  is  attempting  to  develop  efficient 
techniques  to  purify  native,  immunologically  active  herpes  virus  type  2 
(HSV-2)  glycoproteins.  Such  a  viral  subunit  would  have  significant  potential 
as  a  candidate  vaccine.  Dr.  Dreesman  demonstrated  that  HSV-2  glycoproteins 
of  119,000  MW  will  induce  virus  neutralizing  antibodies  in  immunized  rabbits 
and  guinea  pigs.  A  new  technique  was  developed  for  fractionation  of  viruses; 
distinct  peaks  were  isolated,  with  MW  ranging  from  20,000-200,000.  These 
were  tested  for  HSV  type-specific  antibody  by  a  microsolid  phase  radio- 
immunometric  assay.  The  peak  containing  protein  with  100,000-140,000  MW 
reacted  preferentially  with  an  HSV-2  antibody  positive  human  serum,  but  not 
with  an  HSV-1  (oral  herpes)  serum.  This  material  is  now  being  evaluated  for 
induction  of  protective  antibody  in  guinea  pigs.  Other  studies  by  this 
group  show  that  10%  of  females  with  prior  HSV-2  episodes  shed  virus  during 
their  asymptomatic  periods  long  after  their  overt  disease  phase,  a  finding 
of  significant  epidemiological  importance  relative  to  spread  of  the  viral 
infection. 

AI  14648-02  A.  G.  Plaut  (New  England  Medical  Center,  Boston):  Dr.  Plaut 
previously  reported  the  discovery  of  a  protease  enzyme  in  the  gonococcus  that 
destroys  IgA  immunoglobulin.  Purification  kinetics  of  this  enzyme  are  under 
study.  Antibody  to  the  protease  is  inhibitory  to  the  enzyme;  patients  with 
active  gonococcal  infection  have  a  high  titer  of  antibody  to  this  protease. 
He  has  also  found  that  this  IgA  protease  occurs  in  Streptococcus  pneumoniae 
and  Hemophilus  influenzae,  in  addition  to  the  pathogenic  Neisseria  species. 

Hospital  Associated  Infections  (HAI) 

Nosocomial,  or  Hospital  Associated,  Infections  are  an  extremely  troubling 
national  health  problem,  resulting  in  significant  mortality  and  excessive 
costs.  As  the  immunocompromised  patient  population  increases,  the  HAI 
problem  tends  to  increase  as  well.  The  gram  negative  bacteria  that  are 
most  commonly  involved  in  these  infections  show  a  disturbing  trend  to 
antibiotic  resistance,  causing  ever  great  difficulty  in  patient  therapy. 


7-3 


Research  Highlights 

AI  10108-09  A.  I.  Braude  (University  of  California,  S.D.):  Dr.  Braude 
reported  earlier  on  the  efficacy  of  his  J5  antiserum,  obtained  from  human 
volunteers  immunized  with  the  "core"  glycol ipid  from  an  E.   coli  strain,  in 
lowering  the  death  rate  from  gram  negative  bacteremia.  Newer  data  with 
50  of  90  additional  patients  (the  study  is  still  incomplete)  indicate  that 
the  trend  is  very  favorable,  consistent  with  the  life  saving  therapeutic 
effectiveness  reported  earlier.  In  these  newer  studies  the  prominence  of 
bowel,  lung,  and  urinary  tract  sites  for  gram  negative  infections  was 
significant.  The  bacteremias  were  caused  primarily  by  normal  aerobic 
bowel  inhabitants  (E_.  coli  and  Klebsiella  species)  and  antibiotic  resistant 
opportunistic  microorganisms  (Pseudomonas  and  Serratia) ;  Pseudomonas  was  the 
organism  most  frequently  isolated  from  the  blood.  The  prophylactic  value 
of  J5  antiserum  was  studied  in  agranulocytopenic  rabbits  to  determine  if 
it  could  prevent  bacteremia.  Results  indicate  that  the  intravenous  injection 
of  J5  antiserum  protects  rabbits  against  lethal  Pseudomonas  bacteremia;  this 
protection  lasts  for  at  least  six  weeks.  This  study  is  being  extended  to 
determine  the  potential  prophylactic  efficacy  of  the  J5  serum  in  burn 
patients. 

AI  14533-01  R.  Garibaldi  (University  of  Utah):  This  project's  goal  is  to 
establish  cost-effective  approaches  to  prevent  post-operative  nosocomial 
infections.  A  micropore  membrane  filter  contact  technique  for  quantitating 
bacterial  contamination  of  wound  surfaces  was  developed.  This  proved  to  be 
a  sensitive  and  reproducible  method  for  recovering  bacteria;  it  may  be  an 
accurate  predictor  of  wound  colonization.  In  a  retrospective  comparison 
of  hospitalization  duration  following  post-operative  infections,  the  data 
showed  that  wound  infection  rates  varied  with  the  type  of  surgery  and  likeli- 
hood of  intra-operative  bacterial  contamination.  The  highest  infection  rate 
and  prolonged  hospitalization  occurred  following  colon  surgery  (3.8%,  with 
excess  hospitalization  of  23.8  days).  This  represents  a  significant  addition 
to  the  costs  of  medical  treatment.  It  was  also  shown  that  administration  of 
antibiotics  prophylactically  prior  to  surgery  was  frequently  misused.  Such 
misuse  probably  predisposes  to  colonization  and  infection  with  antibiotic 
resistant  bacteria.  The  routine  use  of  an  in-line  micropore  bacterial  gas 
filter  in  anesthesia  equipment  had  no  effect  on  the  incidence  of  post- 
operative pulmonary  infection.  Use  of  these  devices  is  not  cost  effective 
and  probably  should  be  discontinued. 

AI  13120-03  C.  D.  Cox  (University  of  Iowa):  Dr.  Cox  previously  reported 
investigations  on  the  iron-binding  siderophore,  pyochelin,  of  pathogenic 
Pseudomonas.  He  showed  that  all  clinical  isolates  apparently  possess  this 
iron-binding  pigment.  Knowledge  of  the  pyochelin  structure  is  important 
for  understanding  iron  metabolism  and  how  to  interfere  with  this  effect 
during  an  infection.  Most  of  the  pyochelin  structure  has  now  been 
elucidated  --  it  is  about  650  MW,  composed  of  two  identical  subunits 
each  containing  a  salicylic  acid  and  sulphur.  The  compound  is  also  very 
labile.  The  chelated  iron  is  adsorbed  rapidly  to  the  bacterial  surface; 
the  pyochelin  is  exceptionally  effective  in  stimulating  iron  uptake  by  the 
bacteria  at  extremely  low  concentrations.  Pseudomonas  bacteria  passed 
through  mice  infected  intraperitoneal^  are  most  responsive  to  host  iron 

7-4 


for  increased  lethality;  organisms  harvested  from  lung  tissue  (intrathoracic 
infections),  however,  suppress  phagocytosis  and  cause  increased  lethality. 
This  mouse  model  describes  differences  in  ecological  niches  in  the  host 
as  well  as  mechanisms  of  Pseudomonas  virulence. 

AI  12936-03  J.  W.  Alexander  (University  of  Cincinnati):  Dr.  Alexander  has 
been  studying  immune  responses  in  surgical  infections.  Previous  data 
indicated  that  abnormal  neutrophil  function  was  clearly  associated  as  a 
predisposing  factor  in  post-surgical  infections.  This  group  is  now  studying 
the  close  association  between  nosocomial  infection  and  nutritional  status. 
Further  data  indicate  that  well-nourished  burn  patients  supplemented  with 
plasma  did  very  little  better  than  controls,  but  patients  with  a  "high 
protein"  regime  showed  a  significant  improvement  in  neutrophil  function. 
In  a  mouse  model  system,  administration  of  Corynebacterium  parvum  vaccine 
resulted  in  increased  macrophage  activation.  The  C_.  parvum  vaccine  also 
resulted  in  increased  survival  following  challenge  by  Staphylococcus  aureus 
and  Listeria  monocytogenes.  Additional  study  in  guinea  pigs  showed  that 
skin  grafts  in  cortisone-treated  immunosuppressed  animals  had  enhanced 
survival  times  when  C.  parvum  was  administered.  These  observations  are 
of  clinical  significance  in  establishing  a  rationale  for  use  of  substances 
like  £.  parvum  in  treating  infections  in  certain  immunosuppressed  conditions. 
The  clinical  aspects  of  these  studies  are  now  being  investigated. 

AI  11271-06  L.  S.  Young  (University  of  California,  L.A. ):  This  investigator 
has  focused  on  host  interaction  to  Pseudomonas  infection.  He  has  reported 
that  patients  with  advanced  Cystic  Fibrosis  (CF)  showed  a  high  level  of 
circulating  immune  complexes  against  Ps_.  aeuruginosa.  Further  these  com- 
plexes had  "enriched"  antibody  titers  against  lipopolysaccaride  antigens 
of  the  organism.  No  significant  antibody  level  against  Pseudomonas  exotoxin 
A  in  the  CF  patient  sera  was  observed;  preliminary  data  indicated,  however, 
that  the  immune  complexes  contained  endotoxin-like  activity. 

Conference 

A  workshop  on  Pseudomonas  infections  in  Cystic  Fibrosis  (CF)  was  held  on 
January  9,  1979,  to  explore  the  magnitude  of  this  infectious  disease 
problem.  These  infections  are  responsible,  in  large  part,  for  the  extreme 
disability  and  early  mortality  of  the  young  CF  patient.  Recommendations 
for  expanded  research  in  selected  areas  of  this  disease  problem  were 
developed  as  a  result  of  this  meeting;  publication  of  the  summary  pro- 
ceedings will  be  in  the  Journal  of  Infectious  Diseases. 

Streptococcal  Diseases  and  Sequelae  (STP) 

Group  B  streptococcal  disease  (GBS)  persists  as  a  significant  cause  of 
neonatal  mortality  or  serious  sequelae.  Infants  of  mothers  with  low 
maternal  antibody  levels  are  at  high  risk  of  developing  GBS  infections. 
Research  leading  to  a  better  understanding  of  the  disease  problem  and  to 
development  of  a  candidate  vaccine  is  of  high  priority. 


7-5 


Research  Highlights 

NO!  AI  72538  D.  L.  Kasper  (Peter  Bent  Brigham  Hospital,  Boston):  This 
contract  is  for  study  of  the  antigens  of  the  GBS  and  development  of  a 
candidate  vaccine  to  protect  neonates  at  risk.  The  objective  is  to 
vaccinate  pregnant  women  with  low  maternal  antibody  levels  so  that  the 
neonate  at  birth  will  have  a  high  protective  maternal  antibody.  Research 
is  focused  on  the  capsular  polysaccharide  of  the  type  III  GBS.  This 
purified  polysaccharide  elicited  a  significant  antibody  rise  in  immunized 
human  volunteers,  correlated  with  a  rise  in  the  opsonic  index.  The  antigen 
has  a  sialic  acid  side  chain  attached  to  a  carbohydrate  "core";  the  side 
chain  is  important  in  eliciting  the  protective  antibodies.  Studies  of  GBS 
types  la  and  II  polysaccharide  antigens  have  also  been  initiated.  It  is 
expected  that  eventually  a  tri-valent  GBS  vaccine  will  be  developed,  incor- 
porating these  three  antigenic  polysaccharides.  Investigation  has  also 
begun  on  plasmapheresis  procedures  to  prepare  high  titer  human  immune 
globulins  (IgG)  from  human  volunteers.  If  successful  this  material  will 
be  used  for  passive  immunization  to  protect  high  risk  infants  against  the 
late-onset  type  of  GBS  disease. 

AI  13150-03  H.  R.  Hill  (University  of  Utah):  Host-defense  mechanisms 
against  GBS  infections  are  not  fully  defined.  Dr.  Hill  has  developed  a 
promising  rat  model  for  study  of  GBS  pneumonia.  Newborn  rats  (under  16 
hours  old),  inoculated  with  GBS  type  III  intranasally  (LD50  of  5x106 
organisms)  developed  fatal  pneumonia  and  sepsis.  Adult  animals  with 
similar  challenge  showed  no  bacteremia  or  mortality.  Human  serum  contain- 
ing GBS  opsonic  activity,  when  given  intraperitoneal^,  protected  challenged 
neonatal  animals.  The  routes  of  infection,  rapid  death,  neutrophil  changes, 
and  neonatal  susceptibility  are  important  parallels  to  human  infection. 
When  whole  blood  containing  heat-stable  antibody  to  the  infecting  GBS  was 
given  to  human  neonates,  a  rise  in  neonatal  opsonic  activity  resulted. 
Nine  of  nine  infants  receiving  this  transfusion  survived  their  septic 
episodes,  whereas  three  of  six  infants  receiving  blood  lacking  antibody 
to  their  infecting  strain  died. 

AI  14361-02  J.  B.  Zabriskie  (Rockefeller  University):  This  project  is  a 
study  of  the  streptococcal  and  host  factors  that  are  of  pathogenic  importance 
in  the  sequelae  of  streptococcal  infection.  A  unique  population  in  Trinidad 
is  under  study,  where  both  rheumatic  fever  (RF)  and  acute  glomerulonephritis 
(AGN)  are  common  sequelae  of  the  streptococcal  infections.  Results  show  that 
patients  with  RF  respond  primarily  to  antigens  present  in  the  protoplast 
membrane  of  streptococcal  strains  associated  with  RF  and  do  not  react 
to  antigens  found  in  strains  associated  with  nephritis.  Those  strains 
associated  with  AGN  secrete  a  protein  antigen  found  in  the  streptococcal 
cell  membrane;  this  antigen  is  also  detected  in  patients'  glomeruli. 
Each  patient  group  thus  reacts  specifically  to  those  antigens  present 
in  streptococcal  strains  associated  with  a  particular  disease.  This 
patient  reactivity  may  be  genetically  inherited. 


7-6 


Other  Disease  Problems 

Staphylococcus  aureus  is  an  organism  that  can  cause  a  number  of  distinct 
disease  syndromes  within  the  same  host;  the  organism  also  produces  an 
impressive  array  of  biologically  active  toxins. 

AI  14998-02  M.  E.  Melish  (Kapiolani-Children' s  Medical  Center,  Honolulu): 
Dr.  Melish  is  investigating  the  clinical-pathological  correlates  of  infection 
with  staphylococcal  exfoliatin,  or  epidermolytic  toxin  (ET),  the  cause  of 
staphylococcal  "scalded  skin  syndrome"  (SSS)  in  infants.  A  newly-developed 
antibody  radioimmunoassay  was  1,000  times  more  sensitive  than  earlier  methods 
for  detection  of  ET  in  blood  tissues;  this  method  detected  ET  in  acute  sera 
from  all  SSS  patients  tested.  No  free  ET  was  detected,  however,  in  patient 
sera  after  antibiotic  therapy  was  begun.  In  an  adult  mouse  model,  epider- 
molysis by  ET  could  be  induced  regularly;  this  developed  10-16  hours  after 
peak  serum  levels  of  ET  were  established.  Patients  with  generalized  SSS 
showed  no  evidence  of  pre-existing  ET  neutralizing  antibody.  In  contrast  to 
generalized  toxemic  SSS,  localized  bullous  impetigo  can  develop  and  progress 
despite  high  levels  of  circulating  antitoxin.  Screening  of  normal  adults 
revealed  that  over  85%  of  those  over  age  20  have  ET  antitoxin,  whereas  only 
24%  of  children  under  age  two  years  do  so.  Thus,  lack  of  this  age-related 
antitoxin  appears  to  be  associated  with  the  prevalence  of  SSS  in  children. 

AI  07826-10  F.  A.  Kapral  (Ohio  State  University):  In  this  study  on  SSS, 
Dr.  Kapral  showed  that  the  syndrome  is  the  result  of  the  intercellular 
separation  among  cells  of  the  granular  layer;  it  does  not  involve  actual 
cell  destruction.  In  a  neonatal  mouse  model,  he  showed  that  a  positive 
Nikolsky  sign  (loss  of  skin  elicited  by  gentle  stroking)  cannot  serve  as 
the  sole  criterion  for  exfoliatin  action.  Recent  data  show  that  certain 
S_.  aureus  strains  produce  a  substance  causing  a  positive  Nikolsky  sign 
distinct  from  exfoliatin.  This  substance  caused  destructive  lesions 
beneath  the  granular  layer;  it  also  differed  in  antigenic  specificity  from 
ET.  S_.  aureus  strains  isolated  from  patients  produced  this  new  exfoliatin- 
1  ike  material.  Additionally,  an  extractable  lipid-like  material  found 
within  abscesses  exhibited  marked  bactericidal  activity  against  certain 
S.  aureus  strains. 

AI  03772-19  P.  A.  Murphy  (Johns  Hopkins  University):  The  pathogenesis  of 
fever  is  only  vaguely  understood.  Dr.  Murphy  has  shown,  in  a  goat  model, 
that  purified  macrophages  make  at  least  four  distinct  pyrogens;  these  are 
in  response  to  stimulation  by  labeled  endotoxin,  by  labeled  staphylococcal 
organisms  or  by  influenza  viruses.  The  purified  pyrogens  all  gave  double- 
labeled  peaks  regardless  of  the  stimulating  source.  One  of  these  macrophage 
pyrogens  fs  identical  to  that  from  neutrophils;  the  other  three  are  clearly 
different. 

AI  09366-09  T.  A.  Stamey  (Stanford  University):  Urinary  tract  infections 
in  women  represent  a  costly  public  health  problem.  Dr.  Stamey  has  found 
that  cervicovaginal  antibody  in  female  volunteers  was  directed  against  a 
variety  of  E_.  coli  strains  but  not  against  the  volunteers'  indigenous  flora. 
Antibody  was  uniformly  found  in  salivary  fluid,  eliminating  the  possibility 

7-7 


of  an  immunodeficient  reaction  at  mucous  surfaces;  bacterial  pili,  however, 
played  a  significant  role  in  attachment  to  vaginal  epithelial  cells.  The 
defect  allowing  E_.  coli  attachment  and  disease  initiation  is  most  probably 
a  local  immunologic  one  in  vaginal  secretions. 

Leprosy 

Leprosy  is  found  in  almost  every  country  in  the  world;  it  is  a  significant 
health  problem  in  the  developing  countries  of  the  world.  The  majority  of 
the  estimated  12  to  15  million  cases  of  leprosy  (10  million  cases  are 
recorded  at  WHO)  are  found  in  Asia,  Africa  and  South  America;  as  many  as 
2,000  cases  are  in  the  United  States.  Approximately  150  new  cases  of 
leprosy  are  reported  in  the  United  States  yearly,  with  about  25%  of  these 
occurring  in  native-born  Americans.  More  significant  than  the  actual 
number  of  leprosy  cases  is  the  evidence  that  not  more  than  20%  of  the 
estimated  cases  in  the  developing  countries  are  under  regular  treatment. 
Also,  children  make  up  roughly  one  third  of  the  total  number  of  those  people 
infected  with  leprosy.  Because  of  its  worldwide  importance,  leprosy  research 
continues  to  be  supported  by  NIAID  under  the  auspices  of  the  U.S. -Japan 
Cooperative  Medical  Science  Program. 

Research  Highlights 

AI  10094-09  W.  E.  Bullock  (University  of  Kentucky):  Dr.  Bullock  has  been 
concerned  with  further  studies  on  the  nature  and  function  of  suppressor  cell 
populations  generated  within  spleens  of  mice  experimentally  infected  with 
Mycobacterium  lepraemurium.  Most  recently,  he  has  investigated  the  nature 
of  splenic  suppressor  cells  from  infected  mice  that  suppress  cell  mediated 
lymphocytotoxic  activity  by  normal  spleen  cells.  Through  these  studies  it 
has  been  demonstrated  that  two  such  suppressor  cell  populations  are  generated 
within  the  spleens  of  M.  lepraemurium  infected  mice.  One  suppressor  population 
is  composed  of  T  lymphocytes  and  the  other  of  macrophages  or,  possibly,  B  cells, 
The  activity  of  the  two  populations  acting  together  in  whole  spleen  cell  pre- 
parations usually  is  greater  than  the  activity  of  either  cell  population 
alone. 

AI  08417-10  A.  H.  Fieldsteel  (Stanford  Research  Institute):  Dr.  Fieldsteel 
has  continued  his  studies  utilizing  the  neonatally  thymectomized  Lewis  rat 
(NTLR)  as  a  model  for  detecting  persistent  M.  leprae.  These  experiments 
have  demonstrated  the  superiority  of  the  NTLR  over  mice  for  the  detection  of 
small  numbers  of  viable  M.  leprae.  Normal  mice  are  routinely  utilized  to 
monitor  the  efficacy  of  leprosy  chemotherapy.  However,  the  maximum  number  of 
M_.  leprae  that  can  be  inoculated  into  mouse  foot  pads  to  demonstrate  multi- 
plication is  10^,  and  the  ceiling  of  multiplication  is  approximately  10° 
On  the  other  hand,  the  NTLR  can  be  inoculated  with  up  to  10'  M_.  leprae  and 
the  ceiling  of  multiplication  is  10^  to  109  organisms.  These  experiments  demon- 
monstrate  that  the  NTLR  can  be  used  as  a  model  of  M.  leprae  persistence. 

AI  07801-12  J.  L.  Krahenbuhl  (U.S.  Public  Health  Service  Hospital,  San 
Francisco]":  Triatoma  barberi  (a  blood  sucking  bedbug)  was  evaluated  to 
determine  the  period  of  viability  of  M.  leprae  following  ingestion  of  approxi- 
mately 5  x  107  organisms  suspended  in  rabbit  blood.  Inocula  of  5000  M.  leprae 

7-8 


organisms  recovered  from  the  Triatoma  between  one  hour  and  four  days  after 
ingestion  were  viable  and  found  to  multiply  in  mouse  foot  pads  to  a  maximum 
of  1  x  106  organisms;  M.  leprae  recovered  seven  days  after  ingestion  by 
Triatoma  multiplied  to  only  3  x  104  organisms,  and  after  14  days  no  multi- 
plication was  observed. 

Tuberculosis 


In  1978,  28,521  cases  of  tuberculosis  were  reported  to  CDC.  This  represents 
a  decrease,  since  1977,  of  5.4%  in  the  number  of  cases  reported.  Since 
antibacterial  drug  therapy  for  tuberculosis  was  initiated,  a  shift  in 
tuberculosis  victims  from  young  adults  to  older  people  has  taken  place. 
The  atypical  mycobacteria  are  related,  but  not  identical,  to  Mycobacterium 
tuberculosis;  although  diseases  caused  by  atypical  mycobacteria  have  symptoms 
similar  to  tuberculosis,  the  infections  are  not  transmitted  from  man  to  man. 
Also,  the  atypical  mycobacteria  are  frequently  resistant  to  the  antibacterial 
therapy  employed  in  treating  ordinary  tuberculosis.  The  number  of  cases  of 
tuberculosis  and  atypical  tuberculosis  in  the  U.S.  is  small  in  comparison  to 
the  millions  of  cases  found  in  the  developing  nations  of  the  world.  Tuber- 
culosis in  these  countries  remains  one  of  the  major  causes  of  morbidity  and 
mortality  in  all  age  groups.  Tuberculosis  research  continues  to  be  supported 
under  the  auspices  of  the  U.S. -Japan  Cooperative  Medical  Science  Program. 

Research  Highlights 

AI  13813-03  H.  Gruft  (New  York  State  Department  of  Health):  Dr.  Gruft  is 
surveying  the  estuaries  in  the  eastern  U.S.  to  establish  a  basis  for  under- 
standing the  epidemiology  of  atypical  mycobacteria  in  relation  to  their 
geographical  distribution.  He  has  found  a  higher  concentration  of 
Mycobacteri urn  avi um-i ntracel 1 ul are-scrof ul aceum  (MAIS)  strains  along  the 
Gulf  Coast  and  in  the  waters  of  the  Carol inas  and  Georgia.  The  highest 
concentration  of  MAIS  strains  are  found  in  waters  of  1-2%  salinity,  i.e., 
estuaries  and  mouths  of  rivers.  MAIS  strains  are  found  in  high  concentration 
in  waters  from  regions  where  most  of  the  cases  of  positive  skin  tests  have 
been  found.  They  can  also  be  found  in  the  air  in  droplets  small  enough  to 
enter  the  alveoli . 

AI  11807-04  B.  M.  Sultzer  (SUNY  Downstate  Medical  Center):  Since  Dr.  Sultzer's 
original  discovery  that  PPD- tuberculin  is  a  polyclonal  activator  of  B-lympho- 
cytes  from  uninfected  or  unimmunized  mice,  other  laboratories  have  found  PPD 
to  be  a  useful  probe  for  studying  a  variety  of  basic  immunological  problems. 
Further  studies  on  the  nonspecific  activation  of  lymphocytes  have  shown  that 
PPD  can  act  as  a  polyclonal  activator  (PCA)  of  human  peripheral  blood  lympho- 
cytes; that  PPD  can  act  as  an  adjuvant  and  as  a  PCA  in  vitro  and  in  vivo;  and 
that  PPD  stimulates  B-cell  hyperplasia  when  directly  injected  into  mice. 
Continuing  studies  have  shown  that  this  intrinsic  biological  property  of  PPD 
to  activate  B-cell s  is  due  to  tuberculoprotein  and  not  to  extraneous  materials. 
Fractionation  studies  indicated  that  the  antigenic  and  mitogenic  activities 
reside  together  in  several  molecular  species  of  tuberculin  proteins.  Antigens 
without  mitogenic  activity  can  be  separated  from  PPD;  however,  mitogens  with- 
out antigenic  activity  have  not  been  separated  from  PPD  as  yet. 

7-9 


NOT  AI  02079  J.  K.  McClatchy  (National  Jewish  Hospital):  Laboratories 
throughout  the  world  have  sent  Dr.  McClatchy  in  the  last  year  alone  almost 
1,400  isolates  of  atypical  mycobacteria  for  classification  and  identification. 
In  addition  to  his  work  with  these  cultures  under  the  contract,  he  has  sup- 
plied a  number  of  investigators  in  the  U.S.  and  abroad  with  reagents  to 
perform  their  own  serological  identification.  He  has  also  investigated  the 
chemical  structure  of  the  antigens  which  are  used  to  classify  the  various 
atypical  mycobacteria  into  different  species  and  subspecies.  The  development 
of  this  methodology  has  been  very  helpful;  Dr.  McClatchy  can  now  biochemically 
identify  certain  members  of  the  atypical  mycobacteria  which  could  not  be 
taxonomically  classified  using  serologic  methodology.  Current  work  is  being 
directed  toward  the  identification  of  atypical  mycobacteria  found  in  patho- 
logic specimens  from  diseased  individuals. 

Mycology 

Primary  fungal  pathogens  that  are  directly  involved  in  pulmonary  diseases, 
such  as  Coccidioides  immitis  and  Histoplasma  capsulatum,  are  well  established 
as  significant  causes  of  morbidity  and  mortality,  especially  in  well  defined 
geographic  areas  of  the  U.S.  Other  fungal  pathogens,  such  as  Candida  and 
Cryptococcus,  are  important  as  opportunistic  microorganisms,  particularly  in 
patients  with  impaired  host  defense  mechanisms.  Treatment  of  these  fungal 
infections  requires  prolonged  administration  of  relatively  toxic  drugs  and 
the  therapy  may  not  always  be  effective.  The  Institute  is  presently  funding 
research  in  which  these  pathogenic  fungi  and  others,  including  Blastomyces 
and  Aspergillus  species,  are  being  investigated.  The  Institute  has  established 
a  special  program  in  Mycology  to  intensify  research  efforts  in  this  area. 

Research  High! ights 

TO!  AI  00459-06  G.  Medoff  (Washington  University):  Dr.  Medoff's  group  has 
been  studying  the  control  of  dimorphic  transition  in  fungi.  H_.  capsulatum, 
an  important  human  pathogen,  is  one  of  these  dimorphic  fungi.  Its  saprophytic 
form  is  mycelial  and  is  found  in  nature,  whereas  the  unicellular  yeast  is  the 
parasitic  phase  found  in  infected  tissues.  One  phase  can  be  induced  to  trans- 
form to  the  other  in  culture  when  the  temperature  of  incubation  is  adjusted 
to  an  appropriate  level.  An  understanding  of  the  biology  and  genetic  regula- 
tion of  the  process,  it  is  believed,  will  provide  insights  into  the  pathogen- 
esis of  disease  caused  by  dimorphic  pathogens  and  may  help  in  the  control  of 
these  infections.  The  major  differences  found  between  the  two  forms  of  this 
fungus  were: 

(a)  Discovery  and  purification  of  a  cystine  reductase  present  only 
in  the  yeast  phase  of  H_.  capsulatum.  This  enzyme  and  a  cystine 
permease  appear  when  the  temperature  is  switched  from  25C  to  37C. 
Both  enzymes  may  be  involved  in  providing  the  sulfhydryl  groups 
(in  the  form  of  cysteine)  that  are  necessary  to  initiate  and 
maintain  the  yeast  phase. 

(b)  The  level  of  cyclic  AMP  is  approximately  five  times  higher  in  the 
mycelial  phase  than  in  the  yeast  phase  of  H.  capsulatum.  Further- 
more, addition  of  either  cyclic  AMP  or  inhibitors  of  cyclic  AMP 

7-10 


phosphodiesterase  induces  transformation  of  yeast  to  the  mycelial 
phase  even  at  37C,  the  nonpermissive  temperature  for  mycelial 
growth  in  nature. 

(c)  The  yeast  and  mycelial  phase  each  have  three  RNA  polymerases, 
similar  to  those  in  other  eukaryotic  cells.  In  H_.  capsulatum, 
the  enzymes  in  one  phase  are  clearly  different,  functionally  and 
structurally,  from  the  enzymes  in  the  other.  This  represents  the 
first  case  in  which  a  clear  difference  in  RNA  polymerases  has  been 
shown  to  exist  between  morphologic  phases  of  the  same  eukaryotic 
organism. 

Dr.  Medoff  is  now  testing,  with  several  mutants  of  H_.  capsulatum,  a  working 
hypothesis  related  to  the  sequence  of  events  and  control  mechanisms  in  this 
transition. 

K04  AI  00325-05  R.  D.  Diamond  (Boston  University  School  of  Medicine): 
Previous  studies  demonstrated  that  neutrophils  could  attach  to,  damage,  and 
probably  kill  Candida  albicans  hyphae  by  a  nonphagocytic  mechanism.  In  con- 
trast to  most  phagocytic  systems,  attachment  and  spreading  of  neutrophils 
over  live  hyphal  surfaces  with  activation  of  microbial  mechanisms  occurred 
in  the  absence  of  serum.  Addition  of  IgG  from  human  serum  augmented  the 
process.  In  the  absence  of  serum,  neutrophils  did  not  attach  to  killed 
hyphae.  However,  supernatants  from  actively  growing  hyphae  or  hyphae  killed 
with  ultraviolet  light  inhibited  attachment  of  neutrophils  to  live  hyphae. 
Evidence  from  Dr.  Diamond's  current  work  suggests  that  the  soluble  inhibitor 
acts  directly  on  neutrophils;  however,  the  neutrophils  are  not  significantly 
damaged  by  exposure  to  the  inhibitory  substance. 

AI  13770-03  M.  H.  Weiner  (University  of  Texas):  The  objective  of  Dr.  Weiner's 
project  is  to  develop  antigen  immunoassays  to  diagnose  systemic  fungal  infec- 
tion. Dr.  Weiner  has  developed  radio  labeled  antigen-binding  inhibition 
assays  (RIAs)  to  detect  Aspergillus  and  Candida  antigenemia.  He  has  now 
analyzed  the  effectiveness  of  the  RIAs  with  sera  obtained  from  hospital 
patients  with  systemic  bacterial  and  fungal  infections  and  from  normal  donors. 
Using  the  RIAs  he  was  able  to  detect  accurately  Aspergillus  and  Candida  anti- 
genemia in  the  sera  from  patients  suffering  from  these  fungal  infections. 
This  is  the  first  demonstration  of  Aspergil lus  antigenemia  detected  in  human 
sera  obtained  antemortem. 

AI  05022-15  G.  S.  Bulmer  (University  of  Oklahoma):  Cryptococcosis,  a  fre- 
quently fatal  meningeal  fungus  disease  of  man,  is  caused  by  the  heavily 
encapsulated  yeast  Cryptococcus  neoformans.  Dr.  Bulmer's  goal  is  to  deter- 
mine why  and  how  certain  individuals  contract  this  disease.  His  studies, 
with  the  intragastric  inoculation  (mice)  of  C_.  neoformans,  indicate  that 
medical  personnel  should  be  alerted  to  the  possibility  that  cryptococcosis 
may  begin  in  the  gastrointestinal  tract;  i.e.,  cryptococcosis  may  not  always 
originate  in  the  lungs.  Additionally,  these  studies  indicate  that  the  mouse, 
and  possibly  other  rodents,  may  act  as  vehicles  for  dissemination  of  the 
pathogenic  yeast  in  nature.  Heretofore,  pigeons  have  been  considered  to  be 
the  sole  natural  source  for  this  organism.  In  another  study  Dr.  Bulmer  has 
shown  that  an  aqueous  extract  of  garlic  is  a  potent  killing  agent  of  C_. 

7-1] 


m 


neoformans.  This  material  should  be  investigated  further  as  a  possible 
therapeutic  agent  against  cryptococcosis  and  other  mycoses. 

Virology 

Program  Summary 

Viruses  continue  to  be  a  major  cause  of  human  illness  in  nearly  all  parts  of 
the  world,  but  modalities  for  control  and  treatment  of  these  pathogens  remain 
relatively  limited.  The  large  number  and  diversity  of  viruses  that  impact  on 
human  health  add  to  the  complexity  of  the  problem  of  their  control.  Signifi- 
cant advances  have  been  made  in  unraveling  the  components  of  viral  structure, 
replication  and  interactions  with  the  afflicted  host,  but  much  remains  to  be 
learned  before  a  conquest  of  viral  diseases  can  be  attained.  The  Virology 
program  within  the  BV  Branch  encourages  and  supports  multidisciplinary 
approaches  to  research  on  viruses  and  viral  diseases  through  its  subprograms 
on  Clinical  Virology,  Persistent  Infections  and  Chronic  Viral  Diseases,  and 
Biology  of  Viruses. 

Clinical  Virology 

Clinical  Virology  is  a  program  of  Institute  emphasis  focused  on  pathogenesis 
and  immunopathogenetic  mechanisms  of  viral  diseases  of  man.  Clinically 
relevant  research  on  pathogenetic  viruses  is  encouraged  to  bridge  a  widen- 
ing gulf  between  molecular-oriented  virologists  and  clinical  investigators 
specializing  in  infectious  diseases.  Research  projects  on  herpesviruses  are 
especially  prominent,  a  reflection  of  the  high  incidence  and  severity  of 
human  diseases  caused  by  herpes  simplex  viruses,  varicella-zoster  virus, 
cytomegalovirus  and  Epstein-Barr  virus. 

Research  Highlights 

AI  14341-02  L.  Aurelian  (The  Johns  Hopkins  University):  It  is  well  estab- 
lished that  human  infections  with  herpes  simplex  virus  (HSV)  elicit  both 
specific  humoral  and  cellular  responses.  Dr.  Aurelian  has  obtained  pre- 
liminary data  supporting  the  hypothesis  that  patients  with  a  history  of 
recurrent  lesions  suffer  from  an  impaired  generation  of  effector  lymphoid 
functions  (differentiated  lymphoid  cells  that  react  specifically  in  response 
to  exposure  to  viral  antigens).  The  hypothesis  further  suggests  that  various 
HSV  antigens  may  induce  different  responses,  that  such  responses  are  affected 
by  clinical  status  (vis.,  quiescence  versus  recrudescence),  and  that  various 
in  vitro  assays  measure  the  expression  of  different  populations  of  lymphoid 
cells.  In  support  of  the  hypothesis,  recent  results  indicate  that  maximal 
effector  levels  are  reached  shortly  after  onset  of  recrudescence,  i.e., 
during  convalescence.  They  begin  to  wane  thereafter,  with  borderline  to 
negative  levels  being  maintained  during  quiescence.  New  recrudescences 
occur  on  this  background  of  low  (or  negative)  effector  functions.  The 
phenomenon  is  virus  specific. 

AI  14564-02  R.  R.  McKendall  (University  of  California,  San  Francisco):  In  the 
presence  of  specific  antibodies,  herpes  simplex  virus  (HSV)  becomes  local i zed 
to  specific  sites  in  the  body  and  is  eventually  eliminated  or  reduced  to  a 

7-12 


state  of  latency.  Dr.  McKendall  has  been  investigating  the  characteristics 
of  specific  antibody  molecules  responsible  for  this  defense  against  infection 
in  a  murine  model.  He  has  found  that  the  neutralizing  fragment,  Fab,  of  the 
antibody  failed  to  avert  disease,  which  indicates  that  neutralization  alone 
is  not  the  major  effect  of  antibody  defense  against  herpetic  infections.  The 
results  further  suggest  that  effective  control  of  viral  spread  within  tissues 
is  dependent  upon  the  Fc  fragment  of  the  antibody  molecule.  The  Fc  fragment 
is  not  antigen  specific.  Moreover,  if  destruction  of  the  blood-brain  barrier 
is  a  late  event  in  the  course  of  herpes  encephalitis  or  herpes  myelitis,  then 
serum  neutralizing  antibody  would  not  be  expected  to  be  much  of  a  host  advant- 
age. This  suggests  a  new  therapeutic  approach  through  methods  which  would 
enhance  both  antibody  and  cellular  penetration  into  the  central  nervous  system 
early  in  the  course  of  disease. 

AI  14373-02  J.  A.  Zaia  (Sidney  Farber  Cancer  Institute):  Infections  with 
varicella-zoster  (VZV)  exert  deleterious  effects  on  the  clinical  course  of 
immunosuppressed  patients,  particularly  those  with  lymphoproliferative  dis- 
orders, causing  significant  morbidity  and  occasional  death  as  well  as  inter- 
rupting scheduled  chemotherapy.  Dr.  Zaia's  research  is  aimed  at  understanding 
the  pathogenesis  of  VZV  infection  in  these  patients,  including  an  understand- 
ing of  the  elements  of  immunity  which  may  be  defective  during  cancer  therapy. 
He  has  developed  the  methodology  for  efficient  identification  of  human  plasmas 
containing  VZV-immune  globulin  by  means  of  screening  large  amounts  of  recovered 
plasma  for  specific  antibody  to  VZV.  In  recent  studies  he  has  demonstrated 
that  this  immunoglobulin  (VZIG)  prevents  chickenpox  in  immunosuppressed 
children.  The  VZIG  was  as  effective  as  standard  zoster  immune  globulin  (ZIG) 
in  preventing  severe  complications  due  to  chickenpox.  The  advantage  of  VZIG 
is  that  it  can  be  produced  easier  and  made  more  available  than  ZIG.  He  has 
also  developed  a  new  laboratory  method  for  the  rapid  detection  of  VZV  viremia 
during  zoster  infection.  The  method  utilized  the  Fab  component  of  VZIG  for 
the  detection  of  VZV  membrane  antigen  in  peripheral  blood  mononuclear  cells. 
These  results  also  indicate  that  specific  antiviral  antibodies  constitute 
the  major  defense  against  VZV  infections  in  man. 

AI  01475-22  E.  H.  Lennette  (State  of  California  Department  of  Health):  Anti- 
body  against  varicella-zoster  virus  (VZV)  is  routinely  detected  by  compl ement- 
fixation  (CF),  or  immune  adherence  hemagglutination  (IAHA)  or  neutralization. 
Although  neutralization  is  the  most  meaningful  test  in  terms  of  protection 
against  chickenpox  or  zoster,  it  is  extremely  cumbersome  and  expensive. 
Investigators  in  Dr.  Lennette1 s  laboratory  compared  the  newer  techniques 
of  enzyme  immunoassay  (EIA)  with  neutralizing,  IAHA,  and  CF  and  found  that 
EIA  offers  a  rapid,  sensitive,  and  specific  method  of  antibody  assay  that 
is  applicable  in  a  clinical  setting.  The  EIA  test  had  a  94%  correlation 
with  neutralization  and  could  reasonably  replace  the  neutralization  test 
and  reduce  costs  for  serodiagnosis  of  varicella  or  zoster. 

AI  11788-09  P.  M.  Horstmann  (Yale  University):  In  general ,  the  presence  of 
antibody  to  rubella  virus  detected  by  hemagglutination-inhibition  (HI)  tests 
indicates  that  the  person  has  protective  immunity.  However,  recent  evidence 
obtained  by  Dr.  Horstmann  demonstrated  that,  in  a  few  individuals  naturally 
infected  or  vaccinated,  the  presence  of  HI  antibodies  in  the  absence  of 
detectable  neutralizing  antibodies  (NT)  did  not  prevent  reinfection. 

7-13 


Therefore,  the  NT  test  is  the  one  of  choice  for  assay  of  protective  levels  of 
antibody  in  vaccinees.  The  study  showed  that  NT  responses  to  the  new  RA  27/3 
vaccine  were  higher  titered  and  persisted  longer  at  high  levels  than  with  the 
currently  used  HPV77DE5  and  Cendehill  vaccines.  NT  titers  at  3-5  years  post- 
infection were  lower  in  vaccinees  than  in  natural  immunes.  At  nine  years 
post-vaccination,  11%  of  290  children  who  received  the  HPV77DE5  vaccine  were 
found  to  be  antibody  negative  by  HI,  and  all  of  these  had  NT  titers  below 
protective  levels.  The  results  point  to  the  superiority  of  the  RA  27/3 
vaccine,  which  will  soon  become  available  in  the  United  States. 

Workshop  on  Cytomegalovirus  Infections  during  Organ  Transplantation 

As  the  import  of  cytomegalovirus  (CMV)  infection  has  become  evident,  improved 
techniques  to  study  the  molecular  biology  of  this  agent  have  been  developed, 
and  a  variety  of  possible  therapeutic  interventions  has  been  proposed  that 
might  be  useful  in  preventing  and/or  treating  this  infection.  In  an  effort 
to  coordinate  research  efforts  aimed  at  the  control  of  CMV  infection  during 
transplantation  and  to  evaluate  those  therapeutic  and  preventive  modalities 
currently  available,  a  workshop  was  convened  in  June  1978  by  NIAID  comprised 
of  a  small  group  of  experts  in  the  field  of  CMV  who  were  intimately  involved 
and  concerned  with  clinical  transplantation.  The  goal  of  the  workshop  was  to 
summarize  presently  available  information,  and  to  prepare  an  agenda  which  would 
lead  to  the  systematic  application  and  evaluation  of  each  of  the  therapeutic 
modalities  discussed.  A  summary  of  the  proceedings  has  been  published  in  the 
Journal  of  Infectious  Diseases. 

Persistent  Infections  and  Chronic  Viral  Diseases 

The  mechanisms  are  little  understood  by  which  some  viruses  can  infect  a  host 
and  then  remain  within  the  host  in  a  quiescent  state  for  years  or  slowly 
proliferate,  eventually  to  cause  chronic  disease.  It  is  speculated  that 
many  chronic  diseases  of  undetermined  etiology  may  be  caused  by  viruses  with 
unique  capabilities  of  escaping  the  host's  defenses.  The  Virology  program 
has  encouraged  investigations  on  persistent  and  slow  virus  research  in  efforts 
to  identify  and  characterize  the  putative  agents.  Current  emphasis  in  this 
program  has  shifted  from  the  search  for  additional  agents  to  more  complete 
characterization  of  known  persistent  and  slow  viruses  and  their  pathogenetic 
mechanisms. 

Research  Highlights 

AI  06246-15  J.  G.  Stevens  (University  of  California,  L.A.):  Dr.  Stevens  has 
been  studying  the  pathogenesis  of  herpes  simplex  virus  (HSV)  in  model  labora- 
tory and  animal  systems.  Until  recently,  he  was  not  able  to  establish  a 
latent  infection  in  mice  similar  to  that  which  occurs  naturally  in  man. 
Dr.  Stevens  has  now  characterized  15  mutants  of  HSV  with  respect  to  their 
ability  to  become  latent  in  mice.  Seven  of  the  mutants  did  become  latent 
whereas  eight  did  not.  Latency  did  not  depend  on  the  ability  of  the  mutant 
to  replicate  DNA  within  infected  cells,  but  did  appear  to  be  dependent  upon 
viral  protein  syntheses  beyond  the  immediate-early  polypeptide.  The  discovery 
of  these  latent  mutants  promises  to  make  studies  of  latency  and  reactivation 
at  the  molecular  level  in  model  HSV  systems  more  feasible  than  previously 

7-14 


possible.  It  is  also  significant  that  latency  can  be  determined  by  the  viral 
genome.  It  should  now  be  possible  to  identify  the  locus  and  defect  in  the 
viral  genome  which  regulates  latency  and,  ultimately,  to  explain  the  mechanism. 

AI  12438-05  R.  M.  Welsh  (Scripps  Clinic  and  Research  Foundation):  One  of  the 
prime  models  for  study  of  persistent  infections  is  lymphocyte  choriomeningitis 
virus  (LCMV)  in  mice  and  cell  culture.  Dr.  Welsh  has  been  investigating  the 
relationships  between  the  immune  response  and  defective  interfering  (DI) 
particles  in  the  maintenance  of  chronic  virus  disease.  Particular  emphasis 
has  been  on  the  role  of  natural  killer  (NK)  cells  in  response  to  infections 
with  LCMV.  It  has  been  shown  that  cells  infected  with  DI  synthesize  viral 
proteins  at  a  wery   slow  rate.  The  decrease  in  expression  of  viral  antigens 
could  help  to  explain  how  these  persistently  infected  cells  escape  immuno- 
logic attack  mechanisms  of  the  host.  Dr.  Welsh  has  also  shown  that  acute 
infection  with  LCMV  induces  an  augmented  NK  cell  activity  that  is  correlated 
with  the  synthesis  of  interferon  type  I  (antiviral),  and  that  NK  cells  were 
activated  in  vivo  by  interferon  injections.  Thus,  these  NK  cells  may  be  of 
great  importance  in  controlling  virus  infections  and  contributing  to  immuno- 
pathology  in  acute  and  chronic  infections.  DI  particles  may  be  of  signifi- 
cance in  chronic  infections  by  regulating  viral  antigenic  expression,  thereby 
limiting  the  severity  of  immunopathologic  disease. 

Biology  of  Viruses 

Acquisition  of  knowledge  concerning  virus  structure,  function  and  interaction 
with  the  host  has  required  technologies  developed  by  several  scientific  dis- 
ciplines including  microbiology,  biochemistry,  biophysics,  genetics,  physical 
chemistry  and  immunology.  The  field  of  biology  of  viruses  is  very  fluid,  and 
is  often  found  moving  in  the  direction  of  a  new  technology.  Current  trends 
reveal  a  substantial  movement  toward  such  technologies  as  recombinant  DNA, 
recombinant  RNA,  oligonucleotide  fingerprinting,  and  nucleotide  and  peptide 
sequencing  to  approach  questions  yet  to  be  answered  about  the  composition  of 
viruses  and  how  they  infect  cells,  replicate  and  cause  disease. 

Research  Highlights 

AI  12717-04  E-S.  Huang  (University  of  North  Carolina):  Dr.  Huang,  in  col- 
laboration with  Dr.  C.  Alford,  University  of  Alabama,  has  been  analyzing  by 
restriction  enzyme  cleavage  strains  of  cytomegalovirus  (CMV)  isolated  from 
patients  presenting  various  clinical  manifestations  such  as  recurrent  infection, 
congenital  defect  and  mononucleosis.  The  objectives  of  the  study  are  to  under- 
stand the  mode  of  viral  transmission,  to  classify  viral  strains,  and  to  identify 
the  origin  of  CMV  isolates.  In  comparing  viruses  isolated  from  five  sets  of 
mothers  and  their  offspring  the  restriction  enzyme-fragment  patterns  were  found 
identical  even  though  some  of  the  offspring  viruses  were  isolated  several  years 
after  virus  was  isolated  from  the  mothers.  In  one  recurrent  case  the  fragment 
patterns  of  original  and  recurrent  isolates  were  identical,  indicating  that 
the  two  isolates  were  derived  from  the  same  parental  strain.  In  another  recur- 
rent case,  the  recurrent  isolate  was  distinctly  different  from  the  original, 
suggesting  that  the  recurrent  infection  was  caused  by  a  new  virus  strain. 
In  other  collaborative  studies,  Dr.  Huang  has  demonstrated  that  CMV  virus 
isolated  from  an  immunosuppressed  renal  transplant  patient  is  different  from 

7-15 


the  CMV  virus  used  to  immunize  that  patient  before  the  transplantation.  This 
is  an  important  observation  because  it  indicates  that  the  CMV  used  for  vaccin- 
ation was  not  reactivated  after  transplantation  and  during  immunosuppression. 
Rather  it  suggests  that  the  post-transplantation  infection  was  probably 
derived  from  the  donor  kidney. 

AI  14049-02  L.  C.  Norkin  (University  of  Massachusetts):  SV  40  virus  is  an 
agent  found  to  persistently  infect  kidney  cells  of  rhesus  monkeys.  First 
discovered  as  a  contaminant  of  early  polio  vaccines,  it  has  become  an  excel- 
lent model  for  study  by  molecular  virologists  because  of  its  simplicity,  its 
ease  of  propagation  and  its  oncogenic  properties.  Dr.  Norkin  has  been  inves- 
tigating variants  of  this  virus  that  arise  during  persistent  infections  with 
respect  to  properties  that  might  affect  the  course  of  infection.  He  has 
shown  that  stable  persistent  infections  of  rhesus  monkey  kidney  cells  can  be 
established  with  little,  if  any,  visible  damage  to  cells.  Only  about  half 
of  the  kidney  cells  are  initially  susceptible  to  infection,  but  after  three 
to  11  weeks  all  cells  contain  and  express  virus  genetic  information.  Only 
a  small  percentage  of  persistently  infected  cells  produce  infectious  virus 
and  these  eventually  die.  The  virus-producing  cells  are  eventually  killed 
and  the  productive  infection  is  perpetuated  by  the  emergence  of  new  virus- 
producing  cells  from  the  population  of  nonproducing  cells.  Defective  viral 
particles  eventually  emerge  which  interfere  with  the  replication  of  infec- 
tious virus.  Dr.  Norkin  has  demonstrated  that  this  interference  is  not 
associated  with  interferon. 

AI  12458-05  F.  B.  Bang  (The  Johns  Hopkins  University):  Dr.  Bang's  laboratory 
has  been  investigating  the  effect  of  diet  on  susceptibility  to  viral  infec- 
tions. They  have  focused  their  studies  on  vitamin  A  deficiences  in  chickens 
infected  with  Newcastle  disease  virus  (NDV),  and  on  low  protein  diet  in 
genetically  susceptible  and  resistant  strains  of  mice  infected  with  mouse 
hepatitis  virus  (MHV).  Results  have  demonstrated  a  synergistic  effect  of 
NDV  and  vitamin  A  deficiency  in  producing  massive  destruction  of  the  bursa 
and  thymus  (two  major  organs  of  the  immune  system)  in  the  chicken.  Similar 
effects  on  the  immune  system  were  found  with  low  protein  diets  and  MHV  in 
susceptible  mice.  The  mouse  system  proved  more  amenable  than  the  chicken 
for  study  at  the  cellular  level  in  vitro.  In  this  context,  recent  work  has 
shown  that  in  terms  of  susceptibility  to  MHV,  the  behavior  of  the  macrophages 
in  genetically  resistant  and  susceptible  mice  mirrors  both  the  genetic  and 
phenotypic  susceptibility  of  the  intact  mouse.  These  systems  in  the  chicken 
and  mouse  promise  to  provide  models  for  a  better  understanding  of  the  effects 
of  diet  deficiencies  on  susceptibility  to  infectious  agents. 

AI  09706-09  H.  Koprowski  (The  Wistar  Institute):  The  new  technology  of  using 
hybridoma  cells  to  produce  monoclonal  antibodies  is  opening  new  approaches  to 
the  study  of  viral  proteins.  In  this  laboratory  the  technique  was  applied  to 
the  study  of  proteins  of  rabies  virus.  Cultures  of  hybridoma  cells  prepared 
by  fusing  mouse  myeloma  cells  with  spleen  cells  from  rabies-immunized  mice 
resulted  in  numerous  clones  producing  highly  specific  antibodies.  The  anti- 
bodies produced  by  over  100  clones  were  analyzed  and  divided  into  the  cate- 
gories of  specificity  corresponding  to  the  known  rabies  virus  antigens. 
Because  of  the  highly  specific  properties  of  these  monoclonal  antibodies,  the 
investigators  were  able  to  differentiate  strains  of  rabies  virus  antigenically. 

7-15 


Previous  to  these  observations,  it  was  generally  believed  that  all  strains  of 
rabies  were  antigenically  yery   closely  related.  The  monoclonal  antibodies  have 
also  substantiated  that  antibodies  against  the  glycoprotein  of  rabies  virus  are 
responsible  for  neutralization  of  viral  infectivity  and  for  immune  lysis  of 
infected  cells. 

Virology  Research  under  the  Auspices  of  the  U.S. -Japan  Cooperative  Medical 
Science  Program 

Goals  for  this  program  include  the  encouragement  and  support  of  research  that 
will  advance  our  knowledge  of  and  lead  to  eventual  control  of  rabies,  dengue 
and  other  arthropod  diseases  and  viral  gastroenteritis.  The  program  is  moni- 
tored by  the  Panel  on  Viral  Diseases,  an  advisory  group  to  the  U.S. -Japan  CMSP. 

Research  Highlights 

Rabies 

AI  09706-09  H.  Koprowski  (The  Wistar  Institute):  The  new  WI-38  human  diploid 
cell  rabies  vaccine  (HDCV)  developed  at  the  Wistar  Institute  is  now  produced  in 
both  France  and  West  Germany  and  is  licensed  for  pre-  and  post-exposure  human 
prophylaxis  in  several  European  and  Asian  countries.  The  licensing  by  Wyeth 
Laboratories,  Philadelphia,  of  HDCV  produced  in  the  U.S.  is  still  in  progress. 
The  necessary  clinical  investigations  have  been  completed;  production  will  be 
initiated  immediately  in  newly  constructed  facilities.  The  license  is  expected 
to  be  granted  after  completion  of  the  first  four  lots  of  vaccine.  In  the  mean- 
time, the  HDCV  is  widely  used  as  an  Investigational  New  Drug  (IND)  with  the 
authority  for  distribution  in  the  Center  for  Disease  Control,  Atlanta.  Over 
the  last  year,  several  hundred  people  in  the  U.S.  were  successfully  treated 
with  HDCV. 

Conferences 

Workshop  on  St.  Louis  Encephalitis 

A  workshop  convened  in  June  1979,  under  the  auspices  of  the  U.S. -Japan  Coopera- 
tive Medical  Science  Program,  was  held  at  the  Rocky  Mountain  Laboratory  in 
Hamilton,  Montana.  The  state  of  the  science  of  St.  Louis  encephalitis  (SLE) 
virus  was  reviewed  and  potential  areas  for  future  research  were  discussed. 
It  was  the  consensus  of  the  participants  that  substantial  new  information  has 
been  obtained  on  the  biology  and  biochemistry  of  SLE  virus,  and  exciting  new 
research  on  flavi viruses  is  in  progress.  Research  leading  toward  an  SLE 
vaccine  was  encouraged,  but  reservations  were  expressed  as  to  the  advisability 
of  developing  a  vaccine  now  before  there  is  sufficient  information  on  the  pro- 
tective response  to  SLE  in  man.  Potential  control  of  SLE  through  a  weak  link 
in  its  mosquito  vector  transmission  was  discussed,  i.e.,  the  emerging  female 
mosquito  after  overwintering.  More  complete  information  on  the  ecology  of 
overwintering  vectors  is  needed  to  locate  sites  where  control  measures  would 
be  most  effective.  A  summary  of  the  workshop  will  be  published  in  the  Journal 
of  Infectious  Diseases. 


7-17 


2 

$90,578 

6 

$390,795 

0 

0 

0 

0 

CLINICAL  STUDIES  BRANCH 


The  Clinical  Studies  Branch  (CSB)  serves  as  the  Institute's  focus  for  the 
development  and  processing  of  Investigational  New  Drug  Applications,  and  main- 
tains liaison  with  the  NIH  Human  Research  Review  Panel  and  the  NIAID-Clinical 
Research  Subpanel .  It  provides  and  monitors  a  closed  clinical  facility  for 
Institute  volunteer  studies,  and  conducts,  promotes  and  supports  the  develop- 
ment and  evaluation  of  procedures  for  the  diagnosis,  prevention  and  treatment 
of  infectious  diseases,  particularly  those  procedures  leading  to  improved 
patient  care. 

Approximate  Level  of  Support 

Activity  Number  Amount 

Research  Grants 
Research  Contracts* 
Training  Grants 
Fellowships 

Total   8  $481  ,373 

*  Two  contracts  have  been  forward-funded  with  FY  1978  MIDP 
funds  and  those  funds  do  not  show  here. 

Program  Areas 

Closed  Volunteer  Facilities 

The  CSB  currently  supports  one  research  contract  with  the  University  of  Mary- 
land. This  contract  maintains  and  supports  The  Center  for  Vaccine  Development 
(CVD)  where  volunteers  are  studied  before  and  after  they  are  challenged  with 
potentially  transmissible  infectious  agents  and  candidate  vaccines. 

During  the  past  year,  the  contractor  performed  24  volunteer  studies  which 
were  components  of  six  more  comprehensive  research  protocols  approved  by  the 
Institutional  Review  Boards  of  the  University  of  Maryland  and  NIAID.  The 
challenge  studies  were  distributed  as  follows:  enteropathogenic  E_.  col  i , 
seven  studies,  two  protocols;  cholera,  five  studies,  one  protocol;  parvo-like 
gastrointestinal  virus,  three  studies,  one  protocol;  H3N2  influenza,  four 
studies,  one  protocol;  H-j  N-j  influenza,  five  studies,  one  protocol.  Highlights 
of  the  studies  in  volunteers  are  summarized  below. 

A  volunteer  model  of  El  Tor  cholera  was  established  to  study  mechanisms  of 
immunity  to  El  Tor  and  to  evaluate  cholera  vaccines.  The  clinical  severity 
of  El  Tor  cholera,  Inaba  and  Ogawa  serotypes,  was  distinctly  less  than  that 
previously  seen  with  classical  cholera  strains  of  the  same  serotypes.  Further 
studies  also  suggest  that  although  V.  cholerae  in  endemic  areas  may  be  water- 
borne,  the  contaminated  water  must  be  ingested  with  food  in  order  for  clinical 
disease  to  occur  in  the  normochlorhydric  host.  A  naturally  occurring,  non- 


toxigenic  El  Tor  strain  isolated  from  sewage  in  Brazil  was  shown  to  have  most 
characteristics  demanded  of  an  oral,  attenuated  vaccine  strain;  it  will  be 
tried  as  a  vaccine  in  the  near  future.  Preliminary  trials  with  an  oral,  killed 
Inaba  cholera  vaccine  suggested  that  the  vaccine  was  efficacious;  further 
studies  are  planned.  A  highly  purified  E.  col i  type  1  somatic  pilus  vaccine 
prepared  by  Dr.  C.C.  Brinton,  Jr.,  University  of  Pittsburgh,  was  shown  in  pre- 
liminary studies  to  have  low  local  reactogenicity,  absent  systemic  toxicity, 
excellent  immunogenicity,  and  efficacy  (P  =  .04)  in  protecting  immunized 
persons  against  challenge  with  the  virulent  homologous  E_.  coli  strain.  The 
CVD  found  the  ELISA  for  measurement  of  IgG  cholera  antitoxin  in  human  sera 
to  be  a  simple,  sensitive,  and  reproducible  test  to  carry  out  sero-epidemio- 
logic  studies.   In  collaboration  with  the  Center  for  Disease  Control  they 
found  that  long-lived  cholera  antitoxin  is  stimulated  by  natural  infection 
with  V.  cholerae  or  enterotoxigenic  E.  col i .  In  collaboration  with  the  NIAID 
Laboratory  of  Infectious  Diseases,  studies  were  carried  out  with  small  parti- 
cle (27  nm)  agents  of  gastroenteritis  to  measure  serum  and  intestinal  antibody 
and  resistance  to  rechallenge.  Stools  containing  the  27  nm  Norwalk  agent  and 
Hawaii  agent  were  collected  as  sources  of  virus  for  biophysical  studies; 
results  are  pending.  One  of  twenty-one  volunteers  inoculated  intranasally 
with  1 07.0  TCID50  of  A/Alaska/6/77-ts-l-A2  (H3N2)  attenuated  influenza  vac- 
cine strain  developed  systemic  illness.  No  reactions  occurred  in  18  volunteers 
inoculated  with  A/Alaska/6/77  CR29  (H3N2)  cold-adapted  attenuated  vaccine 
strain.  Two  of  12  volunteers  developed  systemic  illness  after  A/Hong  Kong/ 
123/77  ts-A2  (H-| N-j )  attenuated  influenza  vaccine,  while  one  of  12  volunteers 
inoculated  with  the  cold-adapted  strain  developed  coryza  but  no  systemic  ill- 
ness . 

Collaborative  Clinical  Trials  With  Antibiotic  Therapy 

The  major  aim  of  this  program  is  to  sponsor  collaborative  clinical  trials 
designed  to  improve  medical  care  of  patients  with  bacterial  and  mycotic 
infections.  Approved  antibiotics,  or  promising  new  drugs  that  lack  the  eco- 
nomic potential  to  justify  testing  by  the  pharmaceutical  industry,  are  evalu- 
ated in  patients  with  selected  infections. 

During  FY  ' 79  a  contract  was  initiated  with  the  University  of  Alabama  to  study 
ways  to  improve  therapy  of  cryptococcal  meningitis.  The  first  part  of  this 
three  part  protocol  includes  a  controlled  clinical  trial  by  14  collaborating 
institutions  to  determine  the  shortest  treatment  regimen  utilizing  Amphoter- 
icin B  (AMB)  and  5-fl  uorocytosine  (5-FC)  in  combination.  Four  and  six  week 
treatment  regimens  are  being  compared.  The  Collaborative  Cryptococcal  Study 
Group  during  the  first  year  has  made  limited  progress  in  patient  enrollment, 
but  has  nevertheless  achieved  balanced  randomization  into  the  two  treatment 
groups.  Progress  was  slower  than  projected  because  of  the  unexpectedly  low 
occurrence  of  the  disease  this  year.   It  is  anticipated  this  can  be  offset  in 
future  by  more  aggressive  publicity  and  a  recruitment  campaign  among  the 
medical  community.  The  original  plan  for  part  2  of  the  protocol  was  designed 
to  evaluate  the  toxicology  of  Amphotericin  B  methyl  ester  (AME)  in  patients 
with  selected  disseminated  mycotic  infections.  Preliminary  clinical  and  lab- 
oratory data  from  another  open  trial  suggested  that  the  long-term  administra- 
tion of  AME  may  be  associated  with  the  induction  of  neurological  abberrations 
and  various  gradations  of  a  diffuse  leucoencephalopathy .  Pending  a  thorough 


review  and  determination  by  the  FDA,  an  indefinite  moratorium  has  been  placed 
on  the  clinical  administration  of  this  antimycotic  agent.  Before  the  mora- 
torium, the  University  of  Alabama  subcontracted  with  the  Kresge  Hearing  Insti- 
tute, University  of  Oregon  to  conduct  ototoxicity  studies  with  AME  in  labor- 
atory animals.  This  study  may  help  predict  the  ototoxicity  and  neurotoxicity 
of  AME  and  other  methylated  polyene-! ike  antifungal  agents  now  under  develop- 
ment. 

Following  a  contractor's  meeting  of  the  Mycotic  Disease  Collaborative  Group 
in  June,  1979,  we  decided  to  replace  AME  and  conduct  phase  II  studies  with 
ketoconazole  (R41,400),  a  new,  oral  imidazole  derivative  with  broad-spectrum 
antimycotic  activity  and  low  toxicity  developed  by  Janssen  Pharmaceutical  of 
Belgium.  The  Collaborative  Group  will  evaluate  oral  ketoconazole  for  safety 
and  effectiveness  in  the  treatment  of  the  following  group  of  systemic  mycotic 
diseases:  chronic  cavitary  histoplasmosis,  disseminated  or  localized  blasto- 
mycosis, disseminated  coccidioidomycosis  without  meningitis,  disseminated 
sporotrichosis,  and  non-meningeal  cryptococcosis.  Efficacy  studies  of  keto- 
conazole in  animal  models  of  cryptococcal  meningitis  will  also  be  done. 

In  response  to  an  RFP,  issued  during  FY  '79,  a  new  contract  was  awarded  to  the 
University  of  Illinois  to  perform  a  placebo-controlled,  double-blind  evaluation 
of  the  efficacy  of  penicillin  in  the  prevention  of  early  onset  Group  B  Strept- 
ococcal ( GBS)  sepsis  in  the  newborn.  In  addition,  the  study  is  designed  to 
determine  if  penicillin  prophylaxis  influences  the  rate  of  late  onset  GBS 
disease,  increases  or  decreases  the  likelihood  of  later  penicillin  hypersen- 
sitivity, and  causes  the  emergence  of  penicillin  resistant  GBS  or  other  peni- 
cillin resistant  organisms.  It  is  anticipated  that  this  study  will  require 
at  least  three  years  and  20,000  mother-infant  pairs  before  conclusive  data 
will  be  available  to  demonstrate  efficacy  of  the  prophylactic  regimen. 

Based  on  recommendations  from  the  NIAID  Symposium  on  the  Impact  of  Infections 
on  Medical  Care  in  the  U.S.,  held  May  30-31,  1978,  Staff  prepared  and  issued 
an  RFP  during  FY  '79  entitled  "Improved  Therapy  of  Endocarditis  Due  to  Viri- 
dans  Streptococci."  Response  to  this  RFP  was  limited  to  one  technical  pro- 
posal. During  the  primary  review  process  it  was  determined  that  a  controlled 
clinical  trial  was  not  feasible,  because  a  properly  designed  trial  would  take 
too  long  (seven-ten  years)  and  the  resulting  costs  would  be  excessive.  With 
this  information,  a  determination  was  made  by  Staff  to  cancel  the  RFP  and  not 
award  a  contract  for  the  trial.  Subsequently,  plans  were  developed  to  hold 
a  Consensus  Development  Conference  on  the  optimum  therapy  of  penicillin- 
sensitive,  non-enterococcal  endocarditis.  The  conference  will  be  co-sponsored 
by  the  NIAID,  NHLBI,  the  NIH  Office  of  the  Medical  Applications  of  Research, 
and  the  American  Heart  Association. 

Rapid  Viral  Diagnosis 

The  purpose  of  this  program  is  to  place  greater  emphasis  on  the  rapid  diagnosis 
of  viral  infections.  The  ultimate  goal  is  to  provide  the  physician  with  reli- 
able tests  so  that  a  specific  viral  diagnosis  can  be  made  in  time  to  help  in 
the  management  of  the  patient.  To  do  this,  the  methods  must  be  practical, 
precise,  reproducible  and  inexpensive,  and  simple  enough  to  be  conducted  safely 
in  the  physicians'  offices  or  in  small  hospital  laboratories.  Three  contracts 


8-3 


were  awarded  for  the  development  of  such  tests.  A  contractor  at  the  Univer- 
sity of  California,  San  Diego,  will  further  develop  an  immunological  technique 
that  incorporates  two  important  features.  The  first  is  the  immobilization  of 
the  virus  on  filter  paper  discs  and  washing  using  an  immunofiltration  manifold. 
This  technique  eliminates  the  need  for  primary  capture  antibody,  and  effi- 
ciently immobilizes  infected  cells  and  viral  antigens.  The  second  important 
feature  is  the  use  of  purified,  radiolabeled  or  enzyme  labelled  Staphylococcal 
protein  A,  which  binds  to  the  Fc  portion  of  certain  mammalian  immunoglobulins 
thereby  eliminating  need  for  anti species  detection  sera.  The  investigators 
will  adapt  this  methodology  to  detect  influenza  viruses,  respiratory  syncytial 
virus,  parainfluenza  viruses,  and  cytomegalovirus  in  less  than  one  hour.  A 
contractor  at  the  Harvard  School  of  Public  Health  will  use  sensitive,  solid- 
phase  immunoassays  utilizing  viral  antibodies  covalently  coupled  to  nylon  balls 
or  nylon  powder  in  an  enzyme-linked  immunoassay.  This  increases  the  amount  of 
antibody  that  can  be  immobilized,  and  lessens  the  problem  of  antibody  desorp- 
tion.  The  test  is  now  sensitive  enough  to  permit  rapid  identification  of 
influenza  virus  in  clinical  specimens,  and  will  be  applied  next  to  other  respir- 
atory viruses.  A  contractor  at  Johns  Hopkins  Hospital  will  develop  two  tests 
for  rapid  viral  diagnosis.  The  first  is  an  enzyme-multiplied  radioimmunoassay 
(EMRIA)  which  is  the  standard  enzyme  immunoassay  but  uses  a  tritium  labelled 
substrate.  This  test  has  been  shown  to  detect  small  quantities  of  non-replica- 
ting cytomegalovirus  antigen  in  clinical  specimens.  The  enzyme-linked 
fluorescent  assay  (ELFA)  using  a  fluorescent  substrate  is  somewhat  less  sensi- 
tive than  EMRIA,  but  it  is  more  rapid  and  simpler  to  use.  These  two  tests  will 
be  adapted  to  detect  other  respiratory  viruses.  Coordination  of  activities 
within  the  NIH  and  with  the  Pan  American  and  European  groups  on  Rapid  Viral 
Diagnosis  continues  to  prevent  duplication  of  effort. 

Nutrition,  Infection  and  Immunity 

The  goal  of  this  program  is  to  promote  research  on  the  interaction  of  malnutri- 
tion, infection  and  immunity  in  American  hospitals  and  in  overseas  populations. 
Since  the  malnourished  patient  is  at  high  risk  for  infection,  this  goal  is  con- 
sistent with  the  overall  mission  of  the  CSB  which  is  to  encourage  research 
leading  to  improved  patient  care. 

In  FY  '79  the  NIAID  program  in  Nutrition,  Infection  and  Immunity  consisted  of 
12  grants  which  were  monitored  by  at  least  four  project  officers  in  the  IAID 
and  MID  programs.  The  research  focused  on  four  different  areas;  the  modulating 
effect  of  specific  nutrients  on  immune  function,  mechanisms  of  food  allergies 
and  immune  response  to  ingested  antigens,  the  interaction  of  nutrition  and 
infection  in  the  tropical  environment  and  in  American  hospitals,  and  the  modu- 
lating effect  of  specific  nutrients  on  microbial  virulence. 

During  FY  '79,  the  Director  of  NIH  appointed  Dr.  Edelman  vice-chairman  of  the 
NIH  Nutrition  Coordinating  Committee  (NCC).  Through  this  committee  and  in  an 
effort  to  stimulate  research  in  nutrition,  Dr.  Edelman  helped  design  and  initi- 
ate several  new  nutrition  research  programs.  One  of  these  new  initiatives  was 
the  establishment  of  clinical  nutrition  research  units  (CNRU)  sponsored  by 
NIAMDD,  NIA,  and  NCI.  Infection  and  immunity  research  appeared  in  several 
approved  proposals  that  were  submitted  in  response  to  the  RFA  on  CNRU.  These 
units  should  help  stimulate  research  in  clinical  nutrition,  and  will  serve  as 

8-4 


models  of  excellence  for  many  American  academic  centers.  Another  initiative 
involved  the  establishment  of  institutional  and  individual  fellowship  training 
grants  in  nutrition.  Seven  Institutes  have  agreed  to  fund  these  National 
Research  Service  Awards.  NIAID  has  agreed  to  co-sponsor  Research  Career  Devel- 
opment Awards  in  clinical  nutrition  when  the  NCC  develops  a  joint  NIH 
announcement  for  these  in  the  near  future.  Dr.  Edelman  served  on  ad  hoc  study 
sections  to  critique  applications  submitted  to  the  Fogarty  International  Center 
for  the  support  of  national  and  international  nutrition  meetings. 

Dr.  Edelman  served  as  chairman  of  an  international  workshop  sponsored  by  the 
NCC  entitled  "Nutritional  Support  of  the  Patient:  Research  Directions  for  the 
1980' s."  The  participants  in  this  two  day  workshop  held  in  September,  1979, 
developed  a  set  of  research  priorities  that  will  be  used  for  program  planning 
by  eight  Institutes  of  the  NIH.  The  workshop  focused  on  the  feeding  of  the 
sick  patient  by  means  of  enteral  and  parenteral  nutrition  support.  One  of  the 
six  workshop  panels  was  relevant  to  NIAID  programs,  and  delt  with  trauma  and 
infection.  The  workshop  proceedings  will  be  published  by  the  American  Journal 
of  Clinical  Nutrition. 

Dr.  Edelman  delivered  three  invited  lectures  on  "Nutrition,  Infection  and 
Immunity"  at  the  Walter  Reed  General  Hospital  and  Walter  Reed  Army  Institute 
of  Research.  Staff  of  the  CSB  continued  their  collaboration  with  the  Diet, 
Nutrition  and  Cancer  program  of  the  National  Cancer  Institute  by  helping  assess 
the  effect  of  hyperalimentation  and  improved  nutrition  on  infections  and  the 
immune  response  in  selected  cancer  patients.  The  final  results  of  this  study 
will  be  known  early  in  FY  1980. 

Other  Activities 


Mrs.  Mattheis  collects  and  reviews  the  necessary  documentation  required  to  file 
and  maintain  Investigational  New  Drug  Applications  (INDAs)  with  the  Food  and 
Drug  Administration  (FDA)  for  the  Microbiology  and  Infectious  Diseases  Program. 
Currently  18  active  INDAs  are  filed  with  the  Bureau  of  Biologies,  FDA,  and  four 
with  the  Bureau  of  Drugs,  FDA.  During  the  past  year  new  INDAs  were  filed  for 
the  study  of  "Meningococcal  Group  B  Type  2  Outer  Membrane  Protein  Vaccine", 
"Live,  Attenuated  Respiratory  Syncytial  Virus  Vaccine",  "Amphotericin  B  Methyl 
Ester  Aspartate",  and  "Topical  9-(2-hydroxyethoxymethyl )  guanine."  Staff  sup- 
port is  provided  for  the  NIAID-Clinical  Research  Subpanel  (CRS)  which  reviews 
both  intramural  and  contract  supported  clinical  studies.  All  contract  suppor- 
ted clinical  studies  are  reviewed  by  the  CRS  initially  and  every  three  years 
thereafter,  and  by  the  NIAID-Clinical  Director,  in  the  intervening  years. 

The  CSB  and  the  Bureau  of  Biologies,  FDA,  co-sponsored  an  International  Sympo- 
sium on  Potentiation  of  Immune  Response  to  Vaccines,  held  at  the  NIH  on 
February  20  -  21,  1979.  The  symposium  participants  discussed  the  influence  of 
molecular  structure  on  the  adjuvant! city  of  muramyl  dipeptide  and  other  synthe- 
tic immunological  adjuvants,  including  liposomes  and  polynucleotides.  The 
mechanisms  of  immunoregulation  by  these  synthetic  substances,  and  their  use  as 
immunological  probes  and  as  potential  vaccine  potentiators  in  man  was  reviewed. 
The  possibility  was  discussed  of  certain  adverse  side  effects  attending  their 
use  in  man.  Nevertheless,  synthetic  adjuvants,  including  800  variants  of  the 
muramyl  dipeptide  molecule  alone,  are  emerging  as  exciting  new  substances  with 


8-5 


many  basic  and  applied  applications. 

Dr.  Edelman  served  on  the  following  committees  of  other  Federal  agencies:  1) 
NIH  liaison  member  on  the  Infection  Control  Committee  of  the  Veteran's  Admini- 
stration, 2)  the  Human  Ethics  Committees  (Institutional  Review  Boards)  of  the 
Walter  Reed  Army  Institute  of  Research  and  3)  the  Frederick  Cancer  Research 
Center.  As  ex-officio  voting  member  of  the  National  Commission  on  Digestive 
Diseases,  Dr.  Edelman  participated  in  the  natural  demise  of  the  Commission 
when  it  officially  submitted  its  Report  to  Congress  on  January  31,  1979. 

Dr.  Horton  served  as  liaison  member  of  the  NIH  Coordinating  Committee  on  Cystic 
Fibrosis  and  participated  at  the  National  Scientific  Working  Conference  on 
Cystic  Fibrosis,  March  4-7,  1979,  South  Padre  Island,  Texas.  This  working 
conference,  co-sponsored  by  the  Cystic  Fibrosis  Foundation,  NIAID,  NIAMDD,and 
NICHD  was  highly  productive,  and  provided  for  an  excellent  exchange  of  scien- 
tific information  among  multiple  disciplines.  It  identified  future  research 
opportunities  with  guidelines  for  implementing  these  activities.  A  report  on 
this  conference  will  be  prepared  and  distributed  by  the  Cystic  Fibrosis 
Foundation. 


8-6 


DEVELOPMENT  AND  APPLICATIONS  BRANCH 

It  is  the  primary  objective  of  the  Development  and  Applications  Branch  to 
translate  new  information  derived  from  basic  research  into  methodologies 
appropriate  for  the  control  or  prevention  of  designated  infectious  diseases 
in  humans.  Fundamental  and  applied  research  activities  supported  by  the 
Branch  include:  identification  of  important  infectious  disease  problems  with 
potential  for  control  or  prevention  through  immunization  or  utilization  of 
antivirals,  development  of  appropriate  vaccines,  antivirals,  and  other  control 
measures,  design  and  support  of  appropriate  clinical  trials  for  evaluation  of 
control  measures,  and  basic  research  in  related  areas. 

Approximate  Level  of  Support 

Activity  Number        Amount 

Vaccine  Evaluation  Centers 

Research  Contracts 

Influenza 

Research  Contracts 

Interagency  Agreements 

Research  Grants 

Fellowships 

Young  Investigator  Awards 

Respiratory  Diseases 

Research  Contracts 
Interinstitute  Agreements 
Research  Grants 

Hepatitis 

Research  Contracts 

Interagency  Agreements 

Research  Grants 

Career  Awards 

Young  Investigator  Awards 

Antiviral  Substances 

Research  Contracts 
Research  Grants 
Career  Awards 
Fellowships 

Training  Grants 


5 

$  1,218,351 

11 
2 

24 
2 
2 

2,103,956 
51,138 

2,192,305 
25,237 
83,812 

1 
1 
4 

0 

47,872 

264,492 

7 
2 
8 
2 
1 

1,160,711 

157,497 

877,193 

86,665 

36,195 

13 

29 

2 

2 

1 

2,659,248 

2,337,501 

71 ,432 

20,877 

85,688 

9-1 


Approximate  Level  of  Support  (cont. 

Activity 

Bacterial  Vaccines 

Research  Contracts 
Interagency  Agreements 
Research  Grants 
Career  Awards 
Fellowships 

Enteric  Diseases 

Research  Contracts 
Interagency  Agreements 
Research  Grants 
Career  Awards 
Fellowships 
Program  Projects 
Training 

Viral  Vaccines 


Research  Grants 
Conference  Support 


Number 


Amount 


Totals 


13 

$       816,197 

1 

10,821 

18 

1,076,870 

2 

50,551 

2 

32,400 

7 

762,565 

1 

33,750 

35 

2,105,048 

1 

16,200 

1 

14,200 

1 

355,100 

1 

36,504 

7 

548,327 

1 

29,750 

209 

$19,368,453 

Program  Summary 

The  initial  portion  of  this  fiscal  year  (through  March)  was  heavily  involved 
in  completing  studies  directed  at  the  control  of  influenza  A/USSR/77  virus, 
the  most  recent  strain  shift.  Clinical  studies  to  determine  vaccine  safety, 
reactogenicity,  and  antigenicity,  as  well  as  its  sensitivity  to  amantadine, 
were  performed.  Two  other  areas  of  concentrated  effort  in  the  influenza 
program  were  further  development  and  study  of  attenuated  vaccine  candidates 
and  determination  of  the  role  of  amantadine  in  influenza  disease  control. 
A  meeting  was  held  in  February  at  the  Influenza  Research  Center  to  compare 
results  with  Soviet  scientists  on  respective  experiences  with  amantadine 
and  rimantadine.  Based  on  results  from  investigations  supported  by  this  pro- 
gram, there  is  a  revived  interest  in  amantadine.  A  consensus  development 
exercise  on  the  role  of  amantadine  in  the  prevention  and  treatment  of  influ- 
enza is  currently  being  planned. 

Following  the  advice  of  a  Workshop  on  the  Development  of  Antiviral s,  the 
Antiviral  Substances  Program  (ASP)  developed  a  request  for  proposals  for 
the  development  of  targeted  antivirals,  and  a  contract  has  been  awarded. 
Having  determined  that  adenine  arabinoside  monophosphate  (ara-AMP)  was 
not  effective  as  a  topical  drug  against  herpes  labial  is,  clinical  studies 
were  initiated  using  acyclovir  which  appears  to  hold  greater  promise 


9-2 


based  on  animal  studies.  Protocols  have  also  been  developed  to  evaluate 
systemic  ara-AMP  against  herpes  infections,  and  phase  1  studies  are  proceeding 
with  acyclovir.  If  these  latter  studies  prove  satisfactory,  this  drug  will 
be  considered  for  clinical  efficacy  studies.  Final  determination  on  the 
establishment  of  interferon  standards  and  their  distribution  has  been  made 
with  WHO.  This  is  particularly  important  based  on  the  renewed  efforts  in 
clinical  application  of  interferon  against  infectious  diseases  and  cancer. 
Through  the  efforts  of  this  program,  the  first  complete  summary  of  all 
clinical  studies  with  interferon  was  published  (Journal  of  Infectious  Diseases, 
139:109-123,  1979).  One  of  the  most  exciting  findings  of  the  ASP  during  the 
past  year  was  the  apparent  efficacy  of  adenine  arabinoside  in  neonatal 
herpes.  Although  the  data  are  still  being  analyzed,  the  results  appear 
similar  to  the  efficacy  of  this  drug  against  herpes  encephalitis. 

A  new  initiative  for  the  viral  vaccine  program  was  the  release  of  a  request 
for  proposals  to  evaluate  candidate  varicella  vaccines.  Herpes  varicellas 
produces  a  highly  contagious  but  mild  disease  (chicken  pox)  in  healthy 
children;  however,  it  causes  severe  disease  with  increased  morbidity  and 
even  death  in  immunosuppressed  children.  At  a  Workshop  on  Experimental 
Herpesvirus  Vaccines,  it  became  clear  that  varicella  vaccines  with  clinical 
potential  were  available  and  definitive  clinical  studies  should  be  performed. 

Two  other  new  areas  in  the  Branch's  vaccine  program  are  based  in  the  Bacterial 
Vaccine  Program.  The  current  Rocky  Mountain  Spotted  Fever  vaccine  was  with- 
drawn from  the  market  based  on  lack  of  efficacy,  this  created  a  need  for 
accelerated  work  with  a  new  vaccine  candidate  developed  by  USAMRIID.  Studies 
are  underway  on  epidemiology  and  animal  studies  in  anticipation  of  future 
clinical  studies.  An  International  Symposium  on  Pertussis,  principally  co- 
sponsored  by  this  program  and  the  BoB,  raised  many  questions  concerning  the 
pertussis  vaccine  and  considerations  on  possible  improvement  of  this  vaccine 
are  underway.  Another  major  effort  of  this  program  was  the  release  of  a 
request  for  applications  for  studies  on  infant  (less  than  two  years  of  age) 
infections.  This  is  an  offshoot  of  the  studies  on  meningitis  vaccines.  In 
these  studies,  it  was  shown  that  bacterial  polysaccharide  antigens  were  not 
as  effective  in  this  target  population  as  desired. 

The  Hepatitis  Program  is  completing  phase  1  and  2  studies  prior  to  efficacy 
studies  of  the  hepatitis  B  (HBV)  vaccine.  It  has  been  determined  that  the 
HBV  vaccine  containing  alum  adjuvant  is  preferable  to  the  aqueous  preparation. 
The  alum  vaccine  will  be  used  in  the  proposed  hemodialysis  center  study. 
Clinical  studies  to  evaluate  the  possible  effect  of  hepatitis  B  immune  globu- 
lin to  prevent  transmission  from  hepatitis  antigen  positive  mothers  to  newborns 
are  underway.  The  woodchuek  appears  to  be  a  useful  animal  model  for  hepatitis 
pathogenesis  studies;  attempts  to  develop  this  model  are  underway. 

Epidemiologic  studies  on  diarrheal  diseases  initiated  in  FY77  are  beginning 
to  yield  interesting  results.  Trends  related  to  age,  crowding,  and  etiological 
agents  are  emerging.  Studies  with  cholera  vaccine  in  endemic  areas  continue. 
Currently  two  vaccines  (provided  by  the  Wellcome  Foundation),  an  alum  precipi-  , 
tated  whole  cholera  cell  and  a  purified  toxoid  complexed  with  aluminum  hydrox- 
ide, are  being  tested  individually  and  in  combination.  A  major  breakthrough 


9-3 


in  the  treatment  of  cholera  has  been  the  use  of  chlorpromazine  in  reducing 
fluid  loss  in  these  patients.  Of  considerable  interest  was  the  finding  of 
cholera  vibrios  in  U.S.  estuaries.  This  is  being  followed  with  great  care. 

In  FY79  the  DAB  was  involved  in  the  organization  and  conduct  of  six  major 
meetings:  International  Symposium  on  Pertussis,  Workshop  on  Collaborative 
Pneumococcal  Vaccine  Studies,  Workshop  on  Hepatitis  B  Vaccine,  Experimental 
Herpesvirus  Vaccine  Workshop,  Conference  on  Cholera,  and  Workshop  on  Influ- 
enza B  Viruses.  Summaries  on  each  of  these  meetings,  with  the  exception  of 
the  Pneumoccal  Vaccine  meeting,  will  be  published. 

Research  Highlights 

Influenza 

The  Influenza  Program  has  experienced  another  very  active  year.  As  with  the 
previous  year,  the  major  development  in  influenza  has  been  the  reappearance 
of  viruses  which  are  antigenically  very  closely  related  to  those  which  pro- 
duced epidemic  disease  during  the  1947-1957  period.  The  prototype  of  this 
new  era  was  the  A/USSR/77  (HINT)  virus  which  has  been  shown  to  be,  during 
the  last  year,  antigenically  and  biochemically  quite  similar  to  the  A/FLW/50 
(HINT)  strain  which  circulated  in  1950.  Recent  data  suggest  that  the  differ- 
ence between  A/USSR/77  (HINT)  and  A/FLW/50  (H1N1),  a  1950  strain,  may  be  only 
eight  base  pairs  in  the  nucleic  acid.  Although  it  was  widely  anticipated 
that  a  change  in  the  predominant  type  could  be  expected  later  in  the  1970s, 
the  reemergence  of  the  HINT  strains  was  totally  unexpected.  These  new 
variants  have  been  very  restricted  in  their  ability  to  produce  disease  and 
virtually  all  cases  have  been  seen  in  individuals  born  after  1955  or  age  0 
to  24  years.  The  new  variants  also  appear  to  have  supplanted  the  earlier 
H3N2  strains  as  the  predominant  virus.  Only  scattered  isolates  of  H3N2 
viruses  have  been  reported  during  the  last  winter,  none  from  the  United 
States. 

During  the  last  year,  the  Influenza  Program  completed  the  evaluation  of  the 
A/USSR/78  inactivated  vaccines.  Over  2100  subjects  participated,  including 
approximately  1300  under  the  age  of  24  years  and  300  subjects  considered  at 
high  risk  to  influenza.  Final  reports  of  these  studies  are  being  completed 
and  will  be  submitted  for  publication  during  this  fiscal  year.  Field  trials 
of  three  other  vaccines  under  development  were  also  conducted.  The  two 
attenuated  vaccines  were  subjected  to  extensive  tests.  These  include  the 
preliminary  clinical  evaluation  of  these  vaccines  in  approximately  200 
subjects  under  closed  conditions,  200  subjects  to  determine  transmissibility, 
and  over  2100  subjects  during  an  open-field  trial  at  Texas  A  &  M  University. 
However,  the  test  population  did  not  experience  a  large  enough  natural 
challenge,  and  it  will  not  be  possible  to  determine  vaccine  efficacy.  The 
vaccines  did  not  produce  any  significant  reactivity.  The  subjects  will  be 
followed  to  determine  their  antibody  status  and  their  clinical  experience 
after  natural  challenge  with  related  H1N1  viruses.  In  addition,  some  of 
these  vaccinees  will  be  challenged  by  related  and  homologous  viruses  to 
obtain  information  on  the  duration  of  immunity  induced  by  these  vaccines. 
The  third  vaccine,  a  purified  subunit  vaccine,  was  also  tested  and  a  formula- 
tion was  developed  which  induced  excellent  antibody  response  in  subjects 
with  HAI  titers  less  than  1:8. 

9-4 


The  utility  of  amantadine  as  a  prophylactic  and  therapeutic  agent  in  influ- 
enza was  clearly  demonstrated  last  year.  Studies  will  continue  to  explore 
the  use  of  amantadine  in  high-risk  patients  and  in  the  therapy  for  pneumonia. 
Rimantadine  will  also  be  studied.  In  collaboration  with  the  Antiviral  Sub- 
stances Program,  pilot  studies  on  the  possible  synergistic  effect  of  riman- 
tadine and  ribavirin  have  been  started.  Soviet  investigators  had  earlier 
reported  a  synergistic  effect  with  these  drugs.  Studies  using  a  plaque- 
reduction  assay  have  shown  that  these  drugs  are  remarkably  effective  when 
used  in  combination. 

The  results  of  the  studies  presented  above  should  contribute  directly  to  the 
control  of  influenza.  The  basic  scientists  supported  by  the  NIAID  and 
working  with  influenza  viruses  have  continued  to  make  significant  contri- 
butions to  our  understanding  of  myxo  and  paramyxoviruses.  Recent  data 
suggest  the  presence  of  a  ninth  viral  protein.  This  same  investigator  has 
also  shown  that  antibody  to  the  F  protein  of  Sendai  virus  inhibits  syncytia 
formation.  This  may  have  important  implications  with  regard  to  infections 
produced  by  related  viruses,  such  as  respiratory  syncytial  virus  infection, 
and  to  the  possible  design  of  vaccines  for  these  viruses.  Other  investigators 
have  shown  that  certain  genes  in  influenza  are  not  randomly  reassorted 
during  recombination.   In  fact,  two  pairs  appear  to  be  "linked."  Since  these 
pairs  involve  the  RNA  polymerase  genes,  this  observation  may  be  quite  important 
in  attenuated  vaccine  development.  This  group  has  also  found  that  resistance 
to  amantadine  is  linked  to  the  M  protein.  Persistent  viral  infections  of 
influenza  viruses  have  also  been  obtained  independently  by  two  investigators. 
The  significance  of  these  observations  is  not  yet  clear,  but  these  in  vitro 
systems  should  enable  a  variety  of  studies  to  examine  the  biochemical  aspects 
of  viral  interference  and  viral  persistence. 

The  supplemental  funds  provided  to  the  Influenza  Program  during  the  swine 
flu  and  Russian  flu  era  have  been  expended,  and  there  is  a  resulting  reduc- 
tion in  directed  program  activities.  There  are  currently  12  contracts 
compared  with  15  last  year.  This  includes  one  new  contract  awarded  this 
year  to  identify  alternate  cell  substrates  for  diagnosis  and  cultivation  of 
influenza  virus.  Among  the  projects  which  were  terminated  was  the  influenza 
surveillance  network  which  had  effectively  served  as  a  sentinel  system  since 
1976.  In  contrast,  the  number  of  investigator-initiated  projects  has  dramat- 
ically increased  with  the  addition  of  four  grants  to  a  total  of  24,  two 
young  investigator  awards  and  two  fellowships. 

Respiratory  Diseases 

The  magnitude  of  infection  and  illness  from  parainfluenza  viruses  types  1, 
2,  and  3,  and  respiratory  syncytial  virus  (RSV)  is  difficult  to  document, 
but  estimates  from  several  epidemiologic  studies  indicate  that  these  viruses 
cause  severe  morbidity  in  pediatric  populations.  Of  all  severe  lower  re- 
spiratory diseases  in  infants  and  young  children,  perhaps  40  percent  could 
be  prevented  if  effective  vaccines  against  these  agents  were  available  and 
used. 


9-5 


Efforts  to  develop  an  attenuated  respiratory  syncytial  virus  vaccine  have 
produced  a  temperature  sensitive  mutant  that  has  been  satisfactorily  tested 
in  animal  models  and  human  adults.  Studies  in  children  are  underway.  The 
testing  of  the  live  vaccine  developed  by  Merck  Sharp  and  Dohme  has  been 
completed  in  animal  models.  These  studies  were  not  promising,  and  results 
from  an  efficacy  trial  (R.  Bel  she,  personal  communication)  indicate  no 
Benefit.  The  NIAID  does  not  plan  any  further  tests  with  this  product. 

Studies  on  the  epidemiology  and  pathogenesis  of  RSV  and  parainfluenza  infec- 
tions continue  at  Baylor  University  and  the  University  of  Rochester.  Longi- 
tudinal family  studies  have  shown  a  positive  correlation  between  serious  RSV 
disease  and  age  at  time  of  first  infection.  These  studies  have  also  shown 
that  maternal  antibodies  are  protective  in  the  first  months  of  life.  Several 
factors  such  as  crowding  of  living  quarters,  hygienic  practices  and  number 
of  persons  having  regular  contact  with  the  infants,  may  be  related  to  the 
likelihood  of  developing  disease.  Month  and  year  of  birth  are  other  obvious 
variables. 

Viral  Hepatitis 

The  goal  of  the  Hepatitis  Program  is  the  control  of  hepatitis  A  (HAV),  B 
(HBV),  and  non  A-non  B  (NANB)  infections.  Precise  data  on  morbidity,  mor- 
tality, and  economic  burden  to  the  country  are  difficult  to  obtain;  however, 
as  investigations  continue  on  the  etiology,  epidemiology,  and  long  term 
sequelae  of  viral  hepatitis,  it  has  become  apparent  that  many  cases  have 
been  misdiagnosed  or  underreported.  In  1977,  approximately  56,000  cases  of 
viral  hepatitis  were  reported  to  the  CDC.  Allowing  for  misdiagnosis  and 
underreporting,  it  can  be  estimated  that  560,000  cases  of  viral  hepatitis 
occurred  in  the  U.S.  in  1977,  with  a  distribution  of  approximately  50  per- 
cent HBV,  25  percent  HAV,  and  25  percent  NANB.  Estimates  of  death  from  HBV 
range  from  1,500-3,000  (approximately  1  percent  fatality  rate)  per  year. 
Costs  incurred  from  hospitalization,  professional  care,  laboratory  work,  and 
days  lost  from  employment  are  estimated  to  exceed  $600  million  annually. 

Type  B  hepatitis  has  an  incubation  period  of  60-180  days,  and  may  exhibit 
an  acute  and  chronic  phase.  Type  A  hepatitis  has  a  shorter  incubation 
period  of  20-40  days  and  does  not  exhibit  a  chronic  infection.  NANB  hepa- 
titis, with  an  incubation  period  similar  to  HBV,  is  responsible  for  approxi- 
mately 90  percent  of  the  post-transfusion  hepatitis  remaining  in  the  U.S. 
Although  the  virus(es)  responsible  for  this  type  of  hepatitis  has  not  been 
identified,  the  disease  has  been  successfully  transmitted  to  chimpanzees. 

Because  of  its  more  serious  clinical  manifestations,  type  B  is  a  disease  of 
major  public  health  importance  in  the  U.S.  However,  the  global  impact  of 
the  disease  is  more  significant  when  176,000,000  chronic  carriers  remain  as 
a  reservoir  for  infection.  In  addition  to  serving  as  reservoirs  of  virus,  a 
close  association  of  chronic  HBV  and  primary  hepatocellular  carcinoma  (PHC) 
has  been  demonstrated.  The  role  of  the  HBV  in  PHC  remains  to  be  defined, 
but  cause-and-effect  will  be  difficult  to  ascertain. 


9-6 


During  the  past  year,  efforts  have  continued  in  evaluating  experimental 
hepatitis  B  vaccines  in  humans.  As  noted  last  year,  aqueous  formalin- 
inactivated  vaccines  prepared  by  the  NIAID  were  shown  to  be  non-reactogenic 
and  non-infectious  in  volunteers.  To  date,  16  volunteers  have  been  immunized 
with  the  adw  vaccine  and,  although  most  have  developed  humoral  antibody,  the 
long  delay  between  boosters  and  appearance  of  antibodies  indicates  that 
these  vaccines  would  not  be  practical  for  clinical  use. 

Further  testing  of  the  ayw  vaccine  has  not  proceeded  since  one  of  the  five 
volunteers  participating  in  the  initial  safety  tests  developed  NANB  hepatitis. 
At  the  present  time,  evidence  suggests  that  the  vaccine  was  not  infectious, 
but  further  definitive  analyses  must  await  the  development  of  serologic 
markers  for  NANB  virus. 

Independently,  Merck  Sharp  and  Dohme  has  been  exploring  the  development  of  a 
type  B  hepatitis  vaccine.  Their  procedure,  similar  to  the  NIAID  procedure, 
employs  isopycnic  banding  and  rate  zonal  centrifugation  of  the  22  nm  surface 
antigen  with  subsequent  inactivation  with  formalin.  They  have  evaluated  two 
types  of  vaccines  in  chimpanzees  and  humans  --  aqueous  and  adjuvanted  (alum- 
inum hydroxide).  Their  data  with  aqueous  vaccine  are  comparable  to  the  NIAID 
data,  safe,  but  not  adequately  immunogenic.  However,  the  alum  absorbed  vac- 
cine did  appear  highly  immunogenic  with  approximately  65  percent  of  the  vol- 
unteers developing  antibody  three  and  one-half  months  following  a  two  dose 
regimen  and  90  to  95  percent  presenting  antibody  following  a  booster  at  six 
months.  Merck  has  offered  their  vaccine  for  testing  for  efficacy  by  the 
NIAID.  The  independent  evaluation  by  the  NIAID  of  a  potential  vaccine  that 
is  earmarked  for  licensure  will  lend  additional  credence  to  data  generated 
in  efficacy  trials,  and  represents  the  testing  of  vaccine  in  a  population 
that  is  at  extremely  "high  risk"  for  B  virus  infection  and  disease,  i.e., 
hemodialysis  patients  and  staff. 

In  preparation  for  an  HBV  efficacy  trial,  the  New  York  Blood  Center  has 
been  documenting  the  incidence  of  viral  hepatitis  in  patients  and  staff 
within  selected  hemodialysis  centers.  The  results  of  this  study  show  that 
the  current  attack  rates  appear  low  compared  with  rates  observed  in  the 
early  1970s,  yet  are  still  10-20  times  higher  than  in  other  groups  of  hos- 
pitalized patients  and  staff.  Based  on  incidence  of  HBV  infection,  avail- 
ability of  the  test  population,  and  ability  to  give  informed  consent,  this 
population  represents  a  feasible  group  for  vaccine  efficacy.  The  efficacy 
study  will  start  approximately  September,  1979,  and  will  be  a  randomized, 
double-blind  placebo-controlled  trial.  It  is  estimated  that  1,000  patients 
and  900  staff  will  be  randomized  into  the  trial  in  a  two  year  period.  The 
vaccine  to  be  employed  will  be  a  monovalent  ayw,  alum-absorbed  preparation 
provided  by  Merck  Sharp  and  Dohme.  It  is  expected  that  efficacy  data  should 
become  available  in  the  Fall-Winter,  1982. 

Another  approach  to  prevention  and  control  of  HBV  infection  and  sequelae  is 
to  interrupt  maternal -fetal  transmission.  Neonatally  acquired  infection  is 
of  major  magnitude  in  parts  of  Africa  and  the  Far  East.  For  example,  85 
percent  of  newborns  born  of  HBsAg+  HBeAg+  mothers  in  Taiwan  become  infected 
within  the  first  six  months  of  life.  The  NIAID  and  BoB/FDA  are  collaboratively 

9-7 


supporting  a  study  to  evaluate  Hepatitis  B  Immune  Globulin  (HBIG)  for  interrup- 
tion of  this  transmission  route.  During  the  past  year,  115  babies  have  been 
randomized  into  the  double-blind,  placebo-controlled  trial.  Seventy  more  babies 
will  be  entered  into  the  study  prior  to  January,  1980.  Follow-up  of  the  babies 
for  one  year  will  be  required  before  the  data  can  be  interpreted.  Thus  far, 
there  have  been  no  adverse  reactions  to  the  HBIG,  but  since  the  code  has 
not  been  broken,  there  are  no  results  available. 

The  virus  of  hepatitis  B  has  not  been  cultivated  in  vitro.  Attempts  to 
develop  continuous  hepatocyte  cultures  of  human  and  chimpanzee  origin  have 
been  unsuccessful,  and  contractual  support  for  this  activity  is  terminating 
this  year.  Hopefully,  investigator  initiated  approaches  to  this  requirement 
will  be  forthcoming.  Successful  cultivation  of  hepatitis  A  virus  was  reported 
during  the  past  year  by  investigators  at  Merck  Sharp  and  Dohme. 

Exciting  observations  have  been  made  on  a  virus  isolated  from  woodchucks 
(Marmota  monax).  This  virus  has  many  characteristics  in  common  with  human 
HBV,  including  morphology,  DNA  of  similar  size  and  structure,  presence  of 
endogenous  DNA  polymerase,  and  association  with  hepatitis  and  PHC.  Infection 
of  woodchucks  with  this  virus  may  represent  the  best  and  only  animal  model 
for  hepatitis  and  its  relation  to  PHC,  and  studies  are  now  underway  to 
better  characterize  the  animal-virus  system.  A  continuing  problem  will  be 
the  availability  of  woodchucks,  an  animal  that  hibernates  from  late  fall  to 
early  spring.  It  is  clear  that  research  will  be  severely  hampered  until 
young  animals  of  known  serologic  status  are  readily  available  to  investigators. 

Recent  breakthroughs  in  DNA  recombinant  research  have  the  potential  for 
rapidly  chanqinq  approaches  to  molecular  aspects  of  HBV.  At  least  three 
laboratories  have  reported  the  successful  cloning  of  the  HBV  genome  in 
Escherichia  col i ,  with  one  laboratory  reporting  expression  of  HBcAg.  The 
ease  of  which  this  technology  has  been  applied  to  HBV  bodes  well  for  develop- 
ing nucleic  acid  probes  for  studies  of  viral  persistence  and  hepatoma  and, 
perhaps,  the  elaboration  of  HBsAg  or  its  component  polypeptides  as  a  source 
of  future  vaccine  antigen.  An  NIAID  grantee,  Dr.  William  Robinson,  Stanford 
University,  is  a  pioneer  in  this  area  of  investigation. 

The  use  of  antiviral  drugs  in  eradication  of  the  HBV  carrier  state  is  also 
being  assessed  and  is  described  in  the  Antiviral  Substances  Program  section. 

Antiviral  Substances 


Virus  diseases  are  a  leading  cause  of  morbidity  and  mortality  throughout  the 
world,  and  the  use  of  antivirals  is  an  important  means  of  treating  and 
preventing  these  illnesses.  Although  development  of  host  resistance  remains 
the  first  line  of  defense  against  viral  diseases,  there  are  many  viral 
diseases  for  which  vaccines  are  not  available  or  cannot  feasibly  be  given  to 
all  people  at  risk.  Thus,  chemotherapeutic  agents  with  low  toxicity  and 
high  efficacy  are  needed. 


9-8 


Herpes  infections  are  a  major  cause  of  disease  with  the  symptoms  varying 
from  mild  labial  is  infection  to  fatal  encephalitis.  Carefully  controlled 
clinical  trials  were  supported  to  determine  the  efficacy  of  adenine  arabinoside 
(ara-A,  Vidarabine)  for  treatment  of  herpes  encephalitis,  and  the  use  of 
this  drug  reduced  the  mortality  from  70  percent  to  28  percent.  The  results 
from  these  trials  led  to  the  licensure  of  adenine  arabinoside  for  herpes 
encephalitis  in  October,  1978.  The  use  of  adenine  arabinoside  for  other 
types  of  herpes  infections  is  still  under  study,  including  studies  with 
herpes  zoster  and  neonatal  herpes.  Preliminary  results  indicate  that  adenine 
arabinoside  will  also  have  some  beneficial  effect  in  these  diseases.  The 
code  for  the  neonatal  herpes  study  was  recently  broken,  and  preliminary 
analysis  of  the  data  indicates  a  reduction  in  mortality  of  approximately  40 
percent. 

Systemic  herpes  infections  can  cause  devastating  illnesses  and  a  wide  range 
of  antivirals  is  needed  to  treat  these  diseases.  Adenine  arabinoside  is  a 
difficult  drug  to  administer  because  of  its  low  solubility  in  water.  Better 
antivirals  should  be  developed.  Animal  model  studies  supported  by  the 
Antiviral  Substances  Program  have  demonstrated  that  adenine  arabinoside 
monophosphate  (ara-AMP)  and  9-(2-hydroxyethoxymethyl )guanine  (acyclovir, 
ACV)  also  hold  promise  for  treatment  of  systemic  herpes  disease.  These 
drugs  are  being  considered  as  candidates  for  evaluation  in  future  clinical 
studies  against  serious  herpes  infections  in  protocols  similar  to  those 
utilizing  ara-A. 

In  addition  to  systemic  herpes  infections,  topical  herpes  infections  are  a 
major  cause  of  disease  in  the  United  States,  with  type  two  herpes  being  the 
second  most  frequent  genital  infection  in  the  United  States,  and  type  one 
herpes  (herpes  labialis)  causing  recurrent  infections  in  over  30  percent  of 
the  adult  population.  Work  to  define  the  natural  history  of  herpes  labialis 
has  been  supported  by  the  Program.  This  work  led  to  a  subsequent  clinical 
trial  which  demonstrated  that  ara-AMP  was  not  effective  as  a  topical  treatment. 
During  1979,  a  double-blind  clinical  trial  to  evaluate  the  effectiveness  of 
topical  application  of  ACV  for  herpes  labialis  was  started.  These  studies 
are  also  designed  to  evaluate  the  general  use  of  ACV  in  humans,  including 
analysis  of  any  potential  antiviral  resistance  to  the  drug  and  drug  absorption 
through  the  skin. 

Interferon,  a  broad-spectrum  antiviral,  is  a  candidate  antiviral  for  both 
RNA  and  DNA  virus  infections.  Through  grant  sponsored  research,  we  are 
learning  more  about  the  mechanism  of  action  and  the  antigenic  and  biochemical 
nature  of  interferon.  There  are  three  types  of  interferon:  fibroblast  and 
leukocyte  (type  one  interferons)  and  immune  interferon  (type  two  interferon). 
Much  of  our  understanding  on  the  nature  of  interferon  has  come  about  through 
the  use  of  NIAID  reference  interferon  preparations,  which  have  allowed  the 
comparison  of  results  from  laboratories  throughout  the  world.  During  the 
past  year,  the  World  Health  Organization  has  designated  the  NIAID  reference 
reagents  for  human  fibroblast,  rabbit,  and  mouse  interferons  as  international 
standards.  The  use  of  NIAID  antisera  to  human  leukocyte,  human  fibroblast,   ; 
and  mouse  interferons  has  also  enabled  investigators  to  identify  specific 
biochemical  events  brought  about  by  this  antiviral,  and  has  played  an  important 

9-9 


part  in  developing  better  methodologies  for  its  purification.  A  number  of 
studies  on  purification  have  been  initiated,  and  considerable  headway  has 
been  made  in  the  characterization  of  this  elusive  moiety. 

Human  leukocyte  interferon  is  being  evaluated  in  clinical  trials  for  the 
treatment  and  prevention  of  herpes,  hepatitis  B  and  respiratory  viral  infections 
Recent  clinical  trial  data  indicate  that  interferon  can  reduce  the  reactivation 
of  herpes  infection  after  cranial  nerve  surgery  by  43  percent.  The  total 
incidence  of  virus  shedding  was  reduced  from  41  percent  in  placebo  patients 
to  8  percent  in  interferon  patients.  These  studies  are  continuing  to  deter- 
mine optimal  dose  schedules.  Interferon  has  also  been  shown  to  be  efficacious 
in  the  treatment  of  herpes  zoster  infections  in  the  immunosuppressed  patient. 
When  administered  in  combination  with  antihistamines,  it  appears  to  hold 
promise  for  treatment  of  respiratory  viral  infections. 

Currently,  there  are  over  175  million  carriers  of  hepatitis  B  for  which 
there  are  no  satisfactory  therapies.  The  Antiviral  Substances  Program  is 
conducting  studies  to  determine  optimal  regimens  for  modifying  or  eliminating 
the  carrier  state.  Recent  results  suggest  that  the  best  treatment  for 
hepatitis  B  will  be  combination  therapy  with  interferon  and  another  antiviral, 
such  as  adenine  arabinoside. 

Since  1969,  the  Antiviral  Substances  Program  has  been  the  primary  source  of 
funding  and  support  for  interferon  studies  in  the  United  States,  and  these 
studies  have  led  to  an  understanding  of  the  potential  of  interferon  in  the 
clinic.  Based  on  the  results  from  this  Program,  the  National  Cancer  Institute 
and  American  Cancer  Society  have  initiated  carefully  controlled  clinical 
trials  with  interferon  in  a  variety  of  cancers.  Cooperation  among  interferon 
researchers  continues  to  be  an  important  objective  of  the  Program. 

A  critical  part  of  the  effort  to  develop  clinically  effective  antivirals  has 
been  the  establishment  of  a  series  of  animal-viral  disease  models  which 
closely  mimic  some  common  viral  diseases  of  man.  During  the  past  year,  the 
effectiveness  of  acyclovir  against  herpes  infections,  including  genital  and 
systemic  infections,  has  been  demonstrated  in  these  animal  models.  A  new 
antiviral,  phosphonoformate,  is  being  studied  to  determine  efficacy  and 
relative  toxicity.  These  animal  studies  are  important  in  determining  optimal 
dose  schedules  and  routes  of  administration,  information  necessary  for 
planning  successful  clinical  trials  in  man. 

A  new  initiative  to  develop  targeted  antivirals  was  started  by  the  Program 
during  1979.  Based  on  our  knowledge  of  the  nucleic  acid  sequences  in  virus 
genes,  antivirals  will  be  developed  which  will  specifically  interact  with 
the  nucleic  acid  without  interfering  with  host  cell  metabolism.  Targeted 
antiviral  development  is  a  field  of  the  future  and  will  be  supported  by  both 
the  grant  and  contract  mechanisms. 

Bacterial  Vaccines 

Pneumococcal  Diseases:  Studies  sponsored  by  the  NIAID  during  the  past  decade, ; 
along  with  studies  by  manufacturers,  have  led  to  the  development  and  licensure 
of  a  14-valent  pneumococcal  polysaccharide  vaccine  (Merck  Sharp  &  Dohme).  It 

9-10 


is  expected  that  a  second  manufacturer  (Lederle)  will  have  a  licensed  product 
by  the  Fall  of  1979. 

It  is  currently  recommended  that  pneumococcal  vaccine  be  used  in  those  indi- 
viduals over  two  years  of  age  considered  at  high  risk  for  pneumococcal 
infections.  There  are  a  number  of  disease  conditions  which  predispose  to 
pneumococcal  infections;  however,  there  are  little  data  on  the  immunogenicity 
and  efficacy  of  pneumococcal  vaccine  relating  specifically  to  these  populations. 
The  Bacterial  Vaccines  Program  has  developed  a  collaborative  effort  through 
which  pneumococcal  vaccines  are  being  studied  in  patients  with  the  following 
conditions:  sickle  cell  disease,  splenectomy,  bone  marrow  transplantation; 
Hodgkin's  disease,  renal  transplantation,  chronic  dialysis,  lymphoma,  myeloma, 
diabetes,  lupus,  lymphoblastic  leukemia,  alcoholic  cirrhosis  and  chronic 
obstructive  pulmonary  disease.  There  are  approximately  25  studies  underway 
in  various  stages  of  completion.  Although  no  direct  funding  is  being  given 
to  the  investigators  by  the  NIAID,  these  studies  are  being  coordinated  by 
staff  and  all  antibody  assays  are  being  done  through  the  contract-supported 
serology  laboratory  at  SUNY,  Downstate  Medical  Center.  Although  these 
studies  have  not  been  completed,  some  important  information  is  already 
evident:  (1)  Sickle  cell  and  splenectomy  patients  appear  to  respond  well  to 
the  vaccine;  (2)  Hodgkin's  patients  who  have  undergone  splenectomy  and 
chemotherapy  respond  poorly  to  vaccine;  however,  Hodgkin's  patients  given  vaccine 
prior  to  therapy  appear  to  respond  normally;  (3)  antibody  response  is  diminished 
in  renal  dialysis  patients;  and  (4)  simultaneous  administration  of  pneumococcal 
and  influenza  vaccines  does  not  have  a  negative  effect  on  response  to  the 
individual  antigens.  When  the  collaborative  studies  are  completed,  they 
should  allow  for  specific  recommendations  on  the  use  of  pneumococcal 
vaccine  in  various  patient  populations. 

Pneumococci  are  estimated  to  be  responsible  for  40  to  50  percent  of  acute 
otitis  media  in  children.  This  is  an  important  childhood  disease  which  often 
causes  hearing  disorders  and  subsequent  learning  disabilities.  Almost  every 
child  will  have  at  least  one  bout  of  otitis  media;  20  to  30  percent  will  have 
six  or  more  occurrences. 

A  study  of  the  value  of  pneumococcal  polysaccharide  vaccines  in  preventing 
otitis  media  was  initiated  in  1975.  Two  double-blind  efficacy  trials  of  an 
octavalent  vaccine  for  the  prevention  of  disease  in  children  who  have  had 
previous  middle  ear  infections  are  now  underway.  The  code  has  not  been  broken 
on  these  studies;  therefore,  no  data  on  efficacy  are  available  at  the  present 
time.  Data  from  one  of  the  trials  (Huntsville  Hospital)  will  be  analyzed  in 
the  S ummer  of  1979  and  the  other  trial  (Boston  City  Hospital)  will  be  completed 
in  the  Spring  of  1980.  A  third  project  is  designed  to  establish  the  optimum 
dose  and  booster  regimen  for  maximum  antibody  response  of  infants  and  young 
children.  Certain  polysaccharides  (i.e.,  type  3)  are  good  immunogens  in  infants 
when  given  in  one  dose.  Some  cause  measurable  responses  when  given  in  two  doses 
(i.e.,  types  7  and  18),  while  most  of  the  others  are  poorly  immunogenic  in  infants 
with  any  dose  regimen. 

To  better  understand  the  pathogenesis  and  immunology  of  pneumococcal  otitis 
media  and  the  efficacy  of  pneumococcal  polysaccharide  vaccine  in  preventing     • 


9-11 


experimentally- induced  middle  ear  disease,  an  animal  model  system  has  been 
developed.  Utilizing  this  chinchilla  model,  investigators  have  been  able  to 
correlate  vaccination  with  protection  from  disease,  even  in  cases  in  which 
there  has  been  no  measurable  serum  antibody  response  to  the  polysaccharides. 
These  data  suggest  that  pneumococcal  vaccines  may  prevent  some  otitis  media  in 
young  infants  in  which  serum  antibody  response  is  poor. 

Meningitis:  It  is  estimated  that  approximately  20,000  cases  of  bacterial 
meningitis  occur  in  the  U.S.  each  year,  most  in  young  children  and  infants.  A 
pure  polysaccharide  vaccine  for  H_.  influenzae  type  b,  developed  and  tested 
under  our  program,  has  been  shown  ineffective  in  children  under  18  months  of 
age.  Because  most  of  the  H_.  influenzae  meningitis  occurs  in  this  younger  age 
group,  program  efforts  are  now  directed  toward  developing  vaccines  effective  in 
infants.  For  the  past  few  years,  considerable  laboratory  and  clinical  effort 
has  been  concentrated  on  a  new  H.    influenzae  complex  vaccine.  This  preparation 
is  a  complex  of  polyribose  phosphate  (PRP)  and  cell  wall  material,  primarily 
polypeptides  and  1 ipopolysaccharide.  Designated  PRPc,  this  material  showed 
promise  in  animal  studies.  Adult  human  studies  have  shown  the  new  vaccine  to 
be  potentially  a  better  antigen;  however,  it  is  considerably  more  reactive  than 
pure  PRP  and  efforts  to  significantly  reduce  the  toxicity  have  met  with  limited 
success.  Plans  are  being  made  to  continue  studies  of  this  in  children;  however, 
alternate  vaccine  development  approaches  are  also  being  followed.  The  alternate 
approaches  include:  (1)  extraction  and  characterization  of  outer  membrane  pro- 
teins from  H_.  influenzae  to  determine  their  role  in  disease  and  their  potential 
as  immunogens;  (2)  preparation  of  a  complex  of  PRP  with  a  pure  protein  such  as 
the  diptheria  toxoid;  (3)  preparations  of  complexes  of  highly  immunogenic 
bacterial  polysaccharides  with  determinant  groups  of  PRP;  and  (4)  preparation 
of  very  high  molecular  weight  molecules  of  PRP. 

In  addition  to  efforts  to  improve  the  infant's  response  to  bacterial  meningitis 
vaccines,  protection  of  the  neonate  and  young  infant  by  hyperimmunization  of 
the  mother  is  being  attempted.  This  is  being  done  in  humans  by  giving  H_. 
influenzae  PRP  to  women  who  plan  pregnancies  and  to  a  control  group  of  women 
who  do  not  plan  to  become  pregnant.  Serum  samples  from  mothers  and  children 
will  be  studied  to  determine  if  the  level  of  infant  antibody  to  H.  influenzae 
can  be  increased  during  the  crucial  first  few  months  of  life.  Hyperimmunization 
of  the  mother  is  also  being  studied  in  primates;  pregnant  females  will  be 
immunized  to  determine  the  possible  harmful  effects  of  vaccine  administration 
during  pregnancy  as  well  as  to  follow  antibody  levels  in  the  mother  and  offspring. 

Group  A  and  C  meningococcal  polysaccharide  vaccines  have  been  licensed  for  use 
in  adults  and  older  children.  The  group  A  vaccine  has  been  shown  to  be  effective 
in  children  down  to  three  months  of  age  in  an  efficacy  trial  in  Finland  sponsored 
by  the  NIAID.  Group  C  meningococcal  polysaccharide  vaccine  has  been  shown  to 
be  poorly  immunogenic  in  very   young  children.  A  capsular  polysaccharide  extracted 
from  a  variant  group  C  strain  has  been  prepared  at  Rockefeller  University. 
This  new  material  has  a  slightly  different  chemical  structure  from  the  licensed 
group  C  vaccine,  but  it  is  antigenically  cross-reactive.  Through  the  NIAID      i 
program,  this  variant  C  vaccine  has  been  shown  superior  to  the  licensed  material 


9-12 


in  adults  and  in  children  over  two  years  of  age.  Studies  in  infants  will  begin 
in  the  immediate  future. 

During  the  course  of  the  Finnish  trial  of  the  efficacy  of  group  A  meningococcal 
vaccine  in  children  (mentioned  above),  it  was  discovered  that  persistence  of 
antibody  and  the  importance  of  the  booster  regimen  on  persistence  of  antibody 
appear  to  be  age  related.  The  Finnish  study  is  being  continued  to  further  study 
persistence  of  antibody  as  a  function  of  the  type  of  dose  and  booster  regimen 
used. 

Group  B  meningococcal  polysaccharide  is  non-antigenic  in  humans.  A  protein 
antigen  vaccine  candidate,  from  the  outer  membrane  layer  of  the  organism,  has 
been  developed  by  the  BoB.  Phase  I  safety  and  antigenicity  tests  in  adults 
showed  reactions  to  the  vaccines  to  be  acceptable,  but  antibody  responses  were 
somewhat  disappointing.  Plans  are  underway,  however,  for  studies  in  teenage 
children  and  children  over  age  2.  A  second  cell  envelope  protein  antigen  pre- 
pared at  the  Naval  Medical  Research  Institute  will  be  tested  in  adults  in  the 
near  future. 

Rocky  Mountain  Spotted  Fever:  The  presently  licensed  vaccine  for  RMSF  is  not 
considered  effective  and  is  no  longer  commercially  available.  A  new  inactivated, 
whole-cell  vaccine  has  been  developed  at  the  U.S.  Army  Research  Institute  for 
Infectious  Diseases  (USAMRIID).  Because  of  our  mutual  interest,  the  NIAID  has 
assisted  with  support  of  phase  I  clinical  trials  of  the  vaccine  in  adults  at 
the  USAMRIID  facilities  at  Fort  Detrick.  The  vaccine  appears  to  be  superior  to 
the  commercial  material  in  these  studies  and  additional  clinical  studies  may  be 
sponsored  by  NIAID.  Three  new  contracts  have  been  awarded  this  fiscal  year  for 
additional  animal  model  studies  of  RMSF  and  the  USAMRIID  vaccine  and  for  studies 
of  the  epidemiology  of  RMSF. 

Enteric  Diseases 

The  Enteric  Disease  Program  of  the  NIAID  is  concerned  with  reducing  the  morbidity 
and  mortality  of  infectious  diarrheas  in  this  country  and  around  the  world. 
This  includes  viruses,  bacteria  and  their  products,  and  the  unicellular  parasites. 
The  Program  is  seeking  to  define  the  scope  of  the  diarrheal  problem  in  infants, 
children,  and  adults,  to  identify  the  etiologic  agents  and  elucidate  their 
pathogenesis  and  to  gain  knowledge  for  developing  practical  means  of  control. 

The  Institute  funded  three  enteric  disease  study  centers  in  FY  1977  (D.  C. 
Children's  Research  Hospital,  The  University  of  Michigan,  and  the  University  of 
Texas  in  Houston)  for  epidemiological  studies  leading  to  finding  methods  of 
controlling  diarrheal  diseases.  Several  trends  are  beginning  to  emerge:  (1) 
Younger  children  have  more  diarrhea  episodes  than  older  children,  i.e., 
57.4  visits  to  the  doctor's  office/100  persons/year  in  the  0-4  year  olds;  13.9 
visits  to  the  doctor's  office/100  persons/year  in  the  over  20  year  olds  in  a 
small  town  and  rural  population  and  1.7  episodes  of  diarrhea  in  the  first  12 
months  of  life  in  an  urban  area  and  approximately  0.6  episodes  in  the  second  12 
months.  (2)  Overcrowding  of  children  in  day  care  centers  produces  a  higher     , 
attack  rate  of  diarrhea,  even  in  the  more  affluent  populations.  There  were 
several  instances  of  secondary  cases  among  family  members.  Three  of  five 
outbreaks  were  associated  with  Shigella  infections  and  two  were  associated 

9-13 


with  both  rotavirus  and  Giardia  infections.  (3)  Rotavirus  is  a  common  cause  of 
infant  diarrheas  with  a  peak  attack  rate  in  the  winter  as  high  as  71  percent 
during  a  one  month  period.  (4)  Some  families  (16  percent)  with  infants  did  not 
report  any  episodes  of  diarrhea  in  the  first  12  months  of  life;  about  the  same 
number  (15  percent)  reported  two  or  more  episodes  of  diarrhea.  (5)  Type  2 
rotavirus  was  detected  about  three  times  more  often  than  type  1.  (6)  There  is 
•a  suggestion  that  uncultivatable  adenoviruses  play  a  role  in  acute  diarrheal 
diseases. 

The  seventh  world  pandemic  of  cholera  continues  into  its  18th  year.  There  were 
40  countries  reporting  cholera  in  1978  as  compared  to  34  in  1977.  This  includes 
the  United  States  where  the  first  multicase  outbreak  of  cholera  since  1911 
occurred  in  Louisiana  in  August.  Crabs  were  implicated  as  the  source,  with 
their  contamination  coming  from  the  environment.  The  Institute  program  on 
cholera  is  part  of  the  U.S. -Japan  Cooperative  Medical  Science  Program.  Cholera 
studies  remain  important  per  se,  but  they  also  serve  as  an  excellent  model  for 
studies  on  enterotoxin  mediated  diarrheas,  including  studies  of  the  local 
immune  response. 

The  intensive  activity  in  cholera  research  has  produced  the  following:  (1)  Two 
laboratory-modified  live  vaccines  for  testing  in  volunteers;  (2)  Two  isolates 
from  the  environment  identified  as  candidates  for  a  modified  live  vaccine; 
(3)  Evidence  that  cholera  toxin  produces  long  lasting  immunity  in  the  mucosal 
IgA  system  of  dogs;  (4)  A  flagellar  vaccine  which  produces  better  protection  in 
rabbits  than  licensed  whole  cell  vaccine.  This  protection  is  enhanced  1,000- 
fold  when  combined  with  cholera  toxoid;  (5)  Chlorpromazine  is  capable  of  drasti- 
cally reducing  fluid  loss  in  patients  suffering  from  cholera;  and  (6)  Non  0 
Group  1  cholera  vibrios  are  present  in  U.S.  estuaries  as  well  as  in  the  Ganges 
River  delta,  the  perennial  seat  of  cholera  infection.  These  vibrios  produce  no 
detectable  toxin;  however,  some  activate  adenylate  cyclase  in  a  manner  similar 
to  cholera  toxin. 

The  treatment  of  cholera  with  fluids  has  been  refined  many  times  in  the  past 
decade.  This  past  year  provided  a  major  new  breakthrough  in  treatment  when  it 
was  determined  that  chlorpromazine  was  capable  of  greatly  reducing  the  fluid 
loss.  This  is  the  first  example  of  a  safe,  practical,  pharmaceutical  agent 
with  this  capability.  The  implications  are  far-reaching. 

The  Wellcome  Foundation  Ltd.,  Beckenham,  Kent,  England,  has  provided  an  alum 
precipitated  whole  cell  cholera  vaccine,  a  cholera  toxoid  complexed  with  adju- 
vant, and  the  two  products  combined  for  field  testing  in  Dacca,  Bangladesh.  The 
trial  is  being  designed  to  determine  the  protective  role  of  an  alum  precipitated 
whole  cell  vaccine  and  the  additive  or  synergistic  effect  of  the  toxoid  combined 
with  the  vaccine. 

Studies  on  the  interaction  of  cholera  vibrios  with  the  intestinal  mucosa  of 
mice  indicate  chemotaxis  and  protease  (mucinase)  contribute  to  virulence.  This 
suggests  there  are  other  antigens  which  may  be  useful  in  new  cholera  vaccines, 
(e.g.,  mucinase)  and  also  characteristics  which,  when  absent,  may  render  live 
mutants  avirulent  and  thus  of  potential  value  as  living  oral  vaccines.  Vibrio 
ecology  studies  are  of  increasing  importance  with  the  report  of  cholera  in 


9-14 


Louisiana  traced  to  seafood.  V_.  cholerae  isolates  in  marine  estuaries  are 
usually  the  avirulent  non  0  Group  1  vibrios.  Selected  isolates  will  be  tested 
for  virulence  in  volunteers.  Laboratory  tests  to  date  indicate  no  toxin  pro- 
duction; however,  there  is  an  indication  of  ability  to  activate  adenylate  cy- 
clase as  cholera  toxin  does.  The  history  of  cholera  in  Louisiana  indicates 
that  cases  may  recur.  Similar  situations  may  exist  in  other  areas  in  this 
hemisphere  and  pose  an  obvious  threat  to  places  with  poor  sanitation.  A  better 
understanding  of  the  role  of  vibrios  in  the  environment  is  needed  to  suggest 
methods  of  control . 

Considerable  progress  has  been  made  in  studies  of  disease  caused  by  toxigenic 
•E_.  col i  by  using  techniques  developed  in  cholera  studies.  Evidence  continues 
to  show  that  these  strains  are  a  major  cause  of  diarrhea  among  travelers  to 
developing  countries  and  among  children  and  adults  in  these  countries.  They 
occasionally  cause  diarrhea  in  the  U.S.,  especially  in  newborns  in  crowded 
areas  with  poor  sanitation.  Both  the  heat  labile  (LT)  and  heat  stable  (ST) 
enterotoxins  of  E_.  col  i  have  been  purified.  The  LT  has  a  subunit  structure 
(A&B)  very  similar  to  cholera  toxin,  and  they  are  immunologically  related  to 
A&B  cholera  toxin  subunits.  There  are  minor  differences  in  both  E_.  col i  sub- 
units  compared  to  the  cholera  toxin  subunits;  however,  the  biologic  activity  is 
comparable  on  a  weight-for-weight  basis.  The  structure  and  mechanism  of  action 
of  ST  are  being  studied.  Diarrhea  from  E_.  col  i  appears  to  be  related  to  the 
organism's  ability  to  attach  to  the  intestinal  mucosa.  Two  distinct  pilus-like 
colonizing  factors  have  been  identified  on  human  pathogens  (CFS  I  and  CFA  II). 

Antibody  studies  indicate  there  may  be  a  protective  immune  response  to  the  CFAs, 
and  this  is  being  pursued.  It  appears  there  are  other  factors  of  E_.  coli  which 
contribute  to  diarrheas.  One  is  a  S.  dysenteriae-1 ike  toxin  which  is  pathogenic 
in  rabbits.  Also,  some  E_.  coli  without  known  pathogenic  entities  cause  diarrhea 
in  human  volunteers,  indicating  other  virulence  mechanisms.  Genetic  manipula- 
tion of  the  LT  gene  of  E_.  col  i  may  lead  to  the  production  of  a  strain  for 
immunoprophylaxis. 

Viral  Vaccine  Program 

The  viral  vaccine  program  covers  those  areas  where  research  should  be  stim- 
ulated toward  the  goal  of  an  effective  vaccine  but  which  is  not  covered  by  the 
other  specific  programs  of  the  Branch.  During  the  past  year  an  Experimental 
Herpesvirus  Vaccine  Workshop  was  co-sponsored  with  BoB,  NCI,  and  the  Fogarty 
International  Center.  It  became  clear  that  although  attempts  at  vaccine  devel- 
opment against  Herpes  simplex,  cytomegalovirus,  and  varicella  viruses  had  been 
made,  only  the  varicella  vaccine  showed  promise.  Since  varicella  virus  causes 
serious  disease  in  immunosuppressed  children  or  children  with  malignant  diseases . 
an  RFP  to  evaluate  the  efficacy  of  this  candidate  vaccine  was  released.  Work 
with  this  vaccine  has  been  initiated  through  a  grant  with  the  University  of 
Texas  at  San  Antonio  for  phase  1  studies.  The  program  continues  to  support 
research  to  determine  the  antigenic  structure  and  other  characteristics  of  the 
herpes  viruses  in  the  hope  of  developing  other  more  effective  vaccine  candidates. 

The  Development  and  Applications  Branch  also  supports  five  General  Vaccine 
Evaluation  Centers  which  form  the  backbone  of  the  Branch's  efforts  at  control 


9-15 


of  important  disease  problems.  Through  these  groups,  different  populations, 
including  all  age  groups,  normal  and  "at  risk,"  are  available.  These  groups 
are  utilized  for  the  evaluation  of  all  vaccines,  viral  and  bacterial,  and 
antiviral s  of  interest  to  the  Branch.  During  this  past  year  these  Centers  have 
been  overtaxed  and  a  backlog  of  work  has  developed. 

FY  79  has  been  a  successful  year  with  significant  progress  in  vaccine  develop- 
ment, licensure  of  adenine  arabinoside  for  herpes  encephalitis,  promising 
results  in  the  efficacy  of  ara-A  for  neonatal  herpes,  and  interferon  for 
trigeminal  neuralgia,  zoster,  and  chronic  hepatitis  B. 


9-16 


EPIDEMIOLOGY  AND  BIOMETRY  BRANCH 

The  Epidemiology  and  Biometry  Branch  personnel  supports  the  research  program 
of  the  Institute  through  it's  own  projects  and  through  collaboration  with 
other  units  in  the  MIDP  and  the  Institute,  Collaborative  arrangements  included 
consultation  in  study  design,  evaluation  of  the  epidemiological  approach  of 
studies,  advice  on  management  of  large  research  data  sets,  analysis  of  research 
data  and  monitoring  data  from  multicenter  clinical  trails. 

Approximate  Level  of  Support 

Activity  Number         Amount 

HLA  and  Infectious  Disease  Epidemiology 

Research  Contracts  2         $  52,452 

Nosocomial  Infectious  Epidemiology 

Research  Contracts  1  500,000 

HLA  and  Infectious  Disease  Epidemiology 

The  EBB  program  which  addresses  questions  about  HLA  and  Infectious  Disease 
Epidemiology  was  supported  by  two  projects  in  FY79.  The  study  of  the  associ- 
ation between  HLA  phenotype  and  recurrent  herpes  virus  type  1  infection,  which 
is  sponsored  jointly  in  Framingham,  MA,  by  NIAID,  The  Bureau  of  Biologies  and 
NHLBI,  continued  through  the  collection  of  clinical  data  and  the  collection 
of  serum  specimens.  These  data  may  contribute  to  our  understanding  of  the 
relationships  host  factors  such  as  HLA  as  circulating  antibody  have  on  a  not 
uncommon  expression  of  a  universal  infection. 

A  second  project  in  this  program  was  started  in  FY79  when  investigators  at  the 
Immunogenetics  Laboratory  of  the  Johns  Hopkins  University  were  awarded  a  con- 
tract in  The  Influence  of  HLA  on  the  Immune  Response  to  Viral  Vaccines  in 
Humans.  This  study  is  being  conducted  among  families  of  the  Old  Order  Amish 
which  Dr.  Wilma  Bias,  the  Principal  Investigator,  has  characterized  previously 
with  regard  to  HLA,  An  association  between  any  differential  in  the  dynamics 
of  the  various  cellular  and  humoral  components  of  the  immune  response  to  polio, 
mumps,  rubella  or  measles  vaccines  and  HLA  will  be  sought.  Should  crude 
associations  be  noted  it  is  anticipated  the  extensive  and  reliable  geneologic 
information  and  the  access  to  family  members  for  study  may  allow  the  investi- 
gators to  identify  genes  separate  from  but  closely  linked  to  HLA  genes  which 
modify  the  immune  response  to  vaccines. 

Influenza 


The  entire  EBB  staff  participated  actively  in  influenza  research  activities  in 
FY79.  Data  from  the  clinical  trials  of  inactivated  A/USSR  vaccine  was  vali- 
dated, organized  and  provided  to  the  various  investigators, 

10-1 


Dr.  Blackwelder,  Dr.  David  Ailing  of  the  LCI  and  Sir  Charles  Stuart-Harris,  a 
Fogarty  International  Scholar,  conducted  an  investigation  of  relationships 
among  total  and  cardiovascular  mortality  and  influenza  activity  as  measured 
by  acute  respiratory  mortality  and  influenza  mortality.  This  is  an  effort  to 
fit  U.S.  data  to  a  model  of  these  relationships  reported  by  British  workers, 
If  U.S.  data  produce  a  sufficiently  good  model  it  is  anticipated  that  quanti- 
tative analysis  of  patterns  of  death  among  the  elderly  will  identify  clearly 
an  influenza-related  portion  of  mortality  which  will  allow  preventive  strate- 
gies to  be  brought  to  bear  more  efficiently  on  truly  high-risk  persons.  Policy 
decisions  relating  to  influenza  immunization  of  all  people  over  65  require 
reliable  estimates  of  morbidity  and  mortality,  and  what  proportion  may  be  pre- 
vented by  various  strategies.  Epidemiology  is  central  to  thorough  considera- 
tion of  program  options,  and  the  EBB  expects  to  continue  to  contribute 
significantly  to  the  broadened  scope  of  the  influenza  program. 

Clinical  Trials 

The  expanding  clinical  trials  programs  within  the  NIH  has  stimulated  develop- 
ment of  new  administrative  and  research  technologies  in  study  design,  manage- 
ment of  data  and  data  monitoring  and  analysis.  This  is  reflected  in  MIDP 
as  well,  and  the  EBB  has  been  actively  supporting  several  new  initiatives, 
particularly  two  sponsored  by  CSB.  In  a  multicenter  study  of  treatment 
regimens  for  cryptococcal  meningitis,  EBB  staff  will  be  monitoring  research 
data  periodically  for  adherence  to  protocol  and  outcome.  In  another  CSB- 
sponsored  clinical  trial,  EBB  staff  consulted  in  the  study  design  and  expect 
to  be  involved  with  monitoring  clinical  data  in  a  multicenter  trial  of 
penicillin  prophylaxis  of  group  B  streptococcal  sepsis  in  premature  newborns. 

EBB  staff  assisted  the  DAB  bacterial  vaccine  program  officer  in  two  areas  con- 
cerning pneumococcal  vaccines.  In  two  separate  but  similar  trials  of  vaccine 
in  prophylaxis  for  pneumococcal  otitis  media,  EBB  staff  conducted  a  preliminary 
analysis  of  efficacy  data  and  will  assist  in  the  final  analysis.  The  larger 
commitment  in  pneumococcal  vaccines,  however,  was  Dr.  Blackwelder 's  work  with 
Dr.  Gerald  Schiffman  to  devise  a  sound  but  simple  way  to  express  pneumococcal 
polysaccharide  antibody  titers  in  the  myriad  of  clinical  trials  for  which 
Dr.  Schiffman  is  providing  the  serological  data.  Dr.  Blackwelder  also  assisted 
intramural  scientists  in  the  NIAMDD  with  the  analysis  of  data  from  a  clinical 
trial  of  pneumococcal  vaccine  in  patients  with  systemic  lupus  erythematosis, 

EBB  staff  provided  consulation  to  IAIDP  in  two  areas  in  FY79,  including  parti- 
cipation in  the  design  of  a  study  of  the  efficacy  of  skin  tests  to  the  major 
and  minor  group  determinants  of  penicillin.  The  material,  which  was  developed 
by  Dr.  Bernard  Levine,  can  detect  persons  who  are  allergic  to  penicillin,  but 
it  is  not  known  how  many  patients  in  whom  penicillin  is  the  drug  of  first  choice 
are  denied  the  drug  in  the  basis  of  an  unconfirmed  history  of  allergic  reaction 
to  it.  This  study  should  help  to  place  the  history  of  penicillin  allergy  and 
routine  use  of  the  skin  test  in  clinical  perspective, 

Ms.  Jacqueline  Smith  continued  to  program  for  computerized  analysis  the  Kidney 
Transplant  Histocompatibility  Study  (KTHS)  data.  This  multicenter  project 
sponsored  by  IAIDP  has  collected  a  variety  of  clinical  data  judging  the  impact 


10-2 


of  histocompatibility  typing  on  the  survival  of  kidney  transplants,  A  pre- 
liminary analysis  prepared  in  March  showed  a  surprising  differential  in 
survival  rates  among  participating  centers  which  persisted  when  controlling 
for  other  factors  such  as  the  extent  of  tissue  matching,  age,  and  proportion 
of  living  related  donors.  Ms.  Smith  continues  to  be  a  consultant  in  computer 
programming  for  IAIDP  staff  and  the  contractors  who  are  responsible  for  the 
KTHS. 

Impact  of  Infections 

In  FY79  the  EBB  staff  began  with  the  consultant  advice  of  Dr.  Fred  J,  Payne  a 
process  of  accessing  on  a  routine,  ongoing  basis  the  morbidity,  mortality  and 
cost-of-illness  data  of  allergic,  immunologic  and  infectious  diseases.  This 
continuing  project  will  for  the  first  time  develop  these  data  from  the  same 
sources  annually  to  allow  a  demonstration  of  trends  and  comparisons  between 
years.  Earlier  results  showed,  as  expected,  the  infectious  diseases  to  be  a 
tremendous  burden  to  society  in  terms  of  morbidity  but  not  mortality.  This 
project  is  attempting  to  dissect  out  the  the  contribution  of  infectious  disease 
complications  to  the  high  mortality  rates  of  heart  disease,  cancer  and  diabetes. 
Previously  only  the  underlying  cause  of  death  data  were  analyzed  in  studies 
of  hospital  records,  but  the  extent  to  which  infections  effect  adversely  the 
course  of  chronic  illness  has  not  been  established.  A  clearer  understanding 
of  the  magnitude  of  the  impact  of  allergic,  immunologic  and  infectious  diseases 
throughout  the  clinical  spectrum  expressed  in  terms  of  episodes  of  illness, 
direct  cost  of  care,  indirect  costs  such  as  days  out  of  work  or  school  and 
rates  of  complications  in  chronic  illness  is  fundamental  to  the  process  of 
allocating  research  resources  and  planning  research  initiatives  in  these 
diseases. 

Epidemiology  of  Nosocomial  Infections 

EBB  negotiated  partial  funding  of  the  large  Study  of  the  Efficacy  of  Nosocomial 
Infection  Control  Program  (SENIC),  a  CDC  sponsored  study  which  is  in  the 
final  stages  of  data  acquisition.  This  comprehensive  investigation  of  the 
effectiveness  of  standard  hospital  infection  control  procedures  is  expected 
to  identify  more  precisely  patterns  of  infections  acquired  in  modern  hospitals. 

Trans-NIH 

In  the  Trans-NIH  areas  covered  by  EBB  staff,  the  Epidemiology  Committee  com- 
pleted the  draft  for  an  NIH  Epidemiology  Associates  Program,  When  implemented 
this  program  will  provide  three  years  of  training  in  epidemiology  to  fifteen 
physicians  per  year.  The  EBB  will  be  a  preceptorship  for  one  associate  per 
year  who  will  be  trained  in  infectious  disease  epidemiology.  Dr.  Curl  in 
represented  the  Institute  in  the  NIH  Diabetes  Coordinating  and  NIH  Digestive 
Diseases  Committees, 


10-3 


MOLECULAR  MICROBIOLOGY  AND  PARASITOLOGY  BRANCH 


The  Molecular  Microbiology  and  Parasitology  Branch  plans  and  conducts  research 
grant,  program  project  grant,  contract,  training  grant,  fellowship  and  career 
award  programs  in  molecular  microbiology,  biochemistry,  genetics,  DNA  recombin- 
ants, physiology,  parasitology  and  medical  entomology.  It  also  coordinates 
the  activities  of  the  Parasitic  Diseases  Panel  of  the  U.S. -Japan  Cooperative 
Medical  Science  Program. 

Appropriate  Level  of  Support 

Molecular  Microbiology  Section 

Activity  Number  Amount 


Research  Grants  — ' 

202 

$14,289,517 

Research  Contracts 

1 

337,903 

Training  Grants 

4 

322,748 

Fellowships 

Sub- 

■  total 

24 
231 

285,635 

$15,235,803 

1/  Includes  13  Career  Awards  ($440,792) 

Parasitology  and  Medical  Entomology  Section 

Research  Grants  — ' 

Program  Project  Grant 

Research  Contracts 

Training  Grants 

Fel lowships 


184 

$11 

,991,405 

1 

349,238 

5 

338,199 

10 

569,576 

11 

150,100 

Sub-total 

211 

$13,398,518 

2/  Includes  6  Career  Awards  ($197,921 


11-' 


Branch 


Number 


Amount 


3/ 


Research  Grants  — 
Program  Project  Grant 
Research  Contracts 
Training  Grants 
Fellowships 


Sub-total 


386 

$26,280,922 

1 

349,238 

6 

676,102 

14 

892,324 

35 

435,735 

442 

$28,634,321 

3/  Includes  19  Career  Awards  ($638,713) 

Molecular  Microbiology 

Subsequent  to  the  submission  of  the  FY  1978  Annual  Report,  Dr.  Delappe  was  a 
student  in  a  course  at  Aspen,  Colorado  where  he  learned  the  Maxam-Gilbert 
method  of  DMA  sequencing,  including  isolation  of  restriction  fragments, 
labeling  with  polynucleotide  kinase,  specific  chemical  cleavages,  and  gel 
display  of  the  sequence. 

During  the  year  a  number  of  scientific  meetings,  both  foreign  and  domestic, 
relevant  to  the  research  supported  by  this  Section  were  attended.  A  meeting 
on  Recombinant  DNA  and  the  Eukaryotic  Genome,  sponsored  by  the  French  National 
Institute  of  Health  and  Medical  Research  (INSERM),  was  held  in  the  village  of 
Blois,  France  in  November,  1978.  A  great  many  aspects  of  recombinant  DNA 
research  and  their  impact  on  eukaryotic  genome  structure  and  function  were 
discussed  in  the  presence  of  an  international  audience.  The  meeting  was  a 
productive  and  profitable  one  to  attend. 

In  June,  1979,  Dr.  Delappe  gave  an  opening  lecture  at  the  Fourth  International 
Symposium  on  Antibiotic  Resistance  at  the  Castle  Smolenice  near  the  city  of 
Bratislava,  Czechoslovakia.  The  proceedings  will  be  co-published  by  AVICENUM 
(Czechoslovak  Medical  Press,  Prague)  and  Springer-Verlag,  (Heidelberg,  Germany' 

In  May,  Dr.  Delappe  was  asked  to  become  a  member  of  the  Project  Advisory  Group 
of  the  Bureau  of  Drugs,  FDA.  This  is  the  immediate  advisory  group  for  BOD 
research  contracts.  He  was  also  asked  to  become  a  member  of  an  FDA  study 
section,  but  elected  not  to  accept  the  appointment. 

In  October,  an  announcement  was  made  of  the  forthcoming  award  of  a  Nobel  Prize 
to  Dr.  Hamilton  Smith  of  The  Johns  Hopkins  University.  Dr.  Smith  shared  the 
prize  with  two  other  scientists.  The  award  recognized  the  development  of 
restriction  endonucleases,  enzymes  that  can  be  used  to  study  genetic  organi- 
zation and  to  manipulate  DNA.  This  was  one  of  the  origins  of  the  recombinant 
DNA  technology.  At  the  time  he  made  his  contribution  (in  1969),  Dr.  Smith 
was  supported  by  a  Research  Career  Development  Award  and  a  research  grant, 
both  of  which  were  funded  by  the  National  Institute  of  Allergy  and  Infectious 


11-2 


Diseases.  He  was  the  tenth  grantee  of  the  branch  to  receive  the  Nobel  Prize. 

Program  Summary 

In  addition  to  the  basic  free-ranging  research  of  relevance  to  the  Institute 
there  are  two  structured  programs  supported  by  this  section  of  the  Branch, 
one  of  which  involves  mechanisms  of  resistance  to  antimicrobial  agents  and 
the  other,  recombinant  DNA. 

(1 )  Mechanisms  of  Resistance  to  Antimicrobial  Agents 

During  the  past  ten  years,  the  problem  of  microbial  resistance  to  therapeutic 
agents  has  become  increasingly  apparent.  The  relevance  of  this  phenomenon  to 
other  interests  of  this  Institute  such  as  hospital-associated  infections  due 
to  staphylococci,  gram  negative  bacteria,  and  other  organisms,  together  with 
investigations  of  gonococci  and  pneumococci,  is  obvious. 

At  this  point,  there  is  substantial  clinical  and  epidemiological  evidence  that 
development  of  antibiotic  resistance,  and  especially  plasmid  (extrachromosomal ) 
mediated  drug  resistance,  represents  a  major  problem  in  medical  care.  Most  of 
the  projects  are  concerned  with  defining  the  molecular  and  biochemical  basis 
for  resistance,  the  principal  goal  being  to  elucidate  the  fundamental  biolog- 
ical mechanisms  involved  in  the  development  of  drug  resistance  by  microorgan- 
isms. Specific  goals  involve  investigation  of  the  origin,  development, 
evolution,  expression  and  mechanism  of  drug  resistance  in  a  variety  of  specific 
microorganisms.  Examples  of  microorganisms  of  particular  interest  are  (but 
not  limited  to):  Enterobacteriaceae,  Pseudomonas,  Neisseria,  staphylococci, 
streptococci,  mycobacteria,  mycoplasmas,  and  pathogenic  fungi . 

Research  Highlights 

AI  14938-02  C.  Cech  (University  of  Colorado):  This  investigator  has  studied 
the  mechanism  of  rifampicin  inhibition  of  Escherichia  coli  RMA  polymerase  with 
a  newly  developed  steady  state  assay  for  RNA  chain  inititation  and  by  analysis 
of  the  products  formed  with  several  5 '-terminal  nucleotides.  The  major  effect 
of  rifampicin  was  found  to  be  a  total  block  of  the  translocation  step  that 
would  ordinarily  follow  formation  of  the  first  phosphodiester  bond.  These 
effects  were  incorporated  into  a  steric  model  for  rifampicin  inhibition. 
Additional  minor  effects  of  the  enzyme  bound  inhibitor  were  to  increase 
slightly  the  lifetime  of  RNA  polymerase  on  the  \Pr  promoter  and  to  increase 
by  two  the  apparent  Michaelis  constants  of  the  initiating  triphosphates.  The 
products  formed  by  RNA  polymerase  in  the  presence  of  rifampicin  belong  nearly 
exclusively  to  the  class  triphosphate  purine  phosphodiester  nucleotide.  No 
evidence  for  the  accumulation  of  such  molecules  was  obtained  in  vivo. 

AI  10971-08  M.  Hiqqins  (Temple  University  School  of  Medicine):  Dr.  Higgins 
has  examined  the  morphological  effect  of  the  antibiotic  cerulenin  on  cell 
shape  of  Streptococcus  faecal  is.  This  drug,  which  inhibits  fatty  acid 
synthesis,  causes  an  elongation  of  growth  zones  and  a  blockage  of  cell  division 
similar  to  that  observed  when  DNA  synthesis  is  inhibited  in  these  cells.  This 
is  an  interesting  observation  in  that  he  had  predicted  that  a  signal  at  the 


11-3 


end  of  a  round  of  DNA  synthesis  is  necessary  for  an  inhibition  of  autolytic 
activity  and  a  concomitant  stimulation  of  cell  division.  Recently,  it  has 
been  shown  that  a  lipid  intermediate  is  involved  in  a  reduction  of  autolytic 
activity  is  this  organism,  and  it  is  supposedly  at  this  level  that  cerulenin 
works  to  block  cell  division. 

AI  12835-05  R.  Martinez  (University  of  California,  Los  Angeles):  Studies 
involving  the  bactericidal  capabilities  of  normal  rabbit  serum  (NRS)  have 
revealed  the  presence  of  two  principal  components  capable  of  reducing  the 
viability  of  Bacillus  subtil  is,  in  vitro.  These  components,  rabbit  lysozyme 
and  a  non-lysozyme  factor,  comprise  the  classical  8-lysin  system  in  rabbits, 
long  noted  for  its  heat-stability  and  principal  activity  aqainst  gram  positive 
bacteria.  Both  of  these  factors  have  been  Durified  to  homogenietv.  examined 
for  bactericidal  activity  (singly,  and  in  combination),  and  have  been  (or  are 
in  the  process  of  being)  characterized  at  the  molecular  level.  The  results 
indicate  that  the  primary  bactericidal  component  of  NRS  active  against  a 
variety  of  organisms  is  a  single,  low  molecular  weight,  proteinaceous  factor, 
presumably  released  from  platelets  during  the  coagulation  process.  Although 
the  secondary  factor,  lysozyme,  has  also  been  shown  to  exhibit  bactericidal 
activity,  it  has  been  demonstrated  that  when  examined  at  concentrations  found 
in  dilute  NRS,  the  presence  of  this  enzyme  can  account  for  only  a  minor  portion 
of  the  antibacterial  action  observed  for  NRS.  These  results  have  been  confirmed 
in  whole  serum  studies  to  which  specific  inhibitors  of  lysozyme  enzymatic  and 
bactericidal  activity  have  been  added. 

AI  12500-05  J.  Murphy  (Harvard  Medical  School):  During  the  past  year  he  has 
published  a  series  of  experiments  that  have  demonstrated  for  the  first  time 
that  iron  has  a  direct  effect  on  inhibition  of  diphtheria  toxin  production  by 
cells  that  were  "derepressed"  for  toxin.  In  addition,  it  has  been  demonstrated 
that  the  decay  kinetics  of  toxin  release  were  identical  following  either  iron 
or  rifamoicin.  By  preventing  reinitiation  of  RNA  polymerase,  the  decay 
kinetics  imposed  by  rifampicin  block  mimic  transcriptional  control.  Thus,  the 
similarity  of  inhibition  kinetics  of  toxin  release  imposed  by  iron  and  rif- 
ampicin suggested  that  the  control  of  toxin  production  was  at  the  transcrip- 
tional level.  Further  evidence  to  support  this  hypothesis  was  provided  by 
RNA:DNA  hybridization  experiments.  He  has  shown  that  there  is  a  marked 
stimulation  of  hybridization  of  total  ^H-RNA  extracted  from  iron  starved 
corynebacteria  to  e-phage  DNA.  Total  labeled  RNA  extracted  from  cells  that 
were  in  iron  excess  or  from  the  non-lysogenic,  non-toxigenic  C_.  diphtheriae 
C7  (-)  hybridized  to  the  same  extent. 

AI  11756-12  C.  Gilvarg  (Princeton  University):  This  investigator  has  isolated 
the  major  component  of  the  cell  wall  of  Bacillus  megaterium  M46,  and  his  studies 
indicate  that  it  is  a  complex  polysaccharide  over  twenty  times  the  size  of 
previously  reported  teichuronic  acids.  The  molecular  size  and  shape  of  this 
unusual  teichuronic  acid  were  elucidated  by  a  combination  of  hydrodynamics, 
chemical  and  electron  microscopic  studies.  This  should  contribute  to  a  greater 
understanding  of  the  synthesis  and  assembly  of  the  bacterial  cell  wall  which 
plays  an  important  role  in  resistance  to  antimicrobial  agents. 


11-4 


AI  10311-10  D.  Dubnau  (Public  Health  Research  Institute  of  the  City  of  New 
York) :  This  investigator  has  reported  the  construction  of  several  plasmid 
chimeras  by  molecular  cloning,  thus  demonstrating  the  utility  of  the  B_.  subtil  is 
system  for  recombinant  DNA  experiments  and  providing  a  collection  of  new 
plasmids  for  studies  on  replication,  incompatibility,  and  transformation,  as 
well  as  for  the  engineering  of  better  cloning  vectors.  A  plasmid  pEl 94 , 
obtained  from  Staphylococcus  aureus  confers  resistance  to  macrolide, 
lincosamide,  and  streptogramin  type  B  ("MLS")  antibiotics.  For  full  express- 
ion, the  resistance  phenotype  requires  a  period  of  induction  by  subinhibitory 
concentrations  of  erythromycin.  A  copy  number  in  the  range  of  10  to  25  copies 
per  cell  is  maintained  during  cultivation  at  32°C.  It  is  possible  to  transfer 
pE194  to  Bacillus  subtil  is  by  transformation.  In  B_.  subtil  is,  the  plasmid  is 
maintained  at  a  copy  number  of  approximately  10  per  cell  at  37°C,  and  resistance 
is  inducible.  Tylosin,  a  macrolide  antibiotic  which  resembles  erythromycin 
structurally  and  to  which  erythromycin  induces  resistance,  lacks  inducing 
activity.  Two  types  of  plasmid  mutants  were  obtained  and  characterized  after 
selection  on  medium  containing  10  yg  of  tylosin  per  ml.  One  mutant  class 
appeared  to  express  resistance  constitutively  and  maintained  a  copy  number 
indistinguishable  from  that  of  the  parent  plasmid.  The  other  mutant  type  had 
a  5-  to  10-f old-elevated  plasmid  copy  number  (i.e.,  50  to  100  copies  per  cell) 
and  expressed  resistance  inducibly.  Both  classes  of  tylosin-resistant  mutants 
were  shown  to  be  the  result  of  alterations  in  the  plasmid  and  not  to  modifi- 
cations of  the  host  genome. 

(2)  Recombinant  DNA  Molecular  Research 

During  the  past  few  years,  the  development  of  certain  techniques  in  the  area 
of  molecular  biology  has  made  it  possible  to  construct  functional  DNA 
molecules  in  vitro  which  contain  segments  derived  from  diverse  biological 
sources.  The  scientific  innovations  which  led  to  this  technological  break- 
through were  mostly  derivative  from  basic  studies  on  the  mechanism  of 
restriction,  which  normally  acts  as  a  barrier  to  gene  flow  among  microorganisms, 
and  on  the  molecular  biology  and  genetics  of  bacterial  plasmids,  especially 
those  specifying  drug  resistance.  Grantees  of  this  Institute  played  a  pre- 
ponderant role  in  both  of  these  areas.  Whereas  DNA  recombination  in  nature  has 
depended  on  random  processes,  the  experimental  techniques  now  available  enable 
the  in  vitro  construction  and  subsequent  replication  of  DNA  molecules  needed 
for  specific  experimental  goals.  This  basic  advance,  coupled  with  refinements 
of  the  existing  technology,  should  provide  greater  knowledge  of  the  mechanisms 
of  pathogenicity  (at  the  molecular  level)  of  viral,  bacterial,  mycotic  and 
parasitic  agents.  This  information,  in  turn,  may  lead  to  improved  prevention, 
diagnosis  and  treatment  of  infectious  diseases.  Notwithstanding  the  potential 
benefits  of  recombinant  DNA  molecule  research,  there  may  be  associated  poten- 
tial biohazards  which  must  be  avoided  by  the  design,  construction  and  testing 
of  safer  host-vector  systems  for  use  in  these  studies. 

The  primary  goal  of  this  research  is  the  development  and  utilization  of  recom- 
binant DNA  molecule  technology  to  increase  fundamental  knowledge  and  ultimately 
enhance  control  of  the  etiological  agents  of  infectious  diseases.  Another 
goal  is  the  synthesis  of  a  variety  of  biologically  useful  substances  through 
the  construction  of  bacterial  cells  containing  functional  DNA  of  either  plant 


11-5 


or  animal  origin.  An  equally  important  goal  is  the  identification,  assessment 
and  elimination  of  any  and  all  potential  biohazards  encountered  in  the 
exploitation  of  this  technology. 

Research  Highlights 

AI  10311-10  P.  Dubnau  (Public  Health  Research  Institute  of  New  York) :  The 
ability  to  carry  out  molecular  cloning  in  Bacillus  subtil  is  would  be  useful 
for  a  variety  of  studies  on  sporulation,  transformation,  and  gene  expression. 
In  addition,  such  a  capability  might  be  industrially  significant,  because 
Bacillus  species  are  of  considerable  commercial  importance.  Antibiotic 
resistance  chimeric  plasmids  have  been  constructed  by  in  vitro  enzymatic 
manipulation  and  introduced  into  Bacillus  subtil  is  by  transformation.  The 
parental  plasmids  used  had  been  introduced  into  B_.  subtil  is  from  Staphylococcus 
aureus  by  transformation.  Seven  recombinant  plasmids  have  been  constructed 
using  restriction  endonucleases.  Although  all  of  the  recombinant  plasmids 
replicate  and  express  their  antibiotic  resistance  characters,  three  of  them 
have  suffered  a  loss  of  DNA,  either  in  vitro  or,  more  likely,  in  vivo.  The 
deletion  event  in  all  cases  involved  one  of  the  two  termini  used  to  join  the 
parental  plasmids.  The  plasmid  chimeras  reported  should  prove  useful  for  the 
study  of  plasmid  replication,  incompatibility,  and  recombination.  In  addition, 
the  utility  of  the  B_.  subtil  is  system  for  molecular  cloning  has  been  clearly 
illustrated. 

AI  8619-12  S.  Cohen  (Stanford  University  Medical  School):  DNA  fragments 
generated  by  the  EcoRl  or  Hindi II  endonucleases  from  the  low  copy  number 
antibiotic  resistance  plasmids  R6  and  R6-5  were  separately  cloned  using  the 
high  copy  number  ColEl  or  pML21  plasmid  vectors.  The  hybrid  plasmids  that 
were  obtained  were  used  to  determine  the  location  of  the  EcoRl  and  Hindlll 
cleavage  sites  on  the  parent  plasmid  genomes  by  means  of  electron  microscope 
heteroduplex  analysis  and  agarose  gel  electrophoresis.  Ultracentrifugation 
of  the  cloned  fragments  in  cesium  chloride  gradients  localized  the  high 
buoyant  density  regions  of  R6-5  to  fragments  that  carry  the  genes  for  resis- 
tance to  streptomycin-spectinomycin,  sulfonamide,  and  mercury  and  a  low 
buoyant  density  region  to  fragments  that  carry  the  tetracycline  resistance 
determinant.  Functional  analysis  of  hybrid  plasmids  localized  a  number  of 
plasmid  properties  such  as  resistance  to  antibiotics  and  mercury  and  several 
replication  functions  to  specific  regions  of  the  R6-5  genome.  Precise 
localization  of  the  genes  for  resistance  to  chloramphenicol,  kanamycin, 
fusidic  acid  and  tetracycline  was  possible  due  to  the  presence  of  identified 
restriction  endonuclease  cleavage  sites  within  these  determinants. 

AI  8619-12  S.  Cohen  (Stanford  University  Medical  School) :  Messenger  RNA 
that  encodes  the  common  peptide  precursor  for  the  hormones  corticotropin 
and  e-lipotropin  was  purified  from  the  neurointermediate  lobe  of  bovine 
pituitaries,  and  double-stranded  complementary  DNA  species  synthesized  from 
this  template  were  cloned  in  Escherichia,  col j  x1776  by  inserting  them  into 
the  Pst  I  endonuclease  cleavage  site  of  the  pBR322  plasmid.  Certain  of  the 
cloned  cDNA  inserts  contain  nucleotides  corresponding  to  the  complete  amino 
acid  sequence  of  bovine  corticotropin  and  a  coding  sequence  that  corresponds 
to  at  least  the  first  portion  of  bovine  8-1 ipotropin.  The  nucleotide  sequences 


11-6 


coding  for  corticotropin  and  s-lipotropin  are  separated  on  the  cDNA  by  a 
6-base-pair  sequence  encoding  lysine  and  arginine,  indicating  that  the  carboxyl 
terminus  of  corticotropin  is  connected  on  the  precursor  peptide  with  the 
amino  terminus  of  8-1  ipotropin  by  these  two  amino  acids.  In  addition,  the 
cloned  cDMA  insert  is  characterized  by  an  usually  high  C+G  (cytosine  and 
guanine)  nucleotide  base  content  as  well  as  by  a  number  of  DMA  sequence 
duplications. 

K04  AI  119-03  P.  Lovett  (University  of  Maryland):  The  technique  of  molecular 
cloning  involves  the  in  vitro  insertion  of  fragments  of  DNA  into  small  repli- 
cons,  plasmids,  or  phage  genomes,  followed  by  selection  of  chosen  recombinant 
molecules  by  transformation  of  appropriate  recipient  cells.  Direct  application 
of  recombinant  DNA  technology  to  the  study  of  Bacillus  subtil  is  wil 1  ultimately 
provide  a  general  method  for  constructing  partial  diploid  strains  which  will, 
in  turn,  permit  genetic  complementation  analyses  of  specific  mutations  and 
provide  a  source  of  easily  obtainable  DNA  highly  enriched  for  genes  of  chromo- 
somal origin  whose  in  vitro  expression  may  be  of  special  interest  such  as 
sporulation  genes.  Plasmid  pUBllO,  originally  detected  in  Staphylococcus 
aureus,  specifies  resistance  to  neomycin  and  has  been  transformed  into  Bacillus 
subtil  is  168.  In  B_.  subtil  is,  pUBllO  is  stably  maintained  at  about  50  copies 
per  chromosome  and  renders  the  host  resistant  to  neomycin  sulfate  at  5  pg/ml . 
pUBllO  was  transferred  by  transduction  from  B_.  subtil  is  to  strains  of  B_. 
pumilus  and  B_.  licheniformis. 

Parasitology 

On  September  18-20,  1978  the  Branch  was  represented  at  a  "Symposium  on  Water- 
borne  Transmission  of  Giardiasis"  sponsored  by  the  U.S.  Environmental 
Protection  Agency.  The  goal  of  the  symposium  was  to  discuss  the  state  of  the 
art  regarding  Giardia  species  and  the  disease  as  they  relate  to  water  supplies, 
and  to  identify  areas  where  further  research  is  needed.  The  topics  discussed 
included  the  organism,  the  disease,  epidemiology,  detection  methodology  and 
water  treatment  technology.  It  was  concluded  that  more  research  on  al 1 
aspects  of  giardiasis  is  needed. 

The  13th  Joint  Conference  of  the  U.S. -Japan  Parasitic  Diseases  Panel  was  held 
in  Okayama,  Japan  on  October  4-6,  1978.  Forty-seven  Japanese  and  ten  American 
scientists  attended  this  conference  at  which  42  papers  were  presented  on  a 
broad  range  of  studies  of  both  schistosomiasis  and  filariaris.  In  addition 
to  the  formal  scientific  sessions,  many  of  the  U.S.  participants  took  advantage 
of  opportunities  to  develop  potential  collaborative  research  relationships 
with  their  Japanese  counterparts. 

The  Branch  was  also  represented  at  a  conference  on  "Pharmaceuticals  for 
Developing  Countries"  sponsored  by  the  National  Academy  of  Sciences  on 
January  29-31,  1979.  Particularly  relevant  to  the  Parasitology  program  were 
the  presentations  on  cellular  regulatory  processes  in  parasitic  helminths  and 
those  on  the  pharmacological  exploitation  of  biochemical  differences  between 
parasites  and  hosts. 


11-7 


Late  in  1978,  a  Request  for  Application  for  Tropical  Disease  Research  Units 
program  project  grants  was  issued.  The  basic  objective  of  this  new  initiative 
is  to  bring  together  relevant  biomedical  knowledge  and  technology  in  a  multi- 
disciplinary  attack  on  the  world's  tropical  and  parasitic  diseases.  Nine 
applications  were  received  and  reviewed  in  June,  1979  and,  following  the 
October,  1979  Council,  it  is  hoped  that  funds  will  be  available  to  permit 
the  funding  of  2  or  3  of  these  program  projects. 

On  June  15,  1979,  an  RFA  was  issued  for  New  Investigator  Research  Awards  in 
Tropical  Diseases.  To  help  bridge  the  transition  from  training  status  to  that 
of  a  productive  investigator,  this  special  grant  program  will  provide  support 
for  young  scientists  and  physicians  with  meritorious  ideas  for  research  on 
the  world's  tropical  diseases.  Emphasis  will  be  placed  on  research  on  malaria, 
schistosomiasis,  filariasis,  trypanosomiasis,  leprosy,  leishmaniasis  and  the 
immunology  of  these  diseases.  The  first  of  these  awards  will  be  made  in 
July,  1980. 

Program  Summary 

In  addition  to  the  basic  free-ranging  research  of  relevance  to  the  Institute, 
there  are  two  structured  programs  supported  by  this  section  of  the  Branch-- 
biological  regulation  of  vectors  and  immunology  of  parasitic  infections. 

(1 )  Biological  Regulation  of  Vectors 

This  program  has  as  its  goal  the  advancement  of  fundamental  studies  which 
might  lead  to  effective  methods  of  biological  regulation  of  vectors.  For  the 
past  30  years,  control  of  pests  and  disease  vectors  has  been  based  primarily 
on  the  use  of  synthetic  organic  compounds  which  had  the  "advantages"  of  long 
residual  action  and  toxicity  to  a  broad  spectrum  of  target  organisms.  It 
has  now  been  shown  that  because  of  these  very  characteristics,  many  of  these 
pesticides  are  more  deleterious  than  beneficial,  when  all  effects  on  man  and 
his  environment  are  considered.  Furthermore,  resistance  to  broad  spectrum 
chemical  pesticides  has  reduced  their  effectiveness  in  many  vector  control 
programs.  For  these  reasons  the  search  for  alternative  methods  of  pest  control 
has  become  imperative,  and  it  is  generally  agreed  that  the  best  approaches  will 
consist  of  integrated  pest  management  programs  which  combine  biological 
control,  in  the  broadest  sense,  with  the  judicious  use  of  more  specific 
chemicals  and  management  of  the  physical  environment.  This  approach  to  vector 
control  must  be  based  on  adequate  information  about  the  ecology  of  the  target 
organism,  the  environment  in  which  the  control  program  is  to  be  conducted, 
effects  of  control  measures  on  non-target  organisms  in  the  environment,,  and 
the  biology  of  the  disease  organisms  being  transmitted. 

Research  Highlights 

AI  10986-06  P.  Sonenshine  (Old  Dominion  University) :  The  identity  of  the 
female  sex  pheromone  (2,6-dichlorophenol )  of  the  ticks  Dermacentor  andersoni 
and  D_.  variabilis  was  confirmed.  Dr.  Sonenshine  also  demonstrated  the  role 
of  fovea!  glands  as  the  source  of  this  compound.  Pheromone-impregnated 
materials,  especially  dusts,  caused  delay  of  mating  and  confusion  of  mate- 


1-8 


seeking  males.  The  use  of  sex  pheromones  in  an  integrated  control  program 
would  provide  a  negative  pressure  effect  on  the  reproductive  capacity  of  the 
tick  population  surviving  the  direct  effects  of  insecticides. 

AI  11847-11  R.  Barr  (University  of  California,  Los  Angeles):  Dr.  Barr  has 
found  that  Holbachia  pipientis,  a  rickettsia-1 ike  symbiont  of  Culex  pipiens, 
affects  the  sperm  of  male  mosquitoes  in  such  a  way  that  the  sperm  are  not  able 
to  fertilize  eggs  unless  the  eggs  are  also  infected  with  Wolbachia.   If  the 
eggs  are  infected  with  Wolbachia  of  different  geographic  origin,  the  cross  may 
be  incompatible.  Research  is  continuing  to  determine  whether  Wolbachia  might 
be  cultured  and  used  as  a  means  of  controlling  mosquito  reproduction. 

AI  11123-07  N.  Alger  (University  of  Illinois):  Dr.  Alger  has  been  studying 
the  use  of  the  microsporidian  Nosema  algerae  as  a  biocontrol  agent  against 
Anopheles  mosquitoes.  Her  most  recent  studies  in  Pakistan  have  shown  that 
it  is  possible  to  infect  field  populations  of  mosquito  larvae,  and  that  the 
resultant  adult  mosquitoes  will  produce  fewer  eggs,  some  of  which  will  be 
infected  and  will  not  live  long  enough  to  transmit  malaria.  Non-target  aquatic 
invertebrates  are  not  infected;  thus,  the  use  of  N_.  algerae  in  mosquito 
biocontrol  appears  to  be  safe. 

AI  13295-03  C.  Bayne  (Oregon  State  University):  In  a  study  of  the  suscep- 
tibility of  the  vector  snail  Biomphalaria  glabrata  to  Schistosoma  mansoni , 
Dr.  Bayne  has  discovered  that  sporocyst-kill ing  cells  isolated  from  the  snails 
are  actually  amebae  of  the  genus  Nuclearia  which  attach  to  the  surface  and 
penetrate  the  schistosome  sporocysts.  These  amebae-sporocyst  interactions 
may  be  important  determinants  of  snail  susceptibility  in  nature. 

(2)  Immunology  of  Parasitic  Infections 

The  complexity  of  structure  and  function  of  parasites  has  made  the  study  of 
the  immunology  of  these  infectious  agents  exceptionally  challenging  and 
rewarding.  Exciting  opportunities  for  the  elucidation  of  mechanisms  and 
manifestations  of  immunological  responses  to  parasites  now  exist  as  the 
result  of  the  impressive  developments  in  immunology  in  recent  years.  Major 
ultimate  goals  of  studies  on  the  immunology  of  parasitic  infections  are  the 
development  of  effective  vaccines  for  the  prevention  of  parasitic  diseases 
(such  as  malaria,  schistosomiasis  and  filariasis),  the  intervention  in  the 
host  response  to  prevent  or  ameliorate  disease  processes  which  are  immuno- 
logically mediated,  and  the  development  or  improvement  of  immunodiagnostic 
procedures  for  parasitic  infections,  especially  as  they  relate  to  the  immune 
status  of  the  host. 

A  related  goal  of  these  studies  is  to  contribute  to  an  understanding  and 
solution  of  basic  and  clinical  problems  associated  with  other  disease  entities, 
especially  immunological  disorders  and  hypersensitivity  states.  A  number  of 
parasitic  infections  are  excellent  models  for  such  studies  as:  (a)  the 
mechanisms  of  intracellular  immunity,  (b)  the  enhancement  or  suppression  of 
concurrent  infections  or  tumor  development,  (c)  immunopathological  mechanisms, 
(d)  development  of  disease  processes  in  immunosuppressed  or  immunostimulated 
hosts,  (e)  the  biochemical  and  genetic  mechanisms  for  the  development  of 


11-9 


pathogen  variants  with  different  immunological  characteristics,  (f)  the 
genetic  basis  for  variations  in  host  response  and  (g)  the  role  of  IgE  and 
other  cytotropic  antibodies  in  hypersensitivity. 

Research  Highlights 

AI  15235-02  H.  Shear  (New  York  University) :  In  a  study  of  macrophage  acti- 
vation  in  experimental  malaria  (P.  berghei ) ,  Dr.  Shear  showed  that  early  in 
an  infection  macrophages  appear  to  be  activated  and  parasitized  erythyocytes 
are  ingested;  later,  the  macrophages  appear  to  be  "blocked."  Indirect 
evidence  points  to  the  possibility  of  inhibitory  immune  complexes  in  the 
serum  of  infected  animals.  An  analysis  of  this  blocking  activity  should  help 
clarify  the  animals'  inability  to  cope  with  the  disease  ultimately. 

AI  12770-04  M.  Wittner  (Albert  Einstein  College  of  Medicine) :  In  a  study 
of  mechanisms  of  immune  resistance  to  Trypanosoma  cruzi ,  Dr.  Wittner  has 
investigated  the  role  of  macrophages  in  the  in  vivo  resistance  to  T.  cruzi 
infection.  Mice  genetically  resistant  to  T.  cruzi  were  depleted  of  peritoneal 
macrophages  prior  to  infection  with  the  parasite.  Increased  parasitemia  and 
mortality  in  this  group  are  consistent  with  the  hypothesis  that  the  macrophage 
constitutes  an  early  line  of  defense  in  T.  cruzi  infection. 

AI  12663-05  J.  Farrell  (University  of  Pennsylvania):  Using  the  golden  hamster 
as  a  model  for  the  study  of  Kala-azar,  Dr.  Farrell  has  shown  that  hamsters 
inoculated  intracardially  with  L_.  donovani  develop  fatal  visceral  infections 
whereas  animals  injected  intradermal ly  usually  exhibit  self-limiting,  non- 
ulcerating  cutaneous  infections  and  subsequent  resistance  to  reinfection.  He 
is  currently  investigating  the  immunological  parameters  of  these  different 
manifestations  of  the  disease. 

AI  08989-09  W.  Trager  (Rockefeller  University):  In  a  series  of  preliminary 
experiments,  Dr.  Trager  and  his  colleagues  have  developed  in  vitro  techniques 
for  the  collection  of  relatively  pure  merozoites  of  Plasmodium  falciparum  to 
be  used  in  antigen-antibody  studies.  Aotus  monkeys  immunized  with  this  in 
vitro  culture-derived  antigenic  material  are  protected  from  challenge  infec- 
tion. 

AI  12913-03  D.  Boros  (Wayne  State  University):  Dr.  Boros  has  shown  that  the 
granulomatous  inflammatory  response  around  schistosome  eggs  is  mediated  by 
inflammatory  T  lymphocytes.  During  the  chronic  phase  of  the  infection  certain 
lymphocytes  assume  a  suppressive/regulatory  role  and  exert  their  influence  on 
the  inflammatory  lymphocytes  to  diminish  their  activity  which  results  in 
smaller  granulomas  and  hence  diminished  host  pathology. 

(3)  General  Parasitology  Research  Highlights 

AI  14294-03  J.  Bennett  (Michigan  State  University):  In  a  study  of  the  effects 
of  chemotherapeutic  agents  on  schistosome  physiology,  Dr.  Bennett  has  demon- 
strated that  the  effective  schistosomacide,  praziquantel,  causes  a  massive 
contraction  of  the  worm  and  a  large  influx  of  calcium.  Apparently  the  drug 
acts  by  stimulating  the  influx  of  calcium  which  causes  the  muscle  contractions. 


11-10 


AI  15919-01  J.  Dubey  (Montana  State  University):  In  an  investigation  of  a 
human  toxoplasmosis  outbreak  in  a  stable,  Dr.  Dubey  found  that  of  the  37  cases 
reported,  none  had  had  common  meals;  thus  raw  meat  could  not  be  implicated  in 
the  transmission.  Apparently  the  infections  occurred  either  via  the  hand-to- 
mouth  or  dust  inhalation  route,  and  the  source  of  infection  was  a  group  of 
Toxoplasma  positive  cats  which  lived  in  the  stable  area. 

AI  10250-08  L.  Ash  (University  of  California,  Los  Angeles) :  Further  progress 
has  been  made  in  the  development  of  a  primate  host  for  the  human  filarial 
parasite,  Wuchereria  bancrof ti .  In  addition  to  establishing  patent  infections 
in  nine  monkeys,  one  of  these  infections  represented  the  first  serial  passage 
of  the  parasite  from  monkey  to  monkey. 

AI  14718-02  R.  Cypess  (Cornell  University):  There  has  long  been  a  need  for 
a  specific  and  reliable  diagnostic  test  for  toxocaral  visceral  larval  migrans 
(VLM)  since  this  syndrome  presumably  may  be  caused  by  a  variety  of  helminths. 
Dr.  Cypess  has  demonstrated  that  the  enzyme-linked  immunosorbent  assay  (ELISA) 
is  superior  to  indirect  hemagglutination,  bentonite  flocculation  and  double 
diffusion  in  agar  tests.  Based  on  specificity,  sensitivity  and  positive  and 
negative  predictive  values,  the  ELISA  appears  to  be  the  serodiagnostic  method 
of  choice  for  toxocaral  VLM. 

AI  11942-05  M.  Mu'ller  (Rockefeller  University):  Dr.  Miiller,  in  a  study  of 
the  biochemical  cytology  of  Trichomonas  vaginalis,  has  shown  that  drug- 
resistant  strains  of  the  parasite  do  not  take  up  metronidazole  in  the  presence 
of  oxygen.  These  studies  suggest  that  the  mechanism  of  resistance  is  connected 
with  alteration  in  the  effect  of  oxygen  on  the  intracellular  reduction- 
oxidation  system. 

Contract  Activity 

The  schistosomiasis  supply  contract  service,  located  at  the  University  of 
Lowell,  has  continued  to  be  utilized  extensively  by  most  schistosomiasis 
researchers  in  this  country.  This  invaluable  service  can  provide  all  the 
human  schistosome  parasites  and  their  vector  snails. 

The  filariasis  supply  contract,  located  at  the  University  of  Georgia,  has 
continued  to  provide  filariasis  research  workers  with  various  filarial  para- 
sites and  their  vectors.  The  complexity  of  filarial  life  cycles  and  the 
special  facilities  needed  for  the  housing  and  rearing  of  arthropod  vectors 
makes  this  service  contract  especially  valuable  to  researchers  who  otherwise 
might  not  be  able  to  pursue  their  research  interests.  Acceleration  of  research 
in  filariasis  is  in  part  directly  attributable  to  this  service. 

Two  research  contracts  involving  the  development  of  techniques  for  the  cryo- 
preservation  of  various  stages  of  human  and  animal  filariae  are  currently  in 
progress.  Short-  and  long-term  cryopreservation  of  microfilariae  and  infective- 
stage  larvae  of  both  sheathed  and  unsheathed  microfilaria!  species  has  been 
accomplished,  and  this  now  provides  unique  opportunities  to  transport  rare 
and  scarce  materials  to  laboratories  capable  of  working  with  various  aspects 


11-11 


of  these  parasites.  In  particular,  work  on  Wuchereria  bancrofti  and  Brugia 
malayi  infections  has  been  enhanced  considerably. 

As  a  result  of  a  recommendation  made  at  an  NI AID  workshop  on  the  "Radiobiology 
of  Schistosomes  and  Filariae"  held  at  NIH  on  April  17-18,  1978,  an  RFP  was 
issued  for  a  contract  for  the  development  of  techniques  for  the  radio-labelling 
of  schistosome  and  filarial  larvae.  Six  proposals  were  received  and  reviewed, 
and  a  contract  was  awarded  to  Cornell  University  on  June  29,  1979  for  two 
years. 


11-12 


EXTRAMURAL  ACTIVITIES  PROGRAM 
NIAID 
TABLE  OF  CONTENTS 

REPORT  OF  THE  DIRECTOR 12-1 

A.  Introduction 12-1 

B.  The  Review  of  Research  Proposals 12-1 

C.  Implementation  of  the  Multi-Axis  Coding  System  (MACS) 12-1 

D.  Personnel 12-1 

E.  Manpower  Development 12-3 

F.  Decline  of  Physicians  in  Research  Training 12-4 

G.  Survey  of  American  Society  for  Microbiology  Membership 12-5 

H.  Committee  Management 12-5 

I.  Conference  Proposal  Review 12-5 


I.   CONTRACT  MANAGEMENT  BRANCH 


II.   GRANTS  MANAGEMENT  BRANCH 


III.   PROGRAM  ANALYSIS  AND  EVALUATION  BRANCH- 


,13-1 


.14-1 


,15-1 


A.  Fiscal  Management  and  Analysis  Section 15-1 

B.  Program  Analysis  Section 15-4 

C.  Data  Control  Section 15~5 

IV.  PROGRAM  AND  PROJECT  REVIEW  BRANCH 16-1 


A.   Introduction 


,16-1 


B.  Allergy  and  Clinical  Immunology  Research  Committee 16-2 

C.  Transplantation  Biology  and  Immunology  Committee 16-2 

D.  Microbiology  and  Infectious  Diseases  Advisory  Committee 16-2 

E.  Review  Services  Unit  xo  J 


F.   Clinical  Trials. 


V.  RESEARCH  RESOURCES  BRANCH 


.16-3 


,17-1 


A.  Special  Activities *-' 

B.  Asthma  and  Allergic  Diseases  Program  - 

17  1 
Allergen  Reagents ±/  -± 

C.  Immunological  Reagents  and  Resources 17-2 

1  7  —  ? 

D.  Microbiology 

E.  Research  Resources  -  Research  Reagents 17-3 

F.  Molecular  Anatomy  Program 17-4 

G.  Processing  and  Distribution 17-6 

H.  Individual  Contract  and  Agreement  Tabulation 17-10 

I.  Contract  Publications 17-16 


REPORT  OF  THE  DIRECTOR,  EAP 

A.  Introduction 

The  Extramural  Activities  Program  (EAP)  of  the  NIAID  ended  the  decade 
of  the  70' s  by  awarding  the  largest  number  of  regular  research 
projects  of  any  year  during  the  decade,  although  the  number  of  trainees 
supported  was  smaller  during  the  early  1970 's.   The  EAP  workload, 
associated  with  the  increased  number  of  applications  and  proposals 
received,  was  increased  by  about  25%.   This  overload  was  handled  by 
part-time  employees,  overtime,  and  the  increased  productivity  of  all 
the  employees.   Although  there  were  stressful  times  when  it  seemed 
we  would  not  meet  our  assigned  schedules,  it  was  an  exciting  time  to  be 
in  the  EAP. 

B.  The  Review  of  Research  Proposals 

The  Program  and  Project  Review  Branch  was  fully  staffed  for  the  first 
time  since  the  reorganization  of  the  Institute.   To  assist  in  the 
tremendous  workloads  of  the  winter  and  spring  rounds,  reinforcements 
were  received  from  the  Office  of  the  Director,  EAP  and  from  the  Office 
of  the  Director,  NIAID.   This  workload  is  described  later  in  the 
report  from  that  Branch.   All  of  these  reviews  have  resulted  in 
funding  of  those  applications  and  contract  proposals  which  were  approved 
with  good  priority  scores.   The  staff  in  this  Branch  has  been 
congratulated  for  their  efforts . 

C.  Implementation  of  the  Multi-Axis  Coding  System  (MACS) 

In  Fiscal  Year  1979,  we  introduced  and  began  accumulating  data  by  the 
new  multi-axis  coding  system.   This  new  and  more  complex  system  of 
record  keeping  was  brought  about  at  the  recommendations  of  the  Data 
System  Planning  Committee  to  meet  Institute  requirements  for  broader 
scientific  data  and  to  meet  NIAID,  NIH  and  other  requirements  for 
administrative  data.   This  activity  also  required  considerable  extra 
effort  on  the  part  of  staff  because  it  was  a  new  system  which  was 
implemented  during  the  year  in  which  NIAID  received  and  reviewed  the 
largest  number  of  application  and  contract  proposals  to  date. 

This  system  recommended  by  the  Data  Systems  Planning  Committee,  as 
reported  in  last  year's  annual  report  now  includes  four  axes  of 
biological  data;  administrative  subjects  such  as  basic,  applied  and 
development  (BAD);  scientific  base,  applied  technology  transfer,  and 
training  (SATT) ;  and  coding  for  human  subjects,  clinical  trials 
and  prevention.   This  is  a  management  data  system  and  will  eventually 
be  evaluated  on  that  basis. 

D.  Personnel 

There  were  fourteen  departures  from  the  EAP  and  fourteen  new  appointments 
during  FY  1979.   Some  of  these  new  appointments  were  temporary  appoint- 
ments which  terminated  in  1979.   The  lag  time  for  the  process  of 

12-1 


appointing  new  employees  remains  about  the  same  as  it  was  last 
year,  meaning  that  there  were  approximately  forty  man  months  during 
which  there  were  employee  vacancies  in  the  EAP.   This  represents  over 
three  man  years  and  considerably  hampers  the  accomplishment  of  EAP 
objectives. 

Retirements 


Mildred  Applestein 
Gwen  Northcutt 
Eleanor  Wyatt 

Employees  who  have  left  EAP 

Virginia  Alary 
Mary  Baldwin 
Kathy  Cherry 
Sue  Connors 
Maria  Decker 
Melvin  Joppy 
Cynthia  Jose 
Carol  Kimmel 
Harry  Levine 
Janice  Pusey 
Earle  Vance 

New  Appointments 

Todd  Ball 

Betty  Bucher 

Bonnie  Dunning 

Diane  Edwards 

Melo  Ellis  (Stay-in-School) 

Debbie  Frazier  (Temp) 

Carla  Goldblum 

John  Hamill 

Fran  Hisaoka 

Annette  Hobbs  (Expert  Consultant) 

Anne  Rabon 

Sara  Spencer 

Gary  Thompson 

Daryl  Thornton  (Stay-in-School) 

Delores  Walton  (Temp) 

Awards  (Quality  Increase) 

Louis  Bourgeois 
Gertrude  Cohen 
Grace  Ellis 
Fran  Schlademan 
Hubert  Sumner 
Rick  Wiener 


12-2 


Training 

Fifty-five  training  courses  at  a  cost  of  just  over  $5,000  were  taken 
by  staff. 

Figure  I  shows  the  Organizational  Chart. 

E.   Manpower  Development 

The  traditional  training  program  increased  insofar  as  postdoctoral 
awards  were  concerned.   The  trend  in  predoctoral  awards  continued  at 
a  slight  decline.   There  was  a  continued  decline  in  the  number  of 
physicians  who  were  interested  in  research  training  and  this  loss  of 
potential  clinical  investigators  remains  the  major  problem  in  the 
NIAID  manpower  development  program  as  well  as  in  other  similar 
programs  at  the  NIH. 

The  amendment  to  the  National  Research  Service  Act  (NRSA)  which  shifts 
more  support  to  institutional  grants  and  less  to  individual  fellowships 
has  not  made  a  significant  impact  this  fiscal  year.   There  are  81 
T32  training  grants  now  being  administered  by  NIAID.   Of  these,  34 
are  in  the  IAID  program  and  47  in  the  MID  program. 

Table  I  estimates  the  total  number  of  trainees  being  supported  by 
NIAID  on  the  authority  of  NRSA  and  on  the  old  T01  programs  of  which 
there  are  18  still  operating  on  unexpended  funds  and  support  70 
fellows. 

TABLE  I 

All  Trainees  -  Estimated 

PROGRAM 

IAIDP        MIDP        TOTAL 


Predoctoral 

40 

65 

105 

Postdoctoral 

119 

202 

321 

M.D. 

57 

48 

105 

TOTAL 216 315 531* 

*Includes  T01 

Table  II  estimates  the  number  of  individual  fellows  being  supported 
in  each  of  the  major  programs.   The  number  of  M.D.'s  in  comparison 
to  the  number  of  Ph.D.'s  is  also  shown. 


12-3 


TABLE  II 
Individual  Fellowships  (F32) 
PROGRAM 


Degree IAIDP MIDP TOTAL 

Ph.D.  55  94  149 

M.D. 9 8 17 

TOTAL 64 102 166 

Table  III  estimates  the  number  of  trainees  both  predoctoral  and 
postdoctoral  in  the  LAID  and  MID  programs. 

TABLE  III 

Institutional  Training  (T32) 

PROGRAM 

IAIDP  MIDP  TOTAL 


Predoctoral      37  68  105 

Postdoctoral     97 145 262 

TOTAL 134 213 367 

F.   Decline  of  Physicians  in  Research  Training 

The  decline  in  M.D.'s  selecting  research  as  a  career  still  remains  of 
concern  to  those  involved  with  biomedical  training.   One  aspect  which 
appears  not  to  have  been  examined  is  the  long-range  effect  of 
legislation.   Legislation  focusing  on  the  production  of  more  physicians 
for  medical  service  began  in  1963.   It  evolved  into  a  federal  initiative 
that  became  increasingly  focused  on  training.   The  history  of  this 
legislation  was  as  follows: 

1963,  Health  Professional  Educational  Assistance  Act  provided 
for  construction  grants  and  loans  for  undergraduate  students, 

1965,  amendments  to  the  Act  provided  aid  for  scholarship  and 
medical  schools  operating  costs, 

1968,  the  Health  Manpower  Act  emphasized  absolute  increase  in 
medical  school  enrollments, 

1971,  Comprehensive  Health  Manpower  Training  Act  designed  to 
increase  enrollment  through  capitation,  and  to  increase 
practitioners  in  rural  and  shortage  areas  and  family  medicine, 


12-4 


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1976,  Health  Professions  Educational  Assistance  Act,  P.L.  94-484, 
established  a  target  of  50%  of  graduating  M.D.'s  as  primary  care 
specialists,  and  to  encourage  medical  care  institutions  to 
establish  residencies  in  family  practice,  and  primary  care 
specialties. 

These  federal  actions,  plus  the  leveling  of  research  budgets, 
the  low  level  of  stipends,  and  a  pay  back  requirement  for  research 
training  have  made  research  much  less  attractive  than  service 
for  the  graduating  M.D.   In  the  1980' s,  the  NIH  must  reverse 
this  trend. 

G.   Manpower  Survey  being  Conducted  by  the  American  Society  for 
Microbiology 

To  date,  the  Society  has  received  about  17,000  responses  to  the  25,000 
questionnaires  sent  out.   This  was  7,000  more  than  was  anticipated 
by  the  designers  of  the  survey.   These  have  been  coded  and  they  are 
being  keypunched  and  computerized.   Dr.  William  Epstein,  the  technical 
writer,  and  Mr.  John  Dirkse  the  statistician,  are  working  to  examine 
the  data  and  prepare  a  draft  of  the  report.   The  work  on  the  survey 
is  ahead  of  schedule.   A  recent  supplement  has  been  awarded  to  assist 
in  anslysis  of  the  data  acquired  from  the  additional  responses  received. 

H.   Committee  Management 

In  Fiscal  Year  1979,  the  Committee  Management  Officer  retired  and  her 
assistant  left  for  another  position.   The  Committee  Management  Officer's 
position  was  filled  just  prior  to  the  May  Council  by  a  very  capable 
employee  and  the  position  of  Assistant  to  Committee  Management  Officer 
could  not  be  filled  in  the  fourth  quarter  because  of  a  freeze  on 
personnel.   This  meant  that  this  office  also  had  to  operate  with  a 
series  of  temporary  measures  for  accomplishing  typing,  filing,  review 
of  vouchers  and  travel  claims  and  travel  activities.   It  is  a  tribute 
to  the  new  committee  management  —  council  secretary  that  no  major 
problems  resulted  from  operating  an  office  as  important  as  that  of 
committee  management  and  council  services  in  this  manner. 

I.   Conference  Proposal  Review 

The  EAP  review  of  all  "unsolicited"  conference  proposals,  i.e.,  those 
arising  outside  the  program  areas  of  NIAID,  which  was  successfully 
initiated  and  implemented  in  FY  '78,  has  continued  at  about  the  same 
level  in  FY  '79.   Prospecti  were  received  from  the  Fogarty  International 
Center,  as  well  as  directly  from  the  proposers.   Those  which  were 
accepted  for  review,  primarily  on  the  basis  of  Institute  relevance, 
were  subjected  to  a  primary  review  by  an  ad_  hoc  scientific  group 
and  a  secondary  review  by  the  Research  Contract  Review  Group,  the 
latter  being  scheduled  once  each  quarter.   A  maximum  amount  of  funds 
was  set  aside  for  the  support  of  these  conferences.   Eight  R-13 
conference  grant  applications  were  also  received  and  reviewed  in  FY  '79. 


12-5 


They  received  a  primary  No  Study  Section  (NSS)  assignment  but  were 
peer  reviewed  by  the  EAP;  these  were  presented  at  the  appropriate 
Council  meetings  for  final  review  and  approval.   A  total  of  69 
conference  prospecti  and  R-13  grant  applications  were  received  by 
EAP  in  FY  '79.   Of  this  number,  39  were  reviewed  and  35  of  these  were 
approved  for  funding  and/or  sponsorship  for  a  maximum  of  $276,796. 
Eleven  of  the  69  will  be  reviewed  for  possible  FY  '80  funding. 
(See  Table  IV). 


12-6 


TABLE  IV 

CONFERENCE/WORKSHOP  PROSPECTI  REVIEWED  BY  NIAID 
FY  '79 


Received. 


.69 


From  Fogarty  International  Center  (FIC) 59 

Sponsorship  &  Funds 58 

Sponsorship  Only 1 

Directly  by  Institute 2 

From  DRG  (R13's) 8 

Not  Reviewed 19 


Returned  to  FIC  due  to  lack  of  Institute  relevance. . 15 

Returned  to  FIC  due  to  Institute  policy 3 

Returned  to  FIC  due  to  lack  of  justification 

for  meeting  site 1 


Reviewed. 


,39 


Approved 35 

Sponsorship  &  Funds 30* 

Sponsorship  Only 1 

Approved  (No  Funds) 4 

Disapproved  (NIAID  and/or  FIC) 4 

Sponsorship  &  Funds 4 

Sponsorship  Only 0 

To  Be  Reviewed 11 


FUNDED  OR  SCHEDULED  FOR  FY  '79  FUNDING  (Thru  May  '79)  ** 

Other 
IAIDP         MIDP         NIAID 


TOTAL 


# 

$ 

// 

$ 

// 

$ 

// 

$ 

R-13  Grant 
Other  Extramural 

1 
8 

$18,125 
$75,050 

6 

9 

$80,983 
$76,638 

0 
5 

0 
$26,000 

7 
22 

$  99,108 
$177,688 

TOTAL 

9 

$93,175 

15 

$157,621 

5 

$26,000 

29 

$276,796 

^Includes  8  R-13's  some  of  which  will  not  be  paid 
'^Includes  some  which  were  reviewed  in  FY  '78  but  funded  in  FY  '79 


12-7 


I.   CONTRACT  MANAGEMENT  BRANCH 

The  Contract  Management  Branch  provides  management  services  to  the 
Institute's  Research  Program  including  solicitation,  negotiation,  award, 
and  administration  of  all  Institute  research  contracts.   The  CMB  will 
continue  to  implement  contract  policies  and  procedures  promulgated  by 
higher  procurement  authority. 

During  FY  1979,  Mr.  Harry  LeVine,  Contract  Specialist,  resigned  in 
order  to  further  his  education.   His  replacement  was  Ms.  Sara  Spencer 
formally  of  NINDS.   Mrs.  Mildred  Applestein,  Contracting  Officer, 
retired  after  30  years  of  Government  service.   Her  replacement  was 
Mr.  John  Hamill  formally  of  NCI. 

In  accordance  with  Secretary  Califano's  initiative  to  correct  major 
deficiencies  in  the  contracting  process,  CMB  is  required  to  prepare 
the  following  reports: 

1.  Justification  for  Non-Competitive  Procurement  (JNCP)  approved 
monthly. 

2.  Non-competitive  Procurement  and  Scheduling  of  R&D  contracts  - 
monthly. 

3.  Degree  of  competition  (quarterly). 

4.  Even  distribution  of  contract  awards  within  the  fiscal  year 
plus  variances  (quarterly) . 

5.  Major  contracts  over  $500,000  (semi-annual). 

6.  Close-out  of  contracts  (quarterly). 

One  of  the  principal  objectives  CMB  achieved  during  FY  '79,  was  to 
spread  the  workload  more  evenly  through  the  fiscal  year  by  shifting 
contract  renewal  dates  and  new  contracts  as  equally  as  possible  in 
each  quarter. 

It  is  estimated  that  there  will  be  190  active  contracts  with  a  FY  '79 
allocation  of  $16,000,000. 


13-1 


II.  GRANTS  MANAGEMENT  BRANCH 

Fiscal  Year  1979  was  a  year  of  considerable  hardship  for  the  GMB  as 
the  Branch  lost  the  equivalent  of  one  full-time  professional  and 
one  half-time  administrative/award  clerk,  resulting  from  vacancies 
left  unfilled  for  appreciable  periods  of  time.   Nonetheless  the 
Branch  was  responsible  for  approximately  1,700  awards  with  a  dollar 
value  in  excess  of  $115,000,000  during  this  reporting  period.   The 
GMB  is  responsible  for  the  fiscal  and  administrative  management  of 
all  grants  and  awards  issued  by  the  NIAID.   The  GMB  works  very  closely 
with  the  programmatic  divisions  and  provides  the  fiscal  and  administrative 
expertise  necessary  to  more  effective  program  management.   The  GMB  also 
serves  as  an  interpreter  of  grant  policy  and  procedure  issued  by  the 
several  echelons  within  the  DHEW. 

There  were  many  policy  and  procedural  changes  that  impacted 
significantly  on  the  operations  of  the  Branch.   Perhaps  two  of  the  more 
significant  changes  were,  1)  the  loss  of  the  categorical  Report  of 
Expenditures,  which  will  be  replaced  by  the  non-categorical  Financial 
Status  Report;  and  2)  in  a  more  favorable  change,  the  initiation  of  the 
new  project-period  concept  which,  simply  stated,  enables  the  awarding 
component  to  carryover  unexpended  direct  cost  funds  from  one  project- 
period  to  another,  and/or,  to  use  the  total  unexpended  direct  cost 
balance  from  one  project-period  as  a  funding  offset  to  another. 

The  staff  of  the  GMB  has  made  significant  contributions  as  members  and 
chairpersons  of  policy  and  procedure  work  groups,  committees  and 
subcommittees  both  at  the  NIAID  and  at  the  NIH.   Examples  of  such 
activity  include:   NIH  grants  management  self -appraisal,  NIH  change  of 
institution  policy,  PHS  grants  policy  statement,  refinement  of  procedures 
for  development  of  recommended  grant  budgets  on  those  grants  assigned  to 
PPRB  (NIAID)  for  competitive  review,  and  various  other  grant  policy 
and  procedure  drafting  committees.   Additionally,  Branch  personnel 
have  participated  as  faculty  members  or  attendees  at  seminars  where 
grantee  personnel  were  in  attendance,  such  as  the  NIH-AAMC  Workshop 
on  Business  Affairs,  the  AAMC-group  on  Business  Affairs-Continuing 
Education  Program,  and  the  several  Grants  Management  Workshops  conducted 
by  the  GMAC  for  grantee  institutions  and  NIH  personnel  alike. 

Generally  speaking  the  Branch  performed  very  well  in  FY  '79,  even 
under  the  most  adverse  circumstances.   As  a  service-oriented  Branch 
whose  "cradle-to-grave"  involvement  in  the  NIAID  encompasses  all  grant 
programs,  it  is  the  goal  of  the  GMB  to  more  fully  develop  the  partnership 
role  that  exists  between  the  management  specialist  and  their  health- 
scientist  counterpart,  enhance  the  interaction  between  the  management 
specialists  and  the  grantee  community,  and  to  encourage  and  foster 
greater  participation  of  the  entire  GMB  staff  in  the  "total"  NIH 
grants  management  arena. 


14-1 


III.   PROGRAM  ANALYSIS  AND  EVALUATION  BRANCH 

The  title  "Program  Analysis  and  Evaluation  Branch"  replacing  "Review 
and  Evaluation  Branch"  indicates  a  branch  emphasis  on  development  of  an 
analytical  base  to  develop  reports  and  support  the  Office  of  the  Director, 
NIAID  Program  Directors,  and  the  NIAID  staff  data  and  information 
requests.   A  new  management  data  base  is  being  developed  by  branch 
personnel  augmented  by  three  one-time  non-tenured  personnel.   All 
competing  projects  (996)  and  non-competing  applications  (1,812)  will 
be  entered  into  the  new  multi  axis  coding  system,  MACS. 

The  Branch  serves  as  the  focal  point  for  management  and  budgetary 
data  for  Extramural  programs;  referral  liaison  with  the  Division  of 
Research  Grants,  ADP  Liaison  with  other  Institutes  and  NIH,  and 
reporting  of  non-program  specific  reports  such  as  Prevention  and 
Toxicology.   The  Branch  was  able  to  add  age  parameters  to  the  revised 
PHS  #398  (the  grant  application  form)  for  inclusion  in  the  MACS  system 
and  thus  eliminate  a  search  for  this  data. 

A.   Fiscal  Management  and  Analysis  Section 

In  FY  1979,  the  Fiscal  Management  and  Analysis  Section  developed 
detailed  operating  plans  for  the  use  of  all  research  and  training  funds 
and  implemented  the  final  plans.   Awards  were  paid  through  both  "Central" 
and  "Program"  accounts  to  assure  payment  of  the  highest  quality  research 
projects  and  programs  of  special  interest  to  the  aims  of  the  Institute. 
Tables  V  and  VI  show  final  operating  plans  of  EAP,  LAID,  and  MID  for 
contracts  and  grants  in  FY  '79. 

Due  to  the  increase  in  the  overall  appropriation  for  research  grants  in 
FY  '79,  NIAID  was  able  to  pay  about  43%  of  all  research  grant  approvals 
and  61%  of  the  approved  career/academic  award  applications.   In  the 
absence  of  an  appropriation  for  training  and  fellowships,  this  budget  was 
subject  to  the  conditions  of  a  continuing  resolution.   Under  this 
resolution,  NIAID  was  able  to  fund  47%  of  approved  individual  fellowship 
requests  and  24%  of  approved  training  applications. 

Requests  for  fiscal  reports  increased  significantly  during  the  year 
to  over  three  reports  per  week.   Several  studies  were  also  undertaken 
by  this  Section  to  assess  the  impact  of  proposed  changes  in  NIH  policy. 
One  such  study,  "Thinner  Grant  Applications  on  Longer  Awards",  was 
cited  by  NIH  as  an  important  factor  in  making  their  final  decision. 


15-1 


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15-2 


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15-3 


B.   Program  Analysis  Section 

Since  October  1978,  the  efforts  of  the  Program  Analysis  Section  have 
been  almost  exclusively  directed  toward  an  orderly  and  timely  transition 
from  the  old  scientific  classification  system  (three-digit  code)  to  the 
new  multi-axis  coding  system  (MACS).   MACS  is  a  complex  four-digit 
hierarchial  coding  system  consisting  of  four  main  axes; 

Axis  I   -  Organisms/Diseases/Study  Area 

Axis  II  -  Approaches/Methodologies 

Axis  III  -  Anatomical  Systems/Organs 

Axis  IV  -  Hosts 

The  scientific  information  in  every  active  and  pending  grant  application 
received  in  the  NIAID,  including  all  research  and  training  mechanisms, 
are  presently  being  reviewed  and  coded  into  the  above  system.   Other 
areas  of  special  interest  which  are  also  identified  include: 

1.  Trans-NIH  health  problems  (arthritis,  blood-related 
diseases,  bronchopulmonary  disorders,  digestive 
diseases,  etc.); 

2.  Human  subjects  (clinical  or  non-clinical  environment); 

3.  Special  groups  of  human  subjects  (minors,  pregnant  women, 
etc. ) ; 

4.  Age  of  subject; 

5.  Objectives  of  research/training  (diagnosis,  prevention, 
treatment,  etc.); 

6.  R&D  conducted  at  NIH  (NIAID):   Basic,  Applied,  and 
Development; 

7.  Entire  spectrum  of  R  &  D  activities  at  the  NIH  (NIAID): 
^cience  base,  Applications,  Transfer,  and  Training,   Clinical 
trials,  one  part  of  Applications  in  SATT,  is-  also  included 
under  Development.   This  information  forms  the  basis  of  data 
which  is  developed  into  the  Annual  Inventory  of  Clinical  Trials. 

Analysis  of  the  information  captured  by  this  newly  developed  intricate, 
multi-faceted  coding  system,  coordinated  with  our  assignment  of  all 
grants/grant  applications  to  one  of  twenty-four  program  areas  within 
the  MIDP/IAIDP  will  be  used  to  answer,  more  efficiently  and  accurately, 
repetitive  and  non-recurring  types  of  queries  from  within  NIAID 
(scientific  management  information  for  program  planning  and  evaluation) , 
NIH  (e.g.,  Trans-NIH,  BAD/ SATT) ,  members  of  Congress  (problems  of 
special  legislative  concern,  e.g.,  Cystic  Fibrosis,  Sexually-Transmitted 
Diseases),  other  government  agencies,  and  the  public. 

15-4 


C.   Data  Control  Section 

The  Data  Control  Section,  using  the  NIH  and  Parklawn  computer  facilities, 
reports  management,  budgetary,  and  programmatic  activities  for  the 
Extramural  Activities  Program.   In  addition,  special  and  repetitive 
reports  are  provided  for  the  Immunology,  Allergic  and  Immunologic 
Diseases  Program  and  the  Microbiology  and  Infectious  Diseases  Program. 

During  fiscal  year  1979,  a  new  scientific  information  data  base  has 
been  captured  into  NIAID's  computer  records  and  the  system  is  nearing 
completion.   With  this  implementation,  computer  generated  reports  are 
particularly  explicit  and  will  minimize  selective  interpretation  in 
responding  to  requests  for  scientific  data.   The  system  allows  for 
determining  percentages  of  support  in  given  areas,  for  retrieving 
TRANS-NIH  and  Programs  of  Institute  Emphasis  as  identified  by  the 
Program  staff  involved.   The  new  system  has  the  capability  of  reporting 
areas  of  Basic,  Applied,  Developmental  Research,  and  Scientific, 
Application,  Training,  and  Technology  Transfer  in  response  to  NIH 
concern. 

The  ongoing  Contract  system  of  data  capture  and  retrieval  of  NIAID 
contract  information  services  the  contract  office,  budget  office, 
O.D.'s  office  and  continues  to  be  well  received. 


15-5 


IV.  PROGRAM  AND  PROJECT  REVIEW  BRANCH 

A.   Introduction 

In  FY  1979,  there  was  an  enormous  increase  in  the  workload  of  the  Branch, 
primarily  involving  scientific  review  by  the  Microbiology  and  Infectious 
Diseases  Advisory  Committee  (MID).   It  was  estimated  that  the  MID  review 
workload  alone  was  at  least  2.2  times  the  optimal  workload  of  an  average 
DRG  Study  Section.   The  heavy  workload  was  a  direct  consequence  of  the 
release  of  numerous  RFAs  and  RFPs  by  the  MIDP  in  anticipation  of  a  good 
budget  in  1979  and  a  lean  budget  in  1980.   In  the  March  meeting  alone  a 
total  of  68  program  project,  exploratory-developmental,  and  training 
grant  applications  were  reviewed,  as  were  55  new  competing  contract 
proposals  in  response  to  6  RFPs  and  one  PL  480  proposal.   It  would  have 
been  humanly  impossible  for  one  executive  secretary  to  have  handled  the 
review  load.   The  Committee  was  therefore  divided  into  subcommittees, 
each  to  review  a  bloc  of  grants  or  proposals,  and  then  to  meet  on  the 
fourth  day  as  a  full  Committee  for  en  bloc  actions.   This  worked  out 
reasonably  well,  with  commendable  teamwork  and  cooperation  among  review, 
grants  management  and  contracts  management,  and  other  EAP  staff.   The 
Committee  Chairman  performed  an  outstanding  job  in  chairing  the  two 
separate  sessions  of  the  full  MID  as  well  as  two  subcommittees.   Still 
it  is  to  be  hoped  that  in  the  future  more  prudence  be  exercised  in 
programming  efforts  that  result  in  nearly  unmanageable  workloads  for 
both  EAP  review  staff  and  the  Committee. 

The  Allergy  and  Clinical  Immunology  Research  Committee  and  the 
Transplantation  Biology  and  Immunology  Committee  both  had  reasonable 
review  workloads  and  gave  advice  to  the  IAIDP  on  a  variety  of  program 
issues.   The  AIRC  advised  on  increased  flexibility  in  acceptance  of 
Allergic  Diseases  Academic  Award  (K07)  applications.   It  also  indicated 
that  site  visits  should  be  conducted  in  a  timely  manner. 

The  increased  workload  of  the  MID  impacted  on  the  Review  Services  Unit, 
which  serves  the  dual  purpose  of  serving  both  the  Council  and  the  Review 
Committees,  preparing  the  appropriate  books  of  summary  statements  or 
applications  and  proposals  for  extramural  staff  and  Council  or  Committee 
members . 

Through  all  these,  the  Branch  has  been  operating  with  a  skeleton  staff. 
A  detailed  manpower  analysis  of  the  Branch  indicates  that  there  is  a 
very  real  need  for  an  additional  full-time  (assistant)  executive  secretary 
as  well  as  a  clerk-typist  to  handle  the  workload  in  MID.   The  assistant 
executive  secretary  need  not  occupy  a  slot  but  may  be  a  consultant. 
There  is  a  need  for  another  part-time  worker  in  the  Review  Services  Unit, 
or  for  one  of  the  current  part-time  employees  to  be  converted  to  full-time. 
Unless  these  needs  are  met  in  the  immediate  future,  the  quality  of 
scientific  review  and  the  service  offered  by  the  Review  Services  Unit 
will  suffer. 


16-1 


B.  Allergy  and  Clinical  Immunology  Research  Committee 

The  AIRC  met  three  times  during  the  period  of  November  1978  through 
September  1979,  reviewing  two  program  projects,  four  allergic  disease 
center  applications,  seven  training  grant  applications,  and  two  Allergic 
Diseases  Academic  Award  applications  for  a  total  first  year  requested 
amount  of  $1,914,788.   In  addition,  an  ad  hoc  contract  proposal  review 
was  conducted  on  an  amount  of  $2,265,794  for  a  two-year  period.   The 
Committee  gave  advice  to  the  NIAID  on  policies  regarding  the  Allergic 
Diseases  Academic  Award  and  on  the  program  project  mechanism.   On  the 
AADC  program,  the  Committee  advised  on  a  much  more  flexible  policy, 
which  would  allow  multiple  applications  from  multi-campus  institutions 
and  their  clinical  affiliates  to  be  awarded.   It  is  hoped  that  this 
liberal  policy  would  move  the  program  ahead.   If  the  policy  is  accepted 
by  Building  1,  this  would  mean  an  escalation  of  the  review  activities  of 
AIRC  in  the  future. 

Recommendations  were  made  on  program  activities  including  food  allergy, 
drug  allergy,  allergens,  and  fungal  antigens.   Concept  clearance  was 
given  on  the  production  and  evaluation  of  horseradish-peroxidase-labelled 
antihuman  globulin  as  an  ELISA  standard. 

C.  Transplantation  Biology  and  Immunology  Committee 

The  TIC  met  three  times  from  November  1978  to  July  1979,  reviewing  three 
training  grants,  one  unsolicited  contract  proposal  and  four  contract 
proposals  submitted  in  response  to  an  RFP  for  a  total  first  year  requested 
amount  of  $651,253.   In  addition,  an  ad  hoc  contract  proposal  review  was 
conducted  on  a  first  year  amount  of  $964,804.   The  Committee  gave 
advice  to  the  NIAID  on  the  analysis  and  publication  of  the  kidney 
transplant  histocompatibility  study  data,  organization  and  policies  of  the 
serum  bank,  and  the  formation  of  a  hybridoma  banking  and  distribution 
system.   It  gave  recommendations  on  the  development  of  RFPs  for  1)  design 
and  evaluation  of  clinical  tray  configuration,  2)  evaluation  of  alternative 
tissue  typing  techniques,  and  3)  absorption  of  anti-DR  antisera,  and 
RFAs  for  1)  post-transplant  immunologic  monitoring  and  2)  the  role  of 
non-MHC  antigens  in  transplantation. 

Dr.  Harley  Sheffield  joined  the  Branch  in  FY  '79  and  has  served  as 
Executive  Secretary  of  both  the  AIRC  and  the  TIC.   He,  along  with  other 
professionals  in  EAP,  has  acted  as  executive  secretary  on  ad  hoc  reviews 
of  contract  proposals  within  the  IAIDP  as  well  as  the  MIDP.   The  review 
workload  of  the  AIRC/TIC  is  summarized  in  Table  VII. 

D.  Microbiology  and  Infectious  Diseases  Advisory  Committee 

The  Microbiology  and  Infectious  Diseases  Advisory  Committee  met  four 
times  from  November  1978  through  September  1979,  reviewing  101  grant 
applications  (39  program  projects,  39  exploratory  developmental  grants, 
23  training  grants)  for  a  first  year  requested  amount  of  $24,964,364 
direct  costs;  and  109  new  competing,  renewal,  unsolicited,  and  sole 
source  contract  proposals  for  a  total  requested  amount  of  $55,924,888 


16-2 


(Table  I).   Of  the  39  program  projects,  two  (STD  program)  were  in  fact 
reviewed  by  special  review  committees  and  site  visited;  the  others 
(ICIDRs,  mycology  fungal  disease  research,  arthropod-borne  viral 
infections  and  tropical  disease  research  units,  and  an  unsolicited  program 
project)  were  not  site  visited,  but  an  ample  number  of  consultants  expert 
in  the  specific  fields  participated  in  the  review  and  attended  the 
meetings.   Of  the  109  contracts,  71  were  reviewed  by  the  full  MID  and  38 
by  ad  hoc  review  groups  with  two  committee  members  participating  in 
each  review.   Of  the  38  reviewed  ad  hoc,  12  were  non-competing  renewals 
and  26  were  new  competing  proposals  in  response  to  one  RFP  of  which  one 
was  from  an  MID  Committee  member. 

Advice  and  concept  clearance  was  sought  from  the  full  Committee  on  the 
following  initiatives:   Rocky  Mountain  Spotted  Fever  (epidemiologic 
studies,  animal  model),  the  role  of  Streptococcus  viridans  in  bacterial 
endocarditis,  penicillin  prophylaxis  by  group  B  streptococcal  sepsis  in 
the  newborn,  and  potentiation  of  immune  responses  to  vaccines.   Except 
for  the  last  item,  these  have  been  implemented  by  RFPs  and  the  proposals 
reviewed  by  the  Committee. 

E.  Review  Services  Unit 

For  Council-related  work,  the  Unit  processed  2,045  applications  and 
their  respective  summary  statements  for  FY  1979.   This  compares  with 
2,049  applications  and  respective  summary  statements  for  FY  1978 
(Table  II).   Although  this  difference  is  insignificant,  Committee-related 
work  has  increased  the  volume  of  work  for  the  RSU  during  this  fiscal 
year,  the  applications  processed  increasing  from  173  in  1978  to  222  in 
1979,  an  increase  of  28%  (Table  VIII).   All  grant  applications  for  program 
projects,  Centers,  training  and  Allergic  Diseases  Academic  Awards  as 
well  as  all  new  contract  proposals  reviewed  by  the  three  NIAID  Committees 
are  processed  by  the  Unit,  with  books  made,  reviewed  for  conflict  of 
interest,  and  mailed  to  consultants  as  well  as  distributed  to  concerned 
staff.   As  was  stated  previously,  the  heavy  workload  of  the  MID  impacted 
on  the  work  of  the  Unit. 

F.  Clinical  Trials 

The  reporting  to  DRG  of  clinical  trials  active  in  1977  and  1978  was 
completed  on  June  5,  1979,  far  beyond  the  initial  deadline  set  by  DRG.   Of 
the  128  trials  reported,  51  were  from  the  IAIDP,  65  from  MIDP,  and  12  from 
the  intramural  programs .   The  MIDP  reported  14  new  trials  supported  by 
contracts  and  4  supported  by  grants.   The  IAIDP  reported  4  new  trials 
supported  by  grants  and  none  supported  by  contracts.   According  to  DRG, 
a  formal  publication  of  1978  trials  will  be  made  (but  not  of  the  1976 
and  1977  trials,  although  printouts  of  data  are  and  can  be  made  available 
respectively).   It  is  noted  that  the  number  of  trials  reported  in  1976 
was  142.   The  reasons  for  the  seeming  drop  in  number,  from  142  to  128, 
could  be  the  transitional  quarter  in  1976;  or  because  of  possible  uneven 
reporting  by  program  staff  of  grant-supported  trials.   Whereas  in  the  past, 
a  member  of  the  Program  Analysis  Section  screened  the  grants  and  identified 
clinical  trials,  in  consultation  with  the  NIAID  Clinical  Trials  Coordinator, 
this  year  program  staff  have  been  asked  to  undertake  this  activity. 

16-3 


TABLE  VII 
REVIEW  WORKLOAD,  PPRB,  1978-1979 


Category 


1978 
Number        Amount 


1979 
Number     Amount 


AIRC/TIC/AD  HOCS 

Grant  Applications      59 

Contract  Proposals      45 
Subtotal 
MID/AD  HOCS 

Grant  Applications 

Contract  Proposals 
Subtotal  106 

CONTRACTS  FOR  OSD,  OTHER   5_ 

TOTAL  215 


$13,079,077 
13,550,639 


18 
24 


104 

49 
57 


12,594,364 

16,342,567 


42 


101 
109 


6,111,861 


210 

5 


257 


Grant  amounts  are  first  year  direct  costs  only. 
Contract  amounts  are  total  costs. 


TABLE  VIII 
REVIEW  SERVICES  UNIT  WORKLOAD 


$2,409,163 
5,835,232 


24,964,364 
55,924,888 


4,861,363 


Categorv 


1978 


1979 


For  Council: 

Applications  Received 
"Pink  Sheets"  Received 

For  Committees: 

Applications  Processed 
Proposals  Processed 
Total  Processed 


2,049 
2,049 


108 
65 

173 


2,045 
2,045 


119 

103 
222 


16-4 


V.  RESEARCH  RESOURCES  BRANCH 

A.  Special  Activities 

ASM  Exhibit  -  The  Research  Resources  Branch  handled  arrangements  for  an 
NIAID  exhibit  at  the  American  Society  for  Microbiology  Meeting  held  on 
May  5-8,  1979,  in  Los  Angeles,  California.   The  RRB  made  the  arrangements 
to  take  the  Institute  exhibit  to  Los  Angeles  while  manpower  used  during 
the  exhibit  hours  was  supplied  by  the  Office  of  Research  Reporting  and 
Public  Response.   Additional  manpower  was  volunteered  from  the  NIAID 
Extramural  Activities  Program,  Microbiology  and  Infectious  Diseases 
Program  and  from  Intramural  Laboratories.   The  exhibit  was  modified 
from  the  exhibit  previously  shown  at  ASM.   An  NIAID  exhibit  of  different 
origin  was  repainted  and  new  pictures  and  video  tape  installed  to 
present  the  activities  currently  being  carried  out  by  NIAID.   The 
exhibit  was  well  received  and  gave  many  visitors  knowledge  of  the 
various  activities  being  supported  by  the  Institute. 

Dr.  Robert  Byrne  has  continued  to  serve  as  Branch  Chief  with  Sylvia 
Cunningham  responsible  for  management  of  operations  including  the 
distribution  and  cataloging  of  the  microbial,  allergen  and  some  of  the 
immunological  reagents. 

The  need  for  well  characterized  reference  reagents  as  an  adjunct  to 
research  is  well  recognized,  and  in  support  of  the  concept,  the  Research 
Resources  Branch  (RRB)  conducts  a  program  which  distributes  a  wide 
range  of  reagents  to  research  scientists.   Reagents  for  which  a  critical 
need  exist  are  identified  and  arrangements  for  their  production  are 
made  by  the  pertinent  Institute  Program  area.   The  RRB  then  arranges 
for  the  packaging  of  the  bulk  products,  catalogs  the  item  and  distributes 
the  reagent  with  the  information  and  technical  advice  on  reagent 
characteristics  and  use.   The  Branch  is  also  sponsoring  a  contract  for 
the  development  of  a  procedure  for  the  processing  and  packaging  of 
recombinant  DNA  Vector-Host  Systems.   In  addition,  the  Branch  assists 
with  the  contract  for  the  establishment  of  a  Plasmid  Reference  Center. 

B.  Asthma  and  Allergic  Diseases  Program-Allergen  Reagents 

A  procurement  contract  has  been  awarded  to  the  American  Type  Culture 
Collection  to  process  the  minor  determinant  penicillin  products.   The 
RRB  has  made  all  the  arrangements  to  have  these  products  packaged  and 
lyophilized.   The  actual  processing  is  expected  to  begin  the  week  of 
July  30;  it  is  expected  that  approximately  4  months  will  be  required 
to  complete  all  lots  of  material.   After  an  IND  is  submitted,  these 
products  will  be  supplied  to  approximately  six  contractors  who  will  be 
responsible  for  conducting  clinical  trials  to  ascertain  the  penicillin 
sensitivity  of  patients. 

The  contract  with  Johns  Hopkins  University  for  the  preparation  of 
polyvalent  ryegrass  antiserum  was  completed  this  year.   This  material 


17-1 


has  been  processed  by  the  American  Type  Culture,  undergone  certification 
testing  by  Mayo  Clinic  and  is  now  available  for  distribution. 

Although  the  contract  located  at  University  of  Pennsylvania  for  the 
establishment  of  an  allergic  dog  colony  is  sponsored  by  IAIDP,  the 
Chief,  RRB  has  continued  to  serve  as  a  project  officer  on  this  contract. 
The  contract  has  been  highly  productive  in  the  breeding  of  dogs  and 
is  ahead  of  schedule  in  producing  progeny  of  atopic  breeding  stocks. 

In  addition  to  the  activities  listed  above,  the  Branch  has  continued 
to  distribute  allergenic  products  such  as  Ragweed  Antigen  E,  K,  and 
RA3,  Ryegrass  I,  II,  and  III  and  venoms  of  honey  bee,  yellow  hornets, 
white-faced  hornets,  yellow  jackets,  paper  wasps  and  hymenoptera  venom 
diluent;  these  venom  products  are  available  for  distribution  under  an 
IND  as  well  as  for  in  vitro  use. 

C.  Immunological  Reagents  and  Resources 

The  new  contract  awarded  to  Flow  Laboratories  during  FY  1978  for  the 
Maintenance  and  Breeding  of  Rabbits  of  Known  Genotype  for  Use  in 
Immunological  Studies  has  continued  to  serve  a  useful  purpose  in 
maintaining  a  colony  of  500  specially  bred  rabbits  for  use  in  NIAID 
immunological  studies.   In  addition,  these  facilities  provide  reference 
quantities  of  reagents  and  limited  numbers  of  rabbits  to  other 
responsible  investigators  doing  immunological  studies.   During  this 
period,  seven  investigators  have  been  supplied  with  a  total  of  18 
rabbits;  twenty-six  shipments  of  rabbit  antisera  have  also  been  made 
to  other  investigators. 

During  this  period,  the  rabbits  had  to  be  moved  from  their  building  in 
Rockville  to  make  room  for  the  new  Metro  station.   The  rabbits  are 
currently  being  housed  in  Fairfax,  Virginia,  in  a  building  being  leased 
by  Flow  from  Litton-Bionetics .   Flow  expects  their  new  animal  building 
which  is  being  built  in  McLean,  Virginia,  to  be  completed  in  September; 
the  animals  will  be  moved  again  when  the  new  building  is  ready  for 
occupancy. 

D.  Microbiology 

During  the  latter  part  of  FY  1977,  two  contracts  were  awarded  and  have 
continued  during  FY  1979.   The  University  of  Alabama  is  performing 
research  and  developmental  work  to  determine  optimal  conditions  for 
the  preservation,  retrieval,  storage  and  distribution  of  fragile 
bacterial  host  strains.   Three  basic  methods  of  storing  have  been 
studied,  i.e.,  preservation  in  1)  paraffin-sealed  agar  stabs,  2)  by 
freezing  in  peptone-glycerol  broth  and  3)  by  lyophilization.   Results 
to  date  indicate  that  lyophilized  material  stores  better  at  -30°C  or 
-70°C  than  at  4°C  or  room  temperature.   The  peptone-glycerol  broth 
freezing  method  appears  to  cause  some  reversion  frequencies  of  some 
products.   This  problem  is  now  being  carefully  analyzed;  similar 


17-2 


studies  are  being  conducted  on  the  lyophilized  materials  to  determine 
if  the  reversion  frequencies  are  also  occurring  in  these  samples. 
The  MIDP  is  sponsoring  the  Stanford  University  contract  for  the 
establishment  of  a  plasmid  reference  center.   During  the  year  a 
modification  was  made  to  the  contract  work  scope  to  allow  the  contractor 
to  continue  storing  and  shipping  the  bulk  cultures.   RRB  will  therefore, 
not  be  responsible  for  the  packaging,  storing  and  distribution  of  the 
strains  collected  under  this  project.   However,  backup  set  of  these 
strains  are  being  maintained  by  RRB  (to  date  90  sets  have  been 
received  for  storage).   In  addition,  RRB  will  assist  with  the 
publication  and  printing  of  the  Catalog  of  Plasmid  Strains  once  a 
suitable  format  has  been  developed.   To  date,  the  format  for  the  strains 
data  sheet  has  been  revised  three  times. 

E.   Research  Resources  -  Research  Reagents 

While  responsibility  for  the  actual  production  of  reagents  is  with  the 
various  Institute  program  areas,  RRB  has  continued  to  sponsor  certain 
service  type  contracts  which  serve  the  needs  of  the  various  program 
elements . 

The  Ohio  State  University  has  continued  to  serve  as  the  viral  reagent 
testing  laboratory.   In  addition  to  verifying  feedback  data  from 
reagent  users  concerning  product  viability,  the  contractor  has  verified 
certain  results  obtained  by  the  American  Type  Culture  Collection  on 
the  reagent  transfer  contract.   During  this  year,  the  contractor  has 
conducted  tests  on  15  rhinovirus  seeds  and  antisera,  4  enterovirus 
seeds  and  has  confirmed  the  contamination  of  reovirus  type  1  with  SV40. 
The  contractor  is  now  initiating  cross-neutralization  testing  on 
adenovirus  types  10  and  13  to  see  if  he  can  verify  a  report  from  a 
reagent  user  that  our  adenovirus  type  13  rabbit  serum  neutralizes 
adenovirus  type  10. 

After  a  category  of  reagents  has  become  established,  and  the  area 
of  research  that  it  supports  is  well  defined,  it  is  of  questionable 
value  for  RRB  to  continue  storing  and  distributing  that  category  of 
reagents  for  a  prolonged  period  of  time.   During  FY  1975,  it  was 
determined  that  four  viral  reagent  groups  fall  into  this  category. 
RRB  therefore,  entered  into  a  five  year  contractual  arrangement  with 
the  American  Type  Culture  Collection  (ATCC)  to  transfer  the  enterovirus, 
adenovirus,  rhinoviruses  and  arboviruses  reagent  collections  to  the 
ATCC  whose  prime  function  is  to  store  and  distribute  these  types  of 
reagents. 

Under  the  contractual  arrangement  with  ATCC,  samples  of  each  reagent 
type  will  be  preserved  as  a  museum  function.   The  viability  or 
homologous  antibody  activity  of  a  parallel  collection  of  reagents 
will  also  be  assayed.   After  each  viral  group  has  been  assayed  and  the 
results  reported  and  reviewed,  RRB  will  discontinue  that  reagent  group 
from  its  catalog  with  a  notation  that  they  are  available  from  ATCC 
and  all  remaining  stock  of  that  group  of  reagents  will  be  transferred 


17-3 


to  ATCC  for  its  custodianship.   NIH  scientists  and  NIAID  contractors 
will  be  provided  reagents  without  cost  until  RRB  supply  is  depleted. 

During  the  first  four  years  of  this  project,  50  samples  of  each  reagent 
type  for  the  four  viral  reagents  groups  have  been  transferred  to  ATCC 
for  the  museum  function.   The  assay  of  the  enterovirus,  adenovirus  and 
rhinovirus  groups  has  been  completed;  these  three  groups  have  now  been 
transferred  to  the  ATCC.   During  FY  1979,  ATCC  has  primarily  been 
involved  in  the  testing  of  the  arbovirus  reagent  collection  which 
is  the  last  of  the  four  collections  to  be  re-assayed  before  being 
transferred  to  ATCC.   The  schedule  for  assaying  and  transferring 
reagents  originally  prepared  in  1975  is  still  as  projected;  it  is 
planned  that  the  assay  of  arbovirus  reagents  will  be  completed  during 
FY  1980  with  the  inventory  of  arbovirus  reagents  being  transferred  to 
ATCC  upon  completion  of  review  of  the  data. 

The  Branch  has  also  been  involved  in  assisting  with  plans  to  have  the 
rotavirus  reagents  labeled  and  packaged.   These  materials  are  being 
prepared  under  an  Interagency  Agreement  with  Plum  Island,  USDA  which 
is  being  supported  by  MIDP. 

The  Branch  has  also  given  assistance  with  the  preparation  of  other 
contract  documentation,  such  as  work-scope,  reporting  requirements, 
evaluating  criteria,  etc. ,  to  have  additional  arbovirus  reagents 
prepared.   During  the  latter  part  of  FY  1979,  an  interagency  agreement 
will  be  made  with  the  USDA  for  the  preparation  of  an  arbovirus  grouping 
fluid  comprised  of  African  swine  fever,  bovine  ephemeral  fever, 
African  horse  sickness  and  Rift  Valley  fever.   During  FY  1980,  requests 
for  proposals  will  be  issued  to  have  other  arbovirus  reagents  prepared. 
The  reagents  resulting  from  these  projects  will  ultimately  be  received 
by  RRB  for  processing  (if  necessary),  cataloging  and  distribution. 

Prior  years  of  intensive  work  have  resulted  in  completed  work  on  the 
enterovirus,  adenovirus,  rhinovirus,  myxovirus  and  the  agents  and 
antigens  of  hepatitis  A  &  B.   In  most  cases,  seed  virus  preparations 
and  corresponding  antisera  are  now  available  for  most  of  the  viruses 
of  public  health  interest.   The  reagents  which  have  resulted  from 
the  various  projects  have  been  most  useful,  particularly  the 
hemagglutinins,  neuraminidases,  ribonucleoproteins  and  the  matrix 
proteins  of  the  influenza  viruses  of  man  and  animals.   Since  completion 
of  this  production  effort  in  FY  1978  and  listing  in  the  1978  -  1980 
Catalog  of  Research  Reagents,  these  materials  have  become  a  very 
popular  distribution  item.   Information  received  thus  far  from  the 
reagent  users  indicate  that  these  materials  are  very  valuable. 

F.   Molecular  Anatomy  Program 

The  Molecular  Anatomy  Program  (MAP)  during  FY  1979  has  been  evaluating 
for  safety  and  efficacy  in  human  vaccine  trials,  subviral  forms  of 
hepatitis  B  surface  antigen  (HBsAg) ,  purified  from  the  plasma  of  chronic 
carriers  and  inactivated.   Thus  far,  limited  studies  in  volunteers  have 
shown  the  vaccines  to  be  free  of  infectious  hepatitis  B  virus  and  local 


17-4 


and  systemic  side  reactions.   MAP  is  currently  evaluating  different 
vaccine  formulas  and  inoculation  schedules  in  volunteers  to  optimize 
the  humoral  anti-HBs  response.   Four  vaccine  lots  have  been  prepared 
from  the  original  inactivated  HBsAg/adw  preparation;  (1)  untreated, 
aqueous,  (2)  untreated,  alum-adsorbed,  (3)  ether-tween  80  treated, 
aqueous,  and  (4)  ether-tween  80  treated,  alum-adsorbed.   The  four 
lots  passed  safety  tests  and  are  currently  being  evaluated  in  volunteers 
at  the  NIH.   A  large  lot  (1000  doses)  of  the  untreated,  alum-adsorbed 
vaccine  and  an  alum  placebo  were  also  prepared  for  more  extensive 
testing  by  Dr.  Hollinger  at  Baylor  Medical  School  under  an  NIAID 
contract;  the  vaccine  and  placebo  lots  are  currently  on  safety  tests. 

The  hybridoma  technology  offers  great  promise  for  analyzing  the  antigenic 
determinants  of  HBsAg.   Hybridomas  from  spleen  cells  of  Balb/c  mice 
immunized  with  HBsAg/adw  and  mouse  myeloma  cells,  P3  X  63Ag8,  have  been 
developed. 

Many  laboratories  are  now  engaged  in  efforts  to  detect  antigen-antibody 
systems  related  to  the  non-A,  non-B  hepatitis  agents.   MAP  has  involved 
in  defining  the  delta  antigen  and  its  antibody  (6/anti-  5).   This 
specificity  is  found  in  carriers  of  HBsAg  and  appeared  to  be  related  to 
the  HBV  system.   A  sensitive  RIA  for  anti-6  has  been  developed  and 
found  predominately  in  chronic  HBsAg  carriers,  although  a  few 
asymptomatic  carriers  were  also  positive  for  anti-6;  low  and  transient 
titers  of  anti-6  were  found  in  acute  HBV  hepatitis  patients.   The 
evidence  to  date  indicates  that  6  represents  a  marker  of  an  agent, 
not  HBV,  which  requires  HBV  infection  to  provide  certain  helper 
functions  for  its  replication. 

The  eastern  woodchuck  (Marmota  monax)  has  an  HBV-like  virus  and  this 
animal  may  represent  an  inexpensive  model  of  virus-induced  liver 
disease  including  hepatocellular  carcinoma.   MAP  has  examined  approximately 
50  wild-caught  animals  and  based  on  endogenous  DNA  polymerase  activity 
and  virus-like  particles,  approximately  30%  are  chronically  infected 
with  this  virus.   We  showed  that  the  woodchuck  virus  cross-reacts  with 
HBV  in  both  HBsAg  and  HBcAg  components.   Two  of  the  carrier  woodchucks 
have  recently  been  diagnosed  with  hepatocellular  carcinoma.   Further 
evaluation  of  the  woodchuck  as  a  model  of  HBV  infection  and  disease 
will  continue. 

Efforts  in  respiratory  syncytial  virus  research  have  concentrated  on  the 
isolation  and  identification  of  surface  antigens  of  the  virus.   The 
known  instability  of  the  virus  has  been  a  major  problem.   However,  1  M 
MgS04  has  been  very  effective  in  stabilizing  the  infectivity  of  the 
virus  throughout  the  purification  procedures.   Large-scale  isolation 
of  virus  under  stabilizing  conditions  are  now  in  progress.   Several 
lines  of  mouse  Balb/c  cells  have  been  established  that  are  persistently 
infected  with  RSV.   These  cells  produce  low  levels  of  infectious 
virus,  contain  large  amounts  of  ribonucleoprotein  and  express  RSV- 
specific  antigens  on  the  cell  membrane.   These  cells  will  be  useful  in 
the  development  of  murine  hybridomas  for  the  production  of  monoclonal 
antibodies  to  surface  antigens  of  the  virus. 


17-5 


G.   Processing  and  Distribution 

In  addition  to  the  various  program  elements  detailed  above,  the  Research 
Resources  Branch  also  distributes  coronaviruses ,  herpes  viruses, 
interferons,  mycoplasmas  and  reoviruses. 

Due  to  the  limited  number  of  reagents  to  be  processed  during  FY  1979, 
there  was  not  a  formal  research  and  development  contract  to  handle 
this  activity.   However,  the  American  Type  Culture  Collection  did 
process  four  (4)  mycoplasma  seeds  and  one  antiserum  lot  by  the 
purchase  order  mechanism. 

The  Research  Resources  Branch  reagent  collection  (exclusive  of  the 
viral  reagents  transferred  to  ATCC)  now  consists  of  over  650  individual 
reagents.   The  repository  and  distribution  contract  remains  at  Flow 
Laboratories.   During  this  period,  the  repository  activity  was  moved 
from  its  Rockville,  Maryland,  to  a  new  building  in  McLean,  Virginia. 
A  tabular  record  of  distribution  by  this  facility  since  FY  1972  follows: 

DISTRIBUTION  OF  VIRAL,  MYCOPLASMAL, 
AND  ALLERGEN  REAGENTS 


Fiscal  Year 

1972 

1973 

1974 

1975 

1976 

1976  (TQ) 

1977 

1978 

1979  (3/4  year) 


Total  amps.  &  vials 

Total  Transactions* 

Distributed 

572 

21,801 

575 

19,181 

500 

9,932 

592 

6,751 

762 

10,188 

192 

3,126 

613 

7,633 

605 

6,851 

561 

6,907 

*Does  not  include  the  shipments  made  for  testing  and  packaging  purposes 
and  reagents  transferred  to  the  American  Type  Culture  Collection. 

In  addition  to  the  shipments  tabulated  above,  Flow  has  also  made  141 
shipments  for  DAB,  28  shipments  consisting  of  whole  rabbits  or  rabbit 
serum  and  30  interferon  shipments  for  the  National  Cancer  Institute. 
All  of  this  was  conducted  under  the  NIAID  contract  with  the  NCI 
contributing  funds  to  NIAID  for  their  activities.   In  addition, 
American  Type  Culture  Collection  has  made  the  following  shipments  under 
the  reagent  transfer  contract: 

(U.S.  Investigators  and  WHO  Laboratories) 

#  of  Shipments  #  of  Ampoules 

FY  1978  96  1,622 

FY  1979  (3/4  year)  87  1,068 

17-6 


During  this  year,  the  printing  of  the  new  1978  -  1980  Catalog  of 
Research  Reagents  was  completed  by  the  Government  Printing  Office. 
Over  1900  of  the  2200  copies  printed  have  been  mailed  out  to  reagent 
users. 

The  Branch  has  continued  its  efforts  to  publicize  the  availability  of 
these  valuable  reagents  to  the  scientific  community.   The  availability 
of  reagents  was  incorporated  into  the  NIAID  exhibit  which  was  shown 
at  the  American  Society  for  Microbiology  meeting  held  in  Los  Angeles, 
California. 

The  following  two  (2)  tables  describe  the  distribution  of  reagents  by 
groups  and  also  by  institutional  affiliation  during  the  first  three 
quarters  of  FY  '79. 


17-7 


RESEARCH  RESOURCES  BRANCH 

DISTRIBUTION  OF  SPECIFIC  VIRUS  GROUPS 

FY  1979  (3/4  year) 

OCTOBER  1,  1978  -  JUNE  30,  1979 


//  of  Items 
Avail.  (%  of 
Inventory) 

Virus  Group 

SHIPMENTS 
If   of     %  of 

it   of 

AMPOULES 
%  of  // 
Distribution 

9***(1.78) 

Adenovirus 

3 

.5 

18 

.3 

17 

(3.36) 

Allergen 

81 

14.4 

1345 

19.5 

224 

(44.27) 

*Arbovirus 

43 

7.7 

647 

9.4 

4 

(.79) 

Coronavirus 

3 

.5 

9 

.1 

20 

(3.95) 

Recombinant  DNA 

27 

4.8 

244 

3.5 

20 

(3.95) 

**Enterovirus 
(Typing  Pools) 

80 

14.4 

2294 

33.2 

18 

(3.56) 

Hepatitis 

40 

7.1 

240 

3.5 

18 

(3.56) 

Herpes 

2 

.4 

3 

.1 

5 

(.99) 

Interferon 

133 

23.7 

237 

3.4 

86 

(17.00) 

Mycoplasma 

49 

8.7 

837 

12.1 

79 

(15.60) 

Myxovirus 
Paramyxovirus 
and  related 

63 

11.2 

575 

8.3 

6 

(1.19) 

Reovirus 

8 

1.4 

37 

.5 

Combinations 

29 

5.2 

421 

6.1 

Figures  based  on  561  shipments  and  6907  ampoules  shipped  (excludes  testing 
packaging  and  reagents  transferred  to  ATCC). 

* Included  in  this  figure  are  11  shipments  of  Arbovirus  Grouping  fluids 
totaling  201  ampoules. 

**Included  in  this  figure  is  1  shipment  to  a  WHO  Laboratory  totaling 
1130  ampoules . 

***Seed  and  antisera  for  adenovirus  types  1  thru  31  have  been  transferred 
to  the  ATCC. 


17-8 


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17-9 


H.   Individual  Contract  and  Agreement  Tabulation 

PROGRAM:   IMMUNE  SYSTEM  AND  DISEASE  -  REAGENTS  AND  RESOURCES  BRANCH:   RRB 


Principal  Investigator 
Contract  Number 
Contractor 
FY  '79  Obligations 


Objectives  and  Findings 


Sullivan,  Rudolph  R. 
AI  82565 

Flow  Laboratories 
$158,700 


To  maintain  a  genetically  bred 
rabbit  colony  of  known  genotype 
for  use  in  immunologic 
investigations.   A  maximum  of 
500  adult  rabbits  will  be  bred 
and  maintained  by  expert 
veterinary  care.   This  is  for 
support  of  high  priority  research 
on  genetic  control  of  the  immune 
response  and  to  supply  reagents 
and  rabbits  to  other 
investigators . 


17-10 


Individual  Contract  Agreement  Tabulation 


PROGRAM:   INFECTIONS  -  VIRAL  REAGENTS 

Principal  Investigator 
Contract  Number 
Contractor 
FY  '79  Obligations 


BRANCH:   RRB 


Objectives  and  Findings 


Hughes ,  John  R. 
AI  62513 

Ohio  State  University 
$17,313  (FY  1978  Funds) 


This  laboratory  serves  as  a 
shelf-life  viral  reagents 
testing  laboratory  for  members 
of  the  enteroviruses,  rhino- 
virus,  adenovirus,  myxovirus 
and  herpevirus  groups.   This 
contract  will  ensure  the 
integrity  of  these  reference 
standard  reagents  by  assessing 
the  potency  and  purity  of  viral 
reagents  after  long  term  storage. 
This  contract  will  terminate 
this  year. 


17-11 


Individual  Contract  Agreement  Tabulation 
PROGRAM:   INFECTIONS  -  MOLECULAR  ANATOMY  PROGRAM         BRANCH:   RRB 


Principal  Investigator 
Contract  Number 
Contractor 
FY  '79  Obligations 


Objectives  and  Findings 


Gerin,  John 

AI  82572 

Georgetown  University 

$928,952 


The  major  objective  is  to  develop  and 
apply  methods  of  isolating  viruses  and 
viral  antigens  and  the  application  of 
those  techniques  to  research  on 
associated  diseases.   During  FY  1979, 
this  laboratory  has  been  involved  in 
purifying  and  inactivating  subviral 
forms  of  hepatitis  B  surface  antigen 
(HBsAg)  from  the  plasma  of  chronic 
carriers.   These  materials  are  currently 
being  evaluated  for  safety  and  efficacy 
in  human  vaccine  trials.   Limited 
studies  in  volunteers  have  shown  the 
vaccines  to  be  free  of  infectious 
hepatitis  B  virus  and  local  and 
systemic  side  reactions. 

The  contractor  has  also  developed 

hybridomas  from  spleen  cells  of  Balb/c 

mice  immunized  with  HBsAg/adw  and 

mouse  myeloma  cells,  P3  X  63Ag8. 

He  also  has  been  involved  in  defining 

the  delta  antigen  and  its  antibody 

(6/anti-5).   This  specificity  is 

found  in  carriers  of  HBsAg  and 

appears  to  be  related  to  the  HBV  system. 

Evidence  to  date  indicates  that  6 

represents  a  marker  of  an  agent,  not  HBV, 

which  requires  HBV  infection  to  provide 

certain  helper  function  for  its 

replication. 

The  contractor  is  also  now  involved  in 
the  large-scale  isolation  of  RSV  under 
stabilizing  conditions  which  have 
been  developed. 


17-12 


Individual  Contract  Agreement  Tabulation 
PROGRAM:   INFECTIONS  -  PROCESSING  AND  DISTRIBUTION         BRANCH:   RRB 


Principal  Investigator 
Contract  Number 
Contractor 
FY  '79  Obligations 


Objectives  and  Findings 


Stanley,  Thomas 

AI  82542 


Flow  Laboratories 
$184,506  (MID) 
$276,758  (IAIDP) 


When  the  reagents  program  was  given 
the  responsibility  for  production 
of  reference  reagents,  other  responsi- 
bilities were  also  included,  i.e., 
the  development  and  maintenance  of 
appropriate  means  to  receive,  process, 
store  and  distribute  these  research 
materials.   The  contractor  fulfills 
these  responsibilities.   During  the 
past  year,  the  contractor  has  been 
supplying  the  storage  and  shipping 
services  for  the  RRB,  Developmental 
Applications  Branch,  the  Enteric 
Diseases  Branch  and  the  NCI.   As 
a  result  of  these  combined  activities, 
the  contractor  has  made  2548 
shipments  totaling  106,614  vials  + 
38,670  trays  and  570  shipments  of 
bulk  material.   Approximately  95 
incoming  shipments  have  been  handled. 


17-13 


Individual  Contract  Agreement  Tabulation 
PROGRAM:   INFECTIONS  -  PROCESSING  AND  DISTRIBUTION         BRANCH:   RRB 


Principal  Investigator 
Contract  Number 
Contractor 
FY  '79  Obligations 


Objectives  and  Findings 


Clark-Curtiss,  Josephine 
AI  725333 

University  of  Alabama 
$171,657  (FY  1977  funds) 
Contract  awarded  for  a 
3-year  period 


This  contractor  is  determining  the 
optimal  processing,  packaging 
and  storage  conditions  for  vector- 
host  systems  for  recombinant  DNA 
molecule  research.   To  date,  the 
contractor  has  investigated 
various  methods  of  preserving 
these  materials.   The  methods 
employed  to  date  include  1) 
preservation  in  paraffin  -  sealed 
gelatin  slabs  2)  preservation  by 
freezing  in  peptone  -  glycerol, 
preservation  by  lyophilization. 
In  addition,  a  study  is  being 
conducted  comparing  the  effects 
of  various  storage  temperatures. 


17-14 


Individual  Contract  Agreement  Tabulation 
PROGRAM:  INFECTIONS  -  PROCESSING  AND  DISTRIBUTION        BRANCH:   RRB 


Principal  Investigator 
Contract  Number 
Contractor 
FY  '79  Obligations 


Objectives  and  Findings 


Donovick,  Richard 

AI  75-2526 
American  Type 

Culture  Collection 
$149,823 


After  a  category  of  reagents  has 
become  established  and  the  area 
of  research  that  it  supports  is 
well  defined,  it  is  of  questionable 
value  for  RRB  to  continue  storing 
and  distributing  that  category 
of  reagents.   Therefore,  in  FY  '75, 
RRB  entered  into  a  cooperative 
enterprise  with  ATCC  to  transfer 
the  enterovirus,  arbovirus, 
rhinovirus  and  adenovirus  reagent 
collections  to  the  ATCC  for  their 
custodianship  and  distribution; 
complete  transfer  of  the  four 
viral  reagent  groups  will  be  accom- 
plished over  a  five-year  period. 
During  the  first  three  years  of 
the  contract,  50  samples  of  each 
reagent  type  for  the  four  viral 
reagent  groups  have  been  transferred 
for  museum  storage  purposes. 
Under  the  terms  of  the  contract, 
each  of  the  four  collection  of 
reagents  is  to  be  assayed  for 
viability  or  homologous  antibody 
before  the  complete  transfer  is 
made,  therefore,  the  contractor 
has  been  assaying  the  enterovirus, 
adenovirus,  rhinovirus  groups 
during  the  initial  period.   The 
assay  of  these  groups  has  been 
completed,  the  test  results  have 
been  reviewed,  and  the  total 
enterovirus,  adenovirus,  rhinovirus 
collections  have  been  transferred  to 
ATCC.   The  contractor  is  now 
performing  the  required  testing  on 
the  arbovirus  reagents . 


17-15 


CONTRACT  PUBLICATIONS 

Gerin,  J.  L.  and  J.  W.  -K.  Shih.   Structure  of  HBsAg  and  HBcAg.   In: 
Viral  Hepatitis  (Eds.,  G.  N.  Vyas,  S.  N.  Cohen  and  R.  Schmid) .    pp. 
147-153.   Franklin  Institute  Press,  Philadelphia.   1978. 

Feinstone,  S.  N. ,  Y.  Moritsugu,  J.  W.  -K.  Shih,  J.  L.  Gerin  and 

R.  H.  Purcell.   Characterization  of  hepatitis  A.  virus.   In:   Viral 
Hepatitis  (Eds.,  G.  N.  Vyas,  S.  N.  Cohen  and  R.  Schmid).    pp.  41-48. 
Franklin  Institute  Press,  Philadelphia.   1978. 

Purcell,  R.  H.  and  J.  L.  Gerin.   Hepatitis  B.  vaccines:   A  status  report. 
In:   Viral  Hepatitis  (Eds.,  G.  N.  Vyas,  S.  N.  Cohen  and  R.  Schmid). 
pp.  491-505.   Franklin  Institute  Press,  Philadelphia.   1978. 

Dreesman,  G.  R.  and  J.  L.  Gerin.   Summary  of  workshop  on  structure  of 
hepatitis  viruses.   In:   Viral  Hepatitis  (Eds.,  G.  N.  Vyas,  S.  N. 
Cohen  and  R.  Schmid).   pp.  645-648.   Franklin  Institute  Press,  Philadelphia. 
1978. 

Shih,  J.  W.  -K. ,  G.  Hess,  P.  M.  Kaplan  and  J.  L.  Gerin.   The  polypeptides 
of  HBV  (Dane)  particles.   In:   Viral  Hepatitis  (Eds.,  G.  N.  Vyas,  S.  N. 
Cohen  and  R.  Schmid).   pp.  705.   Franklin  Institute  Press,  Philadelphia. 
1978. 

Shih,  J.  W.  -K. ,  M.  Rizzeto,  C.  Langer  and  J.  L.  Gerin.   1979.   Type  B 
hepatitis:   Characterization  of  delta  antigen  and  the  development  of 
radioimmunoassays  for  delta  and  anti-delta.   Abstr. ,  Annl.  Mtg.  Amer. 
Soc.  Microbiol.  S(H)80. 

Rizzetto,  M. ,  D.  Gocke,  J.  Shih,  G.  Verme  and  J.  L.  Gerin.   1979.   A 

solid-phase  radioimmunoassay  for  antibody  to  the  hepatitis  B  associated 
delta  antigen:   Incidence  of  anti-delta  in  HBsAg  carriers.   Abstr. , 
Annl.  Mtg.,  Amer.  Soc.  Microbiol.  S(H)14. 

Rizzetto,  M. ,  J.  W.  -K.  Shih  and  J.  L.  Gerin.   1979.   Characterization 
of  the  hepatitis  B  virus  (HBV)  associated  delta  antigen:   Incidence 
of  anti-delta  in  HBsAg  carriers  by  a  solid-phase  radioimmunoassay 
(SP-RIA).   Fed.  Proc.  38(3). 

Hess,  G.  ,  J.  W.  -K.  Shih,  J.  L.  Gerin,  R.  H.  Purcell  and  K.  H.  Meyer 
zum  Bueschenf elde.  Hepatitis  B.  antigen:  Lack  of  relationship  to 
liver-specific  protein  (LSP) .   J.  Med.  Virol.  (In  Press). 

Parrott,  R.  H. ,  H.  W.  Kim,  C.  D.  Brandt,  M.  0.  Beem,  L.  Richardson, 
J.  L.  Gerin  and  R.  M.  Chanock.  Respiratory  Syncytial  virus.  In: 
Diagnostic  Virology  (Ed.,  E.  Lennette).   (In  Press). 

Hess,  G.,  J.  W.  -K.  Shih,  W.  Arnold,  J.  L.  Gerin  and  K.  H.  Meyer  zum 
Bueschenf elde.  Demonstration  and  partial  characterization  of  22  nm 
HBsAg  and  Dane  particles  of  subtype  HBsAg/adw.   J.  Immunol.   (In  Press). 


17-16 


Shih,  J.  W.  -K. ,  G.  Hess,  P.  M.  Kaplan  and  J.  L.  Gerin.   Characterization 
of  hepatitis  B  virus  (Dane  particle).   J.  Virol,  (submitted). 

Rizzetto,  M. ,  J.  W.  -K.  Shih,  D.  J.  Gocke,  R.  H.  Purcell,  G.  Verme  and 
J.  L.  Gerin.   Incidence  and  significance  of  antibodies  to  delta 
antigen  in  hepatitis  B  infection.   N.  Engl.  J.  Med,  (submitted). 

Moritsugu,  Y. ,  J.  W.  -K.  Shih,  S.  M.  Feinstone,  J.  L.  Gerin  and  R.  H. 

Purcell.   The  hepatitis  A  virion:  Polypeptides  of  hepatitis  A  virus 

obtained  from  stool  of  a  naturally  infected  patient.   J.  Virol. 
(submitted) . 

Shih,  J.  W.  -K. ,  P.  J.  Cote,  Jr.  and  J.  L.  Gerin.   Production  of 

monocolonal  antibodies  against  hepatitis  B  surface  antigen  by  somatic 
cell  hybrids.   AABB  Annl.  Mtg.  (submitted). 

Rizzetto,  M. ,  R.  H.  Purcell  and  J.  L.  Gerin.   Antibody  to  the  hepatitis  B 
virus-associated  delta  antigen  in  hemophiliacs:   Evidence  for  parenteral 
transmission  of  delta  through  superinfection.   AABB  Annl.  Mtg.  (submitted), 

Purcell,  R.  H. ,  R.  A.  Johnson,  V.  J.  McAuliffe,  J.  L.  Gerin,  W.  T..  London, 
E.  Nordenfelt  and  E.  Helgstrand.   Effect  of  antivirals  on  markers  of 
HBV  infection  in  chimpanzees.   Lancet  (submitted). 


17-17 


OFFICE  OF  THE  SCIENTIFIC  DIRECTOR,   NIAID 
1979  Annual    Report 

Table  of  Contents 

Summary  of  Program—Sell  I8-I 

Z01 -Al -0001 3-1 6-OSD  Studies  of  Viral   Antigens   in  18-3 

Virus-induced  Tumors--Lewis 

Z01-A1-00018-13-0SD  Biological    and  Biochemical  18-10 

Characterization  of  Human 
Papovavi  ruses--Takemoto 

Z01-AI-00019-05-0SD  Studies  on  the  Treatment  of  18-14 

Disease  with   the   Interferon 
System — Levy 

Z01-A1-00131-12-0SD  Mechanisms  of  Hypersensitivity  18-19 

in  Histocompatible   Inbred  Guinea 
Pigs--Stone 

Z01-A1-00190-01-0SD  The  Molecular  Genetics  of  18-22 

Eukaryotic  Cells  and  Their 
Viruses— Martin 


SUMMARY  OF  PROGRAM 

Laboratory  and  Clinical  Research,  NIAID 

October  1,  1978  through  October  1,  1979 

Office  of  the  Scientific  Director 

The  Annual  Report  of  the  Intramural  Research  Program  contains  individual  sum- 
maries of  research  projects  in  the  twelve  laboratories  which  constitute  the 
research  component  of  the  National  Institute  of  Allergy  and  Infectious 
Diseases.  Administrative  responsibility  for  the  Intramural  Research  Program 
resides  in  the  Office  of  the  Scientific  Director  (OSD).  The  OSD  has  been  re- 
organized during  the  period  of  the  past  twelve  to  eighteen  months  in  order  to 
provide  for  more  effective  management.  The  reorganization  also  allowed  for 
distribution  of  responsibility  and  authority  to  all  working  levels. 

Mr.  Charles  Cri swell  has  served  as  the  Administration  Officer  in  charge  of 
Intramural  Operations.  Three  Administrative  Assistants  have  been  appointed, 
each  of  whom  report  to  his  office.  These  assistants,  Mrs.  Helen  Bednarek 
(Buildings  5  and  8),  Mrs.  Cathy  Sabo  (Building  7)  and  Mrs.  Bea  McKinley 
(Building  10)  have  assumed  responsibility  for  all  personnel,  budgetary  and 
other  administrative  matters  in  each  of  their  respective  areas.  Assistance 
has  been  provided  to  each  of  the  Administrative  Assistants  through  the  assinn- 
ment  of  one  procurement  technician  and  one  clerk  typist  to  perform  administra- 
tive functions  for  all  laboratories  in  each  of  the  geographic  areas.  Also  re- 
porting to  Mr.  Criswell  is  Mr.  Carter  Smith  who  serves  as  Head  of  the  Animal 
Care  Section  which  currently  consists  of  19  animal  caretakers  among  which  two 
serve  as  team  leaders.  The  animal  care  operations  at  the  Bethesda  campus  have 
been  the  direct  responsibility  of  this  Animal  Care  Section.  Durinn  the  oast 
year  a  new  office  has  been  added  in  OSD.  The  Editorial  Office  headed  by  Mrs. 
Betty  Sylvester  (Editor)  is  now  assisted  by  two  editorial  assistants.  This 
office  utilizes  the  most  modern  of  word  processing  equipment  to  provide  support 
primarily  for  the  production  of  manuscripts.  Currently,  they  have  assumed  a 
workload  sufficient  to  take  care  of  the  needs  of  four  of  the  in-house  Bethesda 
campus  laboratories.  It  is  hoped  that  sufficient  resources  will  be  available 
to  expand  their  services  to  provide  support  to  all  intramural  laboratories. 

A  new  office  of  Special  Assistant  to  the  Scientific  Director  has  been  establish- 
ed to  provide  for  direct  responsibility  for  Safety  and  EEO  programs  in  the  IRP. 
Dr.  Katherine  Cook  Jaouni  has  been  appointed  to  this  position  and  has  performed 
in  a  remarkably  good  manner.  Of  particular  importance  was  the  development  of 
a  Minority  Biomedical  Sciences  Program  which  brought  forty  young  undergraduate 
minority  scientists  to  Washington  for  a  meeting  in  the  spring  of  1979.  From 
amongst  this  group,  ten  were  selected  to  participate  in  research  activities  of 
the  Intramural  Program  in  the  summer  of  1979.  The  program  was  outstandingly 
successful  and  will  be  the  forerunner  of  similar  programs  to  be  developed  in 
future  years. 

The  Rocky  Mountain  Laboratory  in  Hamilton,  Montana  was  reorganized  this  year 

IS-  r 


with   the  plan   finally  approved  formally  on  March   16,   1979.     This   resulted  in 
the  Division  of  this   large  laboratory  complex  at  RML  into  three  scientific 
laboratories,   the  Laboratory  of  Persistent  Viral    Diseases,   the  Laboratory  of 
Microbial    Structure  and  Function  and  the  Epidemioloay  Branch.      In  addition, 
the  Administrative  activities  of  RML  were  assigned  to   the  Operations   Branch  - 
RML.     Mr.    Robert  Steiner  serves   as   Chief  of  the  Operations   Branch.     Currently, 
three  acting  Lab  Chiefs   are  operating  the   three  new  laboratory  facilities   at 
RML  and  a  process  of  search  and  selection   is   underway  to  identify  permanent 
laboratory  chiefs   for  these  three  new  laboratory  activities.     The   reorganiza- 
tion of  RML  into  three  scientific  areas  of  research  will    allow  for  a  focusing 
of  research  endeavor  and  allow  for  the  build-up  of  research  expertise  at  RML. 
It  is  anticipated  that  up  to   15  new  scientists  will    be  added  to  the  Laboratory. 
Because  of  restriction  in   total    number  of  personnel    allowed  within  the  budnet 
for  assignment  at  RML,   this  will    require  a  concomitant  reduction  of  support 
personnel.      It  is  anticipated  that  loss  of  support  personnel   will   be  offset 
through  the  use  of  local   contracts   to  provide  needed  services   to  the  Rocky 
Mountain  Laboratory. 

The   Laboratory  of  Viral    Diseases  was   divided  during  this  year  in  order  to 
allow  Dr.   Wallace  Rowe,   Chief  of  LVD,   to  direct  his  energies   to  the  areas  of 
personal    concern  and   research   interests.     The  Laboratory  had  been  difficult  to 
manage  because  of  the  division  of  its   laboratory  members   into   two   geographic 
areas.      The  majority  of  the  members  of  the  Staff  located  in   Buildinn  7  will   be 
retained  in   LVD  and  work  directly  with   Dr.    Rowe.     The  members  of  the  staff 
which  had  been   located  in  Building  5  have  temporarily  been  assianed  to  the 
Office  of  the  Scientific  Director. 

During  the  past  year,   several    Intramural    Laboratories  were  reviewed  by  the 
Board  of  Scientific  Counselors.     They  reviewed  the  Laboratory  of  Parasitic 
Diseases,   the  Laboratory  of  Infectious   Diseases,   the   Laboratory  of  Viral    Dis- 
eases,  the  Laboratory  of  Streptococcal   Diseases   and  the  Laboratory  of  Biolonv 
of  Viruses.      In  each   instance,   up  to  six  Ad  Hoc  Consultants  were  added  to  the 
Board  of  Scientific  Counselors   in  order  to  provide  greater  expertise   in   the 
review  of  each  of  the   laboratories.      Each   investigator,   including  both   tenured 
and  nontenured  investigators   in  each   laboratory  submitted  a  written   report  of 
their  ongoing  and  future  work.     At  the  time  of  the  on-site  visit  by  the  Board 
of  Scientific  Counselors   and  its  Ad  Hoc  members,   there  was   a  neneral   presenta- 
tion of  Laboratory  activities  but,   in  addition,   each   investigator,  both  tenured 
and  nontenured,  was   interviewed  for  a  period  of  an  hour  by  at  least  two  members 
of  the  Board.      This   format  has   allowed  an  intensive  evaluation  of  overall    pro- 
gram and  individual    contributions   to  programs  within  each  of  the  laboratories. 
The  Board  of  Scientific  Counselors  has  been  instrumental    in  providinq  useful 
suggestions   and  recommendations   following  each  of  their  visits   to  the   labora- 
tories . 

The  scientific  accomplishments  of  the   Intramural    Program  have  been  exciting. 
Many  members  of  the  Staff  have  received  significant  awards   and  recognition 
from  their  peers  and  from  the  appropriate  scientific  organizations.     Perhaps 
the  most  significant  award  of  all   was   the  receipt  of  the  Paul    Ehrlich  prize 
with   its   sizeable  cash  award  given  to  Dr.   "all ace  Rowe   for  his  work   in  the 
fields  of  viral    leukemia  and  immunogenetics . 

13-2 


'SMITHSONIAN  SCIENCE  INFORMATION  EXCHANG E 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl-Al-00013-16  OSD 


PERIOD    COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 


Studies  of  Viral   Antigens   in  Virus-induced  Tumors 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI 

Other 


Andrew  M.   Lewis ,   Jr. 
Hubert  W.   Gerry 


OSD,   NIAID 
Staff  Fellow, 


IIAID 


COOPERATING    UNITS    (if    any) 

Charles   Kirkpatrick   (NIAID);   Arthur  Levine,   Cephas   Patch,  Alan  Rabson   (NCI 
Heiner  Westphal,    (NICHHD)   Robert  Martin   (NIAAMD) 


lab/branch 

Office  of  the  Scientific  Director 


INSTITUTE   AND    LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20014 


TOTAL  MANYEARS: 

6.5 


PROFESSIONAL: 

2.75 


3.75 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 

D  (al)  MINORS   []  (a2)  INTERVIEWS 


rjj  (b)  HUMAN  TISSUES 


•J   (c)    NEITHER 


SUMMARY   OF    WORK    (200   words    or   less   -   underline    keywords)  Jne    initial     Objectives     Of    thl'S     project 

were  to  use  human  adenoviruses  and  adenovirus   -  SV40(Ad2-240)   recombinants   as 
tools   to  study  the  genetics  of  DNA  tumor  viruses,   to  define  the   role  of  viral 
genes  and  viral    antigens   in  viral   oncogenesis  and  to  study  the  biology  of  Ad2- 
SV40  recombinants.     Due  to  the  lack  of  proper  containment  facilities   in  NIAID 
between  January  1973  and  May  1978,   there  has   been  a  5-year  disruption   (see  past 
reports)   in  the  major  thrust  of  this   project.     During  this   interval,   a  study  of 
Ad2-SV40  recombinants  associated  tumor  induction  by  these  agents  with   the  in- 
corporation  into   the  adenovirus   -   2  chromosome  of  a  specific  segment  of  SV40 
DNA.     To  begin   to  understand  the  mechanism  by  which   this  SV40  DNA  segment  conveys 
oncogenicity  to  these   recombinants,   it  was   necessary  to   understand  why  Ad2  was 
nononcogenic  for  hamsters.     Since  Ad2  will    transform  hamster  cells  in   tissue 
culture,  we  initiated  a  study  of  the  oncogenic  properties  of  Ad2  transformed 
""I:*- I?e/e!u!tS   thuS   far.lead.u!  to  suspect  that  unrecognized  host  immune 

1- 

m,,.   ; .,»  m  - - '    -     :■-  -■'■*.  segment 

trie  SV4U  genome  might  induce  functions   in hamc:tor  tumor  calls   ^t   interfere 

PHS-6040 
(Rev.    10-76) 


SrlT  1n  thechamster  reJ'ect  Ad2  transformed  cells  and  transformed  eel 
induced  tumors.  Such  a  concept  suggests  that  the  presence  of  a  specific  s 
or  the  SV40  genome  might  induce  functions   in hamct-or  tumor  tpIIc   tbai   int 

3HS-6040  ltf-d 


with  the   rejection  process.      Future  studies  will   be  directed  toward  more  care- 
fully defining  these  concepts. 

Project  Description 

Major   Findings :     Oncogenicity  of  SV4Q  deletion  mutants   that  induce  altered  17K 
t_  proteins 

The  nondefective  hybrid,  Ad2+ND/,,  which  contains   the  segment  of  the  SV40  genome 
between  map  position  0.11    and  0.59,   induces   tumors   in  hamsters  after  inactiva- 
tion  by  UV  light   (see  1977-1978  Report).     Since  nonhybrid  Ad2  and  the  Ad2+ND2 
hybrid  which  contains   the  segment  of  the  SV40  genome  between  map  position 
0.11    and  0.43  do   not  induce   tumors  after  UV  inacti vation ,   these  results   imply 
that  the  segment  of  the  SV40   genome  between  0.43  and  0.59  is   required  for 
tumor  induction.     To  more  carefully  define   the  region  of  the  SV40  genome  asso- 
ciated with  tumor  induction,  we  studied  the  tumor-inducing  capacity  of  a   number 
of  SV40  mutants   containing  deletions  of  the  early  region  between  map  position 
0.54  and  0.59.      These  mutants   induce  a  normal    92,000  dalton  T  protein,  but 
induce  altered  17,000  t  proteins.     Twenty  to  90%  of  hamsters   inoculated  as   new- 
borns with   SV40  mutants  A883,  A885,  A886     A890,     A2001        andA2005  developed 
tumors  after  a  6   to  12  month   latent  period.      For  this  study,   SV40  wild-type 
virus   produced  tumors   in  90%  of  inoculated  hamsters  within  8  months.     The  dif- 
ferences  in  the   latent  period  prior  to  tumor  development  by  SV40  compared  with 
the  SV40  mutants  was   statistically  significant.     Together  with  the  studies  on 
tumor  induction  by  the  Ad2+ND.   hybrid,   these  studies   imply  that  the  DNA  sequen- 
ces  comprising  the  initial    portion  of  the  early  SV40  genome  between  map  posi- 
tions  0.55  and  0.54  are  not  essential    for  tumor  induction  in  hamsters.     Although 
the  17  k   t-protein  which  is   encoded  by  the  SV40  region  between  map  position 
0.65  and  0.54  is   not  essential    for  tumor  formation,   it  does   appear  to  reduce 
the  latent  period  prior  to   tumor  development. 

Host  response  to  adenovi  rus   2_  transformed  hamster  embryo  eel  1 s 

The  finding  that  a  segment  of  SV40  DNA,   by  recombining  with  the  nononcogenic 
Ad2  genome,   conveyed  tumor-inducing  properties   to  the  Ad2  ND.   raised  questions 
about  the  mechanism  by  which  this   change  to  oncogenicity  was   effected.     Others 
have  shown  that  Ad2     transformed  rat  cells  were   incapable  of  establishing 
tumors   in  immunocompetent  rats.     Since  there  appears   to  be  little  or  no  dif- 
ference between  the  in  vitro  transforming  efficiency  of  the  Ad2  ND„  and  Ad2  ND. 

recombinants   and  nonhybrid  A2,   it  seemed  reasonable  to  assume  that  the  change 
toward  oncogenicity  could  be  a  reflection  of  the  manner  in  which  cells   trans- 
formed in  vivo  by  hybrid  and  nonhybrid  virions   interacted  with   the  host  immune 
system.     As   there  was   little  data  about  the  tumor  inducing  capacity  of  Ad2 
transformed  hamster  cells,   it  was   necessary  to  examine   this   problem  before  the  re- 
combinant could  be  considered. 

Ad2  inactivated  by  UV  light  was   used  to  transform  LSH  hamster  embryo  cells. 
Evaluation  of  transformed  cell    lines   produced  by  Ad2   (strain  adenoid  6)  or 
isolates  of  Ad2  obtained  from  children  in  Washington,   D.    C. ,   or  West  Bengal, 
India,  showed  that  13  of  15  lines   induced  tumors  when  injected  into  newborn 
inbred  LSH  or  randomly  bred  NIH  hamsters    (107  cells/hamster)  with  a  median 
tumor  incidence   in  syngeneic  newborns   of  55%   (range  8  to   100%).      Six  out  of  6 

_    I8-4 


of  these  cell    lines   did  not  induce  tumors  when  10     cells  were  injected  s.c. 
into  weanling  animals  over  21    days  old.      In  contrast  to  the  Ad2- trans  formed 
lines,  similarly  derived  cell    lines   transformed  by  Adl 2  or  SV40  uniformly 
produced  tumors   in  weanlings   following  s.c.   inoculation  of  10?  cells.     Trans- 
plantation of  tumors   from  animals   injected  as   newborns  with  Ad2-transformed 
cell    lines   to  other  newborns  was   readily  accomplished  in  639  of  640,    (99.8%) 
inoculated  newborns.      However,  only  53  of  467   (11.3%)  weanling  hamsters 
challenged  with  these  tumor  lines  developed  neoplasms.     Each  of  these  Ad2- 
transformed  lines  contained  Ad2  T  antigen  detectable  by  complement  fixation 
or  immunofluorescence.     None  of  6  lines  contained  Ad2  by  Sendai    fusion  with 
human  embryonic  kidney   (HEK)  cells   or  other  contaminating  agents  by  a  variety 
of  assays.     The  difference  in  oncogenicity  in  weanling  hamsters  of  Ad2-trans- 
formed  cells  compared  to  Adl 2  -  and  SV40-trans formed  cells  was   not  related 
to  the  dose  of  tumor  injected  or  to  differences   in  in  vitro  growth  properties 
of  the  cell    lines    (e.g.,   doubling  times,  saturation  densities,  growth  in 
spinner,  or  colony  formation  in  soft  agar).     The  tumor  inducing  capacity  of 
Ad2  newborn  tumor  lines   for  newborn  and  weanling  hamsters  was   not  related  to 
the  dose  of  tumor  injected.   With  the  two  tumor  lines   tested,  weanling  rejec- 
tion af  newborn  tumor  transplants  could  not  be  overcome  by  inocula  contain- 
25  times   the  usual    dose  of  tumor  suspension.     These  same  two  newborn  tumor 
lines   produced  progressively  enlarging  neoplasms   in   17-77%  of  syngeneic 
weanling  hamsters  which  had  been   thymectomized  as   newborns.     The  lack  of 
oncogenecity  of  hamster  cells   transformed  j_n  vi tro  by  Ad2  supported  our  spec- 
ulation that  the  SV40  genome  in  cells   transformed  i  n  vi  vo  by  the  Ad2+ND.   re- 
combinant could  be  altering  immunological    determinants   involved  in  tumor  cell 
rejection. 

Viral    DNA  sequences  and  gene  products   in  hamster  cells   transformed  by  Ad2. 

Complementary  strand-specific  adenovirus   DNA  of  full    length  or  from  endonu- 
clease  Bam  HI   fragments  was   used  as  a  probe  to  estimate  the  fractional    repre- 
sentation and  abundance  of  viral   sequences   in  five  hamster  cell    lines 
AD2HEI-5  transformed  with  UV-inacti vated  Ad2.     The  fraction  of  the  viral 
genome  present  in  the  five  transformed  cell    lines  varied  from  44%  in  the 
Ad2HE5  cell    line  to  84%  in  the  Ad2HE3  cell    line.     The  number  of  viral    DNA 
copies  per  diploid  cell    equivalent  ranged  from  1.8  in  the  Ad2HEl    line  to  7.1 
in  the  Ad2HE4  line. 

35 
In  vivo  labeling  with       S-methionine   followed  by  immunoprecipitation  with  an 

antiserum  against  Ad2  early  proteins  revealed  viral-specific  polypeptides 

with  molecular  weights  of  42,000  to   58,000  in  extracts   from  all    five  hamster 

cell   lines.     Several   other  early  viral    polypeptides  were  detected  in  some  of 

the  Ad2  transformed  hamster  cell    lines. 

Studies  are  underway  to  determine   the  specific  regions  of  the  Ad2  genome  that 
are  contained  in  the  Ad2HEl    line  of  Ad2  transformed  hamster  embryo  cells  and 
to  determine  which  of  the  messenger  RNA's   produced  in  these  cells  are  coded 
for  by  these  Ad2  DNA  containing  regions.      Preliminary  results   indicate  that 
RNA  coded  for  by  the  early  region  on   the  left  hand  end  of  the   right  strand 
(the  "transforming"   region)   is   present;   in  addition,   RNAs   representing 
regions  of  the  Ad2  genome  transcribed  late  during  infection   (i.e.   after  viral 
DNA  replication  begins)  are  also  present  but  appear  in  patterns   not  typical 
of  RNA  from  cells   productively  infected  with  Ad2. 

18-5 


Role  of  the  hamster  cellular  immune  response  in  Ad 2  transformed  cell   oncogenesis 

The  tumor-inducing  capacity  of  two  Ad2  transformed  hamster  cell-induced  lines 
(Ad2HTL3  and  Ad2HTL6)  was   tested  in  immunocompromised  hosts.     An  average  of 
35.2%  of  neonatally  thymectomi zed,  syngeneic,  weanling  hamsters  developed 
progressively  enlarging  tumors  when  injected  subcutaneously  with  tumor  suspen- 
sions  prepared  from  these  lines,  while  no  tumors  were  observed  in  normal,   syn- 
geneic, weanling  hamsters  challenged  with   the  same  inocula.     The  susceptibility 
of  neonatally  thymectomi zed  hamsters   to  tumor  challenge  was   directly  related 
to  the  degree  of  immunosuppression  observed  following  thymectomy  as   indicated 
by  the  amplitude  of  the  in  vitro  response  of  whole  blood  cultures   to  concana- 
valin  A.      Pretreatment  of  thymectomi zed  weanlings  with  syngeneic  adult  lymphoid 
cells   resultedin  a  significant  reduction  in  tumor  susceptibility   (p  =  0.03). 
These  findings   suggest  that  the  maturation  of  a   thymus-dependent  cell-mediated 
immune   response  in  hamsters  during  the  first  21    days  of  life   results   in  the 
rejection  of  Ad2- transformed  cells. 

Developmental    hamster  immunobioloqy  related  to   tumor  rejection  in  the  Ad 2 
transformed  hamster  cell   system 

Transplantation  of  the  Ad2HTL  newborn  tumor  line  to  suckling  hamsters   1-15  days 
old  demonstrated  that  maturation  events   in  the  hamsters   immune  system  between 
6  and  7  days  were  responsible  for  tumor  rejection,   \lery  little  data  has  been 
published  concerning   the  ontogeny  of  the  hamster  immune   response;  however, 
based  on  data   from  other  species,   it  has   been  predicted  that  hamster  thymus 
and  spleen   cells   should  respond  to  T  cell   mitogens  within  a  few  days  of  birth 
and  that  suckling  hamsters   should  be  able  to  reject  skin  allografts   at  8  days 
of  age.     To  begin  to  evaluate  the  development  of  cellular  immune   responses   in 
hamsters,   cells   from  bloods  and  spleens   from  different  aged  animals  were  test- 
ed for  their  ability  to  respond  to  the  mitogen  conconavalin  A   (ConA)  and  to 
respond  in   the  mixed  lymphocyte  reaction.     The  results  of  these  studies   indi- 
cate that  hamster  spleen  cells   begin  to  respond  to  ConA  by  age  5  days   and 
attained  100  percent  of  adult  type   responses   by  age  15  days.      Cells   in  the 
blood  did  not  respond  to  ConA  until   age  25  days. 

Hamster  spleen   cells   did  not  begin  to  respond  in   the  mixed  lymphocyte  reaction 
until   18  to  25  days.      To  determine   if  the  lack  of  ConA  responsiveness  of  ham- 
ster spleen  cells   before  age  5  days  was  due  to  T  cell    immaturity  or  immaturity 
of  another  cell    component  which  was   required  for  this   response,   X-ray  inactiva- 
ted adherent  and  nonadhereent   (to  distinguish  between  lymphocytes  and  macro- 
phages)  adult  peritoneal    exudate  cells  were  mixed  with  spleen  cells   from  3  day 
old  hamsters   and  the  mixtures   tested  for  their  ability  to  respond  to  ConA. 
These  results   showed  that,   in  the  presence  of  populations  of  adherent  peritoneal 
exudate  cells,   3  day  old  hamster  spleen  cells   readily  responded   (stimulation 
indices  of  13  to   35)  compared  to  spleen  cells  alone   (stimulation  index  of  1    to 
2)  or  spleen  cells  mixed  with   nonadherent  cells    (stimulation  index  of  1    to   3). 
These   results   imply  that  immaturity  of  cell    populations  which  cooperate  with 
lymphocytes   in  the   response  to  ConA  are  most  likely  responsible  for  the  apparent 
immaturity  of  this   reaction   in  hamsters   less   than   5  days  old.  . 

As  an  in  vivo  corollary  to   the  evaluation  of  immune  cell    responses   to  mitogen 

.    18-6 


and  foreign  antigens,   the  inflammatory  response  to  tumor  development  in  new- 
born, weanlings  and  weanlings   thymectomi zed  during  the  first  24  hours  of  life 
was   examined  histopathologically  in  animals   injected  with  suspensions  of 
Ad2HTL3  and  Ad2HTL6  tumors.      In  newborn  animals  multiple  tumor  nodules   devel- 
oped within  5  days. and  were  quickly  enclosed  by  highly  vascular  connective 
tissue.     New  capillaries  were  seen  entering  these  nodules.     During  the  first 
few  days   inflammatory  cells   consisted  mainly  of  polymorphonuclear  leukocytes 
with  some  lymphocytes  and  histocytes  around  the  forming  nodules.      In  sections 
with  cells   invading  the  surrounding  connective  tissue,  there  was   no  inflam- 
matory response.     The  response  to  tumor  development  in  thymectomi zed  weanlings 
was  similar  to  that  observed  in  newborns.      In  normal   weanlings,   tumor  nodules 
developed  early  but  were  regressing  by  5  days.     Some  new  vessel    formation  was 
present  during  the  first  few  days  of  tumor  nodule  development  but  this  was 
less  impressive  than  the  intense  vascular  response  noted  in  newborns.     Inflam- 
matory cells  consisted  of  polymorphonuclear  leukocytes  during  the  first  day 
followed  by  the  rapid  appearance  of  an   intense  lymphohistiocytic  infiltrate. 
After  7  days,   the  tumor  nodules   consisted  of  mostly  granulation  tissue  with 
many  multinucleated  giant  cells  and  focal   calcification. 

Immunogenicity  of  Ad 2  transformed  hamster  cells  and  transformed  cell-induced 
tumor  li  nes 

To  determine  the  possible  role  of  Ad2  transplantation  antigen   (TSTA)   in  the 
rejection  of  tumors   transplanted  to  weanling  hamsters,   several   types  of 
studies  are  underway.     An  assay  has  been  developed  for  Ad2  TSTA  using  an  Ad2 
tumor  line   (Ad2HTLl )     which  had  been  adapted  to  grow  in  adult  hamsters.      Im- 
munization with  Ad2  significantly  protected   (defined  as  a   resistance  index 
*RI)   greater  than  10)   hamsters   against  a  challenge  with  Ad2HTLl .      Immunization 
with  SV40  did  not  convey  significant  protection   (RI   between   1   to  10)   in  these 
experiments.     This  assay  is   being  used  to  study  the  immunogenicity  of  Ad2 
transformed  cell    lines  and  newborn  and  weanling  tumor  lines   developed  from 
these  transformed  cells.      Results   thus   far  indicate  that  3  Ad2  transformed 
cell    lines    (Ad2HEl  ,  Ad2HE3,  Ad2HE6)   and  the  tumor  lines   established  from  them 
(Ad2HTLl,  Ad2HTL3,  Ad2HTL6)  contain  a  common  Ad2  TSTA  which  during  immuniza- 
tion with  x-irradiated     cells  conveys   protection  to  hamsters   challenged  with 
Ad2HTLl .     Differences   in  RI  did  not  correlate  with  the  differences   in  the 
transplantability  of  the  newborn  tumor  lines   to  newborn  and  weanling  hamsters. 
Adult  hamsters   that  had  rejected  tumors   induced  by  viable  Ad2HTL3  and  Ad2HTL6 
cells  were  also  protected  when  challenged  with  Ad2HTLl .     These  results   show 
that  whether  they  are  accepted  or  rejected  by  weanling  hamsters,   each  of  these 
lines  contains  detectable  Ad2  TSTA.     Such  findings  appear  to  exclude  the 
possibility  that  certain  tumor  lines  can  be  transplanted  to  older  animals 
because  they  lose  detectable  Ad2  TSTA.     To  determine  whether  a  reducation  in 
the  concentration  of  Ad2  TSTA  is   responsible  for  the  transplantability  of 
certain  newborn  tumor  lines   to  adults,   the  amount  of  immunizing  antigen  in 
suspensions  of  different  tumors   is   being  evaluated. 

Difference  i_n  th_e  transplantability  of  virus-induced  neoplastic  cells   i_n 
in-bred  hamsters 

Tumor  cells   behave  like  normal    cells   in  that  they  are  usually  rejected  when 
transplanted  as  allografts   to  histoi ncompatible  hosts. Since  hamsters   are 

-  18-7 


suspected  to  lack   resistance  to  transplantable  tumors  of  viral   origin,  we 
have  been  studying  the  transplantabi lity  of  our  transformed  cells   and  tumor 
lines   to  different  strains  of  inbred  hamsters.      Eleven  Ad2,  Adl2  and  SV40 
transformed  LSH  cell    lines  and  LSH  tumor  lines   established  from  them  are  being 
transplanted  to  syngenic  LSH  hamsters,   CB  hamsters  which  are  histocompatibil- 
ity complex   (MHC)   disparate,  and  PD4  hamsters  which  are  MHC  identical    but  dis- 
parate at  minor  H  loci.      Initial    findings  show  that  Ad2  transformed  cells   pro- 
duced tumors   only  in  newborn   LSH  hamsters.      Ad2   tumor  lines    (Ad2HTLl ,   Ad2HTL3) 
adopted  to  grow  in  LSH  adults   by  serial    passage  in  neonatal    hamsters   produced 
tumors   readily   (TPD.q  1 02 • 3  to   104-97  0.2  ml   of  tumor  suspension)  when  trans- 
planted to  LSH  adults.      These  same  lines   produced  tumors   inefficiently 
(TPD-0  l_0°-°  to  10^-^/0.2  ml)  when  transplanted  to  adult  CB  hamsters  and  of 
intermediate  efficiency   (TPPr-   101-4  to  103-7/0.2  ml)  when  transplanted  to 
PD.   hamsters.     When  10°  to      lb7  Adl2  transformed  LSH  cells  were  inoculated 
into  adult  LSH  hamsters   84%  developed  tumors.     When  the  same  cell   doses  were 
injected  into  CB  hamsters  only  33%  developed  tumors.     Tumor  lines   established 
in  LSH  hamsters   from  Adl2  transformed  cells  produced  tumors   in  a  pattern  sim- 
ilar to   the  Ad2  tumor  lines  when  transplanted  to  adult  LSH,   CB,   and  PD.  ham- 
sters.    When   106  and  107  SV40  transformed  LSH  cells  were  injected  into  adult 
LSH  and  CB  hamsters,   100%  and  97%  respectively  developed  tumors.     SV40  tumor 
lines   produced  tumors  with  equal   efficiency  in  all    3     hamster  strains.     Thus, 
our  Ad2  induced  neoplastic  LSH  cells   produced  tumors   only  in  immunoimmature 
syngeneic  LSH  hamsters;  our  Adl2  induced  neoplastic  LSH  cells   produced  tumors 
efficiently  in  both   immunoimmature  and  immunomature  syngeneic  LSH  hamsters 
but  produced  tumors   less   efficiently  in  allogeneic  hamsters;  our  SV40  induced 
neoplastic  LSH  cells   produced  tumors   efficiently  in  both  syngeneic  and  allo- 
geneic hamsters. 

Current  ideas  about  the  role  of  TSTA  and  allograft  rejection  of  viral    induced 
neoplasms   do  not  account  for  the  finding  above.      In  this   regard,  we  are  con- 
sidering the  possibility  that  the  differences   in   the  transplantabi 1 ity  of 
these  various   cell    lines  are  a  reflection  of  the   unrecognized  manner  in  which 
tumor  and  allograft  determinants  on  the   surface  of  hamster  cells  are  altered 
during  transformation  events  by  a  particular  viral    agent.     Studies  are  under- 
way to  further  clarify  these  findings. 

Signi  fi  cance  to  Biomedi  cal    Research 

The  results   of  our  recent  studies   impinge  on  tumor  immunology  and  the  possible 
role  of  antigenic  determinants  on  cell   surfaces   in  determining  the  outcome  of 
viral-induced  malignancy.     The  availability  of  well-characterized  tumor  lines, 
which  are  transplantable  to  both  immunoimmature  and  immunomature  hamsters  and 
tumor  lines,  which  are  transplantable  to  immunoimmature  but  are  consistently 
rejected  by  immunomature  animals   provides  a  useful    system  for  identifying  and 
studying  the  maturation  of  the  functions   of  the  hamster  cellular  immune  system 
responsible  for  the  rejection  of  viral -induced  tumors.     The  central    theme  of 
tumor  virology  is   the  concept  that  viruses   induce  the  malignant  state   (i.e., 
the  ability  to  produce  tumors   in  a  susceptible  host)   in  cells   by  a  process 
called  transformation.     A  number  of  studies    (including  our  own  work  with   the 
nondefective  Ad2-SV40  hybrids)  with  different  agents   have  associated  trans- 
formation and  tumor  induction  with  the  functioning  of  a   specific  region  of  the 
viral    genome  and  in  some  cases,  with  a  specific  gene  product.      Paridoxi  cal  ly , 

—  18-8 


many  virus-transformed  cells  which  possess   the  properties   ascribed  to  the 
transformed  state  fail   to  produce  tumors  when   injected  into  either  syngeneic 
immunomature  or  immunoimmature  hosts.     The   reasons   for  this   lack  of  transform- 
ed cell    oncogenicity  for  a  host  that  should  be  susceptible  are  poorly  under- 
stood.    Our  studies  with   viral    transformed  hamster  cells   provide  data  which 
pertain   to  this  question.     At  this  juncture,  we   interpret  our  results  with 
the  Ad2  and  SV40  transformed  LSH  hamster  cell   system  as  suggesting  that,   dur- 
ing the  process  of  transformation,   antigenic  determinants  on  cell   surfaces 
in  addition  to  TSTA  are  altered.      Some  of  these  determinants   are  associated 
with  graft  rejection  by  allogeneic  hosts;   others   are  associated  with   the   re- 
cognition and  rejection  of  tumor  cells   by  syngeneic  hosts.     An   understanding 
of  these  cell   surface  modulations   and  the   role  of  the  viral    genome   in   the  al- 
teration of  their  function  would  be  a  significant  step  in   understanding   the 
mechanism     by  which  viruses   convert  normal    cells   to  malignant  ones. 

Publ i cations 


Patch,  C.  T. ,  Levine,  A.S.,  and  Lewis,  A.M.,  Jr.:  The  adenovirus-SV40  hybrid 
viruses.      Comprehensive  Virology,  ]_3:     495-542,   1979. 

Johansson,  K. ,  Persson,  H.,  Lewis,  A.M.,  Jr.,  Petterson,  U.,  Tibbetts,  C.  and 
Philipson,  L.  :  Viral  DNA  sequences  and  gene  products  in  hamster  cells  trans- 
formed by  adenovirus   type  2.   J.   of  Virol.,   27:     628-539,   1978. 

Cook,  J.L.  and  Lewis,  A.  M. ,  Jr.:  Host  response  to  adenovirus  2  -  transformed 
hamster  embryo  cells.      Cancer  Res.,   39:     1455-1461,   1979. 

Lewis,  A.M.,  Jr.  and  Martin,  R.  G. :  The  oncogenicity  of  simian  virus  40  dele- 
tion mutants  that  induce  altered  17k  t-proteins.  Proc.  Nat.  Acad,  of  Sci  .  ,  in 
press. 

Patch,   C.T.,   Hauser,   J.,   Lewis,  A.   M. ,   Jr.,  and  Levine,  A.S.  :     A  method  for 
determining  the  extent  and  copy  number  of  overlapping  and  non-overlapping 
segments  of  integrated  viral    genomes.      J.   of  Virol.,   in  press. 

Cook,  J.L.   and  Lewis,   A.M.,   Jr.:     Age-related  and  thymus-dependent  rejection 
of  adenovirus  2  -  transformed  cell    tumors   in   the  Syrian   hamster.     Cancer  Res., 
in  press. 

Lewis,   A.   M.,   Jr.   and  Cook,   J.L.:     The  association  of  tumor  induction  by  ultra- 
violet light  inactivated  adenovirus   2-SV40  recombinants  with  a  specific  seg- 
ment of  SV40  DNA.      J.   of  Nat.    Cancer  Inst.,   in  press. 


18-9 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZCH-AI-00018-13  OSD 


PERIOD  COVERED 


TITLE  OF  PROJECT  (SO  characters  or  less) 

Biological  and  Biochemical  Characterization  of  Human  Papovaviruses. 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:      Kenneth  K.  Takemoto  OSD,  NIAID 

OTHER:    Sheila  Bond  OSD,  NIAID 

Tatsuo  Miyamura,  Visiting  Fellow   OSD,  NIAID 


COOPERATING  UNITE 


Peter  Howley,  Ming  Fan  Law,  LP,  NCI;  George  C.  Fareed,  UCLA,  Los  Angeles,  CA: 
Hawley  Linke,  UCLA,  Los  Angeles,  CA.,  L.W.  Law,  NCI. 


LAB/ BRANCH 

Office  of  the  Scientific  Director 


SECTION 

Cellular  Virology  Section 


INSTITUTE   AND    LOCATION 


IIAID.   NIH.   Bethesda.   Maryland     20205 


TOTAL  MANYEARS: 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 
G  (a)   HUMAN  SUBJECTS 

D  (al)  MINORS   Q  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


SUMMARY  OF  WORK  (200  words  or 

The  principal  goal 
biochemical  characteriz 
the  past  six  years,  mos 
toward  studies  with  BKV 
of  various  types.  JCV, 
vitro,  and  therefore  ha 


year,  however,  a  major 
different  readily  avail 
This  will  now  enable  us 
this  important  virus. 


less  -  underline  keywords) 

of  this  unit  continues  to  be  the 
ation  of  the  human  papovaviruses, 
t  of  the  research  efforts  of  this 

since  this  virus  could  easily  be 

on  the  other  hand,  has  been  diffi 
s  remained  a  relatively  unknown  vi 
advance  was  made  when  we  were  able 
able  cells,  human  amnion  and  human 

to  conduct  detailed  investigation 


detailed  biological  and 
BKV  and  JCV.  During 
unit  were  directed 
grown  in  cell  cultures 
cult  to  propagate  i_n 
rus.  During  the  past 
to  grow  JCV  in  two 
embryonic  kidney  cells, 
s  on  the  biology  of 


Studies  are  continuing  on  the  mechanism  of  persistent  infection  of  human 
fetal  brain  cells  by  BKV.   It  has  been  established  that  viral  genomes  exist  in 
episomal  form  in  these  cells,  and  that  rare  cells  in  the  culture  spontaneously 
undergo  lytic  cycles  to  produce  infectious  progeny.  This  type  of  virus-cell 
interaction  may  explain  how  thesp  ^a^va viruses  persist  indefinitely  in 

' PHS-6040 
(Rev.    10-76) 


the  human 


PROJECT  DESCRIPTION 

Persistent  BK  virus  infection.  We  reported  last  year  on  the  establishment  of 
a  unique,  heretofore  unrecognized  type  of  persistent  viral  infection  in  cell 
culture.  In  this  system,  BK  virus  was  shown  to  persist  indefinitely  in  trans- 
formed human  fetal  brain  cells.  The  mechanism  whereby  virus  persists  in  the 
cells  has  been  determined.  It  was  found  that  all  cloned  lines  contained  viral 
DNA  in  non-integrated,  episomal  form.  By  restriction  endonuclease  analysis, 
the  episomal  viral  DNA  was  shown  to  be  identical  to  parental  DNA  and  was 
infectious.  The  number  of  epi somes  per  cell  was  between  5  to  10.  It  is 
postulated  that  in  an  occasional  cell,  the  episomal  DNA  is  spontaneously 
"induced"  to  go  into  a  complete  replicative  cycle  and  viral  progeny  is 
produced.  The  persistent  state  is  thus  perpetuated,  even  in  the  presence  of 
potent  antiserum.  We  speculate  that  this  type  of  virus-cell  interaction  can 
perhaps  explain  how  these  viruses  persist  in  their  natural  hosts. 

Another  unusual  feature  of  these  cells  is  that  they  are  transformed  and 
have  most  of  the  properties  of  transformed  cells.  They  grow  in  low  serum 
medium,  form  colonies  in  soft  agar,  are  immortal,  and  produce  tumors  in  nude 
mice.  However,  unlike  the  usual  papovavirus  transformed  cells,  they  do  not 
contain  T-antigens  detectable  by  immunofluorescence.  By  immunoprecipitation 
and  analysis  in  gels,  there  appears  to  be  an  abberant  T-protein  produced  with 
a  molecular  size  in  the  range  of  40K.  Whether  this  protein  is  of  viral  or 
cellular  origin  is  being  determined. 

Replication  of  JC  virus  in  non-fetal  human  cells.  JC  virus  (JCV)  has  now  been 
shown  to  be  the  causative  agent  in  progressive  multifocal  leukoencephalopathy 
(PML),  having  been  isolated  from  or  identified  in  over  30  cases  of  the  disease. 
Studies  on  this  important  virus  have  been  severely  limited  due  mainly  to  the 
difficulty  in  growing  the  virus;  until  recently,  it  has  been  grown  only  in 
human  fetal  glial  cells,  which  are  difficult  to  obtain.  In  the  past  year,  we 
have  successfully  adapted  JCV  to  grow  in  2  types  of  commercially  available 
cells,  human  embryonic  kidney  and  human  amnion  cells.  Both  kinds  of  cells 
support  JCV  replication  with  full  yields  of  virus.  Serological  and  biochemi- 
cal analysis  of  the  adapted  JCV  showed  that  the  virus  was  identical  to  the 
original  strain  grown  in  human  fetal  glial  cells.  Detailed  biological  and 
biochemical  investigations  on  this  virus  can  now  be  performed  and  a  more 
complete  characterization  of  JCV  can  be  expected  within  the  next  few  years. 

BKV  helper  function  for  adenovirus  replication.  Previous  comparative  studies 
on  common  early  viral  functions  between  the  simian  and  human  papovavi ruses, 
SV40  and  BKV,  have  established  a  relatedness  between  the  2  viruses.  Thus, 
they  share  immunologically  related  T-antigens  and  BKV  can  complement  the 
growth  of  temperature  sensitive  SV40  mutants  which  dre   defective  in  early  gene 
functions.  To  understand  more  fully  the  functional  similarities  between  BKV 
and  SV40,  the  helper  function  for  adenovirus  growth  by  BKV  in  non-permissive 
monkey  cells  was  studied.  During  the  course  of  these  experiments,  it  was 
observed  that  replication  of  BKV  in  CV-1  monkey  cells  was  primarily  abortive 
at  37  C.  However,  when  infected  cells  were  incubated  at  high  temperature 
(40  C)  the  cells  became  permissive  for  BKV  replication  and  high  virus  yields 
were  obtained. 

--I8-II 


Based  on  this  finding,  experiments  to  determine  helper  function  for 
adenovirus  growth  in  monkey  cells  by  BKV  were  performed  at  abortive  (37  C)  and 
permissive  (40°C)  temperatures.  Results  of  these  experiments  showed  that 
while  enhancement  of  adenovirus  replication  occurred  in  cells  co-infected  with 
BKV  and  incubated  at  37  C,  there  was  a  10-fold  greater  degree  of  enhancement 
at  40  C.  This  was  probably  due  to  an  increased  production  of  T-antigen  at 
the  higher  temperature.  These  experiments  thus  provide  additional  information 
on  the  high  degree  of  relatedness  between  the  simian  and  human  papovavi ruses 
and  is  in  agreement  with  the  recent  findings  of  extensive  homology  between  the 
genomes  of  the  2  viruses  obtained  by  DNA  sequencing  studies. 

Common  sequences  in  the  genomes  of  JC,  BK,  and  SV40.  In  studies  done  in 
collaboration  with  Drs.  Law  and  Howley  (NCI),  the  DNAs  of  the  primate  papova- 
viruses  SV40,  BK,  and  JC  were  analyzed  for  nucleotide  sequence  homology.  Under 
non-stringent  conditions,  extensive  homology  was  found  throughout  the  genomes 
of  the  3  viruses,  which  correlate  well  with  the  previous  observations  that  the 
structural  (viral)  as  well  as  non-structural  (t-proteins)  antigens  of  the 
primate  papovavirus  are  immunologically  related.  The  region  of  strongest 
homology  among  the  3  genomes  was  localized  in  the  late  region  between  0.76  to 
0.85  map  units  coding  for  VP2. 

Significance  to  Biomedical  Research.  The  human  papovavi ruses  are  ubiquitous 
viruses  which  are  world-wide  in  distribution,  infecting  children  at  an  early 
age  and  persisting  thereafter  throughout  life.  One  of  them,  JCV,  has  clearly 
been  determined  to  be  the  causative  agent  of  PML.  The  papovaviruses  represent 
a  class  of  persistent  viruses  whose  possible  role  in  chronic  diseases,  includ- 
ing cancer,  need  to  be  determined.  They  have  been  demonstrated  to  be  oncogenic 
in  tissue  culture  and  in  animals,  thereby  providing  important  models  for  the 
study  of  viral  oncogenesis. 

Proposed  course.  The  ability  to  propagate  JC  virus  in  non-fetal  cells  will 
now  enable  us  to  conduct  detailed  investigations  on  the  biology  and  biochem- 
istry of  this  virus  which  were  heretofore  not  possible.  An  immediate 
objective  is  to  analyze  the  DNA  of  JCV  virions  since  previous  data  indicated 
extensive  heterogeneity  which  probably  accounted  for  the  poor  growth  and  low 
yields  of  virus.  The  system  of  persistent  BKV  infection  in  T-antigen 
negative,  transformed  human  fetal  brain  cells  will  continue  to  be  investigated. 

Publications 


Israel,  M.A. ,  Takemoto,  K.K.,  Martin,  M.A.,  Soloman,  D.  ,  Howley,  P.M., 
Aaronson,  S.A.  and  Khoury,  G.  Evaluation  of  normal  and  neoplastic  tissue  for 
BK  virus.  Virology  90,  187-196,  1978. 

Bond,  S.B.,  Howley,  P.M.  and  Takemoto,  K.K.  Characterization  of  K  virus  and 
its  comparison  with  polyoma  virus.  J.  Virology  28_,  337-343,  1978. 

Law,  M.F.,  Takemoto,  K.K.  and  Howley,  P.M.  Characterization  of  the  genome  of 
the  murine  papovavirus  K.  J.  Virology  30,  90-97,  1979. 


18-12 


Takemoto,  K.K.,  Linke,  H.,  Miyamura,  T.  and  Fareed,  G.C.  Persistent  BK 
papovavirus  infection  of  transformed  human  fetal  brain  cells.  I.  Episomal 
viral  DNA  in  cloned  lines  deficient  in  T-antigen  expression.  J.  Virology  30, 
1177-1185,  1979. 

Takemoto,  K.K. ,  Howley,  P.M.  and  Miyamura,  T.  JC  human  papovavirus  replication 
in  human  amnion  cells.  J.  Virology  3£,  384-389,  1979. 

Law,  M.F.,  Martin,  J.D.,  Takemoto,  K.K.  and  Howley,  P.M.  The  co-linear 
alignment  of  the  genomes  of  papovaviruses  JC,  BK,  and  SV40.  Virology  (In 
Press) 

Miyamura,  T.  and  Takemoto,  K.K.  Helper  function  for  adenovirus  replication 
in  monkey  cells  by  BK  human  papovavirus.  Virology  (In  Press). 

Awards  and  Honors 


Invited  lecturer,  Virology  '79  Lecture  Series,  The  Institute  for  Medical 
Research,  Camden,  New  Jersey,  January  4,  1979.  "The  Papovavirus  Group." 

American  Society  of  Microbiologists  Symposium  on  Viruses  and  Human  Cancer, 
May,  1979,  Los  Angeles,  California.  "Human  Papovaviruses:  Search  for 
evidence  of  possible  involvement  in  human  cancer." 


18-13 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01-A1 -0001 9-05  OSD 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Studies  on  the  Treatment  of  Disease  with  the  Interferon  System 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 

OTHER: 


Hilton  B.  Levy 
Freddie  Riley 
Clarence  Corey 


Biochemist 

Chemist 

Bio-lab  technician 


OSD,  NIAID 
OSD,  NIAID 
OSD,    NIAID 


COOPERATING  UNITS:      Dr.    Arthur  Levine,   NCI;    Dr.    King   Engel ,   NIANDS;    Dr.    John 
Hooks,   NIDR;   Maj.    Edward  Stephen,   USAMRIID;   Dr.    Chester  Liu,   USAMRIID;   Maj.   Don 
Harrington,   USAMRIID;   Dr.   Martin  Lerner,   Wayne  State  Univ.;   Dr.   Larry  Crane; 
Wayne  State  Univ;   Drs.    Herbert  Oettgen  and  Susan   Krown,   Sloan   Kettering   Inst.; 

cooperating  units  (if  any)  Dr.  Beatrice  Lampkin,   Children's   Hospital    Center,   Cincinnati, 
Ohio;   Dr.   Mosmorine,   Connaught  Laboratories,   Canada;   Dr.   Goodwin  Hilfenhaus, 
Behringeverke,  Germany;   Dr.   Tagir  Bektemirov,   Gamalaya   Institute,  USSR; 
Dr.    Edward  Lvosky,   Litton  Bionetics. 


lab/branch 

Office  of  the  Scientific  Director 


SECTION 

Molecular  Virology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  Bethesda,  Maryland  20014 


TOTAL  MANYEARS: 


PROFESSIONAL: 

2 


CHECK  APPROPRIATE  BOX(ES) 
&  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


(b)  HUMAN  TISSUES 


[]  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  nuclease-resistant,   primate-effective  interferon  inducer,   poly  inosinic 
polycytidylic  acid,   poly  lysine,   carboxymethyl    cellulose   (Poly  ICLC)   has  a  vari- 
ety of  physiological    activities.      In  addition  to  inducing  interferon  in  primates 
it  is  an  immune  adjuvant  and  a   radioprotective  agent.      It  is   currently  being 
evaluated  in  3  collaborative  clinical    studies,  and  3  more  are  being  developed. 
It  appears   to  have  had  ameliorative  effects   in  4  patients;  one  with   lymphoblasti 
leukemia,   one  cancer  patient  whose  widely  disseminated  Herpes   lesions   disappeared 
after  treatment,   and  2  patients  with  chronic  relapsing  polyneuropathy.     A  drug 
without  carboxymethycellulose  has   been  prepared  and  is   being  evaluated. 


I8-14 


PHS-6040 
(Rev.    10-76) 


Project  Description 

This  past  year  has   been  one  of  preparing  to  do  larger  scale  testing  of  poly 
ICLC  in  man.     This   has   included  actual   clinical   studies   and  studies  of  a  more 
basic  type. 

Programs  are  ongoing  with   the  Sloan   Kettering   Institute,  where  a  phase   I  study 
of  the  drug  in  terminal    cancer  patients  is  being  done.     They  are  using  a  dif- 
ferent dosage  and  administration  schedule   than  that  which  we  used  with  Dr. 
Levine  of  the  NCI.     All    that  can  be  said  at  this  writing  is   that  the  patients 
are  making  good  levels  of  interferon.     One  leukemia  patient  had  widely  dissem- 
inated herpes  continuously  for  1   and  1/4  years.     One  week  after  going  onto  the 
drug,   the  herpes   lesions   disappeared  and  have  not  reappeared. 

A  phase   II  study  has   been  initiated  with  the  Children's   Cancer  Study  Group,  a 
multi-institution   collaborative  organization.     The  present  protocol    is   designed 
to  treat  acute  lymphoblastic  leukemia  at  stages  earlier  than  terminal.     No  data 
are  avai  Table  as  yet. 

The   Policy  Network  of  NCI  accepted  poly  ICLC  as   a  high  priority  drug,  and  is 
undertaking  its  manufacture,   packaging  and  distribution.     They  also  are  begin- 
ning a  long-range  toxicology  study,   the  results  of  which  will   be  available  for 
NI AID  use  in  treating  patients  with  viral   disease.     We  have  developed  a  proto- 
col   together  with  NCI   to  treat  a  variety  of  solid  tumors  and  leukemias.     The 
protocol    has   passed  NCI,   Clinical    Center  and  NIAID  review.     There  are  extensive 
immunological   studies   described  in  this   protocol. 

There  have  been  written  the   following  protocols   for  clinical    studies  which  are 
almost  ready  to  submit  for  NIAID  approval. 

1)  With  Drs.   Alexanian  and  Gutterman  of  M.D.   Anderson  Hospital   -   to  treat 
patients  with  multiple  myeloma   in   comparison  with   their  study  using  exogenous 
interferon. 

2)  With  Dr.    Brian  Durie  of  University  of  Arizona  -   to  treat  multiple  myeloma 
and  to  study  the  effect  of  the  drug  on  a  variety  of  immunological    parameters. 

3)  With  Drs.  Mosely  and  Rakela  of  University  of  Southern  California  Medical 
School  liver  unit  -  to  treat  patients  with  hepatocellular  carcinoma  associated 
with  chronic  hepatitis   B  infection. 

Poly   ICLC,   like  the  parent  poly   I   Poly  C,   is   pleiotropic.      In  addition  to  its 
ability  to  induce  interferon,   it  has   demonstrated  the   following  action:     To 
date,   it  has   been  shown  to  be  even  at  low  doses,  an  effective  adjuvant  with 
the   following  weak  vaccines;   Venezuelan   Equine  encephalitis,   Japanese  B  ence- 
phalitis,  Swine   Flu,   hemophilus   influenzae  polysaccharide,   Rift  Valley  fever 
and  Herpes  envelope  antigens.     With  strong  antigens,   such  as   albumin  and 
pneumococcal   polysaccharide  type   III,   if  anything,   there  is  an  inhibitory  action 
on  antibody  production.     Monkeys   receiving  poly   ICLC  showed  a  marked,  but 
transient  increase  in   the  number  of  small    lymphocytes  in   the  paracortex  of  the 
nodes   draining  the  site  of  injection,   as  well   as   an   increase  in  migration   rate 
of  lymphocytes   from  high  endothelial    venules. 

_     18-15 


Poly   ICLC,   like  a  number  of  other  interferon  inducers   is  a  radio  protective 
agent.     Mice  given  Poly   ICLC  can  tolerate  a  significantly  increased  amount  of 
X-irradiation.     While  the  mechanism  of  protection   is   not  clear,   it  is   probably 
associated  with  a  strong  stimulation  of  marrow  stem  cells  as   shown  by  an  in- 
crease in  endogenous   colonies   in  the  spleen. 

In  a  study  with   Dr.    Engel   of  NIAMDS  a  year  and  a  half  ago,  a  patient  with 
chronic  relapsing  polyneuropathy  was   started  on  poly   ICLC.     After  six  weeks  of 
treatment     the  patient  went  from  a  state  of  almost  complete  paralysis   to  one 
in  which  he  was  able  to  walk  six  miles,   and  lift  weights  over  his   head.      He 
continues   to  be  well,   but  requires  weekly  or  biweekly  injections.     Otherwise, 
weakness   begins   to  set  in  again. 

Another  patient  with  a  similar  illness   also  made  a  dramatic  recovery  but  doesn't 
appear  to  require  continued  treatment.      Immunologic  studies  are  being  done  on 
these  patients. 

Connaught  Laboratories,   the  manufacturer  of  Salk   type  polio  vaccine,   has   had  a 
serious   problem  in  obtaining  enough  monkey  kidney  tissue  culture  to  grow  the 
polio  virus.     The  monkeys  are  chronic  carriers  of  foamy  virus,  and  there  is 
barely  time  to  get  a  harvest  of  poliovirus   from  the  primary  tissue  culture 
growth,  before  the  foamy  virus   destroys   the  tissue  culture.     They  could  increase 
the  amount  of  tissue -ul  ture  by  10  fold  if  they  could  make  secondary  cultures. 
In  two  collaborative  experiments   they  found  that  by  treating  the  monkeys  with 
poly  ICLC  before  removing  the  kidneys,   they  could  reduce  the  virus   titer  suf- 
ficiently so  that  secondary  cultures  could  be  made.     They  plan  to  make  the  poly 
ICLC  themselves  and  try  it  again.      If  successful,   they  will    consider  applying 
it  to  their  production  schedule. 

Commercial    interest  in  poly  ICLC  has   been  expressed  by   Pasteur  Development 
Corporation  in   France,   Ciba-Geigy  in  Switzerland  and  Merck,  Sharpe  and  Dohme 
in  this  country. 

There  has   been  some  concern  over  the  presence  of  carboxymethylcellulose   (CMC) 
in  poly  ICLC.     While  CMC  has   been  used  in  parenteral   medicines   for  years,   the 
fact  is   that  there  is   no  known  enzymatic  pathway  for  its   degradation.     Monkeys 
and  humans  who  have  received  poly   ICLC  over  two  years   ago  have  shown  no  adverse 
reactions,   nor  have  those  who  received  CMC  as   a  vehicle   for  steroid  administra- 
tion.     However,   because  it  would  be  easier  to  formulate  and  might  allow  for  the 
preparation  of  a  more  concentrated  solution,  we  have  tried  to  prepare  a  poly   I 
poly  C  poly  lysine  complex  without  CMC.     By  taking  advantage  of  the  known 
effects  of  ionic  strength  and  temperature  on  the   components,  we  have  succeeded 
in  preparing  a  series  of  such  complexes   that  are   resistant  to  hydrolysis   by 
RNase,  and  which   induce  interferon  in  monkeys   and  mice.     Much  work   needs   to  be 
done  before  we  can  give  this   poly   ICLC  to  people. 

We  have  studied  the  effect  of  changing  the  size  of  the  poly  lysine  and  that  of 
the  poly   I  and  poly  C  on  Tm,   nuclease   resistance  and  capacity  to  induce  inter- 
feron in  monkeys.      Poly  lysine  of  molecular  weight  2000  forms  an  ineffective 
complex,   poly  lysine  of  molecular  weight  27,000  is  close  to  maximally  effective. 
9s   Poly   I  and   Poly  C  form     a  much  more  effective   complex  than  does   smaller  poly 
I   or  poly  C.      Our  standard  poly   ICLC  is   made  with   9s    Poly   I,   9s    Poly  C,    27,000 

18-16 


mol .   wt.    poly  lysine  and  high  viscosity  CMC. 
Future  Plans : 

1)  It  is   hoped  in  the  next  year  to  expand  clinical   work  with  patients  with 
malignancies,   emphasizing  the  following  questions:     a)   Are  there  some  tumors 
that  are  responsive?  b)  What  are  the  effects  on  virus   diseases   in  these 
patients?  c)  What  are  the  effects  of  the  drug  on  the  immune  system  in  man? 

2)  With  NCI   it  is   planned  to  do  long-term  toxicological    studies   in  mice  and 
monkeys. 

3)  Through  the  use  of  the  contract  mechanism  we  plan  to  examine  the  question 
of  stability  of  the  drug,   as  well   as   some  biochemical   and  immunological 
matters   that  bear  on  therapy. 

4)  In  a  collaborative  study  with   Dr.   Liu  of  USAMRIID  we  plan  to  examine  the 
effect  of  poly  ICLC  and  interferon  on  a  large  variety  of  cardiovascular 
functions   in  monkeys. 

Publications 


Harrington,   D.   G. ,   Crabbs,   C.    L. ,   Hilmas,   D.    E.,   Brown,   Jr.    R. ,   Higbee,  S.A., 
Cole,      .    E.,   Levy,   H.B.:     Adjuvant  effects  of  low  doses  of  a   nuclease-resistant 
derivative  of  polyinosmic  -acid  on  antibody  responses  of  monkeys   to  inactivated 
Venezuelan  equine  encephalomyelitis   virus   vaccine.      Infection  and   Immunity, 
Apr.    1979,    p.    160-166.  

Levine,  A.    S.,   Sivulich,  M.,   Wiernik,   P.    H.,   Levy,   H.    B.      Initial    clinical 
trials   in  cancer  patients  of  polyribonosinic-polyribo  cytidylic  acid  stabilized 
with   poly-L-lysine,       Carboxymethylcell ulose  [Poly(ICLC)] ,   a   highly  effective 
interferon  inducer:     Cancer  Research   39:   1645-1650.     May  1979. 

Hilmas,   D.    E. ,   Stephens,   E.    L.  ,   Spertzel  ,   R.   0.,   Levy,   H.   B.     Use  of  PICLC  for 
the  prophylaxis  and  treatment  of  Venezuelan  equine  encephalomyelitis   virus 
infection   in   nonhuman  primates.      Current  Chemotherapy.    1978. 

Levine,  A.    S.,   Levy,   H.    B.      Phase   I- 1 1   trials  of  PIC  stabilized  with  poly-L- 
lysine.      Cancer  Treatment  Reports.,   Vol.   62,   No.    11.     Nov.,   1978. 

Levy,   H.    B.,   Lvovsky,   E.     Tropical    treatment  of  vaccinia  virus   infection  with 
an  interferon  inducer  in   rabbits.      Journal    of  Infectious   Diseases,   Vol.   137, 
No.    1.      January,    1978. 

Levy,   H.    B.,   Hilmas,   D.    E.      Evaluation  of  a   nuclease-resistant  derivative  of 
PLCLC  as  a  radioprotective  agent.     Radiation  Research,    77,   1979. 

Stephens,   E.    L.,   Hilmas,   E.    E. ,   Levy,   H.    B.,   Spertzel,   R.    D.      Protective  and 
toxic  effects  of  a   nuclease-resistant  derivative  of  PIC  on  Venezuelan  equine 
encephalomyelitis   virus   in  Rhesus  monkeys.      Journal    Infectious   Diseases,   Vol. 
139   No. 3,    1979. 


18-17 


Lerner,  M.    E.,   Levy,   H.    B.      Physiological    accompaniments  of  sustained 
interferonemia  induced  in  man  by  poly   ICLC.      lrvf_.    Immun.   Accepted. 


TB-I8 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01-A1-00131-12  OSD 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Mechanisms  of  Hypersensitivity  in  Histocompatible   Inbred  Guinea  Pigs 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:     Dr.   Sanford  H.   Stone 


Chief,  Immunology  Section      QSD,   NIAID 


cooperating  units  (if  any)  Dr.    Cedri c  S.   Raine,  Division  of  Neuropathology,  Albert 
Einstein  College  of  Medicine,  New  York,  N.    V. ;   Dr.   Richard  H.    Quarles,   D.M.N, 
NINCDS,   Bethesda,   MD. ;   Dr.    Robert  B.   Nussenblatt,   CB,   NEI,   Bethesda,  MD. 


lab/branch 

Office  of  the  Scientific  Director 


Immunology  Section 


institute  and  location 

NIAID,   Bethesda,  Maryland     20014 


TOTAL  MANYEARS: 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (a1 )  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


CX(c)  NEITHER 


SUMMARY  OF  WORK  (200  *ords  or  less  -  underline  keywords) 

For  some  years,  we  have  been  using  the  unique  tool 
guinea  pigs  for  investigation  of  mechanisms  of  hypersen 
genesis  of  and  the  protection  against  infectious  and  au 
vantages  lie  in  the  ability  to  perform  viable  adoptive 
and  in  the  capacity  to  study  genetic  factors  in  certain 
autoimmune  phenomena.  We  have  concentrated  recently  on 
age-dependent  induction  of  cataracts  in  immature  guinea 
paralysis  and  atrophy  of  autoimmune  encephalomyelitis  an 
treatment  of  this  condition.  The  cataracts  are  reminis 
experimental  entities  shown  to  be  due  to  nutritional  de 
alteration  and  may  be  secondary  to  the  autoimmune  react 


of  inbred  histocompatible 
sitiv 
toimm 


n'ty  in  the  patho- 
lune  diseases.     Ad- 


transfer  of  lymphoid  cells 
hypersensitivity  and 
the  pathogenesis  of  an 
pigs   undergoing  the 
d  on  the  prevention  and 
cent  of  some  clinical   or 
ficiency  or   enzymatic 
ion  in   the  host. 


-18-19 


PHS-6040 
(Rev.    10-76) 


Project  Descri  ption 
Subproject  I  -   Cataract  formation  in  allergic  encephalomyelitis  of  guinea 
pigs 

An  unusual    pathological    event  occurred  in  immature  guinea  pigs   sensitized 
to  spinal    cord  antigens   either  actively  or  passively  by  lymph   node-cell    trans- 
fer.    A  significant  number  of  these  developed  cataracts   bilaterally  during  a 
severe  acute  attack  of  allergic  encephalomyelitis    (EAE).     The  lens   histology 
was   significant  for  posterior  migration  of  the  epithelium  with  some  eyes  show- 
ing vacuolization  under  the  capsule.     The  vitreous,    choroid      and  retina  did 
not  show  foci   of  inflammation  and  the  intra-ocular  and  intracleral  portion 
of  the  optic  nerve  showed  no  evidence  of  cell    drop-out  nor  round  cell    infil- 
tration.     Histological    preparations   from  the  central    nervous   system  (CNS)  of 
the  guinea  pigs   showed  the  classic  picture  of  acute  EAE. 

The  age-dependency  of  this   eye  lesion  was   quite  evident-     Both  wean- 
lings and  newborns,   but  no  adults,   developed     the  opacities;   the  cataracts 
were  not  strain-specific,   occurring  in  both  strain   13  and  Hartley  guinea  pigs. 
The  evidence  points   to  the  probability  that  these  cataracts   are  not  the  direct 
result  of  an  immunological    response,  but  secondary  to  the  acute  paralytic 
syndrome  of  EAE. 

Subproject   II  -   Protection  against  chronic  EAE  by  injection  of  myelin 
basic  protein  in  incomplete   Freund's   adjuvant 

CNS  lesion  morphology  was  examined  in  detail    in  inbred  strain   13  guinea 
pigs   sensitized  for  chronic   EAE  in  which   the  disease  was   either  allowed  to 
develop  or  was   suppressed  by  injections   of  myelin  basic  protein    (MBP).      Path- 
ologic changes  correlated  well   with   the  clinical    picture;   in  chronic  animals 
clinical    disease  was  accompanied  by  inflammation   in   the  CNS  including  fibrosis 
and  remyel i nation.         Relapses   showed  the  CNS  to  contain   recent  changes   super- 
imposed upon  old  lesions.      In  animals   in  which  the  disease  was   suppressed, 
clinical    signs   did  not  develop,  but  some  early  sub-clinical    changes  were  seen 
morphologically.      These  lesions  were   remyelinated  promptly  and  there  was   no 
progression  in  lesion   formation.     This   contrasted  with  the  progression  in  un- 
treated chronic  animals;   long-standing  disease  was   characterized  by  large, 
burnt-out  plaques,  with  Schwann  cell    invasion  and  peripheral    nervous   system 
myelination,  glial    bridges   between  sub-pial   astrocytes  and  the  leptomeninges , 
and  fenestrated  blood  vessels. 

Therapy  of  established  chronic  disease   (as   distinguished  from  suppression 
experiments)   is   under  investigation  currently  using  MBP  in  paralyzed  strain 
13  guinea  pigs.      This  will    require  an  elaborate  set  of  controls  and  will    be 
cautiously     pursued  in  view  of  its   relevance  to  the  treatment  of  multiple 
sclerosi  s. 

Publ  i  cations 


Raine,   C.    S.  ,   Traugott,   U.   and  Stone,  S.    H.  :     Chronic  relapsing  experimental 
allergic  encephalomyelitis:     CNS  plaque  development  in   unsuppressed  and 
suppressed  animals.     Acta   Neuropathol  .   43:43-53,   1978. 


-18-20 


Raine,   C.   S.   Traugott,   U.   and  Stone,   S.   H.  :     Glial    bridges  and  Schwann  cell 
migration  during  chronic  demyeli nation  in   the  C.N.S.      J.   Neurocytol .    7: 
541-553,   1978. 

Traugott,   U.,   Stone,   S.    H.   and  Raine,  C.   S.:     Chronic  relapsing  experimental 
allergic  encephalomyelitis:     correlation  of  circulating  lymphocyte  fluctua- 
tions with  disease  activity  in  suppressed  and  unsuppressed  animals.      J.   Neurol 
Sci.    41 :    17-29,   1979. 


18-21 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE!     U.S.  DEPARTMENT  OF         PROJECT  NUMBER 
PROJECT  NUMBER  (Do  MOT  use  this  space)   IHEALTH,  EDUCATION,  AND  WELFARE 

PUBLIC  HEALTH  SERVICE         „„,   .,  „„,_„  „,  „_ 

hot  ice  cf  Z01  -Al  -001 90-01  -OSD 

INTRAMURAL  RESEARCH  PROJECT 


PERIOD  COVERED 


j TITLE  OF  PROJECT  (80  characters  or  less; 

The  Molecular  Genetics  of  Eukaryotic  Cells  and  Their  Viruses 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI   :  Malcolm  A.  Martin  OSD,  NIAID 

OTHER:  Hardy  Chan,  Senior  Staff  Associate  OSD,  NIAID 

Dean  Hamer,  Staff  Associate  OSD,  NIAID 

Mark  Israel  ,  Research  Associate  OSD,  NIAID 

Yoshiaki  Ito,  Visiting  Scientist  OSD,  NIAID 

Roy  Repaske,  Microbiologist  OSD,  NIAID 

Kama!  Chowdhury,  Visiting  Fellow  OSD,  NIAID 


COOPERATING  UNITS  (if  any) 

Wallace  P.  Rowe,  LVD,  NIAID;  Edward  Scolnick,  LTVG,  NCI 


LAB/BRANCH 

Office  of  the  Scientific  Director 


SECTION 

Molecular  Biology 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS  Q  (b)  HUMAN  TISSUES  Q   (c)  NEITHER 

(a1  )  MINORS  Q   (a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  principal  goal  of  this  unit  continues  to  be  the  biochemical  characteri- 
zation of  eukaryotic  and  animal  viral  genes.  Attention  has  been  focussed  on  the 
segments  of  DNA  and  RNA  Tumor  virus  genomes  responsible  for  the  establishment  and 
maintenance  of  the  transformed  state.  In  this  regard  we  have  shown  that  the 
large  form  of  polyoma  tumor  antigen  plays  no  role  in  tumorigenesis  mediated  by 
polyoma  DNA.  SV40  DNA  recombinants  have  been  used  to  elucidate  the  mechanisms 
regulating  the  synthesis  and  processing  of  eukaryotic  RNA. 

The  first  phase  of  risk  assessment  experiments  conducted  in  the  P4  facility 
at  Ft.  Detnck,  Maryland,  was  recently  completed.  These  studies  showed  that 
polyoma  virus  DNA  is  not  transferred  out  of  E.  coli  K-12  following  the 
inoculation  of  susceptible  newborn  animals  with  bacteria  containinq  recombinant 
plasmids  or  phage. 


18-22 


PWS-6040 
(Rev.    10-76) 


Z01-A1-00190-01-0SD 
Project  Description: 

The  DNA  Recombinant  Research  Unit,  established  in  the  Summer  of  1978,  has 
continued  to  utilize  recombinant  DNA  procedures  to  investigate  the  regulation 
and  structural  organization  of  eukaryotic  cells  and  their  viruses.  While  the 
main  thrust  of  research  activities  continues  to  be  the  study  of  the  transforming 
region  of  SV40  and  polyoma  virus,  new  projects  which  involve  the  molecular 
cloning  and  biochemical  characterization  of  murine  retraviruses  and  the  use  of 
the  SV40  vector  system  to  evaluate  transcriptional  regulation  of  eukaryotic 
genes  has  been  initiated.  Experiments  evaluating  potential  risks  associated 
with  recombinant  DNA  research  have  also  been  carried  out  at  the  P4  facility 
located  at  Ft.  Detrick  and  P2  laboratories  on  the  NIH  reservation. 

During  the  year  Dr.  Daniel  Simmons  resigned  to  take  a  position  as  Assistant 
Professor  of  Biology,  University  of  Delaware.  Dr.  Dean  Hamer  joined  the  Unit 
last  fall  following  post  doctoral  training  in  Dr.  Phil  Leder's  laboratory  and 
will  use  SV40  recombinants  to  investigate  transcription  of  mammalian  genes. 
Dr.  Yoshiaki  Ito  joined  the  group  in  August  following  a  six-year  tenure  at  the 
Imperial  Cancer  Research  Fund  Laboratories,  London  and  will  continue  to 
characterize  transforming  polypeptides  specified  by  papovavirus  genomes. 
Dr.  Roy  Repaske  became  an  active  participant  in  the  risk  assessment  experiments 
coordinated  by  Drs .  Martin  and  Rowe  and  perfected  the  in  vitro  packaging 
procedure  utilized  in  "shotgun"  cloning.  He  has  recently  mastered  nucleotide 
sequencing  procedures  and  will  use  them  to  characterize  specific  segments  of 
cloned  DNA. 

Research  Accomplishments 

I.  The  Oncogenic  Potential  of  Papovaviruses 

During  the  past  two  years  the  polyoma  (PY)  virus  system  has  been  used  to 
evaluate  the  molecular  events  associated  with  transformation  and  tumori genesis. 
The  PY  virus  system  has  at  least  two  advantages  over  SV40:  1)  tumors  are 
readily  and  rapidly  induced  in  newborn  hamsters;  2)  the  PY  viral  genome  encodes 
three  peptides  which  play  a  role  in  the  oncogenic  process  whereas  SV40  DNA 
contains  the  genetic  information  for  only  two  of  these  three.  We  have  extended 
our  previous  studies  involving  tumorigenesis  in  newborn  hamsters  by  evaluating 
the  biological  activity  of  viral  DNA  preparations  previously  digested  with 
restriction  enzymes.  These  experiments  clearly  indicate  that  tumori geni city 
of  PY  DNA  is  enhanced  when  the  distal  portion  of  the  early  gene  region  is 
interrupted.  This  was  an  unexpected  result  since  in  the  more  extensively 
studied  SV40  system,  the  entire  early  region  must  be  intact  for  the  initiation 
and  maintenance  of  the  transformed  state.  Using  immunoprecipitation  techniques, 
we  demonstrated  that  tumor  cell  lines,  originally  induced  by  PY  virions  or  PY 
DNA,  contained  the  small  and  middle  PY  tumor  (T)  antigens  (Ags)  but  not  the 
large  form  of  viral  T  Ag.  Fifteen  independent  cloned  tumor  cell  lines  derived 
from  hamster  tumors  induced  by  PY  virions  or  PY  DNA  were  analyzed  by  blot- 
hybridization  and  shown  to  contain  viral  DNA  sequences  specifying  the  small 
and  middle  PY  T  Ags.  PY  DNA  sequences  encoding  the  large  T  Ag  were  invariably 
interrupted.  These  data  imply  that  PY  large  T  Ag  is  not  required  for  the 
maintenance  of  the  tumor  cell  phenotype.  Studies  are  currently  in  progress 

"18-23 


Z01-A1-00190-01-OSD 

to  evaluate  whether  PY  large  T  Ag  plays  any  role  in  virus  mediated  tumori genesis. 
An  effort  is  also  being  made  to  clone  the  PY  DNA  sequences  integrated  into  the 
tumor  cell  genome.  (Israel,  Chowdhury,  Martin.) 

II .  The  Molecular  Structure  of  Murine  Retraviruses 

Our  unit  has  initiated  a  major  collaborative  effort  with  Dr.  Wallace  Rowe's 
group  (LVD)  to  clone  and  characterize  a  variety  of  murine  retraviruses.  This 
research  activity  grew  out  of  an  ealier  joint  project  with  Dr.  Edward  Scolnick's 
group  (Laboratory  of  Tumor  Virus  Genetics,  NCI)  in  which  Friend  Leukemia  (FLV), 
and  Harvey  Sarcoma  (HaSV)  viruses  were  cloned  in  derivatives  of  E_.  col i  K12 
using  a  lambdaphage  vector.  After  refining  in  vitro  packaging  procedures  to 
permit  the  "shotgun"  cloning  of  unintegrated  viral  DNA,  large  quantities  of 
FLV  and  HaSV  DNAs  were  prepared  and  extensively  characterized.  We  concentrated 
our  energies  of  the  restriction  mapping  of  HaSV  DNA  and  showed  that  one  of  the 
six  recombinant  phage  we  constructed  contained  a  triplication  of  viral  DNA 
sequences  that  flank  both  ends  of  the  integrated  HaSV  DNA.  Work  is  continuing 
on  the  stability  of  the  reiterated  HaSV  DNA  sequences  in  both  eukaryotic  and 
prokaryotic  host  cells  and  determining  the  fine  structure  of  the  reiterated 
DNA  segment.  (Chan,  Repaske,  Martin.) 

More  recently  we  have  begun  the  molecular  cloning  of  the  endogenous 
retraviral  DNA  sequences  present  in  the  AKR  mouse.  Thus  far,  we  have  obtained 
recombinant  phage  preparations  containing  integrated:  1)  ecotropic  AKV; 
2)  xenotropic;  and  3)  two  MCF  DNA  sequences.  The  cloned  AKV  and  xenotropic 
preparations  appear  to  represent  complete  viral  genomes.  These  retraviral  DNA 
inserts  will  be  biochemically  characterized  and  subgenomic  viral  DNA  segments 
will  be  purified  for  use  as  a  probe  in  hybridization  experiments  for  the 
detection  of  specific  regions  of  viral  DNA.  (Chan,  Repaske,  Martin.) 

Ill  Regulation  of  Eukaryotic  Genes 

In  order  to  provide  a  biological  assay  for  regulatory  sequences  present  in 
eukaryotic  DNA  as  well  as  mutants  derived  from  them,  we  have  developed  SV40 
host  vector  systems  that  allow  us  to  introduce  new  genetic  information  into 
animal  cells.  During  the  past  year,  we  have  constructed  recombinant  molecules 
containing  chromosomal  mouse  globin  genes  linked  to  an  SV40  vector  and  infected 
monkey  cells.  We  find  that  the  mouse  globin  gene  signals  for  transcription, 
processing  and  translation  are  recognized  in  monkey  cells  resulting  in  the 
production  of  substantial  quantities  of  globin.  Further,  by  preparing  mutant 
forms  of  SV40,  we  have  characterized  discontinuous  eukaryotic  genes  and  the 
splicing  of  mammalian  RNA.  Specifically,  the  minimum  size  of  the  recognition 
site,  the  species,  cell  type,  and  distribution  of  the  splicing  enzymes,  and  the 
physiological  role  of  splicing  in  stable  RNA  formation  has  been  investigated. 

We  are  now  attempting  to  extend  this  system  to  the  human  globins  and  are 
particularly  interested  in  using  SV40  recombinants  to  elucidate  the  molecular 
lesions  in  various  hemoglobinopathies.  We  have  also  initiated  a  project  to 
form  SV40  recombinants  carrying  hepatitis  virus  type  B  sequences.  Such  hybrids 
could  be  very  useful  in  mapping  the  hepatitis  genome  and,  eventually,  for 
preparing  diagnostic  and  therapeutic  reagents.  (Hamer) 

-  18-24 


Z01-A1-00190-01-0SD 
IV.  Assessment  of  Risks  Associated  with  Recombinant  DNA  Research 

In  December  1975,  Drs.  Malcolm  Martin  and  Wallace  Rowe  were  asked  by  the 
Recombinant  DNA  Advisory  Committee  to  conduct  experiments  in  NIH  containment 
facilities  to  evaluate  potential  risks  associated  with  recombinant  DNA  experi- 
mentation. The  first  phase  of  such  studies  was  carried  out  in  P4  containment 
facilities  located  in  Frederick,  Maryland  or  on  the  NIH  reservation  and  involved 
the  inoculation  of  weanling  mice  or  newborn  hamsters  with  E.   coli  K12  containing 
polyoma-plasmid  or  polyoma-lambdaphage  recombinants.  In  both  animal  systems, 
the  polyoma-recombinants  were  not  transferred  out  of  their  IE.  coli  hosts  follow- 
ing parenteral  inoculation.  As  controls,  polyoma  DNA,  liberated  from  the  pro- 
karyotic  vector  DNA,  was  infectious  (mice)  or  tumorigenic  (hamsters).  Mice 
inoculated  with  purified  recombinant  DNA  preparations  containing  a  single  copy 
of  viral  DNA  never  developed  a  virus  infection  whereas,  a  few  animals  infected 
with  a  recombinant  phage  DNA  preparation  harboring  a  head-to-tail  dimer  of  viral 
DNA  developed  an  infection.  Intact  recombinant  DNA  preparations  as  well  as 
purified  phage  particles  induced  tumors  in  5-19%  of  inoculated  animals,  a 
value  similar  to  that  observed  following  injection  of  supercoiled  PY  DNA  (19%). 
It  should  be  emphasized  that  potentially  infectious  or  tumorigenic  recombinant 
DNA  molecules  are  not  transferred  out  of  EK2  hosts  into  mammalian  cells  in 
either  of  the  animal  model  systems  we  used.  Even  in  those  cases  in  which  animals 
inoculated  with  recombinant  phage  or  purified  recombinant  DNA  did  develop  a 
viral  infection  or  tumors  in  worst  case  experiments  conducted  in  a  laboratory 
setting,  the  biological  activity  of  recombinant  preparations  was  at  least  a 
million-fold  less  active  than  polyoma  virus  particles. 

We  have  recently  received  polyoma  plasmid  and  polyoma  lambda  recombinants 
in  EK1  E.   coli  host  cells,  which  were  constructed  by  investigators  in  Europe. 
We  have  agreed  to  begin  animal  testing  studies  in  the  fall  of  1979.  In  addition, 
we  plan  to  inoculate  bacteria  containing  cloned  Harvey  Sarcoma  and  Friend 
Leukemia  virus  DNAs  into  appropriate  test  animals  to  extend  our  risk-assessment 
analysis  to  another  virus  (retravirus)  system.  (Drs.  Martin  and  Rowe  and 
their  friends. ) 

Publications 


Israel,  M.A.,  Chan,  H.,  Rowe,  W.,  and  Martin,  M.A.:  Biologic  activity  of 
polyoma  virus  DNA  in  animals.  Journal  of  Virology,  29:990-996,  1979. 

Israel,  M.A. ,  Chan,  H.,  Rowe,  W.  and  Martin,  M.A.:  Molecular  cloning  of 
polyoma  virus  DNA  in  E_.  coli .  I.  Plasmid  vector  system.  Science,  203:883- 
887,  1979. 

Chan,  H.W.,  Israel,  M.A. ,  Garon,  C.F.,  Rowe,  W.P.,  and  Martin,  M.A.: 
Molecular  cloning  of  polyoma  virus  DNA  in  E_.  coli .  II.  Lambda  phage  vector 
system.  Science,  203:887-892,  1979. 

Howley,  P.M.,  Israel,  M.A.  ,  Law,  M.F.,  and  Martin,  M.A.:  A  rapid  method  for 
detecting  and  mapping  homology  between  heterologous  DNAs.  Evaluation  of 
polyomavirus  genomes.  Journal  of  Biological  Chemistry,  254:4884-4891,  1979. 


18-25 


Z01-A1-00190-01-OSD 

Hamer,  D. ,  Smith,  K. ,  Boyer,  S.,  and  Leder,  P.:  "SV40  Recombinants  Carrying 
Rabbit  B-Globin  Coding  Sequences.  Cell  17:725-735,  1979. 

Hamer,  D.  and  Leder,  P.:  SV40  Recombinants  Carrying,a  Functiona  Splice 
Junction  and  Polyadenylation  Site  from  the  Mouse  B   -Globin  Gene.  Cell  17: 
737-747,  1979. 

Simmons,  D.T.  and  Martin,  M.A.:  Common  methionine  tryptic  peptides  near  the 
amino-terminal  end  of  primate  papovavirus  tumor  antigens.  Proc.  Nat.  Acad. 
Sci.  U.S.  75:1131-1135,  1978. 

Simmons,  D.T.,  Takemoto,  K.K.,  and  Martin,  M.A. :  Properties  of  simian  virus 
40  and  BK  virus  tumor  antigens  from  productively  infected  and  transformed 
cells.  Virology  85:137-145,  1978. 

Simmons,  D.T.,  Chang,  C,  and  Martin,  M.A.  :  Multiple  forms  of  polyoma  virus 
tumor  antigens  from  infected  and  transformed  cells.  J.  Virol.  29:881-887,  1979. 

Honors  and  Awards 


Dr.  Malcolm  Martin  was  awarded  the  Public  Health  Service  Superior  Service 
Award  in  May  for  his  activities  involving  recombinant  DNA  research.  Dr.  Martin 
continues  to  serve  on  the  Editorial  Boards  of  Journal  of  Virology  and  Journal 
of  Biological  Chemistry.  In  June,  he  completed  a  four  year  appointment  as  a 
member  of  the  Virology  Study  Section,  DRG,  NIH.  In  April,  Dr.  Martin  was 
invited  to  deliver  a  lecture  at  the  Cogene-The  Royal  Society  of  London  Meeting 
on  Recombinant  DNA  held  at  Wye  College,  Kent,  England. 


^18-26 


LABORATORY  OF  BIOLOGY  OF  VIRUSES 

1979  Annual  Report 

Table  of  Contents 


Z01-AI 
Project  Number 

Summary 

00123-13 

00124-10 

00125-10 

00126-06 

00127-12 

00128-12 
00156-04 


Messenger  RNA:  Regulation  of  Synthesis, 
Modification,  and  Translation  —  Moss 

Replication  of  the  Parvovirus,  KRV  — 
Salzman 

Mechanisms  of  Viral  DNA  Replication, 
Transcription,  and  Integration  —  Salzman 

Restriction  Enzyme  Analysis  of  Vaccinia 
Virus  DNA  —  DeFilippes 

Structure  and  Function  of  Genetic  and 
Protein  Components  of  Defective 
Parvoviruses  —  Rose 

Properties  of  Adenovirus  DNA  —  Garon, 
Rose 

Structural  Characterization  of  DNA  Virus 
Genomes  —  Garon 


Page 
19-1 

19-14 

19-19 

19-23 

19-27 

19-30 
19-33 
19-36 


Annual  Report  of  the  Laboratory  of  Biology  of  Viruses 

National  Institute  of  Allergy  and  Infectious  Diseases,  NIH 

October  1,  1978  to  September  30,  1979 

The  goal  of  the  Laboratory  of  Biology  of  Viruses  is  to  determine 
how  viruses  replicate.  At  the  molecular  level,  this  involves  investigation 
of  the  structure  of  the  virion  and  of  its  purified  component  parts,  of 
mechanisms  of  replication  and  transcription  and  expression  of  the  viral 
genome.  Emphasis  is  also  placed  on  cell-virus  interactions  including 
the  process  of  cell  transformation.  We  believe  that  information  of  this 
kind,  although  difficult  to  obtain,  is  fundamental  to  understanding 
disease  processes  and  vital  to  achieving  a  rational  basis  for  chemotherapy. 

Papovaviruses 

The  current  studies  have  tried  to  define  some  of  the  processes 
responsible  for  the  formation  of  viral  messenger  RNA  molecules.  Thus 
far,  these  studies  have  provided  the  precise  structures  of  the  viral 
mRNA  molecules,  have  defined  the  nature  of  the  templates  used  for  the 
synthesis  of  viral  transcripts,  have  located  promoters  on  the  SV40 
genome  that  are  recognized  by  E.  coli  RNA  polymerase  and  have  compared 
SV40  DNA  I  and  SV40  DNA  I  nucleoprotein  complexes  as  templates  for 
transcription . 

The  Structure  of  Early  and  Late  Viral  mRNA 

The  mechanism  of  splicing  of  the  early  and  late  cytoplasmic  species 
of  SV40  mRNA  has  been  studied  using  a  technique  which  first  involves  the 
isolation  of  specific  regions  of  the  viral  genome.  Restriction  enzyme 
cleavage  of  SV40  DNA  generates  specific  DNA  fragments  which  can  be 
fractionated  using  column  chromatography.  These  fragments  are  then 
annealed  to  viral  mRNA  and  the  reaction  products  are  fractionated  by 
velocity  sedimentation.  The  DNA  fragment  which  is  annealed  to  the  viral 
mRNA  is  used  as  a  primer  for  the  enzyme,  reverse  transcriptase,  and  a 
complementary  DNA  copy  of  the  mRNA  is  made.  The  sequence  and  structure 
of  the  cDNA  has  been  compared  with  the  known  nucleotide  sequence  of 
SV40.  This  procedure  has  been  used  to  characterize  the  late  mRNA  which 
codes  for  the  major  structural  viral  coat  protein  and  has  provided 
direct  physical  evidence  for  two  early  mRNA  species.  The  regions  of  the 
DNA  that  have  deleted  in  both  species  of  early  mRNAs  have  been  determined. 
Also,  the  nucleotide  sequences  across  these  spliced  regions  have  been 
determined  and  correlated  with  the  mRNA  coding  for  the  large  T  and  small 
t  antigens.  These  two  mRNA  species  were  determined  to  have  the  same  51  termini 
which  would  suggest  two  levels  for  control  of  genetic  expression.  One 
would  be  the  regulation  of  initiation  of  transcription  at  a  common 
promoter;  the  other  would  involve  post- transcriptional  splicing. 

To  further  characterize  the  mechanism  of  transcription  of  early 
SV40  RNA,  nascent  RNA  chains  that  are  attached  to  template  DNA  molecules 
are  being  isolated  and  characterized  by  the  same  methodology  employed  to 
study  the  processed  cytoplasmic  species  of  early  viral  RNA.  Transcription 


19-1 


complexes  have  been  isolated  from  infected  cells  which  continue  viral 
mRNA  synthesized  in  vitro.  The  structure  of  these  nascent  chains  gives 
insight  into  the  initial  transcription  products  and  the  mechanisms  that 
operate  to  regulate  transcription.  We  are  investigating  differences 
between  RNA's  formed  in  vivo  and  in  vitro.  The  structure  of  nascent 
chains  will  provide  precise  information  about  the  primary  products  of 
transcription.  We  are  also  studying  the  use  of  isolated  nuclei  for  the 
study  of  the  regulation  of  transcription.  Isolated  nuclei  are  able  to 
sustain  in  vitro  synthesis  of  viral  RNA.  In  this  case,  the  SV40  template 
in  the  cell  nucleus  is  present  as  a  nucleoprotein  complex  containing  the 
five  histones.  This  is  in  contrast  to  in  vitro  synthesis  of  nascent  RNA 
chains  described  above  which  uses  a  less  well  defined  transcriptional 
complex. 

These  nascent  RNA  molecules  will  be  probed  with  specific  SV40  DNA 
fragments  to  determine  their  structure.  In  order  to  acquire  a  library 
of  these  specific  fragments,  recombinant  technology  has  been  employed. 
Cloning  of  SV40  DNA  fragments  using  the  plasmid  pBR322  and  E.  coli 
should  provide  sufficient  amounts  of  specific  DNA  fragments  which  can  be 
used  to  probe  nascent  RNA  molecules  and  determine  the  mechanisms  operating 
during  regulation  of  transcription. 

Since  sufficient  amounts  of  specific  SV40  DNA  fragments  can  be 
obtained  from  cloning,  we  will  be  able  to  study  how  synthesis  of  specific 
RNA  sequences  are  affected  by  mutations  within  the  genome.  Further 
characterization  of  RNA  species  obtained  following  Proflavin  treatment, 
which  blocks  RNA  processing,  will  aid  our  understanding  of  the  mechanism 
that  operated  to  regulate  transcription.   (Thompson,  Bina-Stein,  Salzman) 

Localization  of  RNA  Polymerase  Promoters  on  SV4Q  DNA 

Transcripts  of  SV40  DNA  synthesized  by  Escherichia  coli  RNA  polymerase 
have  been  characterized.  This  model  system  has  been  used  for  the  development 
of  new  methods  applicable  to  the  analysis  of  the  mechanisms  involved  in 
the  synthesis  of  the  viral  messenger  RNAs  in  SV40- infected  cells.   It 
has  been  previously  shown  that  E.  coli  RNA  polymerase  recognizes  specific 
initiation  sites  on  SV40  DNA.  Except  for  one  of  them,  determination  of 
the  location  of  these  sites  on  the  SV40  DNA  map  is  only  approximate.  No 
specific  termination  site  for  transcripts  has  been  identified,  and 
consequently  RNA  polymerase  generates  a  heterogeneous  population  of 
molecules.  The  size  of  some  of  these  RNA  molecules  is  several  times  the 
length  of  the  viral  genome. 

We  have  shown  that,  after  binding  of  the  E.  coli  RNA  polymerase  to 
SV40  DNA,  it  was  possible  to  cleave  such  transcriptional  complexes  with 
"single-cut"  restriction  endonucleases  (Bam  H ,  Eco  R_,  Hpa  II).  The 
addition  of  ribonucleotide  triphosphates  to  these  linearized  complexes 
leads  to  the  synthesis  of  defined  species  of  RNA  which  can  be  analyzed 
by  electrophoresis.  The  determination  of  the  size  of  each  of  these  RNAs^ 
together  with  the  assignment  of  the  DNA  strand  on  which  they  are  transcribed 
will  allow  the  precise  mapping  of  the  various  RNA  promoters  (Reuveni, 
Lavialle,  Salzman) . 

19-2 


Transcriptional  Properties  of  SV40  Nucleoprotein  Cores 

Simian  Virus  40  (SV40)  provides  an  excellent  model  system  for 
investigating  the  process  of  transcriptional  regulation  in  eukaryotic 
cells.  Many  structural  aspects  of  viral  transcriptional  complexes  and 
mRNA  molecules  have  been  determined.  In  addition,  the  entire  nucleotide 
base  sequence  of  SV40  DNA  has  been  determined.  Similar  to  most  eukaryotic 
chromatin,  during  the  SV40  infection  cycle,  cellular  histones  ELA, 
H2B, ,  EL,  and  H.  are  bound  to  the  viral  DNA.  Late  in  the  infection 
cycle,  nistone  H,  also  becomes  associated  with  the  bulk  of  the  viral 
chromatin  in  a  stable  nucleoprotein  complex.  Just  prior  to  encapsidation, 
the  SV40  nucleoprotein  complex  undergoes  a  redistribution  of  proteins 
and  histone  H,  is  replaced  by  viral  proteins.  Since  binding  of  H,  on 
chromatin  may  suppress  transcription  of  DNA  sequences,  we  were  interested 
in  determining  if  the  viral  proteins  also  acted  to  modify  or  regulate 
the  process  of  RNA  synthesis.  Such  regulatory  properties  could  be 
important  in  a  clearer  understanding  of  the  process  of  "early"  mRNA 
synthesis  during  infection  of  the  host  cell.  In  view  of  the  close 
similarity  between  this  SV40  DNA-histone-viral  protein  nucleoprotein 
complex  and  cell  chromatin  structure,  such  studies  would  also  be  of 
interest  in  a  more  complete  understanding  of  the  general  problem  of 
transcriptional  regulation  in  eukaryotic  cells. 

A  nucleoprotein  complex  can  be  isolated  from  purified  SV40  virions 
under  very  mild,  physiological  conditions.  These  nucleoprotein  cores 
are  potentially  active  transcriptional  complexes.  These  core  complexes 
do  not  contain  endogenous  RNA  polymerase  activity;  however,  95-100%  are 
able  to  form  active  transcriptional  complexes.  The  transcriptional 
activity  of  those  complexes  is  extremely  high  and  is  similar  to  the 
activity  obtained  with  purified  SV40  Form  1  DNA.  This  observation 
contrasts  with  published  data,  in  which  the  level  of  transcription  of 
nucleoprotein  complexes  (SV40  DNA-Histone)  is  generally  less  than  20%  of 
the  activity  of  deproteinized  SV40  DNA.  The  results  suggest  that  the 
viral  proteins  may  play  a  role  in  the  "activation"  of  the  nucleohistone 
complex.  Analysis  of  the  composition  and  structure  of  the  SV40  nucleoprotein 
core  are  being  carried  out  and  should  allow  an  understanding  of  the 
unexpectedly  high  efficiency  of  the  SV40  core  as  a  template  for  RNA 
synthesis.   (Brady,  Lavialle,  Salzman) 

Parvoviruses 


Kilham  Rat  Virus 


The  specific  biochemical  mechanisms  involved  in  the  replication 
of  the  autonomous  parvovirus,  KRV,  has  been  examined  in  a  rat  nephroma 
cell  line.  The  virus,  isolated  originally  from  a  rat  sarcoma,  contains 
one  molecule  of  linear,  single-stranded  DNA  and  three  capsid  proteins. 
Little  information  is  available  about  replication  of  this  single-stranded 
DNA  virus  in  a  eukaryotic  cell  or  on  the  transcription  of  this  DNA  to 
make  viral  proteins.  It  was  found  that  the  virion  KRV-DNA  can  self 
prime  in  vitro  to  synthesize  the  double-stranded  replicative  intermediate. 


19-3 


To  define  the  structure  of  this  self  priming  terminus,  which  is  important 
in  DNA  replication,  the  nucleotides  in  the  31  DNA  terminus  have  been 
sequenced.  The  DNA  structure  which  can  be  deduced  from  the  sequence 
contains  a  terminal  hairpin.  This  is  consistent  with  the  finding  that 
DNA  replication  is  a  self  priming  reaction. 

It  had  previously  been  found  that  in  an  infected  cell,  two  KRV 
specific  mRNAs  are  synthesized.  The  DNA  which  is  homologous  to  the 
major  21S  viral  mRNA  has  been  mapped  using  both  the  "Southern"  blotting 
technique  and  measurement  of  the  "R  loops"  seen  in  the  electron  microscope. 
Inhibition  studies  suggest  that  these  KRV  mRNAs  are  synthesized  by 
cellular  RNA  polymerase  II.  Preliminary  studies  also  show  that  there  are 
at  least  three  virus  induced  proteins  synthesized  during  infection. 

Adeno  Associated  Viruses 

The  main  objectives  in  studying  defective  human  parvoviruses  (AAV) 
are  to  define  specific  biochemical  mechanisms  used  in  synthesizing 
their  DNA,  RNA  and  proteins,  to  identify  and  characterize  the  helper 
virus  ^nediated  step(s)  required  for  their  replication,  to  relate  biochemical 
findings  to  normal  cellular  processes,  and  to  determine  conditions  for 
selective  interference  of  both  AAV  and  Helper  virus  (adenoviruses, 
herpesviruses)  infection. 

Factors  Which  Confer  Permissivity  on  Defective  AAV 

Human  adenovirus  (Ad)  serotypes  provide  an  early  factor (s)  that  is 
necessary  for  adenovirus-associated  virus  (AAV)  multiplication  in  human 
cell  lines.  However,  little  if  any  AAV  production  occurs  in  primary 
African  green  monkey  kidney  (AGMK)  cells  co- infected  with  AAV  and  a 
helper  human  Ad  (non-permissive  infection)  unless  cells  are  additionally 
infected  with  SV..  (permissive  infection) .  To  determine  the  basis  of 
the  host  restriction  of  AAV  replication  in  AGMK  cells,  AAV  DNA,  RNA  and 
protein  synthesis  were  analyzed  under  various  conditions  of  infection. 
Hybridization  reactions  revealed  no  detectable  AAV-specific  DNA  or  RNA 
in  infections  with  AAV  alone  or  in  combination  with  SV^q.  In  co- infections 
with  AAV  and  Ad5  or  Ad7,  the  synthesis  of  both  AAV-  and  Ad-specific  DNA 
and  RNA  occurred  without  a  significant  rise  in  titre  of  either  virus. 
During  non-permissive  infection,  however,  AAV  DNA  synthesis  was  abnormal 
in  that  an  expected  accumulation  of  single-stranded  progeny  molecules 
was  not  observed.  Finally,  although  intact  20S  AAV  transcripts  were 
present  in  the  cytoplasm  of  AGMK  cells  during  non-permissive  infection 
(in  amounts  ranging  from  50  to  80%  of  that  found  during  permissive 
infection) ,  AAV-specific  polypeptides  were  not  demonstrable  by  polyacrylamide 
gel  electrophoresis.  Taken  together,  these  experiments  indicate  that 
the  host  restriction  of  AAV  replication  in  AGMK  cells  is  exerted  at  the 
level  of  translation  of  the  single  AAV  messenger  RNA.  In  addition,  it 
appears  that  one  or  more  of  the  AAV  polypeptides  specified  by  this 
message  is  required  for  the  production  of  single- stranded  AAV  progeny 
DNA.  The  fact  that  coinfection  with  a  simian  adenovirus,  SV15,  permits 
complete  replication  of  both  Ad  and  AAV  (analogous  to  coinfection  with 


19-4 


SV40)  strongly  suggests  that  an  Ad(SV15)  gene  product  also  exerts  a 
regulatory  effect  on  expression  of  the  AAV  mRNA  (as  well  as  several  late 
human  Ad  mRNAs)  in  AQVIK  cells.  Isolation  and  characterization  of  this 
gene  product  should  be  relevant  to  possible  approaches  for  selectively 
interfering  with  virus  infection.   (Buller,  Sebring,  Rose) 

AAV  DNA  Replication 

Further  understanding  of  mechanisms  involved  in  DNA  replication  was 
obtained  by  studying  AAV  DNA  synthesis  in  cell- free  extracts.  Extracts 
of  nuclei  from  KB  cells  doubly  infected  with  adenovirus-associated  virus 
type  2  (AAV)  and  adenovirus  type  2  (Ad)  were  examined  for  their  ability 
to  synthesize  various  molecular  forms  of  AAV  DNA.  It  was  found  that 
replicating  AAV  DNA  molecules  elongate  to  yield  genome  length  hairpins 
or  linear,  open-ended  duplexes.  Both  plus  and  minus  DNA  strands  serve 
as  templates  for  this  synthesis  which  apparently  does  not  reinitiate  new 
rounds  in  vitro.  Replicating  Ad  DNA  molecules  also  could  be  identified 
in  these  extracts,  but  incorporation  of  [%]TTP  into  Ad  DNA  was  diminished 
80-90%  in  coinfections  with  AAV  as  compared  to  nuclei  extracts  prepared 
from  cells  infected  with  Ad  alone.  These  results  confirm  our  previous 
in  vivo  studies  which  indicated  that  a  self -priming  mechanism  is  involved 
in  the  replication  of  AAV  DNA,  studies  that  represented  the  first  reported 
evidence  for  this  mode  of  initiating  DNA  synthesis.  Investigations  with 
extracts  will  continue  with  the  objective  of  identifying  and  characterizing 
enzymatic  and  regulatory  components  that  participate  in  viral  and  cellular 
DNA  replication.   (Sebring,  Buller,  Rose) 

Adenoviruses 

The  major  objective  of  these  studies  has  been  the  application  of 
physical  and  biochemical  techniques  to  map  and  to  define  the  structure 
of  specific  genetic  regions  in  the  genomes  of  both  oncogenic  and  non- 
oncogenic  human  adenoviruses,  with  the  ultimate  goals  of  (1)  relating 
these  sequences  to  those  biochemical  activities  which  permit  these 
viruses  to  influence  or  gain  control  of  cellular  functions  (e.g.,  cell 
lysis  and  transformation)  and  (2)  of  defining  basic  mechanisms  for 
regulation  of  genetic  expression  (e.g.,  initiation  of  DNA  synthesis).  A 
number  of  unique  features  have  been  described  which  may  have  implications 
in  viral  replication  or  carcinogenesis  or  both. 

DNA  Terminal  Proteins 

The  DNA  of  adenoviruses  is  normally  isolated  as  a  linear  duplex 
molecule  of  discrete  size  using  standard  procedures  of  extraction  with 
protealytic  enzymes,  phenol  and  SDS.  Recently,  it  has  been  possible  to 
demonstrate  forms  of  DNA  that  are  either  circular  or  oligomeric  by  using 
procedures  which  do  not  involve  proteolytic  enzymes.  In  these  latter 
cases,  the  DNA  molecules  are  always  joined  at  their  ends,  and  it  appears 
that  either  end  of  a  molecule  may  interact  with  the  other  end  of  the 
same  molecule  or  with  either  end  of  another  molecule.  The  circles  and 
oligomers  are  resistant  to  treatment  with  4M  guanidinium  chloride,  4M 
urea,  formimide,  sarkosyl  or  mercaptoethanol.  Treatment  with  proteases, 


19-5 


however,  rapidly  converts  the  circles  and  oligomers  to  linear  double- 
stranded  monomers,  as  does  treatment  with  SDS.  It  had  been  previously 
proposed  that  there  is  a  protein  attached  to  each  end  of  the  DNA  molecule 
and  that  this  protein  is  responsible  for  the  formation  of  the  observed 
complexes.  Subsequent  studies  have  clearly  demonstrated  the  presence 
of  a  55,000  dalton  protein  very  tightly  linked  to  the  DNA  molecule. 
Although  the  function  of  the  bound  protein  is  unknown,  it  has  been 
proposed  that  it  may  be  involved  in  DNA  replication  by  allowing  initia- 
tion or  completion  of  the  51  ends  of  the  progeny  strands.  It  has  also 
been  suggested  that  the  protein  may  have  a  structural  role  by  circularizing 
the  DNA  within  the  virus  particle,  may  protect  the  DNA  from  exonuclease 
digestion  or  may  act  as  an  endonuclease  in  a  hairpin  model  of  DNA  replication. 
Furthermore,  it  has  been  recently  demonstrated  that  the  efficiency  of 
transfection  with  the  Ad  5  DNA  protein  complex  is  about  100-fold  higher 
than  that  of  pronase- treated  Ad  5  DNA. 

Brown  et  al.  (Journal  of  Virology  16,  366)  first  demonstrated  that 
these  DNA-protein  complexes  did  not  migrate  into  agarose  gels  during 
electrophoresis.  This  observation  provided  the  basis  for  a  useful  means 
for  detecting  not  only  the  presence  or  absence  of  terminal  protein 
components  but  the  identification  and  purification  of  terminal  DNA 
sequences  as  well.  We  have  fractionated  the  Ad  2  DNA-protein  complex  on 
hydroxy lapatite  columns  and  analyzed  the  DNA  eluate  by  agarose  gel  electro- 
phoresis. Although  the  initial  DNA-protein  complexed  did  not  migrate 
into  agarose  gels,  >75%  of  the  initial  DNA  molecules  in  the  DNA  eluate 
were  now  able  to  enter  the  gels  in  spite  of  the  absence  of  protease  or 
SDS  treatment.  When  the  protein  in  the  DNA  eluate  was  labeled  in  vitro 
with  125if  protein  was  not  seen  associated  with  the  DNA  that  migrated 
into  the  gels.  When  the  DNA  eluate  was  examined  in  the  electron  microscope, 
DNA  was  found  to  be  essentially  linear.  The  re-addition  of  a  DNA-free 
protein  fraction  to  the  DNA  eluate  produced  circularization  of  about  20% 
of  these  linear  molecules.  We  conclude  that  circularization  of  the 
adenovirus  DNA-protein  complex  as  well  as  its  inability  to  migrate  into 
agarose  gels  is  likely  due  to  an  aggregate  of  a  virion  component (s)  in 
addition  to  the  covalently-attached  55K  protein  previously  described. 

Significant  quantitative  differences  were  noted  when  Ad  18,  a 
highly  oncogenic  serotype,  was  extracted  with  4M  guanidinium  chloride 
and  the  DNA-protein  complexes  assayed  in  similar  fashion.  Purified 
virions  of  adenovirus  serotype  2  and  18  were  applied  to  gradients 
containing  guanidinium,  peak  fractions  were  pooled  and  the  DNA-protein 
complexes  dialyzed  and  compared.  35-55%  of  the  molecules  from  each 
serotype  appeared  circular  in  the  electron  microscope.  While  the  DNA- 
protein  complexes  from  serotype  2  were  effectively  bound  to  glass  fiber 
filters  (99%)  under  the  conditions  employed,  over  45%  of  the  guanidinium 
purified  material  isolated  from  serotype  18  passed  unimpeded  through  the 
filter  under  identical  conditions.  Of  some  interest  was  the  fact  that 
nearly  all  molecules  in  this  fraction  were  circular.  Bound  material 
could  be  effectively  released  from  filters  with  1%  SDS  or  pronase 
digestion.  DNA  molecules  from  either  serotype  when  extracted  with  pro- 
teolytic enzymes  did  not  bind  to  filters.  Approximately  half  of  the 
guanidinium  purified  material  from  serotype  18  was  shown  to  enter 


19-6 


agarose  gels  upon  electrophoresis.  The  structural  implications  of  this 
population  of  molecules  which  apparently  does  not  bind  to  glass  fiber 
filters  and  which  easily  penetrates  agarose  gels,  yet  retains  the 
ability  to  circularize,  is  of  considerable  biological  interest.  (Garon,  Rose) 


Pox  Viruses 


Vaccinia 


Our  primary  objective  is  to  determine  the  molecular  events  that  lead  to 
the  selective  transcription  of  DNA  and  subsequent  processing  and  translation 
of  messenger  RNA  (mRNA) .  Poxviruses  provide  a  unique  and  important  system 
for  such  studies  since  the  enzymes  needed  for  transcription  and  mRNA 
modification  are  packaged  within  the  core  of  infectious  virus  particles. 
A  single  enzyme  complex  containing  three  activities  -  RNA  triphosphatase, 
RNA  guanylyltransf erase,  and  RNA  (guanine-7-)methyltransf erase  -  has  been 
purified  from  vaccinia  virus.  Acting  consecutively,  the  individual  activities 
in  the  complex  are  capable  of  modifying  the  5 '-end  of  nascent  RNA  molecules 
to  form  a  cap  structure  in  vitro.  Extracts  from  uninfected  human  cells 
were  shown  to  carry  out  a  similar  set  of  reactions,  however,  three  separate 
enzymes  were  found  instead  of  a  single  complex.  The  donor  and  acceptor 
substrate  specificities  of  the  different  viral  and  cellular  enzymes 
suggested  mechanisms  used  for  processing  mRNA  in  vivo.  Evidence  for  temporal 
control  of  vaccinia  virus  gene  expression  at  the  transcriptional  level 
has  been  obtained  by  DNA: RNA  hybridization  studies  and  by  translation  of 
viral  mRNA  in  a  cell-free  protein  synthesizing  system.  Transcriptional 
and  translational  maps  of  the  vaccinia  virus  genome  are  being  prepared 
using  DNA  fragments,  produced  by  restriction  endonuclease  digestion, 
that  have  been  cloned  in  phage  lambda  using  approved  DNA  recombinant 
technology. 

Major  Findings 

1.   Post-transcriptional  modification  of  mRNA 

A.  Isolation  and  characterization  of  viral  capping  and  methylating 
enzymes. 

Members  of  this  research  group  previously  identified  the  5 '-terminal 
cap  structure  m7G(5')pppN  consisting  of  a  7-methylguanosine  (m7G)  linked 
by  a  5'  to  5'  triphosphate  bridge  to  one  or  two  consecutive  2 ' -O-methylated 
ribonucleosides  (N)  present  in  viral  and  cellular  mRNAs.  The  mechanism 
of  cap  formation  was  determined  by  purifying  the  appropriate  enzymes  from 
vaccinia  virus.  The  latter,  included  (i)  an  enzyme  complex  containing  both 
mRNA  guanylyltransf erase,  which  is  the  capping  enzyme,  and  mRNA(guanine-7-) 
methyltransf erase  and  (ii)  a  separate  mRNA  (nucleoside-2'-) -methyl transferase. 
Further  studies  of  the  purified  enzyme  complex  during  the  past  year  revealed 
a  third  activity,  RNA  triphosphatase.  On  a  molar  basis,  the  RNa  tri- 
phosphatase is  nearly  100-fold  more  active  than  the  associated  mRNA 
guanylyltransf erase.  Consequently,  both  triphosphate  and  diphosphate- 
ended  RNAs  are  capped  at  similar  rates  and  modification  of  the  5 '-end  of 
incomplete  or  nascent  transcripts  occurs  in  the  following  order: 


19-7 


pppN,-N2.. .  .-*•  ppN-jHSL. . .+  Pi  (i) 

GTP  +  ppN,-N2. .  .+  G(5'  JpppKL-N   . .+  PPi  (ii) 

AdoMet  +  G(5 ' )ppFN,-N2. .  .->  m  G(5'  )pppN, -N2- . .+  MoHcy     (iii) 

AdoMet  +  m7G(5')pppN1-N:,...^  m7G  ( 5 ' )  pppN?-N„ . . .  +  AdoHcy     (iv) 
(Venkatesan,  Mass) 

B.   Isolation  and  characterization  of  cellular  capping  and  methylating 
enzymes. 

Previously,  we  detected  capping  activity  in  crude  extracts  prepared  from 
HeLa  cell  nuclei.  During  the  past  year,  this  enzyme  has  been  purified 
approximately  1,000-fold  and  extensively  characterized  particularly 
with  respect  to  its  donor  and  acceptor  substrate  specificities.  This 
analysis  has  provided  important  information  regarding  the  mechanism  of 
capping  cellular  RNA.  Of  a  variety  of  potential  donor  molecules,  only 
GTP  and  ITP  were  utilized  by  the  purified  enzyme.  These  results  indicated 
that  the  mRNA  guanylyltransf erase  recognizes  the  oxygen  on  the  purine 
ring  of  GTP,  discriminates  between  ribose  and  deoxyribose  sugars,  and 
requires  a  triphosphate.  The  inability  to  use  m  GTP  as  a  donor  indicates 
that  methylation  occurs  after  capping.  With  regard  to  acceptor  molecules, 
the  enzyme  is  specific  for  a  diphosphate-ended  oligo-  or  polyribonucleotide. 
The  inability  to  efficiently  cap  triphosphate-ended  acceptors  is  explained 
by  the  absence  of  an  associated  RNA  triphosphatase.  In  that  respect,  and  also 
by  the  absence  of  an  associated  mRNA( guanine- 7-) methyl transferase,  the 
HeLa  cell  capping  enzyme  differs  from  the  one  isolated  from  vaccinia  virus. 
This  result  is  consistent  with  our  previous  purification  of  a  specific 
HeLa  cell  mRNA  (guanine-7-)methyltransferase.  Although  the  possibility  of  a 
pre-transcriptional  capping  mechanism  has  been  considered  by  others,  our 
finding  that  the  minimum  length  acceptor  is  a  dinucleotide  such  as  ppGpC 
makes  this  mechanism  unlikely.  In  fact,  the  Km  for  a  dinucleotide  is 
higher  than  for  a  polymer  indicating  that  a  longer  oligonucleotide  or 
short  polyribonucleotide  is  a  better  acceptor.  We  suggest  that  in  vivo,  an 
RNA  triphosphatase  removes  the  third  phosphate  from  the  5 '-end  of  short 
unfinished  primary  transcripts  and  that  they  are  then  capped  by  the 
enzyme  that  we  have  described.  If  this  enzyme  also  caps  RNA  at  sites 
of  cleavage,  then  additional  kinases  capable  of  forming  diphosphate- 
ends  must  exist.  (Venkatesan,  Moss) 

c.   Coupled  transcription  and  modification 

One  of  the  major  advantages  of  working  with  vaccinia  virus  is  that 
all  of  the  enzymes  necessary  for  synthesis  and  modification  are  contained 
within  the  purified  virus  particle.  After  irradiating  purified  vaccinia 
virus  particles  with  ultraviolet  light  to  form  pyrimidine  dimers  in  the 
DNA,  only  short  abortive  transcription  products  were  made.  Analysis  of 
these  products  indicated,  however,  that  they  were  capped,  methylated  and 
polyadenylylated.  These  results  suggested  to  us  that  neither  completion 
of  an  RNA  chain  nor  processing  from  a  polycistronic  precursor  was  required 
for  modification  of  either  end  of  the  RNA.  The  presence  of  the  poly (A) 


19-8 


tract  at  the  3 ' -end  of  the  short  transcript  further  suggested  that  a 
slow-down  or  cessation  of  transcription,  rather  than  a  specific  3 '-terminal 
sequence,  serves  as  a  signal  for  polyadenylylation.   (Gershowitz,  Moss) 

2.   Organization  and  regulation  of  the  vaccinia  virus  genome 

A.  In  vitro  translation  of  irrmediate  early,  early  and  late  classes  of 
RNA  from  vaccinia  virus  infected  cells      "  "  "  "'" 

Cytoplasmic  RNA,  isolated  at  various  times  after  vaccinia  virus 
infection,  was  translated  in  a  message-dependent  cell-free  system  prepared 
from  rabbit  reticulocytes.  When  programmed  with  RNA  extracted  at  2  hr  after 
infection,  early  viral  proteins  were  made  and  formation  of  cellular  proteins 
was  diminished.  Primarily  late  proteins  were  synthesized  using  RNA 
extracted  at  4  or  more  hours  after,  infection,  suggesting  that  the  switch 
in  protein  synthesis  is  regulated  principally  by  changes  in  RNA  concentration 
rather  than  by  modification  of  the  translation  apparatus  of  the  cell. 
Immediate  early  RNA  was  obtained  by  infecting  cells  in  the  presence  of 
inhibitors  of  protein  synthesis.  Comparisons  between  immediate  early  and 
early  gene  products  did  not  reveal  a  class  of  early  genes  that  require 
protein  synthesis  for  expression.  On  the  contrary,  seven  polypeptides, 
of  which  a  28,000  dalton  species  was  most  prominent,  were  synthesized  in 
relatively  greater  amounts  with  immediate  early  RNA  than  with  early  RNA. 
This  result  suggests  the  possibility  that  expression  of  certain  immediate 
early  genes  is  regulated  by  a  feed-back  mechanism.   (Cooper,  Moss) 

B.  Analysis  of  the  terminal  repetition  within  the  vaccinia  virus  genome 

Previously  we  reported  that  a  3.54  ym  duplex  was  formed  by  annealing 
the  two  ends  of  the  vaccinia  virus  genome.  This  inverted  terminal  repetition 
was  estimated  to  be  about  10,000  nucleotide  base  pairs  long.  To  further 
characterize  this  structural  feature,  restriction  maps  were  made  of  the 
ends  of  the  genome  using  the  following  enzymes:  Eco  RI,  Hpa  II,  and  Hind  II. 
To  accomplish  this,  we  developed  a  novel  mapping  method,  involving  use  of 
"nick  translated"  probes  prepared  from  the  terminal  fragment,  to  order 
partial  digestion  products.  From  this  study,  we  concluded  that  identical 
restriction  sites  are  present  at  the  2  ends  of  the  genome  for  approximately 
10,000  nucleotides  confirming  our  previous  electron  microscopic  analysis 
and  work  of  other  investigators. 

To  determine  whether  the  inverted  terminal  repetition  is  transcribed 
in  vivo,  32P- labeled  cytoplasmic  RNA  was  obtained  from  infected  cells. 
This  RNA  was  then  annealed  to  restriction  fragments,  prepared  from  the  terminal 
repetition  and  adjacent  regions  of  the  DNA  that  were  immobilized  to  a 
nitrocellulose  sheet.  We  found  that  early  RNA  hybridized  to  the  repetition 
except  for  the  terminal  3,000  nucleotide  base  pairs  indicating  that  it 
is  indeed  transcribed.   (Barbosa,  Moss) 

C.  Formation  of  recombinant  DNA  molecules  containing  portions  of  the 
vaccinTa  virus  genome. 

DNA  recombinant  technology  developed  within  the  past  few  years 
followed  by  a  relaxation  of  the  guidelines  for  carrying  out  such  research 

19-9 


has  allowed  us  to  clone  portions  of  the  vaccinia  virus  genome  in  phage 
lambda.  Our  initial  approach  has  been  to  clone  successive  Eco  RI 
fragments  of  DNA  starting  from  one  end  of  the  genome.  Thus  far,  this 
has  included  a  9,500  nucleotide  base-pair  segment  containing  most  of  the 
terminal  repetition,  a  6,500  nucleotide  base-pair  segment  containing 
unique  sequences  adjacent  to  this,  and  the  next  4,000  nucleotides  base- 
pair  segment.  In  order  to  clone  the  end  piece  of  DNA,  it  was  first 
necessary  to  remove  the  terminal  cross-link  with  a  single-strand  specific 
nuclease  and  then  add  synthetic  Eco  RI  linkers.  Each  of  the  recombinant 
DNA  clones  were  identified  by  restriction  endonuclease  digestion  and 
gel  electrophoresis.  Studies  are  now  in  progress  using  the  cloned  genome 
fragments  to  obtain  a  transcription  and  translation  map.   (Wittek,  Chan,  Moss) 

3.   Action  of  specific  antiviral  agents 

Isatin-S-thiosemicarbazone  is  known  to  specifically  inhibit  vaccinia 
virus  replication  at  a  late  stage.  At  6  hr  after  infection,  viral  protein 
synthesis  was  inhibited  by  about  95%.  We  confirmed  that  a  portion  of 
the  virus-specific  RNA  appears  to  be  degraded  (B.  Woodson  and  W.  K.  Joklik, 
1965,  Proc.  Natl.  Acad.  Sci. ,  USA  54:  946-953).  Nevertheless,  the  amount 
of  viral  RNA  that  was  capped,  properly  methylated,  and  polyadenylylated, 
was  reduced  by  only  about  50%.  Moreover,  RNA  from  IBT-treated  cells 
stimulated  cell-free  protein  synthesis  to  one-half  the  level  obtained  with 
RNA  from  control  cells.  Polyacrylamide  gel  electrophoretic  analysis 
further  demonstrated  that  RNA  from  IBT-treated  cells  was  translated  into 
late  viral  proteins  in  vitro.  Thus,  it  seems  possible  that  the  inhibition 
of  protein  synthesis  in  IBT-treated  cells  does  not  result  entirely  or 
directly  from  either  an  inhibition  of  mRNA  synthesis  or  from  a  depletion 
of  mRNA  caused  by  accelerated  degradation.  An  alternative  possibility, 
that  accelerated  degradation  is  secondary  to  a  more  immediate  effect 
of  the  drug  on  protein  synthesis,  was  considered.   (Cooper,  Moss) 

Structural  Studies  of  the  Viral  Genome 

Vaccinia  DNA  has  covalently-linked-ccmplementary  (CDC)  ends  which 
define  each  end  of  the  physical  map  of  the  genome.  These  CLC  end  segments 
can  be  identified  in  each  set  of  segments  that  are  produced  by  a  particular 
restriction  enzyme.  The  set  of  segments  is  alkali  denatured,  neutralized 
and  passed  through  a  BND-cellulose  column  from  which  only  the  CLC  segments 
elute  as  duplex  chains.  The  end  segments  have  been  identified  for 
several  restriction  enzymes  such  as  Sal  I,  Hind  III,  Kpn  I  and  Xho  I. 
For  Sal  I,  the  end  segments  are  identical  and  small  with  a,-mass  of 
2.1  X  106  daltons.  For  Xho  they  are  larger  (both  3.9  X  10  )  and  for 
Hind  III  they  are  larger  still  (17.6  and  13.5  X  106) . 

The  restriction  enzymes  listed  above  as  well  as  several  others  are 
used  to  digest  vaccinia  DNA  to  obtain  unique  DNA  segments  which  are 
separated  by  agarose  electrophoresis.  To  obtain  a  physical  map,  segments 
produced  by  one  enzyme  are  isolated  from  agarose  and  digested  by  a 
second  enzyme  to  find  overlapping  regions.  The  end  segments  described 
here  serve  as  reference  points  from  which  the  maps  are  continued.  The 
Sal  I  and  Hind  III  map  of  vaccinia  strain  WR  are  now  almost  completely 
known.  Kpn  and  Xho  maps  are  also  near  completion. 

19-10 


A  particular  method  which  has  helped  to  generate  reproducible 
partial  DNA  segments  involves  restriction  enzyme  digestion  in  the  presence 
of  actinomycin  D.  The  original  observation  that  the  drug  blocked 
complete  digestion  with  Hind  III  has  been  extended  to  3  other  restriction 
enzymes,  Sal  I,  Xho  and  Kpn.  None  of  these  digests  produce  as  long  a 
partial  as  the  one  half  length  vaccinia  DNA  molecule  generated  with  Hind 
but  they  do  contain  several  smaller  linked  segments  which  help  to  establish 
the  map. 

Initial  experiments  designed  to  locate  the  origin  of  replication 
involve  pulse  labeling  HeLa  cells  with  H-thymidine  after  infection  with 
high  multiplicities  of  vaccinia  virus.  A  peak  of  synthetic  activity 
occurs  2.5  hours  after  infection.  From  2.5  to  3.5  hours  after  infection 
alkaline  and  neutral  sucrose  gradients  of  cytoplasmic  (viral)  DNA 
reveal  a  spectrum  of  molecular  sizes  with  about  10  -  20%  of  the  DNA  in 
full  length  molecules.  These  DNA  species  will  be  examined  in  a  Dintzis 
(Proc.  Natl.  Acad.  Sci.  £7:  247,  1961)  type  experiment. 

Several  experimental  techniques  have  evolved  during  the  year.  The 
method  of  Vogelstein  and  Gillespie  (Proc.  Natl.  Acad.  Sci.  76_:  615, 
1979)  for  extracting  DNA  segments  from  agarose  with  glass  powder  has 
been  modified  so  that  it  is  now  possible  to  routinely  recover  intact, 
70%  or  more  of  yg  amounts  of  large  vaccinia  DNA  segments.   Also,  poly 
rC  columns  have  been  used  to  hybridize  segments  which  contain  short 
stretches  of  GC  rich  regions  in  duplex  DNA.  Using  this  column  technique, 
we  have  found  that  the  largest  Sal  I  segment  binds  to  the  column.  Since 
GC  rich  regions  may  provide  a  structural  basis  for  a  signal  element,  the 
Sal  I  segment  will  be  digested  with  other  enzymes  to  localize  the  GC 
region.  Finally,  we  have  started  to  use  ..malachite  green  columns  to 
fractionate  sheared  vaccinia  DNA  (6  X  10  daltons)  according  to  base 
composition.  Those  fragments  with  the  least  affinity  for  the  column 
have  the  highest  average  GC  content.  They  will  be  radiolabeled  and 
hybridized  to  Sal  I  digests  to  identify  those  segments  from  which  they 
originated.   (De  Filippes) 

Molluscum  contagiosum 

Molluscum  contagiosum  virions  were  isolated  from  clinically  typical 
skin  lesions  and  the  DNA  extracted  and  characterized  using  techniques  of 
electron  microscopy  and  agarose  gel  electrophoresis.  The  structural  char- 
acteristics determined  in  these  studies  tend  to  place  the  MCV  genome  among 
other  members  of  the  poxvirus  group  in  terms  of  genome  molecular  weight 
and  organization.  The  apparent  denaturability  of  the  NCV  genome  into  a 
continuous,  single-stranded  circle  would  point  to  the  presence  of 
terminal  cross-links  in  the  DNA  molecule.  So  far,  this  structural 
arrangement  appears  to  be  unique  to  members  of  the  poxvirus  group.  The 
objective  of  these  studies  has  been  to  define  structural  features  of 
these  molecules  and  to  relate  them  to  the  biochemical  events  involved  in 
the  replication  and  growth  of  this  virus  in  the  cells  they  infect. 

Extracts  from  molluscum  contagiosum  lesions  when  stained  with  sodium 
phosphatungstate  and  examined  in  the  electron  microscope  appears  to 


19-11 


contain  many  structurally  mature  virus  particles  of  both  C  and  M  forms. 
However,  the  virus  has  not  been  successfully  propagated  in  the  laboratory. 
Molecular  information  about  this  virus  has  been  limited  to  description 
of  size,  shape  and  composition  and  has  been  difficult  to  obtain.  The 
structural  characteristics  determined  in  our  initial  studies  (Virology  81: 
247)  tend  to  place  the  MCV  genome  among  other  members  of  the  poxvirus 
group  in  terms  of  molecular  weight  and  organization.  The  average  molecular 
weight  of  MCV-DNA  was  calculated  to  be  118  X  10  and  appeared  to  be 
extremely  sensitive  to  both  mechanical  shear  and  nuclease  damage. 
Single-stranded  circles  measuring  twice  the  length  of  linear  molecules 
were  observed  following  high  levels  of  denaturation.  This  now  appears 
to  be  a  generalized  feature  of  all  poxvirus  DMAs  so  far  examined.  The 
biological  function,  if  any,  of  the  unique  terminal-cross- links  is 
unknown.  Also  of  special  interest  was  the  fact  that  the  most  easily 
denaturable  regions  of  the  MCV  genome  (and  presumably  that  of  the  highest 
AT  content)  appeared  to  be  the  end  19%  of  the  DNA  molecule.  That  the  MCV 
genome  differs  markedly  from  that  of  vaccinia  is  clear  from  the  visual 
denaturation  profiles  and  from  the  restriction  endonuclease  cleavage 
patterns  obtained  in  our  initial  studies.  Further  differences  were  noted 
in  the  extractability  of  the  various  poxvirus  genomes.  Procedures  used 
for  successfully  extracting  and  purifying  the  DNA  molecules  from  one 
viral  genome  proved  completely  unsuitable  for  the  other. 

Previously,  little  was  known  about  whether  most  isolates  belonged 
to  the  same  or  different  MCV  strains  or  whether  a  given  strain  was 
always  isolated  from  similar  clinical  sources.  A  survey  of  the  restriction 
endonuclease  fragment  patterns  of  several  independent  MCV  isolates  was 
initiated.  Lesions  obtained  from  individual  patients  were  never  pooled 
but  rather  purified,  extracted,  and  assayed  as  a  single  isolate.  Initially, 
eleven  such  isolates  were  processed.  In  these  studies,  lesions  isolated 
from  various  sites  on  an  individual  patient  appeared  to  contain  virus 
whose  DNA  showed  identical  gel  patterns.  Furthermore,  virus  was  isolated 
from  the  lesions  of  patients'  relatives  which,  when  assayed  by  the  above 
restriction  endonuclease  procedure,  showed  gel  patterns  identical  to 
those  of  the  patients  themselves.  Interestingly,  only  3  characteristically 
frequent  patterns  were  observed  among  eleven  independent  isolates. 
Restriction  endonuclease  digestion  produced  11  to  20  fragments  ranging 
in  size  from  4  X  10  to  31  X  10  daltons  depending  on  the  virus  isolate. 
Mixing  experiments  showed  comigration  of  some  fragments  in  all  patterns. 
Mixing  experiments  showed  comigration  of  some  fragments  in  all  patterns. 
Although  comigration  of  DNA  fragments  does  not  necessarily  mean  base 
sequence  identity,  extensive  comigration  would  imply  significant  genetic 
or  organization  similarity  among  the  viral  genomes.  Direct  comparisons 
of  these  gel  purified  fragments  by  hybridization  or  heteroduplex  analysis 
have  not  been  attempted  due  to  limits  in  the  quantity  of  viral  DNA 
obtainable  from  each  MCU  isolate. 

Although  we  have  not  collected  a  large  enough  body  of  data  to  make 
any  reasonable  assessment  of  the  relationship  between  the  clinical 
characteristics  of  the  disease  and  the  restriction  endonuclease 
cleavage  patterns,  it  may  be  possible  once  the  data  are  accumulated,  to 
relate  tehse  observations  and  to  develop  a  useful  molecular  epidemiological 


19-12 


scheme.  Such  a  scheme  would  make  possible  rapid  identification  and 
classification  of  MCV  isolates  without  the  necessity  of  propagating  the 
virus  in  the  laboratory.  Furthermore,  since  the  degree  of  sensitivity 
of  such  a  system  is  limited  to  only  the  number  of  cleavage  sites  recognized 
by  a  given  restriction  enzyme,  other  available  enzymes  may  easily  be 
substituted. 

We  propose  to  continue  to  define  structural  features  of  these 
poxvirus  DNA  molecules  with  the  aim  of  (1)  comparing  genetic  variation 
among  members  of  this  group,  and  of  (2)  relating  these  features  to  those 
biochemical  events  which  are  involved  in  the  replication  of  these  agents. 

Honors  and  Awards 

Dr.  NOrman  P.  Salzman  continued  to  serve  on  the  Editorial  Board 
of  the  Journal  of  Virology  and  on  the  Editorial  Advisory  Board,  Biochemistry, 
and  Scientific  Board  of  the  Coordinating  Council  for  Career  Research. 
He  serves  as  Professorial  Lecturer,  Georgetown  University  School  of 
Medicine,  and  was  an  invited  participant  and  Session  Chairman  at  the 
EMBO/FEBS  Workshop  on  Gene  Structure  and  Formation  of  ENA  of  Viruses 
and  Cells. 

Dr.  Claude  Garon  received  the  N.I.H.  Merit  Award. 

Dr.  James  Rose  was  an  invited  participant  at  the  EMBO/FEBS  workshop. 

Dr.  Bernard  Moss  continued  to  serve  as  associate  editor  of  Virology 
and  on  the  editorial  boards  of  the  Journal  of  Virology,  of  Antibiotics 
and  Chemotherapy,  and  of  Intervirology.  In  addition,  he  received  the 
PHS  commendation  Medal,  was  an  invited  principal  speaker  at  the  Society 
for  General  Microbiology  (U.K.)  Meeting,  was  a  Session  Chairman  at  an 
International  Conference  on  Transmethylation,  was  invited  speaker  at 
the  EMBO/FEBS  workshop  on  Gene  Structure  and  Formation  of  RNA  of 
Viruses  and  Animal  Cells,  and  served  on  a  Poxvirus  Study  Group  for  the 
World  Health  Organization. 

Dr.  Riccardo  Wittek,  a  guest  investigator  in  the  LBV,  received  the 
Forderungspreis  which  is  awarded  annually  to  the  outstanding  young 
microbiologist  in  Switzerland,  and  also  served  on  a  Poxvirus  Study  Group 
for  the  World  Health  Organization. 


19-13 


Smithsonian  science  information  exchange!         u.s.  department  of  project  number 

PROJECT    NUMBER    (.Do    NOT    use    this    space)      [HEALTH,    EDUCATION,    AND    -ELfARE 

PUBLIC   HEALTH   SERVICE 

NOTICE  OF  |   n;m   AT    00123-1^    LP" 

j    INTRAORAL   RESEARCH   PROJECT        I   *UX  ^    UU-L^J    "    LC 

!  PERIOD    COVERED 

:  October  1,  1978  -  September  30,  1979 

TITLE  OF  PROJECT  (30  characters  or  lessy 
I 

Messenger  RNA:  Regulation  of  Synthesis,  Modification  and  Translation 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Principal  Investigator:  Bernard  Moss,  Medical  Director,  LBV,  NIAID 

Other:  Sundararajan  Venkatesan  Senior  Staff  Fellow,  LBV,  NIAID 

Ernest  Barbosa  Research  Associate,  LBV,  NIAID 

Steven  Langberg  Staff  Fellow,  LBV,  NIAID 

Jonathan  Cooper  Visiting  Fellow,  LBV,  NIAID 

Bahige  Baroudy  Visiting  Fellow,  LBV,  NIAID 

Riccardo  Wittek  Guest  Investigator,  LBV,  NIAID 


COOPERATING  UNITS  (if  any) 

Ehud  Katz,  Dept.  of  Virology,  Hebrew  University  Medical  Center,  Jerusalem,  Israel 


LAB/BRANCH 

Laboratory  of  Biology  of  Viruses 


SECTION 

Macromolecular  Biology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


'0TAL  MANYEARS: 
9 


3SI0NAL:  OTHER: 

6.5  4.5 


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□  (a1 )  MINORS   Q  (a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Our  primary  objective  is  to  determine  the  molecular  events  that  lead  to  the 
selective  transcription  of  DNA  and  subsequent  processing  and  translation  of 
messenger  RNA  (mRNA)  .  Poxviruses  provide  a  unique  and  important  system  for 
such  studies  since  the  enzymes  needed  for  transcription  and  mRNA  modification 
are  packaged  within  the  core  of  infectious  virus  particles.  A  single  enzyme 
complex  containing  three  activities  -  RNA  triphosphatase,  RNA  guanylytransf erase, 
and  RNA  (guanine-7-)methyltransf erase  -  has  been  purified  from  vaccinia  virus. 
Acting  consecutively,  the  individual  activities  in  the  complex  are  capable  of 
modifying  the  5 '-end  of  nascent  RNA  molecules  to  form  a  cap  structure  in  vitro. 
Extracts  from  uninfected  human  cells  were  shown  to  carry  out  a  similar  set 
of  reactions,  however,  three  separate  enzymes  were  found  instead  of  a  single 
complex.  The  donor  and  acceptor  substrate  specificities  of  the  different 


19-14 


PHS-6040 
(Rev.    10-76) 


viral  and  cellular  enzymes  suggested  mechanisms  used  for  processing  mRNA 
in  vivo.  Evidence  for  temporal  control  of  vaccinia  virus  gene  expression 
at  the  transcriptional  level  has  been  obtained  by  CNA;RNA  hybridization 
studies  and  by  translation  of  viral  mRNA  in  a  cell-free  protein  synthe- 
sizing system.  Transcriptional  and  translational  maps  of  the  vaccinia 
virus  genome  are  being  prepared  using  DNA  fragments,  produced  by  restriction 
endonuclease  digestion,  that  have  been  cloned  in  phage  lambda  using  approved 
DNA  recombinant  technology. 

Major  Findings 

1.  Post-transcriptional  modification  of  mRNA 

A.  Isolation  and  characterization  of  viral  capping  and  methylating 
enzymes  -  Members  of  this  research  group  previously  identified  the  5'- 
terminal  cap  structure  m  G(5')pppN   consisting  of  a  7-methylguanosine 
(m  G)  linked  by  a  5'  to  5 ' triphosphate  bridge  to  one  or  two  consecutive 
2 ' -O-methylated  ribonucleosides  (N  )  present  in  viral  and  cellular  mRNAs. 
The  mechanism  of  cap  formation  was  determined  by  purifying  the  appropriate 
enzymes  from  vaccinia  virus.  The  latter,  included  (i)  an  enzyme  complex 
containing  both  mRNA  guanylyl transferase,  which  is  the  capping  enzyme,  and 
mRNA (guanine-7-)methyltransf erase  and  (ii)  a  separate  mRNA  (nucleoside-2 ' -) 
methyl transferase.  Further  studies  of  the  purified  enzyme  complex  during 
the  past  year  revealed  a  third  activity,  RNA  tr iphosphase .  On  a  molar 
basis,  the  RNA  triphosphatase  is  nearly  100-fold  more  active  than  the 
associated  mRNA  guanylytransf erase .  Consequently,  both  triphosphate  and 
diphosphate-ended  RNAs  are  capped  at  similar  rates  and  modification  of  the 
5' -end  of  incomplete  or  nascent  transcripts  occurs  in  the  following  order: 

pppN,-N2 . . .  ■>  ppN,-N2 +Pi  (i) 

GTP+ppN,-^  ...  +  G  ( 5 ' ) pppN  -N2 +PPi  ( ii ) 

AdcMet+G  ( 5 ' )  pppN1~N2 ■>  m7G  ( 5 ' )  pppN.-N2 . . .  +AdoHcy  ( iii) 

AdoMet+m7G  ( 5 ' )  pppN  HNL ->  m7G  ( 5 ' )  pppN™-N2 . . .  +AdoHcy  ( iv) 

B.  Isolation  and  characterization  of  cellular  capping  and  methylating 
enzymes  -  Previously,  we  detected  capping  activity  in  crude  extracts  prepared 
from  Hela  cell  nuclei.  During  the  past  year,  this  enzyme  has  been  purified 
approximately  1,000-fold  and  extensively  characterized  particularly  with 
respect  to  its  donor  and  acceptor  substrate  specificities.  This  analysis 
has  provided  important  information  regarding  the  mechanism  of  capping 
cellular  RNA.  Of  a  variety  of  potential  donor  molecules,  only  GTP  and  ITP 
were  utilized  by  the  purified  enzyme.  These  results  indicated  that  the 
mRNA  guanylytransf erase  recognizes  the  oxygen  on  the  purine  ring  of  GTP, 
discriminates  between  ribose  and  deoxyribose  sugars,  and  requires  a  tri- 
phosphate. The  inability  to  use  m  GTP  as  a  donor  indicates  that  methylation 
occurs  after  capping.  With  regard  to  acceptor  molecules,  the  enzyme  is 
specific  for  a  diphosphate-ended  oligo-or  polyribonucleotide.  The  inability 
to  efficiently  cap  triphosphate-ended  acceptors  is  explained  by  the  absence 
of  an  associated  RNA  triphosphatase.  In  that  respect  and  also  by  the  absence 

19-15 


of  an  associated  mRNA (guanine-7-) methyl transferase,  the  Hela  cell  capping 
enzyme  differs  from  the  one  isolated  from  vaccinia  virus.  This  result  is 
consistent  with  our  previous  purification  of  a  specific  Hela  cell  mRNA 
(guanine- 7-)methyltransferase.  Although  the  possibility  of  a  pre-transcrip- 
tional  capping  mechanism  has  been  considered  by  others,  our  finding  that 
the  minimum  length  acceptor  is  a  dinucleotide  such  as  ppGpC  makes  this 
mechanism  unlikely.  In  fact,  the  Km  for  a  dinucleotide  is  higher  than  for 
a  polymer  indicating  that  a  longer  oligonucleotide  or  short  polyribonucleotide 
is  a  better  acceptor.  We  suggest  that  in  vivo,  an  RNA  triphosphate  removes 
the  third  phosphate  from  the  5 '-end  of  short  unfinished  primary  transcripts 
and  that  they  are  then  capped  by  the  enzyme  that  we  have  described.  If  this 
enzyme  also  caps  RNA  at  sites  of  cleavage,  then  additional  kinases  capable 
of  forming  diphosphate-ends  must  exist. 

C.  Coupled  transcription  and  modification  -  One  of  the  major  advantages  of 
working  with  vaccinia  virus  is  that  all  of  the  enzymes  necessary  for 
synthesis  and  modification  are  contained  within  the  purified  virus  particle. 
After  irradiating  purified  vaccinia  virus  particles  with  ultraviolet  light 
to  form  pyrimidine  dimers  in  the  DNA,  only  short  aborative  transcription 
products  were  made.  Analysis  of  these  products  indicated,  however,  that 
they  were  capped,,  methylated  and  polyadenylylated .   These  results  suggested 
to  us  that  neither  completion  of  an  RNA  chain  nor  processing  from  a  poly- 
cistronic  precursor  was  required  for  modification  of  either  end  of  the  RNA. 
The  presence  of  the  poly (A)  tract  at  the  3 '-end  of  the  short  transcript 
further  suggested  that  a  slow-down  or  cessation  of  transcription,  rather 
than  a  specific  3 '-end  terminal  sequence,  serves  as  a  signal  for  poly- 
adenyly lation . 

2.  Organization  and  regulation  of  the  vaccinia  virus  genome. 

A.  In  vitro  translation  of  immediate  early,  early  and  late  classes 
of  RNA  from  vaccinia  virus  infected  cells  -  Cytoplasmic  RNA,  isolated  at 
various  times  after  vaccinia  virus  infection,  was  translated  in  a  message- 
dependent  cell-free  system  prepared  from  rabbit  reticulocytes.  When 
programmed  with  RNA  extracted  at  2  hr  after  infection,  early  viral  proteins 
were  made  and  formation  of  cellular  proteins  was  diminished.  Primarily 
late  proteins  were  synthesized  using  RNA  extracted  at  4  or  more  hours  after 
infection,  suggesting  that  the  switch  in  protein  synthesis  is  regulated 
principally  by  changes  in  RNA  concentration  rather  than  by  modification  of 
the  translation  apparatus  of  the  cell.  Immediate  early  RNA  was  obtained 

by  infecting  cells  in  the  presence  of  inhibitors  of  protein  synthesis.  Com- 
parison between  immediate  early  and  early  gene  products  did  not  reveal  a 
class  of  early  genes  that  require  protein  synthesis  for  expression.  On 
the  contrary,  seven  polypeptides,  of  which  a  28,000  dalton  species  was  most 
prominent,  were  synthesized  in  relatively  greater  amounts  with  imtiediate 
early  RNA  than  with  early  RNA.  This  results  suggests  the  possibility  that 
expression  of  certain  immediate  early  genes  is  regulated  by  a  feed-back 
mechanism. 

B.  Analysis  of  the  terminal  repetition  within  the  vaccinia  virus 
genome  -  Previously  we  reported  that  a  3.54um  duplex  was  formed  by  annealing 
the  two  ends  of  the  vaccinia  virus  genome.  This  inverted  terminal  repetition 

19-16 


was  estimated  to  be  about  10,000  nucleotide  base  pairs  long.  To  further 
characterize  this  structural  feature,  restriction  maps  were  made  of  the 
ends  of  the  genome  using  the  following  enzymes:  EcoRI,  Hpall,  and  Hindlll. 
To  accomplish  this  we  developed  a  novel  mapping  method,  involving  use  of 
"nick  translated"  probes  prepared  from  the  terminal  fragment,  to  order  partial 
digestion  products.  From  this  study,  we  concluded  that  identical  restriction 
sites  are  present  at  the  2  ends  of  the  genome  for  approximately  10,000 
nucleotides  confirming  our  previous  electron  microscopic  analysis  and  work 
of  other  investigators. 

To  determine  whether  the  inverted  terminal  repetition  is  transcribed  in  vivo, 

P- labeled  cytoplasmic  RNA  was  obtained  from  infected  cells.  This  RNA  was 
then  annealed  to  restriction  fragments ,  prepared  from  the  terminal  repetition 
and  adjacent  regions  of  the  DNA  that  were  immobilized  to  a  nitrocellulose 
sheet.  We  found  that  early  RNA  hybridized  to  the  repetition  except  for  the 
terminal  3,000  nucleotide  base  pairs  indicating  that  it  is  indeed  transcribed. 

C.  Formation  of  recombinant  DNA  molecules  containing  portions  of  the 
vaccinia  virus  genome  -  DNA  recombinant  technology  developed  within  the 
past  few  years  followed  by  a  relaxation  of  the  guidelines  for  carrying  out 
such  research  has  allowed  us  to  clone  portions  of  the  vaccinia  virus  genome 
in  phage  lambda.  Our  initial  approach  has  been  to  clone  successive  EcoRI 
fragments  of  DNA  starting  from  one  end  of  the  genome.  Thus  far,  this  has 
included  a  9,500  nucleotide  base-pair  segment  containing  most  of  the 
terminal  repetition,  a  6,500  nucleotide  base-pair  segment  containing  unique 
sequences  adjacent  to  this,  and  the  next  4,000  nucleotides  base-pair  segment. 
In  order  to  clone  the  end  piece  of  DNA,  it  was  first  necessary  to  remove 
the  terminal  cross-link  with  a  single-strand  specific  nuclease  and  then 
add  synthetic  EcoRI  linkers.  Each  of  the  recombinant  DNA  clones  were 
identified  by  restriction  endonuclease  digestion  and  gel  electrophoresis. 
Studies  are  now  in  progress  using  the  cloned  genome  fragments  to  obtain  a 
transcription  and  translation  map. 

3.  Action  of  specific  antiviral  agents. 

Isatin-6-thiosemicarbazone  is  known  to  specifically  inhibit  vaccinia  virus 
replication  at  a  late  stage.  At  6  hr  after  infection,  viral  protein  synthesis 
was  inhibited  by  about  95%.  We  confirmed  that  a  portion  of  the  virus-specific 
RNA  appears  to  be  degraded  (B.  Woodson  and  W.K.  Joklik,  1965,  Proc.  Nat. 
Acad.  Sci.,  USA  54,  946-953) .  Nevertheless,  the  amount  of  viral  RNA  that 
was  capped,  properly  nethylated,  and  polyadenylylated,  was  reduced  by  only 
50%.  Moreover,  RNA  from  TBT-treated  cells  stimulated  cell- free  protein 
synthesis  to  one-half  the  level  obtained  with  RNA  from  control  cells.  Poly- 
acrylamide  gel  electrophoretic  analysis  further  demonstrated  that  RNA  from 
IBT-treated  cells  was  translated  into  late  viral  proteins  in  vitro.  Thus, 
it  seems  possible  that  the  inhibition  of  protein  synthesis  in  IBT-treated 
cells  does  not  result  entirely  or  directly  from  either  an  inhibition  of  mRNA 
synthesis  or  from  a  depletion  of  mRNA  caused  by  accelerated  degradation.  An 
alternative  possibility,  that  accelerated  degradation  is  secondary  to  a  more 
immediate  effect  of  the  drug  on  protein  synthesis,  was  considered. 


19-17 


Publications 

Keith, ,J.M. ,  Ensinger,  M.J.  and  Moss,  B.:  Hela  cell  RNA  (2 ' -O-methyladeno- 
sine-N  -) -methyltransf erase  specific  for  the  capped  5 '-end  of  messenger  RNA. 
J.  Biol.  Chem. ,  253,  5033-5041,  1978 

Keith,  J.M. ,  Muthukrishnan,  S.  and  Moss,  B. :  Effect  of  methylation  of  the 
N  -position  of  the  penultimate  adenosine  of  capped  mRNA  on  ribosame  binding. 
J.  Biol.  Chem.,  253,  5039-5041,  1978 

Cooper,  J. A.  and  Moss,  B. :  Transcription  of  vaccinia  virus  mRNA  coupled 
to  translation  in  vitro.  Virology,  88,  149-165,  1978 

Barbosa,  E.  and  Moss,  B.:  mRNA (nucleoside-2 '-) -methyltransf erase  from  vaccinia 
virus:  purification  and  ;ysical  properties.  J^  Biol.  Chem. ,  253,  7692- 
7697,  1978 

Gershowitz,  A.,  Boone,  R.F.  and  Moss,  B.:  Multiple  roles  for  ATP  in  the 
synthesis  and  processing  of  mRNA  by  vaccinia  virus:  specific  inhibitory 
effects  of  adenosine  (6-y-imido)  triphosphate.  J.  Virol.,  27,  399-408, 
1978 

Garon,  C.F.,  Barbosa,  E.  and  Moss,  B.:  Visualization  of  the  inverted 
terminal  repetition  in  vaccinia  virus  DNA.  Proc.  Natl.  Acad.  Sci.  USA 
75,  4863-4867 

Boone,  R.F.,  Parr,  R.P.  and  Moss,  B.:  Intermolecular  duplexes  formed 
from  polyadenylylated  vaccinia  virus  RNA,  J.  Virol.  30,  365-374 

Moss,  B.,  Barbosa,  E.  and  Keith,  J.M. :  Specificity  of  mRNA  methyl- 
transf erase.   In  Usdin,  E.,  Borchardt,  R.T.,  and  Creveling,  D.  (Ed.): 
Transmethylation,  Elsevier/North-Holland,  N.Y.  1979,  pp.  373-380 

Cooper,  J. A.  and  Moss,  B. :  In  vitro  translation  of  immediate  early,  early 
and  late  classes  of  RNA  from  vaccinia  virus -infected  cells.  Virology,  96, 
368-380 

Cooper,  J. A.,  Moss,  B.,  and  Katz,  E. :  Inhibition  of  vaccinia  virus  late 
protein  synthesis  by  isatin-6-thiosemicarbazone:  characterization  and 
in  vitro  translation  of  viral  mRNA.  Virology,  96,  381-392 

Gershowitz,  A.  and  Moss,  B. :  Abortive  transcription  products  of  vaccinia 
virus  are  guanylylated,  methylated  and  polyadenylylated.  J^  Virol . ,  in  press 

Cooper,  J. A. ,  and  Moss,  B. :  Translation  of  specific  vaccinia  virus  RNAs 
purified  as  RNA-DNA  hybrids  on  potassium  iodide  gradients.  Nucleic  Acids 
Res. ,  in  press 


19-18 


SMITHSONIAN    SCIENCE    INFORMATION    EXCHANGE! uTS.    DEPARTMENT   OF  PROJECT   NUMBER 


[PROJECT  NUMBER    ^Do  MOT   use   this   space) 
I 


HEALTH,    EDUCATION,    AND    WELFARE 
PUBLIC    HEALTH    SERVICE 

NOTICE  OF  I    Z01  AI  00124-10  LBV 

INTRAMURAL   RESEARCH   PROJECT 


PERIOD    COVERED 

October  1,  1978  -  September  30,  1979 


TITLE  OF  PROJECT  (30  characters  or  less) 

Replication  of  the  Parvovirus,  KRV 


NAMES,    LABORATORY    AND    INSTITUTE   AFFILIATIONS,    AND    TITLES   OF  PRINCIPAL    INVESTIGATORS   AND    ALL    OTHER 
PROFESSIONAL   PERSONNEL   ENGAGED   ON   THE   PROJECT 

Principal  Investigator:  Dr.  Lois  A.  Salzman   Research  Chemist   LBV  NIAID 

Other:  Dr.  Tonu  Wali,  Visiting  Fellow        LBV  NTAID 
Ms.  Phyllis  Fabisch,  Microbiologist    LBV  NTAID 


COOPERATING  UNITS  (if  an 


LAB/BRANCH 

Laboratory  of  Biology  of  Viruses 


SECTION 

Biochemical  Virology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS:  PROFESSIONAL: 

3.5  2 


OTHER: 

1.5 


CHECK  APPROPRIATE  30X(E3) 

(a)   HUMAN  SUBJECTS  Q  (b)  HUMAN  TISSUES  2£  (c)  NEITHER 

□  (al)  MINORS   3  (a2)  INTERVIEWS  


SUMMARY  OF  WORK  (200  *ords  or  less  -  underline  keywords) 

We  have  been  studying  the  specific  biochemical  mechanisms  involved  in 

the  replication  of  the  autonomous  parvovirus,  KRV  in  a  rat  nephroma  cell 

line.  The  virus,  isolated  originally  from  a  rat  sarcoma,  contains  one 

molecule  of  linear,  single-stranded  DNA  and  three  capsid  proteins . 

Little  information  is  available  about  replication  of  a  single-stranded 

DNA  virus  in  a  euk^ryotic  cell  or  on  the  transcription  of  this  DNA  to 

make  viral  proteins.  We  have  demonstrated  that  the  virion  KRV-DNA  can 

self  prime  in  vitro  to  synthesize  the  double-stranded  replicative  intermediate. 

In  an  attempt  to  resolve  this  self  priming  terminus,  so  important  in  DNA 

replication,  we  have  sequenced  the  nucleotides  in  the  3'  DNA  terminus 

which  probably  also  contains  the  in  vitro  origin  of  DNA  replication. 


19-19 


PHS-6040 
(Rev.    10-76) 


We  have  previously  found  that  in  an  infected  cell  two  KRV  specific  mRNAs 
are  synthesized.  We  have  mapped  the  major  21S  viral  mRNA  on  the  viral 
DNA  molecule  using  both  the  "Southern"  blotting  technique  and  measurement 
of  the  "R  loops"  seen  in  the  electron  microscope.  Inhibition  studies 
have  shown  that  these  KRV  mRNAs  are  probably  synthesized  by  cellular  RNA 
polymerase  II.  Preliminary  studies  also  show  that  there  are  at  least 
three  virus  induced  proteins  synthesized  during  infection. 

Project  Description 

Parvoviruses  are  the  smallest  DNA  containing  viruses  found  in  vertebrate 
tissues.  They  infect  a  wide  variety  of  species  including  man.  The 
parvoviruses  are  classified  as  autonomous  or  defective  depending  on 
whether  or  not  they  require  a  helper  virus  for  replication.  The  virions 
replicate  best  in  rapidly  dividing  cells  and  produce  a  wide  variety  of 
effects  on  insects,  animals  and  cells  in  culture.  The  feline  parvovirus 
has  been  linked  to  the  naturally  occuring  disease  feline  panleukopenia . 
Rodent  parvoviruses  can  produce  fatal  infections  and  teratogenic  effects 
in  fetal  and  newborn  hamsters.  Parvoviruses  are  able  to  persist  in  a 
latent  state  and  to  inhibit  homologous  and  heterlogous  virus  infection. 
The  viruses  can  also  exhibit  antimitotic  activity  and  interfere  with 
natural  or  virus-induced  tumorigenesis. 

We  have  continued  our  studies  of  the  parvovirus,  KRV,  because  of  its 
genetic  simplicity  and  the  small  size  of  the  virus  gene.  KRV  like  the 
other  parvoviruses  possesses  a  single- stranded  DNA  genome  (1.6  x  106 
daltons  or  4500  to  5000  bases)  with  sufficient  information  to  code  for 
only  a  few  proteins  equvalent  to  about  150,000  daltons.  The  transcription 
process  may  be  very  simple  and  involve  only  one  or  two  messenger  RNAs. 
It  is  probable  that  the  viral  DNA  replication  and  transcriptional  components 
are  carried  out  in  large  part  by  host  functions.  Thus,  we  potentially 
can  gain  insight  into  viral  functions,  host  functions  and  the  interaction 
between  virus  and  host. 

In  the  past  year  we  have  concentrated  our  efforts  into  studying  further 
viral  DNA  replication,  transcription  of  the  viral  genome  and  virus 
induced  proteins.  We  have  found  that  the  single-stranded  DNA  from  KRV 
can  serve  in  vitro  as  a  self-primer  for  the  synthesis  of  a  complementary 
linear  viral  DNA  strand.  A  double  stranded  viral  DNA  molecule  has  been 
proposed  as  an  intermediate  in  the  replication  of  the  viral  genome. 
Little  is  known  about  the  replication  of  single-stranded  linear  DNA  in  a 
eukaryotic  cell.  Because  of  the  importance  of  the  3'  terminus  of  the 
viral  DNA  in  self  priming  viral  DNA  synthesis  and  possibly  in  transcription 
we  sequenced  the  3'  terminal  nucleotides.  This  analysis  has  lead  us  to 
propose  a  3'  terminal  hairpin  structure  with  secondary  structures  and 
the  nucleotide  sequence  of  the  DNA  which  at  least  in  vitro  serves  as  the 
origin  of  replication  of  the  complementary  strand.  We  are  currently 
sequencing  the  5'  terminus  of  the  viral  DNA  which  appears  to  have  a 
unique  structure  and  possibly  an  associated  protein. 

In  our  studies  of  the  transcription  of  KRV,  we  have  previously  found 

19-20 


that  two  KRV-specific  mRNAs  are  synthesized  in  infected  RN  cells.  We 
have  also  found  that  only  the  viral  DNA  strand  is  transcribed.  Two 
techniques  have  now  been  used  to  map  these  mRNAs  to  the  viral  genome. 
We  have  mapped  the  fragments  of  several  restriction  enzymes  to  the  KRV 
viral  genome.  Using  the  mapped  restriction  fragments,  the  major  cytoplasmic 
viral  RNA  (21  S)  has  been  mapped  to  the  viral  genome  using  the  "Southern" 
blotting  technique.  This  has  been  complemented  by  Electron  Microscopy 
of  "R  loops"  or  RNA-DNA  duplexes.  Both  techniques  indicate  that  the  21S 
viral  mRNA  maps  from  approximately  0.38  to  0.98  on  the  viral  DNA  strand. 

Further  studies  are  presently  underway  to  determine  if  longer  transcripts 
are  present  in  the  nucleus,  and  to  try  and  determine  if  posttranscriptional 
processing  occurs  by  simple  cleavage  or  by  "splicing".  We  are  also 
trying  to  elucidate  structural  features  of  the  KRV  mRNA  by  assaying  for 
RNA  cap  structures  at  the  51  end  and  to  determine  translatability  of  the 
KRV  mRNAs  in  in  vitro  assays. 

We  have  used  nuclei  isolated  from  infected  rat  nephroma  cells  to  study 
the  enzymes  involved  in  viral  specific  transcription.  Hybridization  of 
RNA  synthesized  in  isolated  nuclei  indicated  that  viral  specific  RNA 
synthesis  started  at  8  to  9  hours  post  infection.  Viral  specific  RNA 
was  inhibited  by  0.1  g  of  amamitin  per  ml,  suggesting  that  the  viral 
genome  is  transcribed  by  cellular  RNA  polymerase  II.  The  viral  RNA 
synthesized  in  the  isolated  nuclei  was  also  analyzed  on  sucrose  gradinets. 
The  KRV  specific  RNA  varied  in  length  from  26S  (full  DNA  transcript)  to 
4S  in  length.  We  propose  to  further  study  the  components  involved  in 
transcription  of  the  viral  genome  after  gentle  disruption  of  the  nuclei. 


19-21 


Publications 

1.  Salzman,  L.A.  and  Rabisch,  P.  Studies  on  the  Replication  of  KVR 

Single-stranded  Linear  DNA.  J.  Gen.  Virol.  39,  571-574  (1978). 

2.  Salzman,  L.A. ,  Fabisch,  P.,  Parr,  R. ,  Garon,  C.  and  Wali,  T.   In 

vitro  Synthesis  of  Double-Stranded  DNA  from  the  Single-stranded 
KRV  DNA  Genome.  J.  Virol.  27:   784-790  (1978). 

3.  Salzman,  L.A. ,  McKerlie,  L. ,  Fabisch,  P.  and  Koczot,  F.  Studies 

on  a  Protein  Found  Associated  with  Kilham  Rat  Virus.  In  D.  Ward 

and  P.  Tattersall  (eds.)  Parvoviruses.  Cold  Spring  Harbor  Laboratory, 

Cold  Spring  Harbor,  L.I.,  New  York  (1978) . 

4.  Salzman,  L.A. ,  P.  Fabisch.  Nucleotide  sequence  of  the  self -priming 

3 'terminus  of  the  single-stranded  DNA  extracted  from  the  Parvovirus, 
Kilham  Rat  Virus.  J.  Virol.  30:   946-951  (1979). 

5.  Wali,  T.M.  and  Salzman,  L.A.,.  Sedimentation  analysis  of  the  RNA 

synthesized  in  vivo  by  the  autonomous  parvovirus,  Kilham  Rat  Virus. 
J.  Virol.  1979  (in  press) 


19-22 


|SM1  THSON I  AN  SCIENCE  INFORMATION  EXCHANG  E 
PROJECT  NUMBER  (Do  MOT  use  this  space) 


U.S.  DEPARTMENT  Of 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00125-10  LBV 


PERIOD   COVERED 

October  1,  1978  to  September  30,  1979 


i TITLE  OF  PROJECT  (30  characters  or  less) 

Mechanisms  of  Viral  DNA  Replication,  Transcription,  and  Integration 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:  Norman  P.  Salzman 
Walter  Bruszewski 
Minou  Bina-Stein 
Nancy  Chiu 
Yaf f a  Reuveni 
John  A.  Thompson 
John  Brady 
Christian  Lavialle 


Chief            LBV, 

NIAID 

Staff  Fellow      LBV, 

NIAID 

Senior  Staff  Fellow  LBV, 

NIAID 

Staff  Fellow       LBV, 

NIAID 

Visiting  Associate  LBV, 

NIAID 

Staff  Fellow       LBV, 

NIAID 

Staff  Fellow       LBV, 

NIAID 

Visiting  Fellow    LBV, 

NIAID 

COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Biology  of  Viruses 


Biochemical  Virology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

9  .0 


PROFESSIONAL: 


1.0 


4.0 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


2  (b)  HUMAN  TISSUES 


G  (=)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  structure  of  SV40  mRNA  molecules  has  been  determined  by  preparing  _ 
cDNA  copies  of  the  viral  mRNAs  using  reverse  transcriptase.  Characterization 
oFtranscripts  formed  when  SV40  DNA  is  transcribed  by  purified  RNA  polymerases 
has  provided  the  location  of  promoters  of  RNA  transcription.  The  role  of 
viral  proteins  as  regulators  of  transcription  has  been  studied  by  in  vitro 
transcription  of  nucleoprotein  complexes . 


19-23 


PHS-6040 
(Rev.    10-76) 


Papovaviruses 

The  current  studies  have  tried  to  define  some  of  the  processes 
responsible  for  the  formation  of  viral  messenger  RNA  molecules.  Thus 
far,  these  studies  have  provided  the  precise  structures  of  the  viral 
mRNA  molecules,  have  defined  the  nature  of  the  templates  used  for  the 
synthesis  of  viral  transcripts,  have  located  promoters  on  the  SV40 
genome  that  are  recognized  by  E.  coli  RNA  polymerase  and  have  compared 
SV40  DNA  I  and  SV40  DNA  I  nucleoprotein  complexes  as  templates  for 
transcription . 

The  Structure  of  Early  and  Late  Viral  mRNA. 

The  mechanism  of  splicing  of  the  early  and  late  cytoplasmic  species 
of  SV40  mRNA  has  been  studied  using  a  technique  which  first  involves  the 
isolation  of  specific  regions  of  the  viral  genome.  Restriction  enzyme 
cleavage  of  SV40  DNA  generates  specific  DNA  fragments  which  can  be 
fractionated  using  column  chromatography.  These  fragments  are  then 
annealed  to  viral  mRNA  and  the  reaction  products  are  fractionated  by 
velocity  sedimentation.  The  DNA  fragment  which  is  annealed  to  the  viral 
mRNA  is  used  as  a  primer  for  the  enzyme,  reverse  transcriptase,  and  a 
complementary  DNA  copy  of  the  mRNA  is  made.  The  sequence  and  structure 
of  the  cDNA  has  been  compared  with  the  known  nucleotide  sequence  of 
SV40.  This  procedure  has  been  used  to  characterize  the  late  mRNA  which 
codes  for  the  major  structural  viral  coat  protein  and  has  provided 
direct  physical  evidence  for  two  early  mRNA  species.  The  regions  of  the 
DNA  that  have  deleted  in  both  species  of  early  mRNAs  have  been  determined. 
Also,  the  nucleotide  sequences  across  these  spliced  regions  have  been 
determined  and  correlated  with  the  mRNA  coding  for  the  large  T  and  small 
t  antigens.  These  two  mRNA  species  were  determined  to  have  the  same  51 
termini  which  would  suggest  two  levels  for  control  of  genetic  expression. 
One  would  be  the  regulation  of  initiation  of  transcription  at  a  common 
promoter;  the  other  would  involve  post- transcriptional  splicing. 

To  further  characterize  the  mechanism  of  transcription  of  early 
SV40  RNA,  nascent  RNA  chains  that  are  attached  to  template  DNA  molecules 
are  being  isolated  and  characterized  by  the  same  methodology  employed  to 
study  the  processed  cytoplasmic  species  of  early  viral  RNA.  Transcription 
complexes  have  been  isolated  from  infected  cells  which  continue  viral 
mRNA  synthesized  in  vitro.  The  structure  of  these  nascent  chains  gives 
insight  into  the  initial  transcription  products  and  the  mechanisms  that 
operate  to  regulate  transcription.  We  are  investigating  differences 
between  RNA's  formed  in  vivo  and  in  vitro.  The  structure  of  nascent 
chains  will  provide  precise  information  about  the  primary  products  of 
transcription.  We  are  also  studying  the  use  of  isolated  nuclei  for  the 
study  of  the  regulation  of  transcription.  Isolated  nuclei  are  able  to 
sustain  in  vitro  synthesis  of  viral  RNA.  In  this  case,  the  SV40  template 
in  the  cell  nucleus  is  present  as  a  nucleoprotein  complex  containing  the 
five  histones.  This  is  in  contrast  to  in  vitro  synthesis  of  nascent  RNA 
chains  described  above  which  uses  a  less  well  defined  transcriptional 
complex. 


19-24 


These  nascent  SNA  molecules  will  be  probed  with  specific  SV40  DNA 
fragments  to  determine  their  structure.  In  order  to  acquire  a  library 
of  these  specific  fragments,  recombinant  technology  has  been  employed. 
Cloning  of  SV40  DNA  fragments  using  the  plasmid  pBR322  and  E.  coli 
should  provide  sufficient  amounts  of  specific  DNA  fragments  which  can  be 
used  to  probe  nascent  RNA  molecules  and  determine  the  mechanisms  operating 
during  regulation  of  transcription. 

Since  sufficient  amounts  of  specific  SV40  DNA  fragments  can  be 
obtained  from  cloning,  we  will  be  able  to  study  how  synthesis  of  specific 
RNA  sequences  are  affected  by  mutations  within  the  genome.  Further 
characterization  of  RNA  species  obtained  following  Proflavin  treatment, 
which  blocks  RNA  processing,  will  aid  our  understanding  of  the  mechanism 
that  operated  to  regulate  transcription.   (Thompson,  Bina-Stein,  Salzman) 

Localization  of  RNA  Polymerase  Promoters  on  SV40  DNA 

Transcripts  of  SV40  DNA  synthesized  by  Escherichia  coli  RNA  polymerase 
have  been  characterized.  This  model  system  has  been  used  for  the  development 
of  new  methods  applicable  to  the  analysis  of  the  mechanisms  involved  in 
the  synthesis  of  the  viral  messenger  RNAs  in  SV40-infected  cells.  It 
has  been  previously  shown  that  E.  coli  RNA  polymerase  recognizes  specific 
initiation  sites  on  SV40  DNA.  Except  for  one  of  them,  determination  of 
the  location  of  these  sites  on  the  SV40  DNA  map  is  only  approximate.  No 
specific  termination  site  for  transcripts  has  been  identified,  and 
consequently  RNA  polymerase  generates  a  heterogeneous  population  of 
molecules.  The  size  of  some  of  these  RNA  molecules  is  several  times  the 
length  of  the  viral  genome. 

We  have  shown  that,  after  binding  of  the  E.  coli  RNA  polymerase  to 
SV40  DNA,  it  was  possible  to  cleave  such  transcriptional  complexes  with 
"single-cut"  restriction  endonucleases  (Bam  Hj,  Eco  Rj,  Hpa  II).  The 
addition  of  ribonucleotide  triphosphates  to  these  linearized  complexes 
leads  to  the  synthesis  of  defined  species  of  RNA  which  can  be  analyzed 
by  electrophoresis.  The  determination  of  the  size  of  each  of  these  RNAs 
together  with  the  assignment  of  the  DNA  strand  on  which  they  are  transcribed 
will  allow  the  precise  mapping  of  the  various  RNA  promoters  (Reuveni, 
Lavialle,  Salzman) . 

Transcriptional  Properties  of  SV40  Nucleoprotein  Cores 

Simian  Virus  40  (SV40)  provides  an  excellent  model  system  for 
investigating  the  process  of  transcriptional  regulation  in  eukaryotic 
cells.  Many  structural  aspects  of  viral  transcriptional  complexes  and 
mRNA  molecules  have  been  determined.  In  addition,  the  entire  nucleotide 
base  sequence  of  SV40  DNA  has  been  determined.  Similar  to  most  eukaryotic 
chromatin,  during  the  SV40  infection  cycle,  cellular  histones  H2A, 
H2B, ,  H3  and  H.  are  bound  to  the  viral  DNA.  Late  in  the  infection 
cycle,  histone  H]_  also  becomes  associated  with  the  bulk  of  the  viral 
chromatin  in  a  stable  nucleoprotein  complex.  Just  prior  to  encapsidation, 
the  SV40  nucleoprotein  complex  undergoes  a  redistribution  of  proteins 
and  histone  H,  is  replaced  by  viral  proteins.  Since  binding  of  H,  on 
chromatin  may  suppress  transcription  of  DNA  sequences,  we  were  interested 

19-25 


in  determining  if  the  viral  proteins  also  acted  to  modify  or  regulate 
the  process  of  RNA  synthesis.  Such  regulatory  properties  could  be 
important  in  a  clearer  understanding  of  the  process  of  "early"  mRNA 
synthesis  during  infection  of  the  host  cell.  In  view  of  the  close 
similarity  between  this  SV40  DNA-histone-viral  protein  nucleoprotein 
complex  and  cell  chromatin  structure,  such  studies  would  also  be  of 
interest  in  a  more  complete  understanding  of  the  general  problem  of 
transcriptional  regulation  in  eukaryotic  cells. 

A  nucleoprotein  complex  can  be  isolated  from  purified  SV40  virions 
under  very  mild,  physiological  conditions.  These  nucleoprotein  cores 
are  potentially  active  transcriptional  complexes.  These  core  complexes 
do  not  contain  endogenous  RNA  polymerase  activity;  however,  95-100%  are 
able  to  form  active  transcriptional  complexes.  The  transcriptional 
activity  of  those  complexes  is  extremely  high  and  is  similar  to  the 
activity  obtained  with  purified  SV40  Form  1  DNA.  This  observation 
contrasts  with  published  data,  in  which  the  level  of  transcription  of 
nucleoprotein  complexes  (SV40  DNA-Histone)  is  generally  less  than  20%  of 
the  activity  of  deproteinized  SV40  DNA.  The  results  suggest  that  the 
viral  proteins  may  play  a  role  in  the  "activation"  of  the  nucleohistone 
complex.  Analysis  of  the  composition  and  structure  of  the  SV40  nucleoprotein 
core  are  being  carried  out  and  should  allow  an  understanding  of  the 
unexpectedly  high  efficiency  of  the  SV40  core  as  a  template  for  RNA 
synthesis.   (Brady,  Lavialle,  Salzman) 

Publications 

Chiu,  N. ,  Radonovich,  M. ,  Thoren,  M. ,  and  Salzman,  N.  P.,  Selective 
degradation  of  newly  synthesized  non-^nessenger  SV40  transcripts.  J.  Virol., 28: 
590-599,  1978. 

Seidman,  M. ,  Garon,  C. ,  and  Salzman,  N.  P.  The  relationship  of  SV40 
replicating  chromosomes  to  two  forms  of  the  non-replicating  SV40  chromosome. 
Nucl.  Acids  Res.  5_:  2877-2893,  1978. 

Panel  V — Virus  Task  Force.  N.  P.  Salzman,  Chairman.  NIAID  Task  Force 
Report,  DHEW  Publication  No.  (NTH) 79-1835 

Bina,  M. ,  Thompson,  A. ,  Thoren,  M. ,  and  Salzman,  N.  P.  (1979)  Rapid 
sequence  determination  of  the  late  SV40  16S  mRNA  leader  using  inhibitors 
of  reverse  transcriptase.  Proc.  Natl.  Acad.  Sci.  76:  731-735. 

Birkenmeier,  E. ,  Chiu,  N. ,  Radonovich,  M. ,  May,  E. ,  and  Salzman,  N.  P. 
Regulation  of  SV40  early  and  late  gene  transcription  without  viral  DNA 
replication.  J.  Virol.  29:  983-989,  1979. 

Seidman,  M. ,  and  Salzman,  N.  P.  Late  replica tive  intermediates  are 
accumulated  during  SV40  DNA  replication  in  vivo  and  in  vitro.  J.  Virol. 
30:  600-609,  1979 

Thompson,  J.  A. ,  Radonovich,  M. ,  and  Salzman,  N.  Characterization  of  the 
5 '-terminal  structure  of  SV40  early  mRNAs.  J.  Virol. ,  in  press. 


19-26 


SMITHSONIAN    SCIENCE    INFORMATION    EXCHANGE! uTs.    DEPARTMENT   OF  PROJECT   NUMBER 

HEALTH,    EDUCATION,    AND    «£LFARE 

PUBLIC   HEALTH   SERVICE  „„„,.,    „,. 

NOTICE  OF  i     ZOl  AI  00126-06  LBV 

INTRAMURAL  RESEARCH   PROJECT 


PROJECT   NUMBER    (Do   NOT   use   this   space 
I 


a5tobe^0VF,ED1978  to  September  30,  1979 


TITLE  OF  PROJECT  (30  characters  or  less, 

Restriction  Enzyme  Analysis  of  Vaccinia  Virus  DNA 


i NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
\  PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Principal  Investigator:  Frank  DeFilippes,  Research  Physicist   LBV  NIAID 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Biology  of  Viruses 

SECTION 

Macromolecular  Biology  Section 

INSTITUTE   AND    LOCATION 

NIAID,  NIH,  Bethesda,  Maryland     20205 

TOTAL   MANYEARS: 

PROFESSIONAL: 
1 

OTHER: 

.2 

CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS  g  (b)  HUMAN  TISSUES  ~  (c)  NEITHER 

D  (al  )  MINORS   □  (a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  purpose  of  the  project  is  to  study  the  organization  of  genes  in 
large  DNA  molecules  obtained  from  animal  viruses.  The  production  and 
ordering  of  unique  segments  of  vaccinia  virus  DNA  provide  a  physical  map 
of  the  genome.  Physical  maps  have  been  determined  for  several  different 
restriction  enzymes.  I  am  also  working  to  improve  the  techniques  for 
separating  and  isolating  very  large  pieces  of  DNA  which  differ  in  size 
and  base  composition  by  relatively  small  amounts.  In  particular,  I  am 
trying  to  isolate  the  DNA  segments  which  contain  the  origin (s)  of 
replication  of  vaccinia  DNA  and  to  test  for  possible  signal  elements  in 
the  DNA  without  prior  knowledge  of  the  nucleotide  sequence. 


19-27 


PHS-6040 
(Rev.    10-76] 


Project  Description 

Vaccinia  DNA  has  oDvalently-linked-conplementary  (CLC)  ends  which 
define  each  end  of  the  physical  map  of  the  genome.  These  CLC  end  segments 
can  be  identified  in  each  set  of  segments  that  are  produced  by  a  particular 
restriction  enzyme.  The  set  of  segments  is  alkali  denatured,  neutralized 
and  passed  through  a  BND-cellulose  column  from  which  only  the  CLC  segments 
elute  as  duplex  chains.  The  end  segments  have  been  identified  for 
several  restriction  enzymes  such  as  Sal  I,  Hind  III,  Kpn  I  and  Xho  I. 
For  Sal  I,  the  end  segments  are  identical  and  small  with  admass  of 
2.1  X  10  daltons.  For  Xho  they  are  larger  (both  3.9  X  10  )  and  for 
Hind  III  they  are  larger  still  (17.6  and  13.5  X  10  ) . 

The  restriction  enzymes  listed  above  as  well  as  several  others  are 
used  to  digest  vaccinia  DNA.  to  obtain  unique  DNA  segments  which  are 
separated  by  agarose  electrophoresis.  To  obtain  a  physical  map,  segments 
produced  by  one  enzyme  are  isolated  from  agarose  and  digested  by  a 
second  enzyme  to  find  overlapping  regions.  The  end  segments  described 
here  serve  as  reference  points  from  which  the  maps  are  continued.  The 
Sal  I  and  Hind  III  map  of  vaccinia  strain  WR  are  now  almost  completely 
known.  Kpn  and  Xho  maps  are  also  near  completion. 

A  particular  method  which  has  helped  to  generate  reproducible 
partial  DNA  segments  involves  restriction  enzyme  digestion  in  the  presence 
of  actinomycin  D.  The  original  observation  that  the  drug  blocked 
complete  digestion  with  Hind  III  has  been  extended  to  3  other  restriction 
enzymes,  Sal  I,  Xho  and  Kpn.  None  of  these  digests  produce  as  long  a 
partial  as  the  one  half  length  vaccinia  DNA  molecule  generated  with  Hind  III 
but  they  do  contain  several  smaller  linked  segments  which  help  to  establish 
the  map. 

Initial  experiments  designed  to  locate  the  origin  of  replication 
involve  pulse  labeling  HeLa  cells  with  H-thymidine  after  infection  with 
high  multiplicities  of  vaccinia  virus.  A  peak  of  synthetic  activity 
occurs  2.5  hours  after  infection.  From  2.5  to  3.5  hours  after  infection 
alkaline  and  neutral  sucrose  gradients  of  cytoplasmic  (viral)  DNA 
reveal  a  spectrum  of  molecular  sizes  with  about  10  -  20%  of  the  DNA  in 
full  length  molecules.  These  DNA  species  will  be  examined  in  a  Dintzis 
(Proc.  Natl.  Acad.  Sci.  47:  247,  1961)  type  experiment. 

Several  experimental  techniques  have  evolved  during  the  year.  The 
method  of  Vogelstein  and  Gillespie  (Proc.  Natl.  Acad.  Sci.  76:  615, 
1979)  for  extracting  DNA  segments  from  agarose  with  glass  powder  has 
been  modified  so  that  it  is  now  possible  to  routinely  recover  intact, 
70%  or  more  of  ug  amounts  of  large  vaccinia  DNA  segments.   Also,  poly  rC 
columns  have  been  used  to  hybridize  segments  which  contain  short  stretches 
of  GC  rich  regions  in  duplex  DNA.  Using  this  column  technique,  we  have 
found  that  the  largest  Sal  I  segment  binds  to  the  column.  Since  GC  rich 
regions  may  provide  a  structural  basis  for  a  signal  element,  the  Sal  I 
segment  will  be  digested  with  other  enzymes  to  localize  the  GC  region. 
Finally,  we  have  started  to  use  malachite  green  columns  to  fractionate 
sheared  vaccinia  DNA  (6  X  10  daltons)  according  to  base  composition. 

19-28 


Those  fragments  with  the  least  affinity  for  the  column  have  the  highest 
average  GC  content.  They  will  be  radiolabeled  and  hybridized  to  Sal  I 
digests  to  identify  those  segments  from  which  they  originated. 


19-29 


{SMITHSONIAN    SCIENCE    INFORMATION    EXCHANGE!  U.S.    DEPARTMENT   OF 

IPROJECT   NUMBER    (Do    MOT    use    this    space;      IhEALTH,    EDUCATION,    AND    WELFARE 

PUBLIC   HEALTH   SERVICE 
NOTICE   OF 
i    INTRAMURAL    RESEARCH    PROJECT 


PROJECT   NUMBER 


Z01  AI  00127-12  LBV 


PERIOD    COVERED 

!October  1,  1978  to  September  30,  1979 


j TITLE  OF  PROJECT  (80  characters  or  lessy 

Structure  and  Function  of  Genetic  and  Protein  Components  of  Defective 
,  Parvoviruses 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 

James  Rose 

Section  Head 

LBV  NIAID 

Cther: 

Mark  Buller 

Visiting  Fellow 

LBV  NIAID 

John  Janik 

Research  Associate 

LBV  NIAID 

Edwin  Sebring 

Research  Chemist 

LBV  NIAID 

Frank  Koczot 

Research  Biologist 

LBV  NIAID 

Claude  Garon 
James  Garrisor 

Research  Microbiologist 
Bio  Lab  Tech  (Bicchem) 

LBV  NIAID 
LBV  NIAID 

COOPERATING  UNITS  (if  an 


LAB/BRANCH 

Laboratory  of  Biology  of  Viruses 


SECTION 

Molecular  Structure  Section 


INSTITUTE  ANO  LOCATION 

NTATTy  NTH.  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 


PROFESSIONAL: 

4.0 


2.5 


CHECK  APPROPRIATE  30x(E3) 
~  (a)   HUMAN  SUBJECTS 

□  (a1  )  MINORS   :~  (a?)  INTERVIEWS 


r^(b)  HUMAN  TISSUES 


Q  (cj  NEITHER 


SUMMARY  OF  *0RK  (200  words  or  less  -  underline  keywords) 

Main  objectives  in  studying  these  defective  human  parvoviruses 
(AAV)  are  to  (i)  define  specific  biochemical  mechanisms  used  in  synthesizing 
their  DNA,  RNA  and  proteins,  (ii)  identify  and  characterize  the  helper 
virus -^nediated  step(s)  required  for  their  replication  and  (iii)  relate 
biochemical  findings  to  normal  cellular  processes  as  well  as  to  potentials 
for  selective  interference  with  both  AAV  and  Helper  virus  (adenoviruses , 
herpesviruses)  infection.  Among  the  methods  used  are  nucleic  acid 
hybridizations ,  analytical  and  preparative  sucrose  and  CsCl  sedimentations, 
molecular  cleavage  with  restriction  nucleases ,  gel  electrophoresis  and 
electron  microscopy. 


19-30 


?"S-6040 
(Rev.  10-76] 


Project  Description: 

(1)  Most,  if  not  all,  of  the  enzymes  involved  in  the  replication  of  AAV 
DNA  probably  play  corresponding  roles  in  cellular  DNA  synthesis.  Among 
these  activities,  the  enzyme (s)  responsible  for  processing  AAV  concatermeric 
intermediates  is  of  particular  interest  because  of  its  site-specific 
attack  at  the  origin  of  DNA  replication.  We  have  purified  and  partially 
characterized  two  single  strand-specific  endonucleases  from  KB  cells 

that  are  possible  candidates  for  such  a  processing  enzyme.  Both  of 
these  KB  cell  enzymes  (KB,  and  KB„)  have  relatively  high  pH  optima 
(pH  9.2  and  9.5)  and  specifically  cleave  single-stranded  DNA.  However, 
the  KB,  enzyme  differs  from  the  KB-  enzyme  with  respect  to -isoelectric 
point  TKB,  =  10.3;  KB2  =  6-^) ,  absolute  requirement  for  Mg   (KB2 
enzyme  can  also  utilize  M   )  and  relative  rates  of  hydrolysis  with 
homopolymers  (for  KB, :  dG>dT>dA>dC;  for  KB-:  dA>dT>dC>dG) .  Differences 
in  rates  of  hydrolysis  among  the  homopolymers  indicate  some  specificity 
for  different  nucleotide  bonds.  Both  enzymes  hydrolyze  poly(dT)  7-8 
times  more  rapidly  than  denatured  viral  or  cellular  DNA.  In  addition,  we 
have  currently  identified  in  KB  cells  several  other  distinct  endonucleases 
that  hydrolyze  poly(dT)  more  rapidly  than  denatured  DNA.  It  is  planned 
to  continue  the  purification  and  characterization  of  these  endonucleases 
as  well  as  to  attempt  to  identify  their  roles  in  the  cells  and,  possibly, 
the  viral  DNA  replication  process. 

(2)  Human  adenovirus  (Ad)  serotypes  provide  an  early  factor (s)  that  is 
necessary  for  adenovirus-associated  virus  (AAV)  multiplication  in  human 
cell  lines.  However,  little  if  any  AAV  production  occurs  in  primary 
African  green  monkey  kidney  (AGMK)  cells  co- infected  with  AAV  and  a 
helper  human  Ad  (non-permissive  infection)  unless  cells  are  additionally 
infected  with  SV-0  (permissive  infection) .  To  determine  the  basis  of 
the  host  restriction  of  AAV  replication  in  AGMK  cells,  AAV  DNA,  RNA  and 
protein  synthesis  were  analyzed  under  various  conditions  of  infection. 
Hybridization  reactions  revealed  no  detectable  AAV-specif ic  DNA  or  RNA 

in  infections  with  AAV  alone  or  in  combination  with  SV._.  In  co- infections 
with  AAV  and  Ad-  or  Ad_,  the  synthesis  of  both  AAV-  and  Ad-specific  DNA 
and  RNA  occurred  without  a  significant  rise  in  titre  of  either  virus. 
During  non-permissive  infection,  however,  AAV  DNA  synthesis  was  abnormal 
in  that  an  expected  accumulation  of  single-stranded  progeny  molecules 
was  not  observed.  Finally,  although  intact  2 OS  AAV  transcripts  were 
present  in  the  cytoplasm  of  AGMK  cells  during  non-permissive  infection 
(in  amounts  ranging  from  50  to  80%  of  that  found  during  permissive 
infection) ,  AAV-specific  polypeptides  were  not  demonstrable  by  polyacrylamide 
gel  electrophoresis.  Taken  together,  these  experiments  indicate  that 
the  host  restriction  of  AAV  replication  in  AGMK  cells  is  exerted  at  the 
level  of  translation  of  the  single  AAV  messenger  RNA.  In  addition,  it 
appears  that  one  or  more  of  the  AAV  polypeptides  specified  by  this 
message  is  required  for  the  production  of  single-stranded  AAV  progeny 
DNA.  The  fact  that  coinfection  with  a  simian  adenovirus,  SV15,  permits 
complete  replication  of  both  Ad  and  AAV  (analogous  to  coinfection  with 
SV.n)  strongly  suggests  that  an  Ad(SVl5)  gene  product  also  exerts  a 
regulatory  effect  on  expression  of  the  AAV  mRNA  (as  well  as  several  late 
human  Ad  mRNAs)  in  AGMK  cells.  Isolation  and  characterization  of  this 


19-31 


gene  product  should  be  relevant  to  possible  approaches  for  selectively 
interfering  with  virus  infection. 

(3)  Further  understanding  of  mechanisms  involved  in  DNA  replication  was 
obtained  by  studying  AAV  DNA  synthesis  in  cell-free  extracts.  Extracts 
of  nuclei  from  KB  cells  doubly  infected  with  adenovirus-associated  virus 
type  2  (AAV)  and  adenovirus  type  2  (Ad)  were  examined  for  their  ability 
to  synthesize  various  molecular  forms  of  AAV  DNA.  It  was  found  that 
replicating  AAV  DNA  molecules  elongate  to  yield  genome  length  hairpins 
or  linear,  open-ended  duplexes.  Both  plus  and  minus  DNA  strands  serve 
as  templates  for  this  synthesis  which  apparently  does  not  reinitiate  new 
rounds  in  vitro.  Replicating  Ad  DNA  molecules  also  could  be  identified 
in  these  extracts,  but  incorporation  of  [  H]TTP  into  Ad  DNA  was  diminished 
80-90%  in  coinfections  with  AAV  as  compared  to  nuclei  extracts  prepared 
from  cells  infected  with  Ad  alone.  These  results  confirm  our  previous 
in  vivo  studies  which  indicated  that  a  self -priming  mechanism  is  involved 
in  the  replication  of  AAV  DNA,  studies  that  represented  the  first  reported 
evidence  for  this  mode  of  initiating  DNA  synthesis.  Investigations  with 
extracts  will  continue  with  the  objective  of  identifying  and  characterizing 
enzymatic  and  regulatory  components  that  participate  in  viral  and  cellular 
DNA  replication. 


Publications : 

Buller,  R.  M.  L. ,  Straus,  S.  E.  and  Rose,  J.  A.:  Mechanism  of  host 
restriction  of  adenovirus-associated  virus  replication  in  African  green 
monkey  kidney  cells.  J.  Gen.  Virol.  43:  663-672,  1979. 


19-32 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00128-12  LBV 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Properties  of  Adenovirus  DNA 


NAMES,  LABORATORY  ANO  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


Principal  Investigators: 


Other: 


Claude  F.  Garon 
James  A.  Rose 


Research  Microbiologist  LBV  NIAID 
Medical  Officer        LBV  NIAID 


Judy  A.  Sprague   Biologist  LBV  NIAID 

James  W.  Garrison  Bio  Lab  Tech  (Biochem)  LBV  NIAID 


COOPERATING  UNITS  (if  any) 

R.  Padmanabhan  University  of  Maryland  School  of  Medicine,  Baltimore,  Maryland 


lab/branch 
Laboratory  of  Biology  of  Viruses 


SECTION 

Macromolecular  Biology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NTH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 
1.1 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   FJ  (a2)  INTERVIEWS 


HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  major  objective  of  these  studies  has  been  the  application  of 
physical  and  biochemical  techniques  to  map  and  to  define  the  structure 
of  specific  genetic  regions  in  the  genomes  of  both  oncogenic  and  non- 
oncogenic  human  adenoviruses ,  with  the  ultimate  goals  of  (1)  relating 
these  sequences  to  those  biochemical  activities  which  permit  these 
viruses  to  influence  or  gain  control  of  cellular  functions  (e.g.,  cell 
lysis  and  transformation )  and  (2)  of  defining  basic  mechanisms  for 
regulation  of  genetic  expression  (e.g. ,  initiation  of  DNA  synthesis) .  A 


number  of  unique  features  have  been  described  which  may  have  implications 
in  viral  replication  or  carcinogenesis  or  both. 


19-33 


PHS-6040 
(Rev.    10-76) 


Project  Description 

The  DNA  of  adenoviruses  is  normally  isolated  as  a  linear  duplex 
molecule  of  discrete  size  using  standard  procedures  of  extraction  with 
protealytic  enzymes,  phenol  and  SDS.  Recently,  it  has  been  possible  to 
demonstrate  forms  of  DNA  that  are  either  circular  or  oligomeric  by  using 
procedures  which  do  not  involve  protealytic  enzymes.  In  these  latter 
cases,  the  DNA  molecules  are  always  joined  at  their  ends,  and  it  appears 
that  either  end  of  a  molecule  may  interact  with  the  other  end  of  the 
same  molecule  or  with  either  end  of  another  molecule.  The  circles  and 
oligomers  are  resistant  to  treatment  with  4M  guanidinium  chloride,  4M 
urea,  formimide,  sarkosyl  or  mercaptoethanol.  Treatment  with  proteases, 
however,  rapidly  converts  the  circles  and  oligomers  to  linear  double- 
stranded  monomers,  as  does  treatment  with  SDS.  It  had  been  previously 
proposed  that  there  is  a  protein  attached  to  each  end  of  the  DNA  molecule 
and  that  this  protein  is  responsible  for  the  formation  of  the  observed 
complexes.  Subsequent  studies  have  clearly  demonstrated  the  presence 
of  a  55,000  dalton  protein  very  tightly  linked  to  the  DNA  molecule. 
Although  the  function  of  the  bound  protein  is  unknown,  it  has  been 
proposed  that  it  may  be  involved  in  DNA  replication  by  allowing  initia- 
tion or  completion  of  the  5'  ends  of  the  progeny  strands.  It  has  also 
been  suggested  that  the  protein  may  have  a  structural  role  by  circularizing 
the  DNA  within  the  virus  particle,  may  protect  the  DNA  from  exonuclease 
digestion  or  may  act  as  an  endonuclease  in  a  hairpin  model  of  DNA  replication. 
Furthermore,  it  has  been  recently  demonstrated  that  the  efficiency  of 
transfection  with  the  Ad  5  DNA  protein  complex  is  about  100-fold  higher 
than  that  of  pronase- treated  Ad  5  DNA. 

Brown  et  al.  (Journal  of  Virology  16_,  366)  first  demonstrated  that 
these  DNA-protein  complexes  did  not  migrate  into  agarose  gels  during 
electrophoresis.  This  observation  provided  the  basis  for  a  useful  means 
for  detecting  not  only  the  presence  or  absence  of  terminal  protein 
components  but  the  identification  and  purification  of  terminal  DNA 
sequences  as  well.  We  have  fractionated  the  Ad  2  DNA-protein  complex  on 
hydroyapatite  columnes  and  analyzed  the  DNA  eluate  by  agarose  gel  electro- 
phoresis. Although  the  initial  DNA-protein  complexed  did  not  migrate 
into  agarose  gels,  >75%  of  the  initial  DNA  molecules  in  the  DNA  eluate 
were  now  able  to  enter  the  gels  in  spite  of  the  absence  of  protease  or 
SDS  treatment.  When  the  protein  in  the  DNA  eluate  was  labeled  in  vitro 
with  125j;  protein  was  not  seen  associated  with  the  DNA  that  migrated 
into  the  gels.  When  the  DNA  eluate  was  examined  in  the  electron  microscope, 
DNA  was  found  to  be  essentially  linear.  The  re-addition  of  a  DNA- free 
protein  fraction  to  the  DNA  eluate  produced  circularization  of  about  20% 
of  these  linear  molecules.  We  conclude  that  circularization  of  the 
adenovirus  DNA-protein  complex  as  well  as  its  inability  to  migrate  into 
agarose  gels  is  likely  due  to  an  aggregate  of  a  virion  component (s)  in 
addition  to  the  covalently-attached  55K  protein  previously  described. 

Significant  quantitative  differences  were  noted  when  Ad  18,  a 
highly  oncogenic  serotype,  was  extracted  with  4M  guanidinium  chloride 
and  the  DNA-protein  complexes  assayed  in  similar  fashion.  Purified 
virions  of  adenovirus  serotype  2  and  18  were  applied  to  gradients 


19-34 


containing  guanidinium,  peak  fractions  were  pooled  and  the  DNA-protein 
complexes  dialyzed  and  compared.  35-55%  of  the  molecules  from  each 
serotype  appeared  circular  in  the  electron  microscope.  While  the  DNA- 
protein  complexes  from  serotype  2  were  effectively  bound  to  glass  fiber 
filters  (99%)  under  the  conditions  employed,  over  45%  of  the  guanidinium 
purified  material  isolated  from  serotype  18  passed  unimpeded  through  the 
filter  under  identical  conditions.  Of  some  interest  was  the  fact  that 
nearly  all  molecules  in  this  fraction  were  circular.  Bound  material 
could  be  effectively  released  from  filters  with  1%  SDS  or  pronase 
digestion.  DNA  molecules  from  either  serotype  when  extracted  with  pro- 
teolytic enzymes  did  not  bind  to  filters.  Approximately  half  of  the 
guanidinium  purified  material  from  serotype  18  was  shown  to  enter 
agarose  gels  upon  electrophoresis.  The  structural  implications  of  this 
population  of  molecules  which  apparently  does  not  bind  to  glass  fiber 
filters  and  which  easily  penetrates  agarose  gels,  yet  retains  the 
ability  to  circularize,  is  of  considerable  biological  interest. 


19-35 


|SM  I  THSON  I  AN    SCIENCE    INFORMATION    EXCHANGE]  U.S.    DEPARTMENT   OF  j   PROJECT   NUMBER 

.PROJECT   NUMBEP.    [Do    MOT    use    this    space;      JHEALTH,    EDUCATION,    AND    WELFARE    ( 

PUBLIC    HEALTH    SERVICE 
I  NOTICE  Cf 

INTRAMURAL  RESEARCH   PROJECT        |Z01   AI    00156-04    LBV 

PERIOD    COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (50  characters  or  less; 

Structural  Characterization  of  DNA  Virus  Genomes 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Principal  Investigator:  Claude  F.  Garon,  Research  Microbiologist  LBV  NIAID 


COOPERATING  UNITS  (if  any) 

Dr.  J.  W.  Burnett,  University  of  Maryland  Hospital,  Baltimore,  Maryland 
Dr.  H.  B.  Bradford,  Louisiana  Health  and  Human  Resources  Administration 
New  Orleans,  Louisiana 


LAB/ BRANCH 

Laboratory  of  Biology  of  Viruses 


SECTION 

Macromolecular  Biology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20014 


TOTAL  MANYEARS:  j PR0FESSI 0NAL: 

0.2 


CHECK  APPROPRIATE  BOX(ES) 
G  (a)   HUMAN  SUBJECTS  £  (b)  HUMAN  TISSUES  []  (c)  NEITHER 

L  (a1  )  MINORS   2  (a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  »ords  or  less  -  underline  keywords) 

Molluscum  contagiosum  virions  were  isolated  from  clinically  typical  skin 
lesions  and  the  DNA  extracted  and  characterized  using  techniques  of 
electron  microscopy  and  agarose  gel  electrophoresis .  The  structural 
characteristics  determined  in  these  studies  tend  to  place  the  MCV  genome 
among  other  members  of  the  poxvirus  group  in  terms  of  genome  molecular 
weight  and  organization.  The  apparent  denaturability  of  the  MCV  genome 
into  a  continuous,  single-stranded  circle  would  point  to  the  presence  of 
terminal  cross-links  in  the  DNA  molecule.  So  far,  this  structural 
arrangement  appears  to  be  unique  to  members  of  the  poxvirus  group.  The 
objective  of  these  studies  has  been  to  define  structural  features  of 
these  molecules  and  to  relate  them  to  the  biochemical  events  involved  in 
the  replication  and  growth  of  this  virus  in  the  cells  they  infect. 


19-36 


PMS-6040 
(Rev.    10-76) 


Project  Description 

Molluscum  contagiosum  virus  (MCV)  is  a  member  of  the  mammalian 
poxvirus  subgroup  which  causes  a  benign  tumor  of  the  skin  in  humans. 
The  disease  is  most  common  in  children.  Cases  appear  to  occur  world- 
wide and  are  often  associated  with  poverty,  overcrowding  and  poor  hygiene. 
Molluscum  contagiosum  virions  were  isolated  from  clinically  typical  skin 
lesions  and  the  DNA  extracted  and  characterized  using  techniques  of 
electron  microscopy  and  agarose  gel  electrophoresis.  In  some  experiments, 
a  comparison  of  the  structural  features  of  the  MCV  genome  and  other 
members  of  the  poxvirus  group  was  made. 

Extracts  from  molluscum  contagiosum  lesions  when  stained  with 
sodium  phosphatungstate  and  examined  in  the  electron  microscope  appeared 
to  contain  many  structurally  mature  virus  particles  of  both  C  and  M 
forms.  However,  the  virus  has  not  been  successfully  propagated  in  the 
laboratory.  Molecular  information  about  this  virus  has  been  limited  to 
description  of  size,  shape  and  composition  and  has  been  difficult  to 
obtain.  The  structural  characteristics  determined  in  our  initial  studies 
(Virology  81:  247)  tend  to  place  the  MCV  genome  among  other  members  of 
the  poxvirus  group  in  terms  of  molecular  weight  and  organization..  The 
average  molecular  weight  of  MCV-DNA  was  calculated  to  be  118  X  10  and 
appeared  to  be  extremely  sensitive  to  both  mechanical  shear  and  nuclease 
damage.  Single-stranded  circles  measuring  twice  the  length  of  linear 
molecules  were  observed  following  high  levels  of  denaturation.  This  now 
appears  to  be  a  generalized  feature  of  all  poxvirus  DNAs  so  far  examined. 
The  biological  function,  if  any,  of  the  unique  terminal-cross- links  is 
unknown.  Also  of  special  interest  was  the  fact  that  the  most  easily 
denaturable  regions  of  the  MCV  genome  (and  presumably  that  of  the  highest 
AT  content)  appeared  to  be  the  end  19%  of  the  DNA  molecule.  That  the 
MCV  genome  differ  markedly  from  that  of  vaccinia  is  clear  from  the 
visual  denaturation  profiles  and  from  the  restriction  endonuclease 
cleavage  patterns  obtained  in  our  initial  studies.  Further  differences 
were  noted  in  the  extractability  of  the  various  poxvirus  genomes. 
Procedures  used  for  successfully  extracting  and  purifying  the  DNA  molecules 
from  one  viral  genome  proved  completely  unsuitable  for  the  other. 

Previously,  little  was  known  about  whether  most  isolates  belonged 
to  the  same  or  different  MCV  strains  on  whether  a  given  strain  was 
always  isolated  from  similar  clinical  sources.  A  survey  of  the  restriction 
endonuclease  fragment  patterns  of  several  independent  MCV  isolates  was 
initiated.  Lesions  obtained  from  individual  patients  were  never  pooled 
but  rather  purified,  extracted,  and  assayed  as  a  single  isolate.  Initially, 
eleven  such  isolates  were  processed.  In  these  studies,  lesions  isolated 
from  various  sites  on  an  individual  patient  appeared  to  contain  virus 
whose  DNA  showed  identical  gel  patterns.  Furthermore,  virus  was  isolated 
from  the  lesions  of  patients'  relatives  which,  when  assayed  by  the  above 
restriction  endonuclease  procedure,  showed  gel  patterns  identical  to 
those  of  the  patients  themselves.  Interestingly,  only  3  characteristically 
frequent  patterns  were  observed  among  eleven  independent  isolates. 
Restriction  endonuclease  digestion  produced  11  to  20  fragments  ranging 
in  size  from  4  X  10  to  31  X  10  daltons  depending  on  the  virus  isolate. 


19-37 


Mixing  experiments  shewed  comigration  of  some  fragments  in  all  patterns. 
Although  comigration  of  DNA  fragments  does  not  necessarily  mean  base 
sequence  identity,  extensive  comigration  would  imply  significant  genetic 
or  organization  similarity  among  the  viral  genomes.  Direct  comparisons 
of  these  gel  purified  fragments  by  hybridization  or  heteroduplex  analysis 
have  not  been  attempted  due  to  limits  in  the  quantity  of  viral  DNA 
obtainable  from  each  MCU  isolate. 

Although  we  have  not  collected  a  large  enought  body  of  data  to  make 
any  reasonable  assessment  of  the  relationship  between  the  clinical 
characteristics  of  the  disease  and  the  restriction  endonuclease  cleavage 
patterns,  it  may  be  possible  one  the  data  are  accumulated,  to  relate 
these  observations  and  to  develop  a  useful  molecular  epidemiological 
scheme.  Such  a  scheme  would  make  possible  rapid  identification  and 
classification  of  MCV  isolates  without  the  necessity  of  propagating  the 
virus  in  the  laboratory.  Furthermore,  since  the  degree  of  sensitivity 
of  such  a  system  is  limited  only  by  the  number  of  cleavage  sites  recognized 
by  a  given  restriction  enzyme,  other  available  enzymes  may  easily  be 
substituted. 

We  propose  to  continue  to  define  structural  features  of  these 
poxvirus  DNA  molecules  with  the  aim  of  (1)  comparing  genetic  variation 
among  members  of  this  group,  and  of  (2)  relating  these  features  to  those 
biochemical  events  which  are  involved  in  the  replication  of  these  agents. 


19-38 


LABORATORY  OF  CLINICAL  INVESTIGATION 
1979  Annual  Report 
Table  of  Contents 


-Z01-AI 
Project  Number 

Summary 

00043-14 

00045-11 

00046-11 
00047-10 
00048-09 
00049-09 
00050-09 
00051-0? 

00054-0? 

00055-07 
00056-06 

00057-06 
00058-06 


Immunology  and  Chemotherapy  of  Systemic 
Mycoses — Bennett 

Studies  on  the  Interaction  of  Antibody  and 
Complement  on  the  Production  of  Immune 
Damage — Frank 

Pathogenesis  of  Delayed  Hypersensitivity — 
Kirkpatrick 

Clinical  Studies  on  Patients  with  Known 
or  Suspected  Parasitic  Diseases — Neva 

The  Pathophysiology  of  Autoimmune  Hemolytic 
Anemia — Frank 

Initiation  and  Regulation  of  Antigen 
Recognition — Frank 

Clinical  Studies  of  Complement 
Abnormalities  of  Man — Frank 

Host  Defense  Mechanism  Against  Pseudomonas 
Infection  in  Normal  and  Immunosuppressed 
Hosts — Aduan 


Page 
20-1 
20-11 

20-14 

20-18 
20-24 
20-29 
20-34 
20-36 
20-40 


The  Etiology  and  Pathogenesis  of  Viral  20-41 

Gastrointestinal  and  Respiratory  Tract 
Infections  in  Man — Dolin 

Regulation  of  the  Immune  Response  in  Man         20-44 
and  Experimental  Animals — Fauci 

Biochemical  Pathways  of  Mediator  Release         20-60 
and  Mechanism  of  Tissue  Injury  in 
Allergic  Diseases — Kaplan 

Basic  Studies  on  Pathogenic  Fungi —  20-61 

Kwon-Chung 

The  Pathogenesis  and  Chemotherapy  of  Herpes-     20-66 
virus  Infections  in  Man — Dolin 


Table  of  Contents  (Continued) 


Z01-AI 
Project  Number 

00154-04 


00155-04 
00188-01 

00189-01 


Immunologic,  Neurophysiologic,  Biochemical 
and  Cellular  Events  in  Immediate 
Hyp  er s  ens  i  t  ivi  ty — Kal iner 

Phagocyte  Cell  Function — Gallin 

Rapid  Diagnosis  of  Infections  by  Enzyme- 
Linked  Immunoadsorbent  Assays — Straus 

Clinical  and  Biochemical  Studies  of  Human 
Enteral  Adenovirus — Straus 


Page 
20-70 

20-78 
20-86 

20-89 


Summary  of  Program 
Laboratory  of  Clinical  Investigation 
October  1,  1978  -  September  30,  1979 

Michael  M.  Frank,  M. D. ,  Chief  of  Laboratory 
and  Clinical  Director,  NIAID 

Introduction 

This  has  been  a  highly  rewarding  year  for  the  Laboratory  of  Clinical 
Investigation.   It  has  been  a  year  of  continuing  change  and  development 
of  the  new  directions  of  the  program.   Basically,  two  years  ago  a  decision 
was  made  to  continue  to  have  represented  within  the  laboratory  strong 
programs  in  infectious  diseases,  immunology  and  allergy.   Several  of  the 
senior  members  of  the  staff  left  for  other  excellent  appointments  and  an 
opportunity  was  had  to  change  the  directions  of  the  laboratory  at  a  time 
when  there  was  no  possibility  for  further  growth  in  positions  or  space. 
A  decision  was  made  to  allow  for  modest  increases  in  the  size  of  the 
commitment  toward  cellular  immunology  under  Dr.  Fauci,  toward  complement 
research  under  Dr.  Frank,  toward  allergy  research  under  Dr.  Kaliner  and 
toward  research  in  the  phagocytic  cell  in  host  defense  under  Dr.  Gallin. 
Our  commitment  to  mycology  continues  to  be  evident  and  strong  and  our 
commitment  to  virology  was  reaffirmed  with  the  appointment  of  a  new 
leader  of  the  virology  program. 

Dr.  Fauci  had  the  opportunity  to  recruit  a  second  senior  individual  in 
cellular  immunology,  Dr.  Barton  Haynes,  who  joined  the  senior  staff. 
The  Clinical  Immunology  Section  recruited  one  of  its  former  Clinical 
Associates,  Dr.  Steven  Hosea,  to  join  the  staff  as  an  independent  investi- 
gator developing  the  area  of  the  pathophysiology  of  bacterial  infection.   Dr. 
Kaliner  assumed  the  position  of  Chief  of  the  Allergy  group  with  the 
departure  of  Dr.  Allen  Kaplan  and  was  given  a  second  position  to  recruit 
a  second  senior  allergist.   Dr.  Dean  Metcalf,  one  of  our  former  Clinical 
Associates  has  been  recruited  and  will  be  joining  the  laboratory  at  the 
end  of  theis  fiscal  year.   Dr.  Metcalf  served  3  years  as  a  Clinical 
Associate  in  the  Laboratory  of  Clinical  Investigation  and  then  joined 
Dr.  Frank  Austin's  department  at  Harvard  where  he  continued  to  develop 
these  investigative  skills.   He  will  be  starting  a  program  in  food 
hypersensitivity.   Thus,  the  various  groups  have  continued  to  be  rounded 
out  in  the  face  of  very  limited  potential  for  growth. 

The  Clinical  Immunology  group  now  has  extensive  depth  in  the 
general  area  of  complement  protein  interactions  and  contribution  of 
complement  to  medical  illness.   The  group  has  extended  its  depth  considerably 
in  the  general  area  of  immune  complexes  and  the  pathophysiology  of 
immune  complexes  and  has  worked  very  closely  with  members  of  the  Cancer 
Institute  interested  in  these  same  problems.   The  cellular  immunology 
program  has  extended  under  Dr.  Fauci  and  Dr.  Haynes  and  is  deeply  involved 
in  lymphocyte  function  in  autoimmune  diseases.   We  continue  to  study  the 
pathophysiologic  responses  of  the  phagocytic  cell  and  continue  to  develop 
our  capabilities  in  bacterial  infection,  various  aspects  of  mycotic 
infection. 

20-1 


During  this  year  Dr.  Raphael  Dolin  left  to  assume  the  position  of 
Chief  of  the  Infectious  Diseases  at  the  University  of  Vermont.   After  an 
extensive  search  Dr.  Stephen  Straus  was  added  to  the  program  to  continue 
and  develop  our  strength  in  virologic  investigation.   Dr.  Straus   comes 
to  us  from  Washington  University  in  St.  Louis  where  he  received  extensive 
training  in  infectious  diseases.   Before  that  he  was  trained  in  basic 
virology  in  the  NIAID  Laboratory  of  the  Biology  of  Viruses.   As  discussed 
below  he  will  extend  and  develop  our  expertise  in  the  the  use  of  antiviral 
agents  and  in  the  pathophysiology  of  viral  infection. 

Dr.  Charles  Kirkpatrick  has  continued  his  studies  of  mucocutaneous 
candidiasis  and  of  delayed  hypersensitivity  defects  in  a  wide  variety  of 
clinical  diseases.   Dr.  Kirkpatrick  has  accepted  a  position  as  Professor 
of  Medicine  in  Denver  and  will  be  leaving  the  Institute  at  the  end  of 
this  fiscal  year.   Nevertheless,  it  is  hoped  that  our  expertise  in 
cellular  immunology  will  be  maintained  through  Dr.  Fauci's  efforts  in 
this  area.   Dr.  Kirkpatrick  has  made  enormous  strides  in  the  development 
of  rational  treatment  for  patients  with  mucocutaneous  candidiasis  and  in 
our  understanding  of  the  pathophysiologic  basis  of  this  disease.   It 
can  safely  be  said  that  his  efforts  have  been  responsible  for  most  of 
the  available  therapies  which  are  now  in  use  in  treatment  of  this  disease. 
Moreover,  his  contributions  to  the  development  of  our  understanding  of 
transfer  factor  have  been  considerable  and  it  is  expected  that  he  will 
be  able  to  continue  his  efforts  to  understand  the  structure  of  transfer 
factor  and  its  mechanism  of  action  in  his  new  position  in  Denver. 

Research  Accomplishments 

CLINICAL  VIROLOGY  SECTION 

Dr.  Straus   is  continuing  the  ongoing  effort  of  the  viral  disease 
section  in  exploring  the  pathogenesis  and  natural  history  of  herpes 
virus  infections  in  man.   Moreover  studies  of  infections  mononucleosis 
have  been  extended  during  the  year  and  a  number  of  effects  of  the  causative 
agent  the  EB  virus,  upon  the  target  B  lymphocyte  have  been  examined. 
Moreover  effects  of  EB  virus  on  T  cell  responses  and  on  helper  and 
supressor  T  cell  function  have  been  examined.   The  group  continued  and 
confirmed  earlier  observations  on  the  usefulness  of  adenine  aranoside 
in  therapy  of  serious  herpes  virus  infections.   An  enzyme  linked  immuno- 
adsorbant  assay  was  also  developed  for  detecting  influenza  A  hemagglutinin, 
Candida  cell  wall  mannan,  human  adenoviruses,  herpes  simplex  virus  and 
antibodies  to  pneumococci.   These  important  diagnostic  advances  will 
make  rapid  diagnosis  and  patient  evaluation  much  more  practicable  as 
they  are  further  developed.   Dr.  Straus  initiated  studies  of  enteral 
adenovirus  infection  as  a  cause  of  gastroenteritis  in  children.   These 
viruses  are  very  difficult  to  grow  and  a  collaborative  study  has  been 
initiated  to  try  several  methods  of  approach  to  the  culture  of  these 
viruses  and  to  the  study  of  their  pathophysiologic  effects.   It  is  hoped 
to  add  studies  of  this  viral  group  to  the  ongoing  studies  in  NIAID  of 
the  causes  of  viral  gastroenteritis  including  the  addition  of  viral 
challenges  of  normal  volunteers  to  define  the  pathophysiology  of  these 
infections. 

20-2 


CLINICAL  MYCOLOGY  SECTION 

Dr.  Bennett  continued  his  investigations  of  host  defense  reactions 
to  Cryptococcus  neoformans.   The  immunodominant  groups  of  the  type  A 
form  of  the  Cryptococcus  were  studied  in  an  attempt  to  determine  the 
precise  mechanisms  of  antigenicity.   In  this  Cryptococcal  type,  defined 
originally  by  Drs.  Kwon-Chung  and  Bennett,  the  immunodominant  group  was 
found  to  be  1  3  mannan  as  a  backbone  with  Oacetyl  groups  and  to  a 
variable  extent  the  glucuronyl  side  chain.   An  ELISA  assay  was  also 
developed  for  the  detection  of  Aspergillus  fumigatus.  Under  Dr.  Kwon- 
Chung  efforts  were  extended  to  understand  the  various  pathways  utilized 
in  Cryptococcus  neoformans  and  C.  bacillisporus.  Also  the  nuclear  states 
of  basidiospores  in  the  sexual  development  of  C.  neoformans  were  studied 
as  were  the  mating  types  among  the  various  groups  which  have  been  observed 
in  this  laboratory.   The  observation  that  Cryptococci  can  be  divided  into 
distinct  species  is  considered  of  major  importance  in  understanding  the 
biology  of  this  organism  and  perhaps  in  explaining  some  aspects  of  the 
host  defense  mechanisms  engaged  by  these  organisms. 

CLINICAL  PHYSIOLOGY  SECTION 

Dr.  Fauci's  unit  has  continued  to  explore  the  precise  mechanisms 
and  immunoregulatory  events  associated  with  triggering  of  B  lymphocytes 
following  nonspecific  and  antigen  induced  stimulation.   Functionally 
distinct  as  well  as  overlapping  immunoregulatory  lymphocyte  subsets  have 
been  characterized.   The  precise  abnormalities  of  immunologic  reactivity 
have  been  determined  in  several  autoimmune  diseases  such  as  systemic 
lupus  erythematosus,  Sjogren's  Syndrome,  infectious  mononucleosis, 
sarcoidosis,  and  tuberculosis.   Dr.  Fauci's  group  has  succeeded  in 
producing  an  anti-ideotypic  antibody  which  specifically  blocked  the 
function  of  human  B  cells  bearing  the  corresponding  antibody  ideotype. 
Hybridoma  lymphocyte  cell  lines  were  established  which  are  secreting 
antibody  which  binds  specifically  to  functionally  distinct  subsets  of 
human  blood  lymphocytes.   The  availability  of  such  antibodies  should 
prove  of  major  importance  in  further  exploration  of  immunologically 
mediated  effects  in  humans.   Countercurrent  centrifugation  techniques 
were  adapted  from  those  previously  described  and  which  result  in  95% 
purity  and  yield  of  monocytes  from  human  blood.   Again,  the  availability 
of  such  highly  purified  cells  should  greatly  ease  further  developmental 
work  in  cellular  immunology.  Modulation  of  lymphocyte  subsets  by  a 
number  of  clinically  relavant  pharmacologic  agents  has  been  further 
defined.   Clinical  therapeutic  studies  in  this  group  have  yielded  highly 
favorable  results  in  the  approach  to  the  treatment  of  systemic  necrotizing 
vasculitis.   A  number  of  systemic  necrotizing  vasculities  have  yielded  to 
therapy  with  cyclophosphamide  or  cyclophosphamide  and  glucocorticoids. 

CLINICAL  IMMUNOLOGY  SECTION 

This  Section  has  continued  its  studies  in  three  major  areas,  the 
role  of  complement  in  the  production  of  disease,  the  structure  and 
function  of  immune  complexes  in  disease  and  the  role  of  the  reticuloendo- 
thelial system  in  the  development  of  autoimmune  illness  and  in  host 

20-3 


defense  mechanisms.  In  the  first  of  these  areas  great  strides  have  been 
made  in  the  purification  of  large  quantities  of  complement  components 
which  are  available  for  study  in  patients.   This  has  required  an  enormous 
effort  in  terms  of  protein  purification  and  new  methodologies  have  been 
developed  for  the  purification  and  separation  of  functionally  active 
components.   Virtually  all  of  the  components  of  the  classical  pathway 
have  now  been  purified  and  are  now  available  for  study  as  are  certain  of 
the  regulatory  proteins  of  the  complement  system.  A  new  method  has  been 
developed  for  the  evaluation  of  the  opsonic  fragment  of  C3  and  it  has 
been  possible  to  study  the  degradation  of  C3  on  a  molecular  basis  in 
precise  quantitative  detail.  This  complement  protein  is  of  major  importance 
in  bacterial  destruction  and  further  understanding  of  its  mechanisms  of 
action  is  of  great  importance.    Moreover,  methods  have  been  developed 
for  analyzing  these  mechanisms  of  degradation  in  normal  sera.   Studies 
of  complement  receptor  function  and  Fc  immunoglobulin  receptor  function 
have  continued  to  provide  great  insight  into  normal  pathophysiologic 
mechanisms  and  disease  states.   Thus,  for  the  first  time  a  receptor  for 
C5  has  been  identified  unequivocally  on  polymorphonuclear  neutrophils 
and  the  binding  of  C5  to  C3  has  been  studied  extensively.   The  role  of 
phagocytic  cells  in  the  degradation  of  and  decay  of  C3  on  opsonized 
particles  has  also  been  extensively  explored.  Tests  of  in  vivo  reticulo- 
endothelial function  in  man  have  been  developed  which  are  now  coming 
into  wide  use  around  the  world.   It  has  been  demonstrated  for  the  first 
time  that  certain  kinds  of  autoimmune  diseases  associated  with  the 
presence  of  circulating  immune  complexes  are  also  associated  with  defects 
in  the  function  of  the  reticuloendothelial  system,  and  other  autoimmune 
diseases  associated  with  circulating  immune  complexes  are  not.   In 
general  the  autoimmune  diseases  associated  with  clearance  defects  are 
those  which  have  as  part  of  the  clinical  spectrum  tissue  deposition  of 
immune  complexes  and  tissue  damage.   Thus  in  general  the  most  severe 
autoimmune  diseases  are  associated  with  defects  in  immune  clearance. 
Extensive  studies  have  been  performed  in  lupus  erythematosus,  mixed 
connective  tissue  disease,  rheumatoid  arthritis,  mixed  cryoglobulinemia, 
Sjogren's  Syndrome,  primary  biliary  cirrhosis  and  chronic  active  hepatitis, 
etc.  Defects  in  immune  clearance  which  are  specific  for  various  types  of 
receptors  on  phagocytic  cells  have  been  demonstrated  as  well.   Importantly, 
the  standard  test  for  RES  function  in  man,  that  which  measures  clearance 
of  aggregated  human  serum  albumin,  has  been  shown  to  be  useless  in  these 
evaluations.   A  large  population  of  patients  with  Hereditary  Angioedema 
have  continued  to  be  followed  and  evaluated.   New  therapy  is  being 
developed  as  is  further  understanding  of  the  pathophysiology  of  this 
interesting  "experiment  of  nature". 

CLINICAL  ALLERGY  AND  HYPERSENSITIVITY  SECTION 

Dr.  Kirkpatrick' s  laboratory  has  continued  to  study  the  biologic 
basis  of  cellular  immunity.   Continued  studies  of  a  large  group  of 
patients  with  mucocutaneous  candidiasis  has  shown  a  proportion  of 


20-4 


these  patients  have  an  abnormal  ratio  of  helper  T  to  suppressor  T  lymphocytes, 
suggesting  a  problem  in  the  regulation  of  immunologic  function.   Dr. 
Kirkpatrick's  group  has  isolated  a  new  lymphokine  termed  E-RAF  which 
appears  to  induce  the  maturation  of  null  cells  into  E-rosette  forming  T 
cells.  He  has  shown  that  there  are  several  products  which  can  be  elaborated 
from  lymphocytes  which  produces  these  effects  and  that  certain  kinds  of 
patients  with  immunologic  deficiency  do  not  produce  this  factor.   In  Dr. 
Kirkpatrick's  laboratory  several  controlled  clinical  trials  of  new 
agents  for  use  in  treatment  of  mucocutaneous  candidiasis  have  been 
conducted  including  a  control  trial  that  has  shown  that  clotrimazole 
troches  are  effective  therapy  for  oral  candidiasis.   It  has  also  been 
shown  that  transfer  factor  may  induce  more  prolonged  remissions  from 
candidiasis  in  patients  who  receive  this  therapeutic  agent. 

ALLERGIC  DISEASES  SECTION 

The  Allergic  Disease  Section  has  continued  to  widen  its  investigations 
of  the  basic  immunologic  and  physiologic  events  in  immediate  hypersensitivity 
reactions  and  in  the  pathophysiologic  processes  which  take  place  in 
patients  with  a  variety  of  allergic  conditions.   A  number  of  areas  are 
now  being  studied  in  detail  and  although  the  work  is  only  in  its  infancy 
a  wide  variety  of  important  leads  have  already  been  unearthed.   One 
project  involves  characterization  of  mucous  secretion  and  identification 
of  the  factors  which  control  mucous  secretion.   This  is  quite  important 
because  excessive  mucous  secretion  in  such  diverse  diseases  as  asthma 
and  cystic  fibrosis  play  a  major  role  in  the  pathology  of  the  disease. 
Methods  have  been  developed  to  study  mucous  secretion  and  the  mucous 
products  have  now  been  identified  and  radiolabelled.   The  effect  of 
drugs  on  mucous  secretion  is  now  under  study.   The  site  and  control  of 
human  lung  parenchyma  as  opposed  to  airway  prostaglandin  production  has 
also  been  partially  identified  and  the  factors  generated  during  the 
anaphylactic  response  which  cause  prostaglandin  synthesis  have  been 
partially  isolated.   In  another  area  of  investigation  the  histologic 
responses  to  the  injection  of  mast  cell  granules  have  been  characterized 
both  in  rat  and  monkey  skin  and  those  factors  responsible  for  eliciting 
the  inflammatory  response  have  been  partially  isolated  and  characterized. 

Detailed  studies  have  been  performed  on  the  autonomic  nervous 
system  in  patients  with  asthma  and  allergic  rhinitis  and  a  number  of 
important  points  have  been  discovered.   Among  these  is  that  patients 
with  asthma  and  allergic  rhinitis  have  significant  impairment  of  beta 
adrenergic  responsiveness  and  increased  responsiveness  to  cholinergic 
stimuli.   Patients  with  allergic  rhinitis  have  normal  response  to  alpha 
adrenergic  stimulation  while  asthmatics  have  augmented  response  to 
alpha-adrenergic  stimulation.   These  stimuli  control  the  responsiveness 
and  state  of  dilatation  of  the  airways  of  the  human  lung  and  these 
observations  are  of  direct  relevance  in  our  attempts  to  understand 
asthma. 


20-5 


BACTERIAL  DISEASES  SECTION 

Dr.  John  Gallin' s  laboratory  has  continued  to  study  various  aspects 
of  phagocytic  cell  function.   Mechanisms  of  leukocyte  activation  by 
chemotactic  factors  have  been  studied  using  electrophysiologic  techniques 
as  well  as  techniques  which  probe  cell  surface  charge  and  ultrastructure. 
Dr.  Gallin' s  group  has  shown  that  secretion  of  specific  granules  which 
may  accompany  chemotaxis  is  associated  with  increased  cell  adhesiveness 
and  increased  availability  of  chemoattractant  receptors.   Vigorous 
exocytosis  is  associated  with  depressed  chemotaxsis,  decreased  availability 
of  chemoattractant  receptors,  hydrolysis  of  the  chemoattractant  by 
secreted  products  and  markedly  increased  cell  adherence  and  aggregation. 
Human  pyrogen  has  been  shown  to  be  a  potent  stimulator  of  neutrophil 
exocytosis  and  causes  activation  of  the  hexose  monophosphateshunt .   Dr. 
Gallin' s  laboratory  had  previously  shown  that  neutrophils  can  be  divided 
into  two  populations,  those  with  large  numbers  of  Fc  receptors  and  those 
with  small  numbers  of  Fc  receptors.   Recent  work  has  shown  that  both 
sets  of  neutrophils  have  equal  C3b  receptor  activity  and  that  neither 
has  a  demonstrable  C3d  receptor.   Biological  differences  however  between 
the  two  sets  of  granulcytes  have  been  clearly  indicated.   Thus,  in  vivo 
injection  of  endotoxin  into  patients  or  the  hemodialysis  of  patients 
leads  to  loss  from  the  circulation  of  those  neutrophils  with  demonstrable 
Fc  receptors  and  a  marked  increase  in  the  percentage  of  granulocytes 
with  no  demonstrable  Fc  receptors. 

BIOLOGIC  STRUCTURE  SECTION 

Dr.  Allen  Rosenthal  left  the  Laboratory  of  Clinical  Investigation 
to  assume  the  position  of  Director  of  Immunology  of  the  Merck  Sharpe  and 
Dohme  Company  one  year  ago.   At  that  time  he  had  a  number  of  fellows  and 
a  number  of  important  research  projects  which  are  of  interest  to  the 
Institute.  During  this  year  those  projects  were  partially  or  entirely 
completed. 

It  was  shown  that  antiinsulin  antibodies  of  exquisite  antigenic 
specificity  can  be  prepared  and  identified.   Moreover,  it  was  shown  that 
the  cellular  immune  response  to  the  insulin  antigen  does  not  necessarily 
recognize  the  same  antigenic  groupings  as  the  humoral  response.   Precise 
fine  structural  analysis  of  the  actual  amino  acids  recognized  during  the 
cellular  response  to  insulin  has  been  defined  and  initial  experiments 
have  begun  on  determining  the  role  of  histocompatibility  groups  on 
antigenic  recognition  of  portions  of  the  insulin  molecule.   This  is  a 
far  ranging  project  which  may  ultimately  give  us  a  great  deal  of  information 
about  the  biological  basis  of  insulin  allergy.   Dr.  Rosenthal  intends  to 
continue  these  investigations  at  the  Merck  Sharpe  &  Dohme  Company. 

In  all,  this  has  been  an  impressive  year  in  the  Laboratory  of 
Clinical  Investigation.   Both  clinically  and  in  terms  of  basic  science 
great  strides  have  been  made.   Our  research  groups  find  themselves  with 
many  more  problems  than  we  can  possibly  find  time  to  investigate  and  we 
all  look  forward  to  future  developments  with  great  anticipation. 

20-6 


CLINICAL  PARASITOLOGY  SECTION 

The  parasitic  disease  group  has  continued  to  extend  its  basic  studies 
of  Chagas '  disease,  schistosome  and   filariasis.   The  association  of  absence 
of  HLA  tissue  type-Al  with  chronic  Chagas'  disease  was  not  confirmed  by  an 
expanded  study  of  cases,  but  DR  (B  cell)  typing  will  still  be  done  on 
frozen  cells.   The  diversity  of  HLA  haplotypes  encountered  in  this  Brazilian 
patient  population  corroborates  what  anthropologists  have  said  of  this 
population  -  namely,  extreme  genetic  mixing.   Further  study  of  different 
antigenic  fractions  of  schistosome  worms  has  disclosed  one  fraction  to 
which  antibody  levels  in  patients  correlate  quite  well  with  numbers  of 
eggs  being  excreted,  or  intensity  of  infection.   Evidence  for  serum  in- 
hibitory factors  and  for  suppressor  cells  has  been  found  in  filariasis 
patients  characterized  by  unresponsiveness  to  filarial  antigens.   The  role 
of  immune  complexes  in  filariasis  patients  is  also  being  studied.   Serum 
and  cell  factors  were  also  found  that  contributed  to  antigen  unresponsive- 
ness in  patients  with  chronic  schistosomiasis. 


20-7 


20-8 


Honors  and  Awards 

This  has  been  a  year  in  which  the  efforts  of  members  of  the  Laboratory 
have  been  well  recognized.   Members  of  the  Laboratory  have  been  elected 
to  many  learned  societies  and  now  serve  on  the  Editorial  Board  of  many 
major  journals. 

Specifically,  Dr.  Anthony  Fauci  was  elected  President  of  the  American 
Federation  for  Clinical  Research  and  received  the  Public  Health  Service 
Meritorious  Service  Award.   Dr.  Fauci  joins  Dr.  Frank  as  a  member  of  the 
Association  of  American  Physicians.   Dr.  Frank  was  invited  to  give  the 
plenary  lecture  at  the  Japanese  Rheumatism  Association  Meeting  in  Japan  in 
June  of  1979  and  Dr.  Fauci  gave  the  Stanislaus  Jaros  Memorial  Lecture  to 
the  American  Association  for  Clinical  Immunology  and  Allergy  in  1978.   Dr. 
Frank  was  invited  to  speak  at  the  plenary  session  of  the  Infectious  Diseases 
Society  Meeting  in  1979.   Dr.  Michael  Kaliner  was  elected  to  the  American 
Society  for  Clinical  Investigation  bringing  the  number  of  members  of  that 
Society  within  the  LCI  to  five.   Dr.  Jack  Bennett  was  elected  to  the  Council 
of  the  Infectious  Diseases  Society  and  Dr.  Kirkpatrick  was  appointed  to  the 
Education  Committee  of  the  American  Academy  of  Allergy.   The  American 
Academy  of  Allergy  also  appointed  Dr.  Fauci  to  the  Postgraduate  Education 
Committee.   In  addition,  Dr.  Gallin  was  appointed  to  the  Program  Committee 
of  the  American  Association  of  Immunologists.   Dr.  Kwon-Chung  was  elected 
Chairperson  of  the  Medical  Mycology  Division  of  the  American  Society  for 
Microbiology  and  was  also  selected  Society  Lecturer  for  the  British 
Mycopathological  Society  at  the  Annual  Meeting  in  Birmingham,  England. 

The  Laboratory  of  Clinical  Investigation  is  also  well  represented  on 
Editorial  Boards  of  major  journals.   Dr.  Frank  and  Dr.  Fauci  are  now  both 
serving  on  the  Editorial  Board  of  the  Journal  of  Clinical  Investigation. 
Dr.  Fauci  serves  as  Section  Head  of  the  Editorial  Board  in  Clinical 
Immunology  of  the  Journal  of  Immunology  and  Dr.  Gallin  is  a  member  of  the 
Editorial  Board.   Dr.  Fauci  serves  as  a  Section  Head  of  the  Editorial 
Board  in  Allergy  and  Immunology  of  the  American  Journal  of  Medicine.   Dr. 
Frank  serves  on  the  Editorial  Board  of  Blood  and  on  the  Editorial  Board  of 
the  new  Journal  of  Infectious  Disease  Reviews.   Dr.  Bennett  serves  on  the 
Editorial  Board  of  the  Journal  of  Infectious  Disease  and  the  Journal, 
Antimicrobial  agents  and  chemotherapy  and  on  the  Editorial  Board  of  the 
Journal  of  Clinical  Microbiology  as  well.   Dr.  Kirkpatrick  serves  on 
Editorial  Boards  of  three  journals,  Thymus,  The  Journal  of  Allergy  and 
Clinical  Immunology,  and  the  Journal  of  Cutaneous  Pathology.   Dr.  Gallin 
serves  on  the  Editorial  Board  of  the  Journal  of  Immunology  and  also  of  the 
new  journal,  Inflammation.   Dr.  Kaliner  serves  on  the  Editorial  Board  of 
the  Journal  of  Allergy  and  Clinical  Immunology.   Dr.  Fauci  serves  on  the 
Editorial  Board  of  the  Journal  of  Immunopharmacology  and  on  the  Editorial 
Board  of  the  American  College  of  Physicians,  Medical  Knowledge.   Self- 
Assessment,  Audiocassett  Program.   Dr.  Kwon-Chung  serves  on  the  Editorial 
Board  of  the  Journal,  Sabauraudia.   Thus,  many  major  journals  in  modern 
clinical  immunology  in  infectious  disease  and  clinical  research  are 
represented  by  members  of  the  Laboratory. 


20-9 


20-10 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI   00043-14  LCI 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 


Immunology  and  Chemotherapy  of  Systemic  Mycoses 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:      J.E.  Bennett 

OTHER:    A.D.  Hernandez 
D.K.  Henderson 


Head,  Clinical  Mycology  Section 
Guest  Worker 


LCI  NIAID 


COOPERATING  UNITS  (if  any) 

S.  Kim  (FDA) 

A.  Bhattacharjee  (NIAMDD) 

P.  Pizzo  (NCI) 


LAB/BRANCH 


Laboratory  of  Clinical  Investigation 


Clinical  Mycology  Section 


NSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland   20205 


TOTAL  MANYEARS 

3  11/12 


PROFESSIONAL: 

3  H/12 


OTHER: 


2.0 


CHECK  APPROPRIATE  BOX(ES) 
Xj  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   fj  (a2)  INTERVIEWS 


S  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  immunodominant  groups  on  type  A  Cryptococcus  neoformans  were  found  to 
be  the  al,3  mannan  backbone,  particularly  the  0-acetyl  groups  and,  to  a  vari- 
able extent,  the  3-glucuronyl  side  chain. 

Radioimmunoassay  and  ELISA  have  been  developed  to  detect  a  specific 
neutral  polysaccharide  of  Aspergillus  f umigatus . 


20-11 


PHS-6040 
(Rev.  IO-76) 


Project  No.   Z01  AI  00043-14  LCI 
Project  Description: 

The  general  goals  of  the  unit  have  been  to  study  host  defense  in 
systemic  mycoses  and  to  improve  diagnosis  and  treatment  of  these  disorders. 

Objectives: 

1)  Determine  the  role  which  antibody  to  C.  neoformans  plays  in  host 
defense;  i.e.,  the  cause  and  significance  of  absent  antibody  formation  in 
most  patients  with  the  disease. 

2)  Characterize  the  immunodominant  groups  which  contribute  to  serotype 
specificity  in  C_.    neoformans  polysaccharides. 

3)  Purify  polysaccharides  of  Aspergillus  fumigatus,  devise  assays  for 
these  polysaccharides  and  determine  whether  these  antigens  appear  in  body 
fluids  during  infection. 

Methodology,  Results  and  Significance: 

1)  Hapten  inhibition  radioimmunoassay  has  been  used  to  explore  further 
the  immunodominant  positions  on  C_.    neoformans  polysaccharides.   Although 
individual  sera  do  vary,  antibody  binding  sites  on  type  D  polysaccharide  in- 
clude the  oil, 3  mannan  backbone,  particularly  the  0-acetyl  groups  there,  and, 
for  occasional  sera,  the  glucuronyl  side  chain.   On  type  A,  |3-glucuronyl 
mannan  is  one  determinant.   None  of  these  determinants  has  proven  immuno- 
dominant in  type  C;  and  it  seems  likely  that  larger,  more  complex  structures 
are  immunodominant.   Lack  of  0-acetyl  groups  and  extensive  substitution  of 
the  al,3  mannan  backbone  are  the  structural  features  of  type  C  which  account 
for  these  differences  in  immunodominance.   (Bennett  and  Bhattacharjee) 

2)  Young  healthy  volunteers  were  found  to  have  lgM  and  lgG  antibody  to 
type  A  C_.  neoformans  polysaccharide.   Antibody  was  significantly  less  common 
beyond  50  years  of  age,  a  time  in  life  when  cryptococcosis  is  more  common. 
The  majority  of  patients  with  cryptococcosis  do  not  have  anti-cryptococcal 
antibody  during  either  illness  or  in  later  years.   It  remains  to  be  deter- 
mined whether  disease  produces  tolerance  or  inability  to  respond  to  the 
antigenic  challenge  reflects  a  predisposing  cause  of  infection.   (Bennett  and 
Henderson) 

3)  A  neutral  polysaccharide  of  Aspergillus  fumigatus  was  tyraminylated 
and  labeled  with  12^I.   From  40-50%  of  the  radioactivity  was  precipitable 
with  rabbit  antisera.   Although  the  polysaccharide  has  a  fairly  homogeneous 
pattern  on  Sephadex  chromatography  (mol.  wt.  36,000  daltons),  the  portion  of 
the  polysaccharide  not  binding  to  antibody  should  be  able  to  be  removed  by 
affinity  chromatography.   The  final  material  can  be  used  for  structural 
analysis  and  to  search  for  antigenemia  in  aspergillosis. 

The  partially  purified  polysaccharide  can  be  detected  by  ELISA  down 
to  100  ng/ml.   This  assay  also  will  be  used  to  search  for  antigenemia  in 


20-12 


Project  No,   Z01  AI   00043-14  LCI 

aspergillosis.   (Bennett  and  Kim) 

4)   Radioimmunoassay  has  found  antibody  against  C^  albicans  mannan  in 
both  normal  volunteers  and  immunosuppressed  patients,  with  no  substantial 
differences  between  these  groups.   Small  numbers  of  patients  with  fatal  dis- 
seminated candidiasis  have  had  antibody  titers  similar  to  the  other  two 
groups. 

Publications: 

1.  Diasio,  R.B.,  Lakings,  D.E.,  Bennett,  J.E.:   Evidence  for  conversion  of 
5-f luorocytosine  to  5-f luorouracil  in  man:   A  possible  factor  in 
5-fluorocytosine  clinical  toxicity.   Antimicrob.  Agents  Chemother.  14: 
903-908,  1978. 

2.  Feldman,  L.,  Lamson,  M. ,  Gallelli,  J.F.  and  Bennett,  J.E.:  Surveillance 
of  nosocomial  infections  by  antibiotic  monitoring.  J_.  Amer .  Med .  Assoc . 
241:2806,  1979. 

3.  Bennett,  J.E.:   Diagnosis  and  management  of  candidemia  in  the  immuno- 
suppressed host.   Scan.  J_.  Infect.  Pis.  Supp .  16:83-86,  1978. 

4.  Mandell,  G.M.,  Douglas,  R.G.,  Bennett,  J.E.:   Principles  and  Practices 
of  Infectious  Disease.   John  Wiley  and  Sons,  New  York,  N.Y.,  1979. 

5.  Segal,  E.,  Berg,  R. ,  Pizzo,  P.  and  Bennett,  J.E. :  Detection  of  Candida 
antigen  in  the  sera  of  patients  with  candidiasis  by  an  ELISA  inhibition 
technique.   J.  Clin.  Microbiol.  10:116-118,  1979. 

6.  Bennett,  J.E.,  Dismukes,  W.E.,  Duma,  R.J.,  et  al:  A  collaborative  study 
comparing  amphotericin  B  alone  or  combined  with  flucytosine  in  the  treat- 
ment of  cryptococcal  meningitis.   N_.  Engl.  J_.  Med.  301:126-131,  1979. 


20-13 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  Al  00045-11  LCI 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Studies  on  the  Interaction  of  Antibody  and  Complement  on  the  Production 
of  Immune  Damage 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


OTHER: 


M.  Frank 


C.  Hammer 

T.  Gaither 

K.  Katusha 

L.  Renfer 

M.  Santaella 

H.  Gresham 

E.  Brown 

S.  Hosea 


Head,  Clinical  Immunology  Section  NIAID,LC| 
and  Chief 

Senior  Staff  Fellow  NIAID,LCt 

Resident  Biologist  NIAID.LCI 

Microbiologist  NIAID.LCI 

Chemist  NIAID,LCl 

Guest  Researcher  on  IPA  NIAID,LC:[ 

Medical  Technologist  NIAID,LCI 

Clinical  Associate  NIAID.LCI 

Fellow  in  Infectious  Diseases  NIAID,LCI 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Clinical  Investigation 


SECTION 

Clinical  Immunology  Section 


INSTITUTE  AND  LOCATION 

NIH,  NIAID,  Bethesda,  MD   20205 


TOTAL  MANYEARS: 

5   4/12 


PROFESSIONAL: 

2  10/12 


3  6/12 


CHECK  APPROPRIATE  BOX(tS) 
J   (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   rj  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


0  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Techniques  have  now  been  developed  in  the  laboratory  for  the  large- 
scale  purification  of  Clq,  C4,  C3,  5,  6,  7,  8,  9,  and  the  C56  complex. 
New  assays  have  been  developed  for  the  C3b  inactivator  and  for  the 
protein  S1H  and  the  role  of  these  proteins  in  C3b  inactivation  has  been 
further  explored.  Studies  are  ongoing  on  the  characteristics  of  this 
interaction  which  leads  to  the  destruction  of  the  opsonically  active  C3b 
site  on  erythrocyte  surfaces.  Studies  have  also  been  conducted  on  the 
interaction  of  complement  with  bacteria  and  considerable  progress  has 
been  made  in  studies  of  the  role  of  complement  in  producing  the  clinical 
symptoms  of  infectious  disease. 


20-1^ 


JHS-6040 
(Rev.  10-76) 


Project  No.  Z01  AI  0045-11  LCI 

Project  Description: 

Objective: 

The  objective  of  this  program  is  the  longterm  study  of  the  role  of 
antibody  and  complement  in  the  production  of  immune  injury  in  vitro  and 
in  vivo.   To  this  end  the  changes  which  were  initiated  last  year  in  the 
Clinical  Immunology  Section  have  continued.   A  pronounced  emphasis  is 
continuing  on  protein  purification  and  assay  and  on  studies  of  protein 
interaction  within  the  complement  system  with  the  goal  of  developing 
reagents  suitable  for  pathophysiologic  studies  in  patients. 

Methods  Employed: 

The  methods  employed  involved  purification  of  complement  components, 
their  use  in  the  sequential  build-up  of  complement  intermediates  on  cells, 
the  identification  of  sera  of  patients  with  defined  complement  abnormalities 
so  that  these  sera  can  be  used  as  reagents,  and  the  purification  of  immuno- 
globulins of  various  classes  by  standard  techniques.   Methods  for  titration 
of  complement  components  have  been  developed  in  our  laboratory,  and  these 
are  in  routine  use.   Methods  for  cultivation  of  various  types  of  bacteria  as 
well  as  for  bactericidal  assays  have  also  been  developed  in  the  laboratory  and 
methods  for  radiolabeling  of  bacteria.   Disc  gel  polyacrylamide  electro- 
phoresis as  well  as  electrofocusing  techniques  have  been  developed  in  the 
laboratory  and  methods  have  been  developed  to  prepare  proteins  of  very  high 
purity.   Also  large  batch  methods  have  been  developed  for  the  first  time  for 
the  purification  of  the  various  complement  proteins,,.  Methods  have  also  been 
developed  for  the  radiolabelling  of  bacteria  with    I. 

Major  Findings: 

Using  methods  initially  developed  for  the  purification  of  C3 ,  C5,  C6, 
and  C7  from  separate  plasma  pools  we  have  consolidated  the  purification 
protocol  to  allow  for  the  isolation  of  most  of  complement  proteins  in  bio- 
logically active,  homogenous  form  from  one  large  pool  of  human  plasma.   The 
basis  of  success  of  this  purification  scheme  has  rested  on  the  ability  to 
obtain  a  large  number  of  selective  highly  resolved  chromatographic  pools 
early  in  the  purification  protocol  in  which  biological  activity  has  been 
stabilized.   We  have  found  that  the  use  of  specific  protease  inhibitors  and 
agents  which  block  complement  activation  is  requisite  for  recovery  of  bio- 
logical activity  of  many  of  the  complement  proteins.  We  have  demonstrated 
that  in  addition  to  the  use  of  inhibitors  in  preservation  of  complement 
activity  the  ability  to  proceed  rapidly  through  the  first  ion  exchange 
steps  yields  improved  recoveries  of  biologically  active  complement 
proteins.   Using  the  biochemically  homogeneous  components  we  have  begun 
to  prepare  monospecific  antisera  in  goats  and  burros.   These  reagents 
are  of  indispensable  value  in  clinical  studies  to  determine  antigenic 
levels  in  patients  and  to  further  study  the  interaction  of  the  proteins 
and  their  biological  activity. 

20-15 


Project  No.  Z01  AI  00045-11  LCI 

This  year  has  also  seen  the  completion  of  a  new  assay  system  which 
quantitates  the  functional  activities  of  the  alternative  pathway  control 
proteins  glH  and  C3b  inactivator.  Homogeneous  C3  preparations  radio- 
labelled  with    I  iodine  are  used  to  prepare  cellular  intermediates  in 
the  complement  sequence  and  the  use  of  these  intermediates  has  allowed 
us  to  clarify  the  functions  of  glH,  the  C3b  inactivator  and  a  proteolytic 
serum  enzyme  involved  in  inactivation  of  the  biologically  important  C3 
fragment  C3b.   The  assay  is  based  on  the  release  of  radiolabelled  C3c 
which  occurs  only  after  all  of  these  proteins  have  interacted  with  C3b 
bound  to  the  cell  membrane.   The  C3b  cleavage  fragments  formed  during 
inactivation  of  cellbound  C3b  have  been  characterized  by  SDS  polyacrylamide 
gel  electrophoresis.   This  analysis  verified  the  C3b  molecule  undergoes 
two  separate  cleavages,  first  by  the  C3b  inactivator  and  S1H  and, 
second,  by  the  proteolytic  enzyme.   The  assay  is  presently  being  applied 
to  study  S1H  and  C3b  inactivator  activities  in  the  sera  of  patients  with 
a  variety  of  illnesses.   Experiments  are  currently  being  designed  to 
study  the  inactivation  of  C3b  bound  to  the  surface  of  various  strains  of 
bacteria.   The  goal  is  to  determine  if  there  is  relationship  between 
virulence  of  various  bacterial  strains  and  the  susceptibility  of  the 
bacterial  bound  C3b  to  inactivation  by  the  control  proteins.  Preliminary 
studies  have  been  performed  using  S.  typhosa  0901  to  establish  the 
optimal  conditions  for  C3  inactivation  and  C3  binding  and  for  inactivation 
of  the  bound  C3b  by  purified  control  proteins.   Studies  of  interaction 
of  bacteria  and  complement  have  proceeded  with  the  development  of  new 
techniques  for  radiolabelling  of  bacteria.   These  studies  will  be  extended 
to  consideration  of  the  role  of  complement  in  the  development  of  the 
clinical  signs  and  symptoms  associated  with  bacterial  infection. 

Significance  to  Biomedical  Research 

At  present  it  is  clear  that  complement  activation  plays  an  essential 
role  in  protecting  the  individual  from  infection  with  microorganisms.   It 
is  also  clear  that  unregulated  complement  activation  is  a  crucial  component 
of  autoimmune  disease  and  regularly  leads  to  tissue  injury.   Very  little  is 
known  about  how  the  complement  proteins  function  in  disease  states  however. 
The  studies  underway  in  our  laboratory  at  present  will  make  it  possible  to 
explore  each  of  these  questions  in  a  systemic  manner.   They  represent  the 
overcoming  of  formidable  methodologic  barriers  and  are  essential  to  further 
progress  in  theses  areas. 

Proposed  Course: 

We  plan  to  extend  these  studies  to  obtain  a  further  understanding 
of  the  role  of  the  proteins  in  the  regulation  of  complement  function  in  normal 
physiologic   situations  and  in  disease  states.   We  plan  to  examine  the 
role  of  control  proteins  in  infectious  states  and  in  control  against 
bacterial  infection.  In  addition,  we  plan  to  determine  the  role  of 
complement  activation  fragments  in  the  development  of  clinical  signs  and 
symptoms  of  bacterial  disease.   We  plan  to  further  purify  complement  proteins 
and  components  so  that  we  can  study  their  metabolism  and  interaction 
in  a  variety  of  disease  states. 

20-16 


Project  No.  AI  00045-11  LCI 
Publications 

1.  Frank,  M.M. :   The  Complement  System  in  Host  Defense  and  Inflammation. 
Rev.  Infect.  Pis.  1:3  483-501,  1979. 

2.  Gaither,  T.A. ,  and  Frank,  M.M. :   Complement.   In  Todd,  Sanford,  Davidson, 
(Eds) :  Clinical  Diagnosis  and  Management  by  Laboratory  Methods. 
Philadelphia,  PA.   W.B.  Saunders  Company,  16th  Ed.,  1979,  pp.  1245-1261. 

3.  Lawley,  T.J.  and  Frank,  M.M. :  Complement.   In  Dermatologic  Reviews. 
In  press. 

4.  Gelfand,  M.C.,  Frank,  M.M. ,  Green,  I.  and  Shin,  M.L. :   Binding  Sites 
for  Immune  Complexes  Containing  IgG  in  the  Renal  Interstitium. 
Clin.  Immunol,  and  Immunopath.   13:  19-29,  1979. 

5.  Gaither,  T.A. ,  Hammer,  C.  and  Frank,  M.M. :  Studies  of  the  Molecular 
Mechanisms  of  C3b  Inactivation  and  a  Simplified  Assay  for  31H  and 
C3b  Inactivator.   J_.  Immunol.  In  press. 

6.  Lawley,  T.J.,  Moutsopoulos,  H.M. ,  Katz,  S.I.,  Theof ilopoulos ,  A.N. , 
Chused,  T.M.  and  Frank,  M.M. :  Demonstration  of  Circulating  Immune 
Complexes  in  Sjogrens  Syndrome.   J_.  Immunol.  In  press. 

7.  Macher,  A.M.,  Bennett,  J.E.,  Gadek,  J.E.,  and  Frank,  M.M. :  Complement 
Depletion  in  Cryptococcal  Sepsis.   J.  Immunol.  120:  1686-90,  1978. 


20-17 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00646-11  LCI 


PERIOD    COVERED 

October  1,  1978  to  Setember  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Pathogenesis  of  Delayed  Hypersensitivity 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


OTHER: 


Charles  H.  Kirkpatrick 


Eskild  A.  Petersen 


A.I. 


>ata 


M.  Adedin 
J.I.  Gallin 
L.E.  Greenberg 


Head,  Clinical  Allergy 
&  Hypersensitivity  Section 

Guest  Worker 


Visiting  Fellow,  NIAID,  LCI 

NICHD,  LCI 
NIAID,  LCI 
Bio.  Lab.  (Micro.,  NIAID,  LCI 


COOPERATING  UNITS  (if  any) 

J.  Cook  &  A.  Lewis,  OSD,  NIAID,  S.  Shama,  Dermatology  Branch,  NCI,  S.  Gupta, 
Sloan-Kettering  Cancer  Center,  P.G.  Sohlne,  Medical  College  of  WI ,  D.W.  Ailing 
OSD,  NIAID. 


LAB/ BRANCH 

Laboratory  of  Clinical  Investigation 


SECTION 

Clinical  Allergy  and  Hypersensitivity  Section 


INSTITUTE  AND  LOCATION 

NIH,  Bethesda,  MD   20205 


TOTAL  MANYEARS: 

4  9/12 


PROFESSIONAL: 
2  9/1? 


CHECK  APPROPRIATE  BOX(ES) 
£  (a)   HUMAN  SUBJECTS 

D  (a1  )  MINORS   □  (a2)  INTERVIEWS 


HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  nature  of  the  immunologic  defect  that  predisposed  certain  patients  to 
chronic  and  recurrent  fungal  infections  such  as  mucocutaneous  candidiasis  has 
been  further  defined.   Certain  patients  have  abnormal  ratios  of  Tu  to  TQ 
lymphocytes  that  are  compatible  with  a  problem  with  regulation  of  immunocyte 
function. 

A  new  lymphokine,  E-RAF,  has  been  defined.   It  seems  to  induce  maturation  of 
"null"  cells  into  E-rosette-f orming  T-cells.   These  observations  indicate 
that  "null"  cells  are  not  "null",  but  are  pre- T-cells. 

Two  therapies  for  chronic  mucocutaneous  candidiasis  have  been  studied.   A  con- 
trolled clinical  trial  has  shown  that  clotrimazole  troches  are  effective  thera- 
py for  chronic  oral  candidiasis.   Transfer  factor  from  candida-sensitive  donors 
may  restore  the  cellular  immune  responses  to  Candida  to  these  patients  and 
prolong  drug-induced  remissions. 
20-18 

PHS-6040 
(Rev.  10-76) 


Project  No.  Z01  AI  00046-11  LCI 
Project  Description: 
Objectives  (sub-project  A): 

1)  Characterization  of  cellular  immune  responses  in  immunologically 
deficient  patients  with  chronic  infectious  diseases; 

2)  Studies  of  the  pathogenesis  of  acquired  and  congenital  immunologic 
deficiencies; 

3)  Studies  of  lymphokine  production  by  cells  from  normal  and  immuno- 
deficient  subjects: 

4)  Studies  of  developmental  immunology  including  studies  of  the  immuno- 
logic activities  of  human  cord-blood  lymphocytes  and  maturation  of  cell- 
mediated  immune  systems  in  Syrian  hamsters. 

Methods  Employed 

The  major  portion  of  our  studies  of  cellular  immune  deficiency  have  been 
conducted  in  patients  with  chronic  mucocutaneous  candidiasis.   Approximately 
60  patients  with  this  syndrome  have  been  evaluated.   Evidence  of  immunologic 
deficiency  has  been  found  in  66%  of  the  patients. 

The  general  model  for  the  pathogenesis  of  cellular  immunodeficiency  that 
we  presented  in  1971  still  serves  as  valid  outline  in  which  to  conduct  studies 
of  host-defenses.   According  to  his  model,  patients  with  candidiasis  develop- 
ing during  infancy  or  early  childhood  have  a  congenital  basis  for  their 
immunologic  deficits.   Other  patients  are  apparently  normal  until  adult 
years  when  they  develop  candidiasis.   In  these  patients,  the  immunologic  ab- 
normalities are  probably  acquired.   This  problem  has  been  under  study 
and  measurements  of  immuno- regulatory  activities  in  candidiasis  patients 
and  the  subpopulations  of  T-lymphocytes  have  been  measured. 

In  addition,  the  biological  role  of  a  lymphokine,  E-rosette  augmenting 
factor  (E-RAF) ,  that  was  discovered  by  Dr.  Agbata  in  our  laboratory  has 
been  under  study  in  patients  with  immunodeficiency  syndromes  both  before 
and  after  they  were  treated  with  immuno-reconstitutive  measures. 

The  studies  of  development  of  immunocompetence  by  human  and  hamster 
lymphoid  cells  have  continued. 

The  collaborative  studies  (with  J. I.  Gallin)  on  the  relationships  be- 
tween cellular  immunity  and  chemotaxis  in  patients  with  exceptional  suscepti- 
bility to  pyogenic  and/or  fungal  infections  have  continued.   The  studies  of 
effects  of  levamisole  on  chemotactic  responses,  lymphocyte  function  and  resis- 
tance to  infection  have  been  extended  and  a  controlled  clinical  trial  has  been 
designed. 


20-19 


Project  No.  Z01  AI  90046-11  LCI 
Two  evaluations  of  chemotherapeutic  agents  for  chronic  candidiasis  have 
been  conducted. 

A  major  achievement  in  the  laboratory  has  been  the  development  of  a  mur- 
ine model  for  transfer  factor.   The  system  has  been  adapted  for  transfer  of 
delayed-type  hypersensitivity  to  soluble  proteins  and  in  vitro  correlates 
of  cellular  immunity  are  being  compared  with  the  in  vivo  responses. 

Major  Findings: 

The  lymphokine,  E-RAF,  is  produced  by  mitogen-stimulated  cells  and  by 
antigen-stimulated  lymphocytes  if  the  cell  donor  has  cellular  immunity  to 
the  antigen.   We  now  have  evidence  that  antigen- induced  E-RAF  and  mitogen- 
induced  E-RAF  have  different  molecular  weights.   It  apparently  functions 
by  enhancing  maturation  of  "null"  cells  into  T-cells. 

E-RAF  production  has  been  studied  in  a  patient  with  SCID  both  before 
and  after  thymus  transplantation.   Mitogen  and  antigen  stimulated  cells 
did  not  yield  E-RAF  before  the  transplant.   By  the  third  post-transplant 
day,  mitogen-stimulated  lymphocytes  from  the  patient  produced  E-RAF.   On 
the  same  day,  the  patients  cells  did  not  respond  to  mitogens  with  LIF  pro- 
duction or  lymphocyte  transformation  and  he  did  not  have  normal  numbers 
of  circulating  T-cells. 

Most  candidiasis  patients  have  normal  numbers  of  circulating  E-rosette- 
forming  T-cells.   However,  the  majority  of  patients  have  somewhat  low 
numbers  of  T/'cells  (containing  the  helper  cells)  and  excessive  numbers 
of  T-cells  (containing  the  suppressor  cells).   When  the  ratios  of  T/'/T/^ 
are  calculated  6  of  16  patients  had  low  values,  suggesting  an  immunoregulatory 
defect  in  these  patients. 

Human  lymphocytes  may  develop  suppressor  activity  when  they  are  acti- 
vated with  concanavalin-A.   In  contrast,  human  cord  blood-lymphocytes  are 
suppressive  of  responses  by  adult  cells  even  without  concanavalin  activation. 
Fetal  cells  appear  to  be  in  a  "suppressive"  state  under  normal  conditions. 

The  work  on  ontogeny  of  lymphocyte  function  and  resistance  to  oncogenesis 
with  Ad  2-transformed  cells  and  cell  lines  has  been  extended.   All  newborn 
hamsters  are  susceptable  to  these  tumors  until  about  9  days  of  age.   After  this 
time,  all  animals  are  resistant.   At  about  this  age,  spleen  cells  become 
responsive  to  concanavalin  A;  similar  responses  by  blood  lymphocytes  do 
not  appear  until  the  fourth  week  of  life.   Of  particular  interest  was  the 
finding  that  spleen  cells  do  not  become  responsive  to  alloantigens  (in  MLR) 
until  7-10  days  after  development  of  resistance  to  tumor  induction.   Other 
experiments  have  shown  that  the  developmental  delay  is  probably  due  to  immature 
macrophages  rather  than  lymphocytes. 

A  controlled  clinical  trial  showed  that  clotrimazole  buccal  troches  were 
efficacious  in  treating  chronic  oral  candidiasis.   A  study  of  the  systemic 
antifungal,  ketoconazole,  has  been  started. 


20-20 


Project  No.  Z01  AI  00046-11  LCI 

The  murine  transfer  factor  system  has  been  adapted  to  transfer  of 
cell-mediated  responses  to  ovalbumin  and  ferritin.   The  in  vivo  responses  are 
compared  to  MIF  production  by  antigen- stimulated  cells.   Studies  with 
genetically-controlled  antigens  are  underway. 

Significance 

1)   These  experiments  and  those  presented  in  sub-project  B  (below)  further 
define  the  relationship  between  cellular  immune  responses,  inflammation  and 
susceptibility  to  certain  chronic  infectious  diseases.   The  relative  contri- 
bution of  individual  lymphokines  to  cell  mediated  immune  responses  is  unclear. 
For  example,  it  is  not  known  which  or  how  many  lymphokines  are  required  to 
produce  a  "positive"  skin  test.   It  is  also  important  to  define  the  relationship 
of  specific  lymphokine  production  and  "immunity"  to  infection  with  a  micro- 
organism.  E-RAF  appears  to  induce  maturation  of  "null"  cells  into  E-rosette- 
forming  T-cells.   Thus,  the  "null"  cells  are  probably  pre-T  cells. 

Proposed  Course:   These  studies  will  be  continued. 

Objectives  (sub-project  B) : 

1)  Evaluation  of  immunologic  reconstitution  as  a  therapeutic  adjunct  for 
treating  chronic  infectious  diseases; 

2)  Identification  and  characterization  of  the  components  of  dialyzable  transfer 
factor  and  assessment  of  their  functions  in  immunologic  and  inflammatory 
reactions; 

3)  Investigation  of  transfer  factor-like  materials  in  laboratory  animals. 

Methods  Employed: 

The  trial  of  transfer  factor  in  candidiasis  is  being  continued  with  the 
oldest  participants  being  in  the  seventh  year  of  treatment.   These  subjects 
receive  transfer  factor  every  four  months.   A  companion  study  in  which  other 
immunodef icient  candidiasis  patients  are  being  treated  with  transfer  factor 
from  either  candida-sensitive  or  Candida- insensitive  donors  is  underway. 

Major  Findings: 

Thus  far  seven  patients  in  whom  remissions  were  induced  with  amphotericin 
are  receiving  transfer  factor  from  candida-sensitive  donors.   In  each  case 
the  patient  has  developed  in  vivo  and  in  vitro  evidence  of  cellular  immunity 
to  Candida.   This  indicates  that  the  transfer  factor  was  active  and  that  trans- 
fer factor  was  capable  of  "correcting"  the  immunologic  lesion  in  the  patients' 
cells.   Four  of  the  patients  have  maintained  positive  skin  responses  to  Candida 
and  all  of  these  patients  have  remained  in  remission  for  3  to  8  years.   These 
remissions  are  substantially  longer  than  those  expected  with  amphotericin  alone. 
The  three  patients  who  failed  to  maintain  cellular  immunity  to  Candida  also 
suffered  relapses  of  cutaneous  candidiasis. 

20-21 


Project  No.  Z01  AI  0,0046-11  LCI 

Five  patients  have  received  transfer  factor  from  donors  who  do  not  have 
cellular  immunity  to  Candida.   All  patients  were  treated  with  amphotericin  to 
induce  remissions.   None  of  the  recipients  developed  cellular  immunity  to 
Candida,  an  observation  that  suggests  that  transfer  factor  has  immunologic 
specificity.   Two  of  these  patients  have  relapsed.   The  other  three  are  in 
remission  and  have  been  for  periods  of  8  months  to  3  years. 

In  summary,  all  recipients  of  transfer  factor  (from  either  candida-sensi- 
tive  or  Candida- insensitive  donors)  have  remissions  that  are  longer  than  those 
seen  with  amphotericin  B  alone.   It  is  important  to  note  that  the  relapses 
occurred  only  in  persons  who  did  not  achieve  or  maintain  cell-mediated  immunity 
to  Candida. 

The  differences  between  the  numbers  of  remissions  in  the  two  groups 
is  significant  at  the  level  of  p=0.055. 

Publications : 


1.  Kirkpatrick,  C.H.,  Greenberg,  L.E.,  Chapman,  S.W.,  Goldstein,  G. ,  Lewis, 
V.M.  and  Twomey ,  J.J.:  Plasma  thymic  hormone  activity  in  patients  with 
chronic  mucocutaneous  candidiasis.   Clin.  Exp.  Immunol.  34:  311-317,  1978. 

2.  Kirkpatrick,  C.H. :  Transfer  of  Delayed  Cutaneous  Hypersensitivity  With 
Transfer  Factor.  Cell.  Immunol.  41:  62-71,  1978. 

3.  Kirkpatrick,  C.H.  and  Ailing,  D.W. :  Treatment  of  chronic  oral  candidiasis 
with  clotrimazole  troches.   A  controlled  clinical  trial.  New  Engl.  J. 
Med.  299:  1201-1203,  1978. 

4.  Togawa,  A.,  Oppenheim,  J.J.  and  Kirkpatrick,  C.H. :  Ability  of  dialysates 
containing  transfer  factor  to  induce  lymphocyte  activating  factor  by 
human  mononuclear  cells.   Cell.  Immunol. ,  45:-  133-141,  1979. 

5.  Kirkpatrick,  C.H. :  Transfer  of  cellular  immunity  with  transfer  factor. 
J.  Allergy  Clin.  Immunol.  63:  71-73,  1979. 

6.  Agbata,  A.I.  and  Kirkpatrick,  C.H.:   Release  of  E-rosette  augmenting 
factor  (E-RAF)  following  stimulation  of  human  leukocytes  with  mitogens 
or  antigens.  J.  Immunol.  122:  1080-1086,  1979. 

7.  Kirkpatrick,  C.H.  and  Windhorst,  D.W. :  Mucocutaneous  candidiasis  and 
thymoma.  Am^  J.  Med.  66:939-945,  1979. 

8.  Petersen,  E.A.  and  Kirkpatrick,  C.H. :  Nature  and  activities  of  transfer 
factor.  Ann.  N.Y.  Acad.  Sci.  in  press. 

9.  Kirkpatrick,  C.H. :  Immune  Deficiency  Disorders,  in  Immunology  II. 
(J. A.  Bellanti,  ed.),  Saunders,  Philadelphia,  1978,  pp.  644-689. 


20-22 


Project  No.  Z01  AI  00046-11  LCI 

10.  Kirkpatrick,  C.H.  and  Greenberg,  L.E.:  Treatment  of  chronic  mucocutaneous 
candidiasis  with  transfer  factor,  in  Immune  Regulators  in  Transfer  Factor. 
(A.  Khan,  C.H.  Kirkpatrick,  and  N.O.  Hill,  eds.),  Academic  Press,  New  York, 
1979,  pp.  547-559. 

11.  Kirkpatrick,  C.H.  and  Sohnle,  P.G. :  Chronic  Mucocutaneous  Candidiasis, 

in  Immunodermatology .  (B.  Safai  and  R.A.  Good,  eds.)  Plenum  Press,  in  press 

12.  Kirkpatrick,  C.H. :  Transfer  Factor,  in  CRC  Critical  Reviews  in  Clinical 
Laboratory  Sciences,  in  press 

13.  Cook,  J.L.,  Lewis,  A.M.  and  Kirkpatrick,  C.H. :  Age-related  and  thymus- 
dependent  rejection  of  Adenovirus  2-transf ormed  cell  tumors  in  the 
Syrian  hamster.  Cancer  Res.  39:  in  press,  Sept.  1979 


20-23 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  AI  00047-10  LCI 


PERIOD  COVERED 


October    1,    1978    to    September    30,    1979 


TITLE  OF   PROJECT   (30  characters   or  less) 
Clinical   Studies    on    Patients    With   Known   or    Suspected   Parasitic    Diseases. 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Pis:      F.A.Neva  Head,  Clinical  Parasitology  Section,  LCI,  NIAID 

(dual  position) 
I.E.  Nash         Senior  Investigator,  Clinical  Parasitology  Section, 

(dual  position),  LCI,  NIAID 
E.A.  Ottesen       Senior  Investigator,  Clinical  Parasitology  Section, 

(dual  position),  LCI,  NIAID 
D.J.  Wyler         Senior  Investigator,  Clinical  Parasitology  Section, 

(dual  position),  LCI,  NIAID 
Others:    R.  D'A.  Gusmao     Visiting  Fellow,  LPD, NIAID 
T.  Lawley  Medical  Investigator,  NCI 

M.N.  Lunde  Research  Zoologist,  LPD, NIAID 
J.D.  Berman  Clinical  Associate,  LCI, NIAID 
D.M.  Dwyer         Research  Microbiologist,  LPD, NIAID 


cooperating  units  (if  any)  Federal  University  of  Goias,  Goiania,  Brazil  (Profs.  J. 
Rezende  and  A.  Rassi) ;  Duke  University,  Division  of  Immunology  (Dr.  F.  Ward); 
Federal  University  of  Parana,  Brazil  (Dr.  E.  Ferreira) ;  Tuberculosis  Chemotherapy 
Center,  Madras,  India  (Dr.  S.P.  Tripathy) ;  Medical  College  of  Madras,  India 


LAB/BRANCH 

Laboratory  of  Clinical  Investigation 


SECTION 

Clinical  Parasitology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  Bethesda,  Maryland   20205 


! TOTAL  MANYEARS: 
5    3/12 


PROFESSIONAL: 

5  3/12 


I  CHECK  APPROPRIATE  BOX(ES) 
'$%&)      HUMAN  SUBJECTS 

C  (a1 )  MINORS   H  (a2)  INTERVIEWS 


XX (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  *ords  or  less  -  underline  keywords) 

The  interesting  association  of  absence  of  HLA  tissue  type-Al  with  chronic 
Chagas'  disease  was  not  confirmed  by  an  expanded  study  of  cases,  but  PR  (B  cell 


typing  will  still  be  done  on  frozen  cells.   The  diversity  of  HLA  haplotypes 
encountered  in  this  Brazilian  patient  population  corroborates  what  anthropo- 
logists have  said  of  this  population  -  namely,  extreme  genetic  mixing. 

Further  study  of  different  antigenic  fractions  of  schistosome  worms  has 
disclosed  one  fraction  to  which  antibody  levels  in  patients  correlate  quite 
well  with  numbers  of  eggs  being  excreted,  or  intensity  of  infection. 

Evidence  for  serum  inhibitory  factors  and  for  suppressor  cells  has  been 
found  in  f ilariasis  patients  characterized  by  unresponsiveness  to  filarial 
antigens.   The  role  of  immune  complexes  in  f ilariasis  patients  is  also  being 
studied.   Serum  and  cell  factors  were  also  found  that  contributed  to  antigen 
unresponsiveness  in  patients  with  chronic  schistosomiasis . 


20-24 


3HS-6040 
(Rev.  10- 


Project  No.  Z01  AI  00047-10  LCI 

Cooperating  Units,  cont'd: 

(Prof.  K.V.  Thiruvengadam) ;  University  of  Kentucky  Medical  Center, 
Lexington,  Ky.  (Dr.  J.  Burke). 

Summary  of  work,  cont'd: 

Evidence  for  multiplication  of  several  species  of  leishmania  within 
phagolysosomes  was  found  using  a  system  of  macrophages  derived  from  human 
monocytes . 

Project  Description: 

This  report  covers  the  clinical  investigations  carried  out  by  the 
four  investigators  in  the  Clinical  Parasitology  Section  (Drs.  Neva,  Nash, 
Ottesen  and  Wyler) .   Their  experimental  or  non-clinical  research  is  reported 
in  the  Annual  Report  from  the  Laboratory  of  Parasitic  Diseases.   Therefore, 
there  will  be  some  overlap  in  reporting  and  in  publications  listed.   The 
Clinical  Parasitology  Section  provides  consultative  service  in  clinical 
parasitic  diseases  to  the  Clinical  Center  and  several  members  of  the  Section 
also  participate  with  LCI  Staff  for  clinical  infectious  diseases  at  the 
Clinical  Center  and  at  the  Naval  Medical  Center.   As  in  the  past  year,  some 
of  the  studies  were  carried  out  in  collaboration  with  investigators  in 
India  and  in  Brazil. 

Pathogenesis  and  Immunology  of  leishmaniasis.   While  there  have  been 
many  in  vitro  studies  of  paras ite-macrophage  interaction  in  leishmaniasis, 
human  macrophages  have  not  been  used  for  such  work.   A  system  which  would 
use  human  macrophages  would  obviously  be  a  great  advantage  for  research 
related  to  human  disease  with  this  parasite.   Therefore,  it  was  cf  particular 
interest  that  a  system  involving  macrophages  derived  from  human  monocytes 
could  be  developed  in  which  leishmanial  growth  could  be  observed  and  demon- 
strated.  The  parasites  were  shown  to  be  present  and  dividing  within 
phagolysosomes,  as  happens  in  vivo .   Intracellular  multiplication  in  such 
cells  was  found  with  L.  donovani  and  with  L.  tropica,  the  parasites 
associated  with  visceral  and  cutaneous  leishmaniasis,  respectively.   Such 
an  _in  vitro  system  shows  promise  for  studying  effects  of  drugs  as  well  as 
immunological  events  associated  with  leishmanial  infections.  (Wyler,  Berman 
and  Dwyer) . 

Possible  influence  of  immune  response  on  Chagas'  disease.  Collaborative 
studies  with  Brazilian  colleagues  were  initiated  recently  on  Chagas'  disease 
as  reported  previously.   In  a  preliminary  investigation  which  involved 
mainly  the  study  of  blastogenic  response  to  T^  cruzi  antigens  in  patients 
with  various  forms  of  chronic  Chagas'  disease,  tissue  typing  at  the  A,  B, 
and  C  loci  was  also  carried  out.   This  preliminary  study  suggested  that  both 
positive  and  negative  correlations  with  certain  HLA  specificities  occurred 
in  patients  with  the  chronic  disease.   The  most  striking  association  was 
that  none  of  18  megaesophagus  patients  were  A-l,  while  this  type  occurred 
in  23  percent  of  44  seronegative  controls  and  in  28  percent  of  individuals 
with  infection  but  no  disease,  all  from  the  same  area  of  Brazil.   Therefore, 


20-25 


Z01  AI  00047-10   LCI 

a  larger  study  was  carried  out  in  the  past  year  to  include  more  cases  and 
family  members,  and  to  freeze  lymphocytes  for  B-cell  and  possible  Dw  typing 
as  well.   We  have  now  studied  larger  groups  of  40  to  50  in  various  clinical 
categories  and  the  previous  suspected  HLA  association  is  no  longer  present 
in  typing  at  A,  B  and  C  loci.   Therefore,  we  can  only  conclude  that  our 
sampling  groups  were  not  large  enough  for  statistically  significant  results. 
The  B-cell  typing  has  not  yet  been  done  at  the  time  this  report  was  pre- 
pared.  The  geneticists  and  our  collaborators  interested  in  HLA  are 
fascinated  by  the  genetic  diversity  of  the  study  population  and  believe 
they  have  found  some  new  HLA  specificities.   But  we  are  only  sadder  and 
wiser  with  the  intricacies  of  relating  HLA  tissue  types  to  parasitic 
diseases.  (Neva,  Gusmao,  Ward  and  Brazilian  collaborators). 

Clinical  significance  of  schistosome  antigens.   While  antigenic 
complexity  of  parasites  makes  immunologic  studies  more  difficult,  it  also 
provides  more  opportunities  to  dissect  and  examine  the  immunologic  signi- 
ficance of  various  antigenic  components.   Last  year  we  reported  that  the 
immune  response  in  patients  to  the  polysaccharide  gut  antigen  showed  a 
characteristic  time  course,  being  highest  in  recent  infections  and  falling 
rapidly  with  time.   This  antibody  response,  however,  did  not  reflect  or 
correlate  with  intensity  of  infection.   Recent  work  which  has  focused  upon 
individual  fractions  of  the  TCA-soluble  material  from  whole  worms  has 
disclosed  a  relationship  between  response  to  one  of  these  antigenic  compon- 
ents and  numbers  of  eggs  excreted.   This  antigen  fraction,  a  glycoprotein, 
is  but  one  of  several  fractions  that  make  up  a  crude  antigen  reported  by 
others  as  correlating  with  intensity  of  infection.   But  the  antibody 
response  to  this  specific  glycoprotein  fraction  did  not  change  with  time 
after  infection.   Thus,  antibody  response  to  various  but  specific  antigens 
of  parasites,  such  as  schistosomes,  may  assist  in  clinical  evaluation  of 
patients  with  the  infection.   With  the  antigens  just  cited,  antibody  levels 
in  a  patient  can  indicate  whether  the  infection  is  recent  or  more  than  a 
year  in  duration  and  some  indication  of  how  heavy  the  infection  is.  (Nash 
and  Lunde) . 

Immune  response  to  helminth  infections.   Recently  completed  studies 
of  histamine  release  from  cells  of  patients  with  tropical  pulmonary 
eosinophilia,  a  form  of  filarial  infection  without  circulating  microfilariae, 
highlighted  the  specific  nature  of  the  IgE-mediated  allergic  reaction  pro- 
voked by  microf ilarial  antigens  upon  contact  with  sensitized  cells.   But  an 
equally  if  not  more  intriguing  question  is  raised  by  this  observation  - 
namely,  why  is  not  histamine  release  going  on  all  the  time  in  vivo  in  those 
patients  whose  filariasis  is  associated  with  circulating  microfilariae? 
There  are  indications  that  serum  inhibitory  factors  are  involved  in  this 
allergic  hyporesponsiveness .   Many  of  the  patients  with  chronic  filariasis 
and  circulating  microfilariae  also  exhibit  both  serum  and  adherent  sub- 
populations  of  mononuclear  blood  cells  that  contribute  to  cell-mediated 
unresponsiveness  to  filarial  antigens.  (Ottesen) . 

The  role  of  circulating  immune  complexes  is  also  under  study  in  patients 
with  different  clinical  manifestations  of  filariasis.  The  Clq  binding  immune 
complexes  that  were  demonstrated  in  patients  with  acute  schistosomiasis 


20-26 


Z01  AI  00047-10  LCI 

are  being  studied  further  to  better  characterize  antigenic  make-up  of  these 
complexes.  (Ottesen  and  Lawley) . 

Tests  for  specific  antibody  in  strongyloidiasis.  A  long  experience 
with  persistent  strongyloides infection  in  an  immuno-def icient  patient  made 
it  possible  to  obtain  larval  antigens  from  this  parasite.  Preliminary 
results  suggested  that  such  antigens  might  be  useful  for  serologic  tests 
in  patients.  Therefore,  S_;_  stercoralis  as  well  as  other  nematode  larval 
antigens  are  being  investigated  for  possible  usefulness  in  the  diagnosis 
of  human  strongyloides  infection.  (Neva  and  Burke) . 

Publications: 

1.  Ottesen,  E.A.,  Hiatt,  R.A. ,  Cheever,  A.W. ,  Sotomayor,  and  Neva,  F.A.: 
The  Acquisition  and  Loss  of  Antigen-Specific  Cellular  Immune  Responsiveness 
in  Acute  and  Chronic  Schistosomiasis  in  Man.   Clin.  Exp.  Immunol.   33 : 
38-47,  1978. 

2.  Ottesen,  E.A.  and  Weller,  P.F.:   Eosinophilia  Following  Treatment  of 
Patients  with  Schistosomiasis  mansoni  and  Bancroft's  filariasis.   J.  Inf. 
Pis.   139:   343-347,  1979. 

3.  Ottesen,  E.A. ,  Neva,  F.A. ,  Paranjape,  R.S.,  Tripathy,  S.P., 
Thiruvengadam,  K.V.  and  Beaven,  M.A. :   Specific  Allergic  Sensitization  to 
Filarial  Antigens  in  Tropical  Eosinophilia  Syndrome.   Lancet  I:  1158-1161, 
1979. 

4.  Nash,  T.E.,  Ottesen,  E.A. ,  and  Cheever,  A.W. :   Antibody  Response  to  a 
Polysaccharide  Antigen  Present  in  the  Schistosome  Gut.   II.   Modulation  of 
Antibody  Response.   Am.  J.  Trop.  Med.  Hyg.   27:   944-950,  1978. 

5.  Neva,  F.A.  and  Ottesen,  E.A. :   Tropical  (Filarial)  Eosinophilia.   New 
Eng.  J.  Med.   298:  1129-1131,  1979. 

6.  MacQueen,  J.M.,  Ottesen,  E.A. ,  Ottesen,  C,  Amos,  D.B.,  and  Ward,  F.E.: 
HLA  Histocompatibility  Antigens  in  a  Polynesian  Population  -  Cook  Islanders 
of  Mauke.   Tissue  Antigens   13:   121-128,  1979. 

7.  Lunde,  M.N. ,  Ottesen,  E.A. ,  and  Cheever,  A.W. :  Serological  Differences 
Between  Acute  and  Chronic  Schistosomiasis  mansoni  Detected  by  Enzyme  Linked 
Immunosorbant  Assay  (ELISA) .   Am.  J.  Trop.  Med.  Hyg.  28:   87-91,  1979. 

8.  Collidge,  E.,  Weller,  P.F.,  Ramsey,  P.G.,  Ottesen,  E.A. ,  Beaver,  P.C. 
and  von  Lichtenberg,  F.C.:   Zoonotic  Brugia  Filariasis  in  New  England. 
Ann.  Int.  Med.    90:   341-343,  1979. 

9.  Ottesen,  E.A. :   Modulation  of  the  Host  Response  in  Human  Schistosomiasis 
II.   Adherent  Suppressor  Cells  which  Inhibit  Lymphocyte  Proliferative 
Responses  to  Parasite  Antigens.   J.  Immunol.   (In  press). 


20-27 


Z01  AI   00047-10  LCI 
Publications,  cont'd: 

10.  Ottesen,  E.A. :   Filarial  Infection  and  the  Host  Response  in  Man. 
Paradoxes  and  Insights.   In  Escape  from  Immune  Surveillance:   The  Interface 
Between  Immune  Mechanisms  and  Disease.   D.B.  Amos,  R.S.  Schwartz  and  B.W. 
Janicki,  eds.   Academic  Press  (In  press). 

11.  Ottesen,  E.A. :  Visceral  Larva  Migrans  and  Other  Migratory  Helminths 
of  Man.  In  Principles  and  Practice  of  Infectious  Disease,  Mandell,  G.I., 
Douglas,  R.G. ,  and  Bennett,  J.E.,  eds.   J.  Wiley  and  Sons,  New  York 

(In  press) . 

12.  Lawley,  T. J. ,  Ottesen,  E.A. ,  Hiatt,  R.A.  and  Gazze,  L.A. :   Circulating 
Immune  Complexes  in  Acute  Schistosomiasis.   Clin.  Exp.  Immunol.  (In  press). 

13.  Lunde,  M.N  and  Ottesen,  E.A.:   Enzyme-linked  immunosorbent  assay  (ELISA) 
for  detecting  IgM  and  IgE  antibodies  in  human  schistosomiasis.   Am.  J.  Trop. 
Med.  Hyg.   (In  press) . 

14.  Cohen,  S.G.  and  Ottesen,  E.A.:   "Eosinophils  in  immune  function"  in 
Cell  Biology  of  Immunity  and  Inflammation,   Oppenheim,  J. ,  Rosenstreich,  D. 
and  Potter,  M.  ,  eds.   Harvard  Elsevier-North  Holland,  Inc.   (In  press). 

15.  Nash,  T.E.:   Antibody  response  to  a  polysaccharide  antigen  in  schisto- 
somialsi.   I.   Sensitivity  and  specificity.   Am.  J.  Trop.  Med.  Hyg.   27 : 
939,  1978. 

16.  Nash,  T.E.,  Ottesen,  E.A. ,  Cheever,  A.W. :  Antibody  response  to  a 
polysaccharide  antigen  present  in  schistosome  gut.  II.  Modulation  of 
antibody  response.   Am.  J.  Trop.  Med.  Hyg.   27:  944-950,  1978. 

17.  Neva,  F.A. ,  Wyler,  D.J.  and  Nash,  T.E.:   Cutaneous  leishmaniasis  -  A 
case  with  persistent  organism  after  treatment  in  presence  of  normal  immune 
response.    Am.  J.  Trop.  Med.  Hyg.   28:   467-471,  1979. 

18.  Wyler,  D.J.:   Leishmaniasis.   In  Current  Therapy  (Conn,  H.,  ed.) 
W.B.  Saunders  Co.,  Philadelphia,  Pa. 

19.  Wyler,  D.J.  and  Miller,  L.H:   Plasmodium  species  (Malarial)  (Chapter 
227).   In  Principles  and  Practices  of  Infectious  Diseases   (Mandell,  G.L., 
Douglas,  R.G.,  and  Bennett,  J.E.,  eds.)   (In  press). 

20.  Wyler,  D. J. :   Cellular  aspects  of  immune  regulation  in  malaria. 
Bull.  WHO.   (In  press). 

21.  Wyler,  D.J.,  Herrod,  H.  and  Weinbaum,  F.I.:  Response  of  sensitized 
and  unsentized  human  lymphocyte  subpopulations  to  Plasmodium  falciparum 
antigens.   Infect.  &  Immunol.   24:   106,  1979. 


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Z01   AI   00047-10  LCI 

22.  Wyler,  D.J.,  Oppenheim,  J.J.  and  Koontz,  L.C.:  The  influence  of 
malaria  infection  on  the  elaboration  in  vitro  of  soluble  mediators  by 
adherent  mononuclear  cells.  Infect.  &  Immunol.   24 :  151,  1979. 

23.  Wyler,  D.J.,  Weinbaum,  F.E.  and  Herrod,  H. :   Characterization  of  the 
in  vitro  proliferative  responses  of  human  lymphocytes  to  leishmanial 
antigens.   J.  Infect.  Pis.   1979  (In  press) 


20-28a 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00048-09  LCI 


PERIOD  COVERED 

September  30,  1978  to  October  1,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

The  Pathophysiology  of  Autoimmune  Hemolytic  Anemia 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


OTHER: 


M.  Frank 


M.  Hamburger 


Head,  Clinical  Immunology 
Section  and  Chief,  LCI 

Clinical  Associate 


LCI 
LCI 


NIAID 
NIAID 


cooperating  units  (if  any)  T>  Lawley  (Dermatology  Branch,  NCI/NIH)  ;  P.  Plotz  (Arthritis 
Branch,  NIAMDD/NIH) ;  H.  Moutsopoulos,  (NIDR) ;  T.  Chused,  (NIAID);  E.  Franklin, 
(NYU  School  of  Medicine);  G.  Sharp,  (Univ.  of  Missouri  School  of  Medicine). 


lab/branch 
T.^hnrat-nry  of  Clinical Investigation 


SECTION 

Clinical  Immunology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH  Bethesda,  MD 


20205 


TOTAL  MANYEARS: 

2 


PROFESSIONAL: 
2 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 
X 

D  (al)  MINORS   []  (a2)  INTERVIEWS 


(b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Me  have  continued  to  develop  and  extend  the  usefulness  of  our  new  technique  for 
measuring  IgG  Fc  receptors  and  complement  receptors  in  various  groups  of  patients 
with  autoimmune  disease.   These  techniques  have  proven  useful  both  in  diagnostic 
tests  and  for  developing  a  further  understanding  of  pathophysiologic  processes. 
In  the  past  year  we  have  extended  our  studies  to  patients  with  mixed  IgM,  IgG 
cryoglobulins  and  have  shown  that  these  patients  can  be  divided  into  two  groups. 
The  patients  with  Fc  receptor  defects  are  the  ones  who  develop  immune  complex 
mediated  renal  disease.   The  patients  with  normal  Fc  receptor  function  do  not 
appear  to  develop  immune  complex  mediated  renal  disease.   The  follow-up  studies 
have  been  performed  in  our  patients  with  lupus  erythematosus  and  it  has  been 
shown  that  as  the  patients  improve  the  immune  clearance  defect  also  improves. 
Patients  with  mixed  connective  tissue  disease  have  been  studied  and  have  been 
shown  in  general  to  be  free  of  defects  in  immune  clearance  not  withstanding  the 
fact  that  they  have  circulating  immune  complexes.   This  is  in  keeping  with 
previous  observations  that  these  patients  do  not  develop  immune  complex  mediated 
tissue  injury.  20-0Q 

PHS-6040 
(Rev.  IO-76) 


Project  No.  Z01  AI  00048-09  LCI 

Project  Description: 

Obj  ectives : 

To  define  the  pathophysiologic  function  of  antibody  and  complement  on 
the  surface  of  red  cells  in  patients  with  hemolytic  anemia,  and  to  use  the 
findings  which  have  been  developed  from  this  study  to  develop  new  tests  for 
immunologically  specific  receptors  function  in  patients  with  a  variety  of 
disease  states.   The  tests  developed  for  the  study  of  autoimmune  hemolytic 
anemia  have  been  found  to  have  wide  application  to  other  autoimmune  and 
immunologic  problems. 

Methods  Employed: 

These  studies  examine  the  clearance  of  radiochromated  erythrocytes 
from  the  circulation.   The  initial  studies  were  performed  in  a  guinea  pig 
model,  but  all  of  the  recent  studies  have  been  performed  in  human  volunteers 
or  in  patients  with  various  diseases.   In  the  initial  studies  in  humans, 
the  clearance  of  erythrocytes  coated  with  IgM  isoagglutinin  antibodies  were 
examined.   In  later  studies  radiochromated  erythrocytes  were  coated  with 
highly  purified  human  IgG  anti-Rh  antibodies  and  the  survival  of  these  cells 
was  also  studied.   The  third  general  set  of  clearance  studies  examined  the 
clearance  of  radioactive  albumin  which  had  been  aggregated.   This  is  the 
standard  method  for  determining  RES  clearance  and  can  be  used  to  determine 
both  liver  blood  flow  and  the  ability  of  the  reticuloendothelial  system  to 
clear  particulate  materials. 

Major  Findings: 

In  past  years,  we  identified  the  immunologically  active  protein  frag- 
ments which,  when  deposited  on  red  cells,  are  responsible  for  the  clearance 
of  those  cells  from  the  circulation.   It  was  reported  that  receptors  for  these 
immunologically  specific  fragments  were  present  on  the  membrane  of  macrophages 
which  were  responsible  for  removal  of  cells  coated  with  these  fragments  from  the 
circulation.   It  became  clear  that  it  was  possible  to  study  patients  with  a 
wide  variety  of  diseases  to  determine  whether  their  receptors  for  these  immuno- 
logically active  fragments  were  functioning  normally  in  vivo.   It  has  been 
known  for  years  that  the  reticuloendothelial  system  (RES)  plays  a  major  role 
in  removing  foreign  material  from  the  circulation.   These  foreign  materials 
include  bacteria  and  parasites,  as  well  as  endogenously  generated  immuno- 
reactive  materials  such  as  immune  complexes.   It  was  suspected  that  defects 
in  RES  function  might  be  important  in  both  infectious  and  immunologic  diseases. 
However,  when  this  question  was  examined  directly  using  the  techniques  available, 
no  defects  in  RES  function  were  noted  in  any  disease  state  in  which  blood  flow 
to  the  RES  organs  was  normal.   Until  the  present  studies,  the  technique 
available  for  examination  of  RES  function  was  that  of  study  of  clearance  of 
aggregated  albumin  from  the  circulation.   This  study  has  led  to  the  development 
of  new  techniques  for  evaluation  of  RES  function.   Using  these  new  techniques 
developed  within  our  unit  we  have  shown  that  there  are  indeed  diseases  with 
defects  in  RES  function  if  one  examines  this  question  looking  at  specific  re- 

20-30 


Project  No.  Z01  AI  00048-09  LCI 

ceptors  for  immunologically  active  material.   Thus  receptors  for  comple- 
ment and  immunoglobulin  on  a  patient's  macrophages  will  recognize  these 
immunologically  active  materials  coating  foreign  substances  as  they  circu- 
late. 

The  defects  are  quite  specific  for  different  sets  of  receptors.   Our 
test  utilizes  the  patient's  own  red  blood  cells.   The  cells  are  coated  with 
antibody  and/or  complement  fragments  and  are  infused  into  the  patient  and 
their  clearance  is  followed.   Using  this  technique  we  have  found  that  almost 
every  patient  with  active  lupus  erythematosus  has  a  major  defect  in  Fc  re- 
ceptor specific  clearance  although  the  clearance  of  aggregated  materials  is 
normal.   We  believe  that  it  is  quite  likely  that  this  clearance  defect  ex- 
tends to  the  clearance  of  the  circulating  immune  complexes  which  occur  in  these 
patients.   If  such  complexes  are  not  cleared  efficiently  from  the  circulation, 
they  can  be  deposited  in  glomeruli  where  they  can  cause  immune  damage.   We 
find  that  as  the  disease  improves,  the  clearance  defect  also  tends  to  improve 
and  the  comlexes  present  in  the  circulation  tend  to  disappear.  However  it  is 
striking  that  most  patients  with  lupus  erythematosus  have  a  persistence  of 
their  clearance  defect  for  long  periods  of  time,  even  on  treatment.  Our  stud- 
ies in  lupus  erythematosus  have  already  been  confirmed  by  a  group  in  London. 
These  initial  findings  in  lupus  were  published  in  the  past  year  in  the 
New  England  Journal  of  Medicine. 

Extensive  studies  have  been  conducted  of  Sjogren's  Syndrome.  These  stud- 
ies demonstrated  that  patients  with  clearance  defects  in  the  presence  of  cir- 
culating immune  complexes  tend  to  develop  peripheral  manifestations  of  their 
Sjogren's  Syndrome  including  autoimmune  disease  and  extensive  tissue  injury. 
On  the  other  hand  patients  who  do  not  have  clearance  defects  tend  not  to  de- 
velop secondary  manifestations  of  their  Sjogren's  Syndrome  with  peripheral 
tissue  destruction  even  in  the  presence  of  circulating  immune  complexes.   This 
material  is  in  press  in  the  Annals  of  Internal  Medicine.   We  have  now  had  an 
opportunity  to  analyze  the  circulating  immune  complexes  in  patients  with 
Sjogren's  Syndrome  and  we  have  shown  that  a  very  high  proportion  of  patients 
have  immune  complexes  and  that  these  complexes  are  of  multiple  types.   Moreover, 
the  rheumatoid  factor  present  in  these  patients  is  only  one  type  of  complex 
and  is  not  responsible  for  many  of  the  positive  tests  for  immune  complexes 
shown  in  the  studies  of  the  serum  of  these  patients. 

In  the  past  year  with  Dr.  Edward  Franklin  and  his  group  at  NYU  we 
have  had  an  opportunity  to  study  patients  with  IgM-IgG  mixed  cryoglobulinemia. 
These  patients  have  been  shown  to  fall  into  two  groups  with  regard  to 
the  development  of  peripheral  manifestation  of  disease  in  spite  of  the 
fact  that  all  have  circulating  immune  complexes.   One  group  of  patients 
has  been  shown  to  possess  normal  Fc  receptor  function  and  these  patients 
do  not  develop  certain  severe  peripheral  manifestations  of  disease  such 
as  immune  complex  mediated  glomerulonephritis.   A  second  group  of  patients 
have  been  shown  to  have  a  clearance  defect  and  it  is  these  patients  that 
have  evidence  of  glomerulonephritis.   Once  again  the  usefulness  of  the 
clearance  studies  in  determining  the  nature  of  the  pathophysiologic  process 
in  this  disease  is  clear. 

20-31 


Project  No.  Z01  AI  00048-09  LCI 

We  have  also  studied  a  large  group  of  patients  with  mixed  connective 
tissue  disease  with  Drs.  Gordon  Sharp  and  Harry  Moutsopoulos.   These 
patients  tend  not  to  have  tissue  deposition  of  immune  complexes  and  exten- 
sive tissue  damage  although  they  have  circulating  immune  complexes  in 
quantity.   Interestingly,  our  studies  show  little  or  no  evidence  of  a 
clearance  defect  in  these  patients  except  for  those  who  have  many  of 
the  manifestations  of  systemic  lupus  erythematosus.   The  latter  patients 
tend  to  resemble  patients  with  lupus  erythematosus  in  that  they  have  anti-DNA 
etc.  and  interestingly  these  patients  do  have  clearance  defects.   Finally, 
we  have  studied  a  group  of  patients  with  rheumatoid  arthritis.   These  patients 
do  not  have  striking  Fc  receptor  clearance  defects  suggesting  that  much  of 
the  immunopathology  that  occurs  is  not  systemic  immune  complex  mediated 
injury  but  originates  in  the  joints  themselves. 

Significance  to  Biomedical  Research 

These  techniques  allow  for  the  complete  reevaluation  of  reticuloendo- 
thelial system  function  in  man.   Already  they  are  being  widely  applied.   The 
use  of  these  techniques  has  already  allowed  for  the  description  of  the  first 
patients  with  receptor  specific  defects.   C3b  specific  defects  have  been 
demonstrated  in  primary  biliary  cirrhosis  and  Fc  receptor  defects  in  a 
number  of  serious  autoimmune  diseases.   These  techniques  provide  a  powerful 
new  tool  in  the  evaluation  of  these  patient  groups  and  provide  a  new  approach 
to  understanding  the  pathophysiologic  basis  of  these  diseases. 


Proposed  Course: 

We  intend  to  extend  these  studies  to  additional  autoimmune  diseases. 
Moreover,  we  intend  to  attempt  to  determine  whether  patients  with  various 
kinds  of  infectious  disease  may  also  have  clearance  defects  which  contribute 
to  their  inability  to  clear  bacteria  from  the  circulation.   At  the  present 
time  we  are  examining  the  possibility  of  studying  patients  with  sickle  cell 
anemia  to  determine  whether  these  patients  have  a  clearance  defect  that  might 
help  account  for  their  propensity  to  develop  overwhelming  sepsis. 

Publications : 

1.  Jones,  E.A. ,  Frank,  M.M. ,  Jaffe,  C.J.,  and  Vierling,  J.M. :   Primary 

Biliary  Cirrhosis  and  the  Complement  System.   Ann.  Int.  Med. : 
In  press. 

2.  Jaffe,  C.J.,  Vierling,  J.M. ,  Jones,  E.A. ,  Lawley,  T.  and  Frank,  M.M. : 
Receptor  specific  clearance  by  the  reticuloendothelial  system  in 
chronic  liver  diseases:   Demonstration  of  defective  C3b  specific 
clearance  in  primary  biliary  cirrhosis.   J.  Clin.  Invest.   November 
1978:  62:1069-1077,  1978. 

3.  Lawley,  T. ,  Moutsopoulos,  ELM.  ,  Katz,  S.I.,  Theof ilopoulos ,  A.N., 
Chused,  T.M. ,  and  Frank,  M.M. :   Circulating  Immune  Complexes  in 
Sjogren's  Syndrome.   J .  of  Immun .   In  press. 

20-32 


Project  No.  Z01  AI  00048-09  LCI 


4.  Frank,  M.M. ,  Hamburger,  M.I.,  Lawley,  T.J.,  Kimberly,  R.P.,  and 
Plotz,  P.H.:   Defective  Reticuloendothelial  System  Fc  Receptor 
Function  in  Systemic  Lupus  Erythematosus.   N.  Engl.  J.  Med. 
300:  518-523,  March,  1979. 

5.  Moutsopoulos,  H.M. ,  Chused,  T.M. ,  Mann,  D.L. ,  Klippel,  J.H., 
Fauci,  A.S.,  Frank,  M.M. ,  Lawley,  T.J.  and  Hamburger,  M.I.: 
Sjogren's  Syndrome  (Sicca  Syndrome):   Current  Issues.  An  edited 
transcript  of  a  Combined  Clinical  Staff  Conference  of  the  Clinical 
Center,  Bethesda,  Maryland,  April  19,  1979. 

6.  Hamburger,  M.I.,  Moutsopoulos,  H.M. ,  Lawley,  T.  and  Frank,  M.M. : 
A  defect  in  Reticuloendothelial  System  Fc  Receptor  Function  in 
Sjogren's  Syndrome.   Ann.  Int.  Med.   In  press. 

7.  Hamburger,  M.I.,  Gorevic,  P.,  Franklin,  E.C.  and  Frank,  M.M. :   The 
Function  of  the  Reticuloendothelial  System  in  Autoimmune  Disease. 
Transaction  of  the  Association  of  the  American  Physicians.  1979. 
In  press. 

8.  Frank,  M.M. :   The  Function  of  the  Reticuloendothelial  System  in 
Autoimmune  Diseases.   In.   Immune  Mechanisms  in  Renal  Disease. 
Cummings,  N.  (Ed.)   In  press. 

9.  Frank,  M.M. :   Reticuloendothelial  Complement-Specific  Clearance  of 
Circulating  Particles,  p.  77-8  In  Jones,  E.A.  (Moderator).   Primary 
Biliary  Cirrhosis  and  the  Complement  System.   Ann.  Intern.  Med. 

90:  72-84,  1979.  ' 

10.  Frank,  M.M. :   The  Role  of  Complement  in  the  in  vivo  Destruction  of 
Erythrocytes.  In.  Proceedings  of  the  American  Red  Cross  Eleventh 
Annual  Scientific  Symposium.   In  press. 

11.  Frank,  M.M. :   The  Role  of  the  Reticuloendothelial  system  in  the 
Clearance  of  Circulating  Cells  Coated  with  Antibody  and  Complement. 
In:  Proceedings  of  the  WHO  Meeting  on  the  Role  of  the  Spleen  in 
the  Immunology  of  Parasitic  Diseases.   In  press . 

12.  Frank,  M.M.  ,  Atkinson,  J.  and  Jaffe,  C:   The  Role  of  Antibody  and 
Complement  in  Immune  Clearance  of  Erythrocytes  in  Man.   In  Clinical 
Aspects  of  the  Complement  System:  International  Symposium.  Opferkuch,  W. 
Rother,  K.  and  Schultz,  D.R.  (Eds)  Georg  Thieme  Publ.,  1978,  pp.  70-76. 


20-33 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00049-09  LCI 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Initiation  and  Regulation  of  Antigen  Recognition 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


OTHER: 


M.M.  Frank 

J.T.  Blake 
K.U.  Cehrs 
K.  Yokomuro 
J.  Schroer 
J .  Thomas 
R.  Clark 


Acting  Head,  Biologic  Structure 

Section 
Bio.  Lab.  Tech.  (Micro.) 
Bio.  Lab.  Tech.  (Micro.) 
Guest  Worker 
Guest  Worker 
Clinical  Associate 
Clinical  Associate 


LCI  NIAID 
LCI  NIAID 
LCI  NIAID 
LCI  NIAID 
LCI  NIAID 
LCI  NIAID 
LCI  NIAID 


COOPERATING  UNITS  (if  any) 

None 


lab/branch 

Laboratory   of   Clinical    Investigation 


SECTION 

Biologic  Structure  Section 


INSTITUTE  AND  LOCATION 

NIAID/NIH  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

5   11/12 


PROFESSIONAL: 

4       5/12 


1      6/12 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


(c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  interest   of  the  Biologic  Structure  Section  is  to  characterize  the 
role  of  the  macrophage  in  antigen  recognition  by  T  lymphocytes  in  the  expres- 
sion and  initiation  of  cell-mediated  immunity  in  man  and  experimental  animals. 
The  Section  has  been  interested  in  exploring  the  role  of  the  major  histocom- 
patibility complex  in  the  genetic  regulation  of  cell-cell  interactions 


required  for  expression  of  cell  mediated  and  humoral  immunity.   The  Biologic 
Structure  Section  is  also  actively  characterizing  the  physical  state, 
localization,  and  fate  of  macrophage  associated  antigen.   Additional  studies 
of  genetics  of  immune  responsiveness  to  insulin  done  in  man  are  assessed  and 
compared  to  mouse  and  guinea  pig.   Determinant  selection  by  macrophages 
functionally  describes  Ir  gene  control  of  antigen  recognition  by  T  lymphocytes 
in  experimental  animals.   An  understanding  of  the  operation  of  a  similar 
mechanism  in  man  may  have  implications  in  the  pathophysiology  of  insulin 
allergy  in  diabetics. 


20-34 


PHS-6040 

(Rev.  IO-76) 


Project  No.  Z01  AI  00049-09  LCI 
Project  Description: 
Objectives : 

1)  To  characterize  in  a  quantitative  and  qualitative  fashion  the  role 
of  macrophages  and  lymphocytes  in  the  development  of  cellular  immunity. 

2)  To  study  in  experimental  animals  the  mechanism  of  lymphocyte 
antigen  recognition  using  defined  antigens  such  as  insulin. 

3)  To  study  the  cellular  and  molecular  basis  of  the  genetic  control  of 
immune  responsiveness  and  the  role  of  surface  membrane  determinants  in  cell 
cooperation. 

Methods  Employed: 

Standard  assays  have  been  developed  for: 

1)  Determination  of  DNA  synthesis  of  mouse  and  guinea  pig  lymphocyte 
populations . 

2)  Preparation  and  isolation  of  partially  purified  populations  of 
lymphocytes . 

3)  Column  purification  of  cells  and  proteins. 

4)  Binding  of  lymphocytes  to  macrophage  receptors. 

5)  The  uptake  and  fate  in  culture  of  soluble  protein  antigens. 

Major  Findings: 

Dr.  Alan  Rosenthal  left  the  NIH  last  year  to  assume  a  position  as 
Director  of  Immunology  of  Merck,  Sharp  and  Dohme.   This  year's  efforts 
have  been  directed  at  completing  projects  of  his  fellows  who  were  committed 
to  an  additional  years  tenure. 

Members  of  this  Laboratory  have  begun  to  characterize  seven  anti- 
insulin  antibody  secreting  cell  lines  prepared  by  cell  fusion  techniques  in 
collaboration  with  Dr.  K.  Jin  Kin.   Several  interesting  results  were 
obtained  from  these  studies.   An  autoreactive  antibody  which  binds  to  a 
mouse's  own  insulin  was  one  of  the  seven  isolated.   Furthermore,  the 
precise  amino  acid  sequence  of  the  binding  site  on  the  insulin  molecule 
(region  of  A  chain  residues  8-10)  of  another  anti-insulin  fusion  product 
was  identified.   This  last  antibody  can  distinguish  beef  insulin  from  pork 
insulin,  rat  insulin  or  other  insulins  which  differ  in  amino  acid  sequence 
in  this  region  of  the  insulin  molecule.   In  collaboration  with  Dr.  James  W. 
Thomas,  the  kinetics,  specificity  and  Ir  gene  control  of  the  plaque  forming 
cell  response  to  insulin  and  TNP-insulin  in  the  mouse  were  also  completed 
and  submitted  for  publication. 


20-35 


SMITHSONIAN    SCIENCE    INFORMATION 
PROJECT   NUMBER    (Do   NOT    use    this 


EXCHANGE  U.S.    DEPARTMENT   OF 

ioace)      IHEALTH,    EDUCATION,    AND    WELFARE 
PUBLIC   HEALTH    SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH   PROJECT 


PROJECT   NUMBER 

ZOl  AI   00050-09   LCI 


PERIOD    COVERED 

October   1,    1978    to   September   30,    1979 


TITLE   OF    PROJECT   (80  characters   or  less) 

Clinical   Studies    of   Complement  Abnormalities   of  Man 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


OTHER: 


M.  Frank 


S.  Hosea 
K.  Katusha 
M.  Santaella 


Head,  Clinical  Immunology 
Section  and  Chief  LCI 

Clinical  Associate 

Microbiologist 

Guest  Researcher  on  IPA 


LCI 


NIAID 


LCI  NIAID 
LCI  NIAID 
LCI    NIAID 


COOPERATING  UNITS  (if  any) 

D.W.  Ailing,  OSD,  NIAID,  NIH) :  D.  Triantaphylapolis  and  M.  Wickerhauser 
American  Red  Cross,  Bethesda,  MD 


lab/branch 

Laboratory  of  Clinical  Investigation 


SECTION 

Clinical  Immunology 


INSTITUTE  AND  LOCATION 

NIH/NIAID,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 

3   8/12 


PROFESSIONAL: 

3  2/12 


CHECK   APPROPRIATE   BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Hereditary  Angioedema  is  an  autosomal  dominant  disease  characterized 
by  abnormalities  in  CI  esterase  inhibitor.   We  have  previously  shown  that 
danazol  corrected  the  protein  abnormality  in  patients  with  this  disease. 
During  this  year  we  have  further  extended  observations  of  the  usefulness 
of  danazol  and  danazol  toxicity  in  a  large  patient  population.   We  have 
begun  to  characterize  the  incidence  of  autoimmune  diseases  in  this  patient 
group  as  well.   Further,  we  have  extended  our  experience  with  the  use  of 
purified  CI  esterase  inhibitor  in  the  treatment  of  Hereditary  Angioedema 
attacks  and  have  shown  that  the  therapy  can  abort  these  potentially  life 
threatening  attacks. 


20-36 


PHS-6040 
(Rev.  IO-76) 


Project  No.  Z01  00050-09  LCI 

Project  Description: 

Objectives: 

The  general  objective  of  this  program  has  been  to  define  the  role  of 
complement  in  human  illness  and  to  develop  better  methods  of  therapy  for 
immune  damage  mediated  by  complement.   As  in  the  past  year,  major  emphasis 
was  placed  on  our  studies  with  patients  with  hereditary  angioedema.   This 
illness,  which  carries  a  high  mortality  rate,  is  particularly  useful  for 
study  since  it  can  serve  as  a  model  for  complement-mediated  immune  damage 
in  man  and  as  model  for  the  study  of  genetically  controlled  autosomal  domi- 
nant diseases.   We  were  particularly  interested  this  year  in  defining  the 
usefulness  and  toxicity  of  danazol  in  a  large  patient  population.   We  were 
also  interested  in  extending  our  efforts  to  stop  attacks  of  hereditary  angio- 
edema after  they  have  already  begun.   Danazol  does  not  work,  in  this  situation. 

Methods  Employed: 

Purified  complement  components  are  prepared,  and  antisera  to  various 
complement  components  and  component  inhibitors  are  used  for  estimation  of 
component  levels  in  human  sera  and  other  body  fluids.   Double-blind  thera- 
peutic studies  are  employed  to  test  new  methods  of  therapy.   Patients  on 
experimental  drug  protocols  are  followed  to  determine  signs  of  toxicity. 

Major  Findings: 

In  the  past  years,  we  have  developed  and  evaluated  a  new  drug  danazol 
for  treatment  of  hereditary  angioedema.   Earlier  we  showed  that  this  drug 
is  markedly  effective  in  treating  this  disease  and  is  relatively  non-toxic. 
The  drug  has  the  striking  effect  of  causing  patients  with  the  deficient  serum 
inhibitor  protein  to  synthesize  this  protein  so  that  levels  of  the  protein  in 
blood  return  to  normal.   With  the  restoration  of  normal  levels  of  the  protein 
in  blood,  the  patient  corrects  his  biochemical  abnormalities  and  thereafter 
is  free  of  symptoms.   The  long-term  use  of  this  new  drug  has  never  been 
studied  in  any  patient  population  and  it  was  essential  to  begin  to  define 
those  situations  in  which  the  drug  was  useful  or  on  the  other  hand  was  likely 
to  cause  problems.   We  also  were  interested  in  attempting  to  stop  attacks  of 
hereditary  angioedema  once  they  had  begun.   It  takes  days  for  danazol  to  have 
its  effect.   Patients  who  are  not  on  danazol  however,  will  often  present  to  an 
emergency  ward  having  severe  angioedema  which  may  be  life- threatening.   At 
the  present  time  there  is  no  safe  method  of  treating  such  patients.   The  use 
of  fresh,  frozen  plasma  has  been  advocated,  since  fresh  plasma  has  the  protein 
which  the  patients  are  missing;  however,  fresh  plasma  also  has  complement 
components  in  it  and  the  patients  are  depleted  of  these  components  at  the 
time  of  an  attack.   It  has  been  suggested  that  the  infusion  of  complement 
components  will  make  these  patients  much  more  ill  before  they  improve.   Thus, 
fresh  plasma  is  not  advocated  for  use  in  this  disease. 


10-37 


Project  No.  Z01  00050-09  LCI 

Twenty-seven  new  patients  were  seen  with  Hereditary  Angioedema  in  1978 
and  8  additional  new  patients  were  seen  in  1979.   We  now  have  63  patients  on 
danazol  therapy,  7  on  oxymethylone,  4  on  EACA,  9  on  methyltestosterone.   We 
have  developed  considerable  further  experience  with  danazol  toxicity  and  are 
now  the  leading  group  in  developing  methods  to  evaluate  danazol  toxicity.   We 
are  also  now  the  largest  group  following  patients  with  HAE  on  therapy  and  have 
begun  to  pull  together  our  studies  of  the  incidence  of  autoimmune  disease  in 
this  interesting  disease.   We  now  have  several  patients  with  autoimmune  thy- 
roiditis, glomerulonephritis,  regional  enteritis,  ulcerative  colitis,  pan- 
creatitis, etc.   We  are  initiating  studies  to  assess  how  the  complement  de- 
ficiency state  predisposes  to  autoimmune  disease. 

Our  infusion  studies  are  also  progressing  in  a  highly  satisfactory  way, 
however,  we  are  still  having  difficulty  obtaining  large  quantities  of  the  CI 
esterase  inhibitor  from  the  Red  Cross.  We  have  assisted  the  Red  Cross  in  prep- 
aration and  in  evaluation  of  CI  esterase  inhibition.   Moreover,  we  have  now 
completed  a  series  of  infusions  of  purified  CI  esterase  inhibitor  into  patients 
with  HAE  at  times  when  they  were  not  experiencing  attacks  and  also  have  infused 
the  protein  into  patients  in  the  midst  of  attacks.   These  studies  have  shown 
that  CI  esterase  inhibitor  is  quite  benign  and  does  not  carry  the  risk  of  hep- 
atitis.  When  infused  into  patients  free  of  clinical  symptoms  these  patients 
experience  an  elevation  of  CI  esterase  inhibitor  and  a  subsequent  elevation 
of  C4  levels  as  might  be  expected  from  pathophysiologic  mechanism  of  this 
disease  process.   When  infused  into  patients  experiencing  attacks  of  HAE, 
attacks,  have  been  aborted  in  as  short  a  period  as  5  minutes  and  as  long 
a  period  as  several  hours.   It  would  appear  that  this  will  afford  an 
excellent  approach  in  emergency  rooms  for  treating  patients  with  life- 
threatening  attacks  of  Hereditary  Angioedema. 

Significance  to  Medical  Research 

Our  laboratory  has  been  the  focus  for  most  of  the  recent  therapeutic  de- 
velopments in  current  treatment  of  this  disease.   Patients  with  hereditary 
angioedema  experience  a  25%  mortality  and  have  extensive  morbidity  from  their 
constant  attacks  of  the  disease.   As  a  result  of  our  therapeutic  interventions 
these  patients  lead  a  normal  life.   Moreover  our  development  of  drugs  and 
our  study  of  their  mechanism  of  action  has  led  to  a  far  greater  understanding 
of  the  genetic  basis  of  the  protein  abnormality  underlying  the  disease.   At 
present,  this  disease  represents  one  of  the  few  genetically  controlled  dis- 
orders in  which  there  has  been  biochemical  correction  and  thereby  cure  of  a 
severe  illness. 

Proposed  Course: 

We  are  continuing  to  evaluate  the  long  term  use  of  drugs  in  patients  with 
this  particular  disease.   We  are  also  gradually  increasing  the  number  of  other 
patients  with  other  complement-related  abnormalities  which  are  under  study. 
These  include  such  diverse  illnesses  as  autoimmune  hemolytic  anemia  and 
systemic  lupus  erythematosus. 


20-38 


Project   No.    Z01   AI   00050-09   LCI 


Publications: 


1.  Gelfand,  J. A.,  Rosa,  G.R.,  Conley,  C.L.,  Allen,  J.C.,  Reinhart ,  R. , 
Humphrey,  R.L.  and  Frank,  M.M. :  Acquired  CI  Esterase  Inhibitor  Deficiency 
and  Angioedema.   Medicine  58:  321-28,  1979. 

2.  Levinson,  A.I.,  Summers,  R.J.,  Lawley,  T.J.,  Evans,  R. ,  III,  and 
Frank,  M.M. :  Evaluation  of  the  Adverse  Effects  of  Long-Term  Hypo- 
sensitization. J_.  Allergy  Clin.  Immunol .  In  press. 

3.  Atkinson,  J. P.  and  Frank,  M.M. :  The  Complement  System.   In  Parker,  C. 
(Ed.):  Clinical  Immunol.  Philadelphia,  PA,  W.B.  Saunders  Co.  In  press. 

4.  Parillo,  J.E.,  Lawley,  T.J.,  Frank,  M.M. ,  Kaplan,  A. P.  and  Fauci,  A.S.: 
Immunologic  Reactivity  in  the  Hypereosinophilic  Syndrome.  J_.  Allergy 
Clin.  Immunol.   In  press. 

5.  Frank,  M.M. :   The  Effect  of  Sex  Hormones  on  a  Complement  Related 
Clinical  Disorder,  Hereditary  Angioedema.  Proceedings  of  the  Kroc 
Foundation  Meeting  on  Sex  Factors,  Steroid  Hormones  and  the  Host 
Response.  Amacher,  P.  and  Talal,  N.  (Eds.).   In  press. 

6.  Gadek,  J.E.,  Hosea,  S.W.,  Gelfand,  J. A.  and  Frank,  M.M. :  Response 
of  Variant  Hereditary  Angioedema  Phenotypes  to  Danazol  Therapy: 
Genetic  Implications.  J.  Clin.  Invest.  64:  280-286,  July,  1979. 

7.  Frank,  M.M.  and  Hosea,  S.W.:  Complement.  In  Cohen,  A.S.  (Ed.): 
The  Science  and  Practice  of  Clinical  Medicine.  New  York:  Grune 
and  Stratton,  Inc.  1979,  p.  393. 

8.  Hosea,  S.W.  and  Frank,  M.M. :  Differential  Diagnosis  of  Hypocomple- 
mentemia.   In  Cohen,  A.S.  (Ed.):  The  Science  and  Practice  of 
Clinical  Medicine.  New  York:  Grune  and  Stratton,  Inc.  1979,  p.  432. 

9.  Lawley,  T.J.  and  Frank,  M.M. :  Immune  Complexes  and  Immune  Complex 
Mediated  Diseases.   In  Parker,  C.  (Ed.):  Clinical  Immunology. 

In  press. 

10.  Frank,  M.M. ,  Gelfand,  J. A.,  Sherins,  R.J.,  Ailing,  D.W.,  and 
Gadek,  J.:   The  treatment  of  hereditary  angioedema  with  danazol. 
In  Clinical  Aspects  of  the  Complement  System:  International 
Symposium.   Opferkuch,  W. ,  Rother,  K.  and  Schultz,  D.R.  (Eds.) 
Georg  Thieme  Publ. ,  1978,  pp.  134-138. 


20-39 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00051-07   LCI 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less 

Host  Defense  Mechanism  Against  Pseudomonas  Infection  in  Normal  and 
Immunosuppressed  Hosts 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


OTHER: 


R.P.  Aduan 


E .  Harvey 


Medical  Officer 


Bio.  Lab  Tech.  (Micro.) 


LCI  NIAID 


LCI  NIAID 


COOPERATING  UNITS  (if  any) 

A.S.  Levin,  A.B.  Deisserroth,  and  F.R.  Applebaum  (Pediatric  Oncology  Branch, 
NCI,  NIH) 


LAB/BRANCH 

Laboratory  of  Clinical  Investigation 


SECTION 

Bacterial  Disease  Section 


INSTITUTE  AND  LOCATION 

NIAID,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 

2   3/12 


PROFESSIONAL: 

1   3/12 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


3  (c)    NEITHER 


SUMMARY    OF    a/0RK    (200    words    or    less    -    underline    keywords) 

INACTIVE  DURING  CURRENT  YEAR.   TERMINATED. 


20-40 


PHS-6040 
(Rev.    10-76) 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANG 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  000.54-07  LCI 


PERIOD    COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

The  Etiology  and  Pathogenesis  of  Viral  Gastrointestinal  and  Respiratory 
Tract  Infections  in  Man 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 

OTHER: 


R.  Dolin          Head,  Medical  Virology  Section  LCI  NIAID 

(until  January  1,  1979) 

R.  Berg           Clinical  Associate  LCI  NIAID 

R.  Schooley       Clinical  Associate  LCI  NIAID 


COOPERATING  UNITS  (if  any) 

A.Z.  Kapikian  (LID/NIAID/NIH) ;  R.G.Wyatt  (LID/NIAID/NIH) ;  B.R.  Murphy  (LID/ 
NIAID/NIH) . 


lab/branch 

Laboratory  of  Clinical  Investigation 


SECTION 

Medical  Virology  Section 


INSTITUTE  AND  LOCATION 

NIAID/NIH  Bethesda,  MD 


TOTAL  MANYEARS 

6/12 


PROFESSIONAL: 

6/12 


CHECK  APPROPRIATE  BOX(ES) 
£  (a)   HUMAN  SUBJECTS 

D  (aQ  MINORS   []  (a2)  INTERVIEWS 


3  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

TERMINATE  THIS  PROJECT 

Studies  of  the  etiology  and  pathogenesis  of  viral  gastrointestinal  tract 
infections  in  man  continued  until  the  departure  of  the  Principal  Investigator. 


20-41 


PHS-6040 
(Rev.  IO-76) 


Project  No.  Z01  AI  00054-07  LCI 
Project  Description: 
Objectives : 

1)  To  investigate  the  pathogenesis  of  and  host  response  to  viral 
infections  of  the  respiratory  and  gastrointestinal  tracts  in  man.   Experi- 
mentally induced  disease  in  normal  volunteers,  as  well  as  naturally  occurring 
cases  are  studied. 

2)  To  define  the  biologic,  immunologic,  and  epidemiologic  properties 
of  the  etiologic  agents  of  viral  gastroenteritis  in  man. 

Methods  Employed: 

Viral  agents  were  obtained  from  naturally  occurring  outbreaks  of 
gastroenteritis  and  respiratory  infection.   The  pathogenesis  and  host 
responses  to  these  agents  were  evaluated  in  in  vitro  systems  and  in 
volunteer  studies. 

Major  Findings: 

Characterization  of  the  small  27-32nm  gastroenteritis  agents  con- 
tinued in  volunteer  studies.   Investigations  of  the  cell-mediated  immune 
responses  to  influenza  vaccines  were  completed. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

Viral  respiratory  and  gastrointestinal  tract  infections  are  the  most 
common  disease   experiences  in  American  families  and  affect  all  age  groups 
of  a  broad  segment  of  population.   As  yet,  there  are  no  adequate  control 
measures  available.   Identification  and  characterization  of  etiologic 
agents,  as  well  as  studies  of  the  pathogenesis  of  these  diseases  in  man, 
should  form  the  basis  for  rational  development  of  methods  of  prevention 
and  treatment. 

Proposed  Course: 

This  study  has  been  terminated  upon  the  departure  of  the  Principal 
Investigator. 


20-42 


Project  No.  Z01  AI  00054-07  LCI 
Publications : 

1.  Dolin,  R. :   Antiviral  compounds  in  viral  infection  of  the  gastrointesti- 
nal tract.   In  Buchanan,  R. ,  Galasso,  G.  and  Merigan,  T.  (Eds.): 
Antivirals  in  Man.   In  press. 

2.  Dolin,  R.  :   Viral  gastroenteritis.   In  Beeson,  P.B.,  McDermott,  N. 
and  Wyngaarden,  J.B.  (Eds.)  Textbook  of  Medicine  15th  Edition, 
Philadelphia,  Pa.,  W.B.  Saunders.   In  press. 

3.  Dolin,  R. :   Norwalk-like  agents  of  viral  gastroenteritis.   In  Mandell, 
G. ,  Douglas,  R.G.,  Bennett,  J.E.  (Eds.)  Principles  and  Practice  of 
Infectious  Diseases,  New  York,  Wiley  and  Sons.  In  Press. 

4.  Reichman,  R.C.,  Pons,  V.G.,  Murphy,  B.R. ,  Caplan,  E.A.  and  Dolin,  R. : 
Cell  mediated  cytotoxicity  following  influenza  infection  and  vaccination 
in  humans.   J.  Med.  Virol.   In  press. 

5.  Pons,  V.G.,  Reinertsen,  J.R.,  Steinberg,  A.D.  and  Dolin,  R. :   Decreased 
cell  mediated  cytotoxicity  against  virus-infected  cells  in  systemic 
lupus  erythematosus.   J.  Med.  Virol.   In  press. 


20-43 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00055-07  LCI 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Regulation  of  the  Immune  Response  in  Man  and  Experimental  Animals 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Head,  Clinical  Physiology  Section  LCI  NIAID 
Deputy  Clinical  Director  NIAID 

Biologist  LCI  NIAID 

Clinical  Associate  LCI  NIAID 

Guest  Worker  LCI  NIAID 

Senior  Investigator  LCI  NIAID 

Clinical  Associate  LCI  NIAID 

Clinical  Associate  LCI  NIAID 

Clinical  Associate  LCI  NIAID 

Biologist  LCI  NIAID 


PI: 

A. 

S.  Fauci 

Other: 

C. 

Burch 

T. 

Cupps 

A. 

Dimitriu 

B. 

F.  Havnes 

P. 

Katz 

H. 

C.  Stevenson 

D. 

Volkman 

G. 

Whalen 

COOPERATING  UNITS  (if  any) 

None 


LAB/BRANCH 

Laboratory  of  Clinical  Investigation 


SECTION 

Clinical  Physiology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland   20205 


TOTAL  MANYEARS: 


6  5/i: 


PROFESSIONAL: 


4  5/12 


CHECK  APPROPRIATE  BOX(ES) 
3  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   H  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


•  underline  keywords) 

and  immunoregulat 


gulatory  events  associated  with  triggering 


SUMMARY  OF  WORK  (200  ^ords  or  less  • 

The  precise  mechanisms 
of  human  B  lymphocytes  following  non-specific  and  antigen  induced  stimulation  havd 
been  delineated.   Functionally  distinct  as  well  as  overlapping  immunoregulatory 
.-ymphocyte  subsets  have  been  characterized.  The  precise  abnormalities  of  immuno- 


logic reactivity  have  been  characterized  in  several  immunologically  mediated 
diseases  such  as  systemic  lupus  erythematosus,  Sjogren's  syndrome,  acute  infec- 
tious mononucleosis,  sarcoidosis  and  tuberculosis.   For  the  first  time,  an  anti- 
:-diotypic  antibody  has  been  produced  which  specifically  blocks  the  function  of 
iiuman  B  cells  bearing  the  corresponding  idiotype.   Hybridoma  lymphocyte  cell  lines 
have  been  established  which  are  secreting  antibody  which  binds  specifically  to 
functionally  distinct  subsets  of  human  blood  lymphocytes.   Counter-current 
centrifugation  techniques  have  been  adapted  which  result  in  95%  and  greater  purity 
and  yield  of  monocytes  from  human  blood.   These  cells  have  been  characterized 
functionally  and  structurally.   Modulation  of  lymphocyte  subsets  by  a  number  of 
clinically  relevant  pharmacologic  agents  have  been  defined.   Clinical  therapeutic 
studies  have  yielded  extraordinarily  favorable  results  in  the  cytotoxic  drug 
Approach  to  the  systemic  necrotizing  vasculitides  ■ 


PHS-6040 

10-76) 


20-44 


Project  No.  Z01  AI  00055-07  LCI 
Project  Description 
Obj  ectives : 

1)  To  delineate  the  mechanisms  of  regulation  of  immunologic  reactivity 
by  purified  subpopulations  of  human  lymphoid  cells.   To  examine  the  precise 
functional  capabilities  of  these  individual  lymphoid  cell  subpopulations  as 
well  as  the  mechanisms  whereby  they  maintain  the  normal  homeostatis  of 
positive  and  negative  signals  which  regulate  normal  immunologic  reactivity. 

2)  To  determine  the  role  of  these  lymphoid  cell  subpopulations  in  the 
pathogenesis  of  immunologically  mediated  diseases  such  as  the  vasculitides , 
hypersensitivity  diseases,  granulomatous  diseases,  the  spectrum  of  auto- 
immune diseases,  as  well  as  infectious  diseases  characterized  by  aberrations 
of  immunologic  reactivity.   In  particular,  to  delineate  the  alterations  of 
various  subpopulations  of  cells  by  mechanisms  such  as  a)  modulation  of  cell 
surface  receptors  (for  example,  Fc  receptors)  by  immune  complexes,  cryo- 
globulins or  other  serum  factors;  b)  activation  of  cell  subsets  by  viruses 
(such  as  Epstein-Barr)  virus);  and  c)  depletion,  proliferation,  or  redistri- 
bution of  cell  subsets  among  lymphoid  compartments.   In  this  regard,  to 
determine  the  role  of  these  mechanisms  in  the  abnormalities  of  immunoregulation 
characteristic  of  these  diseases. 

3)  To  develop  hybridoma  cell  lines  secreting  monoclonal  antibodies 
directed  against  stable  lymphoid  cell  surface  antigens  which  define  func- 
tionally distinct  subpopulations  of  immunoregulatory  cells. 

4)  To  determine  the  role  and  mechanisms  of  anti- idiotypic  antibodies 
in  the  regulation  of  human  B  cell  responses  in  normal  immune  reactivity  and 
in  diseases  characterized  by  the  production  of  abnormal  serum  components 

(M  components) . 

5)  To  investigate  the  cellular  interactions,  molecular,  and  biochemical 
events  associated-  with  triggering  of  various  mononuclear  cell  subpopulations 
and  to  determine  the  relationship  between  selective  triggering  via  various 
cell  surface  receptors  and  the  kinetics  of  expression  of  various  functional 
capabilities  of  the  given  cell  populations. 

6)  To  continue  to  develop  and  further  perfect  culture  and  assay  systems 
for  primary  in  vitro  activation  of  human  B  lymphocytes  to  antibody  production 
following  polyclonal  activation  or  specific  antigenic  stimulation. 

7)  To  develop  an  in  vitro  model  of  autoreactivity  by  selective 
triggering  or  depletion  of  immunoregulatory  cell  subpopulations  in  order  to 
define  more  precisely  the  primary  or  secondary  role  of  altered  immunoregula- 
tion in  autoimmune  diseases. 


20-45 


Project  No.  Z01  AI  00055-07  LCI 

8)  To  delineate  the  mechanisms  whereby  recognition  of  self  molecules 
(such  as  la-like  determinants)  plays  a  role  in  non-self  antigenic  recog- 
nition.  In  this  regard,  to  determine  the  role  of  autoreactivity  in  the 
normal  immunologic  homeostasis  in  man  and  to  characterize  the  aberrancies  of 
this  otherwise  normal  level  of  autoreactivity  which  are  associated  with 
pathologic  autoimmunity  and  altered  immune  surveillance  in  certain  neo- 
plastic diseases,  particularly  those  of  the  lymphoprolif erative  type. 

9)  To  develop  methodologies  of  fractionating  purified  populations  of 
human  peripheral  blood  monocytes  and  T  cell  subsets  by  elutriation  centri- 
fugation  techniques  in  order  to  avoid  pre-activation  of  the  respective 
populations  which  invariably  occurs  with  positive  selection  methodologies. 

10)  To  induce,  purify  and  characterize  soluble  factors  from  purified 
subpopulations  of  human  lymphocytes  and  monocytes  which  are  involved  in  the 
regulation  of  immunologic  reactivity. 

11)  To  delineate  the  precise  mechanisms  whereby  immunosuppressive  agents 
such  as  corticosteroids  and  cytotoxic  drugs  affect  the  immune  responses  in 
man  specifically  examining  their  selective  effects  on  subpopulations  of 
lymphocytes  and  monocytes  with  regard  to  their  circulatory  kinetics,  cellular 
interactions,  activation,  and  expression  of  in  vivo  and  in  vitro  functional 
capabilities.   In  addition,  to  correlate  the  selective  effects  of  corticoster- 
oids and  cytotoxic  drugs  on  these  various  parameters  with  the  suppression  of 
disease  activity  in  various  inflammatory  and  immunogically-mediated  disease 
states  in  man,  and  by  doing  to  aim  at  a  greater  understanding  and  therapeu- 
tic specificity  in  the  clinical  use  of  these  agents.   Furthermore,  to  study 
the  mechanisms  whereby  manipulations  of  immune  reactivity  as  described  above 
affect  host  defenses  against  infection,  tumor  surveillance  and  propensity 
towards  autoimmune  states. 

12)  To  study  the  functional  capabilities  of  eosinophils  and  mononuclear 
cells  in  the  idiopathic  hypereosinophilic  syndrome  in  order  to  elucidate  the 
mechanisms  whereby  these  cells  cause  invasion  of  tissue  and  subsequent  tissue 
damage  in  this  disease.   To  use  this  information  to  gain  a  greater  under- 
standing of  the  functional  properties  of  eosinophils  in  normal  states,  various 
diseases  and  to  determine  their  sensitivity  to  various  therapeutic  regimens. 

13)  To  extend  the  concept  of  immunoregulation  to  the  effect  of  the  immune 
system  on  other  cell  types.   Specifically,  to  study  the  mechanisms  of  immuno- 
regulation by  lymphoid  cell  subpopulations  on  eosinophil  and  neutrophil 
kinetics  and  function  in  the  hypereosinophilic  syndrome  and  certain  neutro- 
penic states  respectively. 

14)  To  continue  to  study  the  spectrum  of  the  vasculitides  in  man  from  a 
mechanistic,  pathophysiologic,  clinical  and  therapeutic  standpoint. 

Methods  Employed: 

1)   The  predominant  theme  of  this  laboratory  is  the  delineation  of  the 

20-46 


Project  No.  Z01  AI  00055-07  LCI 

mechanisms  of  immunoregulation  in  normal  human  immune  reactivity  and  in  diseases 
characterized  by  abnormalities  of  immunoregulation. 

In  this  regard,  a  major  component  of  the  methodology  relates  to  the 
fractionation,  identification  and  purification  of  immunoregulatory  lymphoid 
cell  subpopulations  and  the  _in  vitro  culturing  of  these  cells  to  determine 
their  diverse  functional  capabilities.   Subpopulations  of  cells  are  identified 
predominantly  by  surface  markers  and  functional  capabilities  are  assessed  by  a 
variety  of  in  vitro  assays.   We  have  focused  on  the  relationship  between  various 
in  vivo  and  in  vitro  triggering  signals  and  the  subsequent  _in  vivo  expression  of 
direct  and  immunoregulatory  functional  capabilities.   This  has  been  focused 
predominantly  in  the  area  of  activation,  proliferation  and  differentiation  of 
human  B  lymphocytes  to  antibody  producing  and  secreting  cells.   In  this  regard, 
we  have  been  utilizing  a  unique  model  of  human  B  cell  function  with  was 
originally  developed  in  this  laboratory.   It  is  a  system  of  primary  in  vitro 
stimulation  of  bone  marrow  derived  (B)  lymphocytes  by  polyclonal  activation 
as  well  as  specific  antigenic  stimulation  with  subsequent  measurement  of 
single  cell  antibody  production  by  a  direct  hemolysis-in-gel  plaque  forming 
cell  (PFC)  assay.   We  have  further  extended  this  system  to  now  measure  indirect 
(IgG)  PFC.   In  addition,  we  also  have  developed  a  system  for  the  measurement  of 
Ig  secreting  cells  of  all  classes  by  use  of  staphylococcal  protein  A  (SPA) 
coated  target  erythrocytes  together  with  class  specific  developing  antisera. 

Using  these  models,  we  have  developed  a  system  for  the  selective  generation 
of  suppressor  cells  or  helper  cells  capable  of  modulating  In  vitro  antibody 
production.   In  addition,  we  have  produced  soluble  mediators  of  B  cell  function 
in  man  and  have  characterized  the  cell  types  of  their  origin. 

Other  assays  employed  to  delineate  the  functional  capabilities  of 
lymphoid  cells  are  the  _in  vitro  blastogenic  responses  to  mitogenic  and 
antigenic  stimulation,  elaboration  of  various  soluble  mediators,  cell 
mediated  cytotoxicity  against  various  autologous,  allogeneic  and  xenogeneic 
targets.   Various  cytotoxicity  assays  include  spontaneous,  mitogen  dependent, 
and  antibody  dependent  cellular  cytotoxicity.   A  particular  aim  is  to  identify 
and  characterize  the  abnormalities  of  lymphocyte  or  monocyte  subpopulations 
involved  in  immunologically  mediated  diseases  to  determine  the  primary  or 
secondary  nature  of  these  abnormalities  and  their  relationship  to  the  altered 
state  of  immune  reactivity,  as  well  as  to  create  _in_  vitro  models  of  altered 
immunoregulation  by  selectively  triggering  or  manipulating  immunoregulatory 
subpopulations  of  cells. 

2)   Highly  sensitive  assays  (enzyme  linked  immunosorbent  -  ELISA)  have 
been  developed  and  are  being  employed  to  measure  the  in   vitro  production  of 
specific  antibody  following  in  vivo  immunization  of  human  subjects  as  well 
as  primary  in  vitro  antigenic  stimulation. 


20-47 


Project  No.  Z01  AI  00055-07  LCI 

3)  Antisera  against  purified  subpopulations  of  human  lymphoid  cells 
have  been  developed  in  order  to  identify,  isolate,  characterize,  inhibit 
functional  capabilities  or  deplete  these  functionally  distinct  subsets  from 
various  cell  suspensions. 

4)  In  relationship  to  #3,  hybridoma  cell  lines  have  been  developed 
which  are  producing  large  quantities  of  monoclonal  antibodies  directed 
against  functionally  distinct  lymphocyte  subsets. 

5)  Recently  developed  elutriation  centrif ugation  techniques  are  being 
employed  to  isolate  purified  (greater  than  95%)  populations  of  human  monocytes 
from  peripheral  blood,  thus  avoiding  adherence  techniques. 

6)  Methods  to  fractionate  purified  populations  of  eosinophils  (90- 
98%  pure)  from  human  blood  have  been  developed  and  are  being  employed. 

7)  Corticosteroids  are  administered  to  normal  volunteers  and  patients 
requiring  this  drug  and  selective  effects  of  these  agents  on  the  compart- 
mentalization  and  functional  capabilities  of  monocytes  and  lymphocyte 
subpopulations  are  determined.   In  addition,  the  differential  effects  of 

in  vitro  corticosteroids  and  _in  vitro  irradiation  on  various  lymphoid  cell 
functional  capabilities  such  as  triggering,  proliferation,  differentiation, 
antibody  production  and  secretion  are  being  studied. 

8)  A  subject's  lymphoid  cells  are  labeled  with   chromium  and  reinfused 
back  into  their  circulation  to  determine  the  effects  of  disease  states  and 

of  chemotherapeutic  agents  on  the  viability  and  circulation  patterns  of 
these  cells. 

9)  Bone  marrow  aspirates  are  performed  in  normal  subjects  and  patients 
with  various  diseases  prior  to,  during  and  following  therapy.   Purified 
populations  of  lymphoid  cells  are  obtained  from  these  bone  marrow  cell 
suspensions  by  various  separation  techniques  in  order  to  delineate  the 
immunocompetence  of  bone  marrow  lymphoid  cells  in  normal  man,  as  well  as 
the  alteration  of  compartmentalization  and  function  of  these  cells  in 
untreated  and  treated  disease  states. 

Major  Findings  and  Significance  to  Biomedical  Research  and  the  Program 
of  the  Institute: 

A  number  of  significant  findings  in  human  immunobiologic  research  have 
emerged  from  this  laboratory  over  the  past  year.   These  have  been  in  several 
general  areas  including  mechanisms  of  triggering  of  human  B  lymphocytes; 
immunoregulation  of  B  and  T  cell  function;  identification  and  purification 
of  functionally  distinct  immunoregulatory  and  effector  lymphocyte  and  monocyte 
subpopulations  in  normal  individuals  and  a  characterization  of  the  aberrations 
of  such  subsets  in  certain  disease  states;  and  finally  studies  involving  the 


20-48 


Project  No.  Z01  AI  00055-07  LCI 

pathogenesis  and  therapeutic  approach  to  diseases  characterized  by  abnormalities 
of  immunologic  reactivity.   Specific  findings  of  particular  relevance  include: 

1)  Major  advances  have  been  made  in  the  delineation  of  the  mechanisms  of 
activation  of  human  B  lymphocytes.   Mitogen  induced  and  antigen  specific  in 
vitro  assays  of  B  cell  function  have  been  originally  developed  and  are  now 
being  extended  in  this  laboratory.   These  include  the  first  polyclonally  in- 
duced and  antigen  induced  direct  and  indirect  hemolytic  plaque  forming  cell 
(PFC)  assay  for  human  B  cells.   In  addition,  a  highly  sensitive  enzyme  linked 
immunosorbent  (ELISA)  assay  has  been  developed  to  detect  minute  quantities 

of  supernatant  Ig  and  specific  antibody  from  ^n  vitro  cultures  of  human  lympho- 
cytes.  These  innovative  assay  systems  are  being  used  to  delineate  the  complex 
mechanisms  of  immunoregulation  of  human  B  cell  function  in  normal  individuals 
and  in  disease  states,  as  will  be  described  below.   Representatives  from 
numerous  laboratories  throughout  the  world  have  visited  our  laboratory  this 
year  to  learn  these  unique  methodologies,  and  have  been  successful  in  instituting 
these  systems  in  their  own  respective  laboratories.   Of  particular  note  in  this 
regard  is  the  fact  that  in  1978  an  international  workshop  on  human  B  cell 
function  was  organized  and  directed  by  the  Head,  Clinical  Physiology  Section, 
LCI,  NIAID.   The  purpose  was  to  bring  together  the  world's  leading  investigators 
in  this  area  and  compile  a  state  of  the  art  publication  with  guidelines  for 
furture  research.   This  led  to  the  extremely  well  received  book  "Antibody 
Production  in  Man.   In  Vitro  Synthesis  and  Clinical  Implications"  Edited  by 
A.  S.  Fauci  and  R.  E.  Ballieux,  Academic  Press,  New  York,  1979. 

2)  Precise  delineation  of  immunoregulatory  lymphocyte  subpopulations  in 
normal  individuals  has  been  accomplished.   We  have  previously  reported  the 
original  description  of  a  delicate  balance  between  helper  and  suppressor  cell 
subpopulations  in  the  modulation  of  normal  B  cell  immunologic  reactivity.   In 
addition,  we  have  demonstrated  that  certain  otherwise  normal  individuals  are 
hypo-responders  in  these  _in  vitro  B  cell  systems,  and  that  this  hyporesponsive- 
ness  resulted  directly  from  overly  active  or  triggered  suppressor  cells.   We 
have  now  extended  these  studies  to  precisely  characterize  the  identity  and 
nature  of  this  naturally  occuring  suppressor  T  cell. 

In  addition,  we  have  characterized  the  precise  identity,  kinetics  and 
mechanisms  of  action  of  the  mitogen  (Con-A)  induced  suppressor  cell  in 
normal  humans . 

3)  Applying  the  abovementioned  systems  of  immunoregulation  we  have  charact- 
erized the  primary  and  secondary  immunoregulatory  abnormalities  in  a  number  of 
immunologically  mediated  diseases. 

We  have  demonstrated  the  presence  of  altered  proportions  and  absolute 
numbers  of  immunoregulatory  T  cell  subpopulations  in  patients  with  systemic 
lupus  erythematosus  (SLE) .   In  addition,  we  have  demonstrated  a  deficiency  of 
suppressor  cell  function  in  SLE  patients.   In  this  regard,  an  in  vitro  model  of 
pre-triggering  suppressor  cells  by  activation  with  immune  complexes  has  been 
developed  to  support  the  hypothesis  of  immune  complex  associated  alterations 


20-49 


Project  No.  Z01  AI  00055-07  LCI 

of  immunoregulation  in  SLE  and  related  syndromes.   We  have  defined  the  spectrum 
of  relative  alterations  of  immunologic  reactivity  by  demonstrating  the  presence 
of  mild  hyperreactivity  of  B  cells  associated  with  reversible  alterations  of 
immunoregulatory  T  cell  subsets  in  Sjogren's  syndrome  (SS)  without  other 
associated  connective  tissue  disorders;  modest  to  severe  B  cell  hyperreactivity 
with  irreversible  alterations  of  immunoregulatory  T  cell  subsets  in  SS 
associated  with  mild  SLE;  and  severe  B  cell  hyperreactivity  together  with 
alterations  and  depletions  of  immunoreguatory  T  cell  subsets  in  frank  SLE. 

Of  major  inportance  is  the  fact  that  we  have  developed  an  anti-idiotypic 
antibody  against  the  surface  IgM  and  serum  monoclonal  IgM  peak  of  a  patient 
with  chronic  lymphocytic  leukemia  whose  leukemic  B  cell  clone  is  secreting 
monoclonal  antibody  against  SRBC  determinants.   The  anti-idiotypic  antibody 
reacts  with  the  patient's  B  cell  surface  IgM  and  serum  IgM  but  not  with  normal 
B  cell  surface  IgM  or  serum  IgM.   Furthermore,  the  anti-idiotypic  antibody 
blocks  the  spontaneous  and  mitogen  induced  secretion  of  anti-SRBC  antibody  by 
the  patient's  B  cells  but  not  by  normal  B  cells.   This  is  the  first  demonstration 
in  man  of  the  inhibition  of  B  cell  function  by  anti-idiotypic  antibody  and 
has  obvious  important  implications  in  the  understanding  of  the  role  of  the 
idiotype  network  in  the  regulation  of  human  immunologic  reactivity. 

We  have  demonstrated  the  presence  of  spontaneously  activated  B  cells 
in  Epstein-Barr  virus  (EBV)-induced  acute  infectious  mononucleosis  (IM) .   We 
have  further  shown  that  this  is  due  to  the  direct  triggering  of  B  cells  by  EBV  via 
surface  receptors.   We  have  characterized  the  atypical  lymphocyte  in  IM  and 
have  demonstrated  for  the  first  time  the  emergence  of  suppressor  T  cells  in 
acute  IM  and  their  subsequent  disappearance  in  the  convalescent  state.   This 
has  important  implications  in  the  suppression  of  B  cell  outgrowth  in  IM  which 
may  be  an  important  mechanism  of  containment  of  disease  activity  and  prevention 
of  evolution  into  a  B  cell  neoplasm. 

In  addition,  we  have  demonstrated  the  alteration  of  immunoregulatory  T 
cell  subsets  as  well  as  the  presence  of  adherent  suppressor  monocytes  in 
sarcoidosis  and  tuberculosis. 

4)   Because  of  the  potential  relationship  between  certain  of  the  abovementioned 
autoimmune  diseases  and  aberrancies  of  normally  occurring  autoreactivity ,  we 
investigated  the  mechanisms  of  regulation  of  autologous  and  allogeneic  reactivity 
in  the  models  of  the  autologous  and  allogeneic  mixed  lymphocyte  reactions  (MLR) . 
In  addition,  we  have  defined  the  relative  stimulatory  and  responder  capabilities 
of  various  lymphocyte  and  monocyte  subpopulations  in  the  MLR.   The  relationship 
between  the  autologous  and  allogeneic  MLR  and  the  development  of  suppressor  and/or 
cytotoxic  T  cells  was  described.   In  this  regard,  the  presence  of  a  potent 
adherent  suppressor  cell  of  the  autologous  MLR  was  demonstrated  in  sarcoidosis. 
Furthermore,  the  modulation  of  the  autologous  and  allogeneic  MLR  in  normal  sub- 
jects by  adherent  cells,  prostaglandins,  irradiation  and  in  vitro  and  in  vivo 
corticosteroids  has  been  delineated. 


^0-50 


Project  No.  Z01  AI  00055-07  LCI 

5)  Because  of  the  potential  role  of  cytotoxic  cells  mediating  either  antibody 
dependent  cellular  cytotoxicity  (ADCC)  or  natural  killer  (NK)  activity  in 
immunologically  mediated  disease,  we  have  precisely  delineated  the  ADCC  and  NK 
capabilities  of  multiple  heterogeneous  subpopulations  of  T  cells,  null  cells 
and  monocytes.   In  these  studies,  we  have  demonstrated  the  overlapping  and 
distinct  cytotoxic  capabilities  of  these  heterogeneous  lympohoid  cell  subsets. 

6)  We  have  adapted  a  unique  cell  fractionation  technique  called  elutriation 
(counter-current  centrifugation)  for  the  purpose  of  purifying  human  blood 
monocytes.   This  technique  provides  a  major  advance  in  our  ability  to  carefully 
and  precisely  study  the  human  monocyte. 

This  technique  has  the  advantage  over  the  current  adherence  techniques  of 
monocyte  isolation  of  negatively  selecting  the  human  monocyte  in  excellent 
purity  (95%)  and  excellent  yield  (95%).   The  yield  of  this  procedure  is  so 
great,  that  up  to  1.5  billion  monocytes  have  been  purified  from  the  blood  of 
a  single  normal  human  at  one  time.   In  addition,  this  technique  has  proved  to 
provide  much  more  effective  monocyte  depletion  of  mononuclear  cell  suspensions 
than  the  current  technique  of  depleting  adherent  cells  on  sephadex  G-10  columns 
since  elutriation  does  not  remove  non-monocyte  adherent  cells  (such  as  B  cells) . 
Thus,  we  have  been  able  to  characterize  large  numbers  of  purified  human  monocytes 
with  regard  to  their  intracytoplasmic  enzyme  activity.   We  have  also  examined 
monocytes  with  scanning  and  transmission  electron  microscopy  for  the  details 
of  their  cytoskeleton  assembly.   In  addition,  we  have  placed  large  numbers 
of  these  cells  into  culture  to  examine  how  these  parameters  are  altered 
when  these  cells  mature  into  macrophages.   We  have  been  able  to  examine  the 
evolution  of  these  functions  on  a  daily  basis  for  one  week  using  a  single 
normal  individual's  monocytes.   Human  monocytes  have  been  examined  for  their 
ability  to  perform  the  interrelated  functions  of  phagocytosis  and  killing  in 
an  ADCC  system.   We  have  shown  that  while  monocytes  have  improved  phagocytic 
function  after  being  placed  in  culture,  they  have  diminished  killing  capability 
in  ADCC.   Of  interest  is  that  we  are  the  first  group  to  demonstrate  spontaneous 
killing  of  monocytes  against  human  red  cells  and  that  this  function  improves 
when  monocytes  mature  into  macrophages.   Hence,  for  the  first  time,  human 
monocytes  in  a  highly  purified,  non-activated  state  have  been  precisely 
characterized. 

7)  We  have  characterized  functionally  distinct  subpopulations  of  T  cells  on 
the  basis  of  relatively  unstable  surface  receptors  such  as  the  Fc  receptor 
for  IgG  or  IgM  (T  or  T  ).   In  addition,  we  have  developed  and  characterized 
a  rabbit  anti-human  T  cell  antiserum  against  stable  cell  surface  components. 

Most  importantly,  we  are  among  the  first  few  laboratories  in  the  world  to 
have  produced  and  fully  characterized  a  variety  of  mouse  lymphocyte  hybrid 
cell  lines  which  are  producing  virtually  unlimited  quantities  of  antibodies 
which  bind  to  a  number  of  distinct  as  well  as  overlapping  subpopulations  of 
human  peripheral  blood  mononuclear  cells.   In  particular,  antisera  have 
been  produced  which  bind  to  a  functionally  distinct  human  peripheral  blood 
T  cell  subset.   In  addition,  another  antibody  is  being  produced  which  binds 
specifically  to  human  blood  monocytes. 


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Project  No.  Z01  AI  00055-07  LCI 

8)  In  line  with  our  interest  in  the  pharmocologic  modulation  of  the  immuno- 
regulatory  apparatus  in  man,  we  have  studied  and  characterized  the  selective 
and  differential  effects  of  a  number  of  agents  directly  on  effector  lympho- 
cye  subsets  as  well  as  on  functionally  distinct  immunoregulatory  lymphocyte 
subsets.   These  agents  include  corticosteroids,  cyclophosphamide,  azathioprlne, 
irradiation,  cyclic  nucleotides,  and  prostaglandin  inhibitors.   A  graded 
differential  sensitivity  of  B  cells,  more  than  suppressor  T  cells,  more  than 
helper  T  cells  was  demonstrated  to  cyclophosphamide,  azathioprine  and  irrad- 
iation.  Cyclic  AMP  directly  inhibited  suppressor  T  cells,  while  prosta- 
glandin inhibitors  blocked  prostaglandin  mediated  suppression  of  certain 
subsets  of  adherent  suppressor  cells.   Corticosteroids  had  the  most  complex 
effects  with  selective  and  differential  effects  on  the  circulatory  kinetics 
and  functional  capabilities  of  T  and  B  cell  subsets. 

We  have  also  delineated  the  relationship  of  corticosteroid  receptors  on 
lymphocyte  subsets  (with  regard  to  receptor  density,  binding  affinity  and 
dissociation  constant)  and  the  differential  effects  of  _in  vivo  and  in  vitro 
corticosteroids  on  these  subsets. 

9)  We  have  continued  our  clinical  studies  of  a  number  of  diseases  of  estab- 
lished or  suspected  immunologic  mediation.   These  comprise  virtually  the  entire 
spectrum  of  vasculitis  including  Wegener's  granulomatosis,  systemic  necrotizing 
vasculitis  (polyarteritis  nodosa  type),  hypersensitivity  vasculitis,  Takayasu's 
arteritis,  temporal  arteritis,  and  lymphomatoid  garnulomatosis .   In  addition, 
we  are  studing  idiopathic  midline  granuloma,  sarcoidosis,  granulomatous 
hepatitis,  juvenile  rheumatoid  arthritis,  Weber-Christian  panniculitis,  a 
heterogeneous  group  of  acquired  immunodeficiency  diseases,  and  host  defense 
defects  (chronic  granulomatous  disease,  Chedick-Higashi  syndrome) ,  and  a  large 
number  of  patients  with  the  idiopathic  hypereosinophilic  syndrome.   The  above 
patient  groups  are  under  our  primary  care.   In  addition,  we  are  carrying  on 
collaborative  studies  with  others  in  SLE,  Sjogren's  syndrome,  mixed  connective 
tissue  disease,  rheumatoid  arthritis,  chronic  lymphocytic  leukemia,  acute 
infectious  mononucleosis,  tuberculosis,  and  Cogan's  syndrome. 

In  addition  to  carrying  on  extensive  investigations  delineating  the  mech- 
anisms of  altered  immunologic  reactivity  in  these  diseases,  the  results  of  which 
are  described  above,  we  have  established  treatment  protocols  for  several  of  these 
disorders.   Major  contributions  have  resulted  from  these  and  dramatic  long-term 
remissions  and  even  cures  have  been  established  by  us  in  several  of  these 
formerly  fatal  diseases  by  the  use  of  chronic  low  dose  cytotoxic  agents  (usually 
cyclophosphmide) ,  particularly  in  the  severe  systemic  necrotizing  vasulitides. 
These  results  are  either  recently  published  or  in  press.   In  particular,  we 
continue  to  prospectively  follow  the  largest  group  of  patients  with  Wegener's 
granulomatosis  in  the  world  and  have  established  a  greater  than  90%  remission 
rate  with  the  use  of  cyclophosphamide.   In  addition,  we  have  most  recently 
published  similar  striking  results  with  cyclophosphamide  in  systemic  necrotizing 
vasculitis  of  the  polyarteritis  nodosa  group. 


20-52 


Project  No.  Z01  AI  00055-07  LCI 

In  addition,  we  are  prospectively  following  the  largest  group  of  patients 
in  the  world  with  the  idiopathic  hypereosinophilic  syndrome  and  have  effected 
striking  remissions  by  the  use  of  hydroxyurea  which  has  been  shown  to  prevent 
and/or  halt  the  eosinophilic  myocardopathy  which  is  the  major  source  of  mor- 
bidity and  mortality  in  this  disease. 

Given  the  striking  therapeutic  results  mentioned-  above,  our  group  has 
been  the  major  referral  center  for  these  diseases  and  the  therapeutic 
protocols  which  we  have  established  are  now  being  adopted  and  employed 
successfully  world-wide. 

Proposed  Course: 

These  projects  will  continue  along  the  lines  which  have  been  described. 

Publications : 

1.  Fauci,  A.  S.:   Immunosuppressive  and  ant i- inflammatory  effects  of 
glucocorticoids.  In  Baxter,  J.  D.,  Rouseau,  G.  G.  (Eds.):   Mechanisms 
of  Glucocorticoid  Hormone  Action.  Springer -Verlag,  New  York- 
Heidelberg-Berlin.  In  press. 

2.  Fauci,  A.  S.:   Midline  granuloma,  In  Textbook  of  Medicine,  Fifteenth 
Edition.  P.  B.  Beeson,  W.  McDermott,  J.  B.  Wyngaarden,  Editors. 

W.  B.  Saunders  and  Co.,  Philadelphia,  1979,  pp.  220-221. 

3.  Fauci,  A.  S.:   Wegener's  granulomatosis.  In  Textbook  of  Medicine, 
Fifteenth  Edition.  P.  B.  Beeson,  W.  McDermott,  J.  B.  Wyngaarden, 
Editors,  W.  B.  Saunders  and  Co.,  Philadelphia,  1979,  pp.  218-220. 

4.  Fauci,  A.  S.:   Familial  mediterranean  fever.  In  Textbook  of  Medicine. 
Fifteenth  Edition,  P.  B.  Beeson,  W.  McDermott,  J.  B.  Wyngaarden, 
Editors.   W.  B.  Saunders  and  Co.,  Philadelphia,  1979,  pp.  2055-2057. 

5.  Hunninghake,  G.  W. ,  Haynes ,  B.  F. ,  Parrillo,  J.  E.,  and  Fauci,  A.  S.: 
Comparison  of  the  relative  effector  cell  capabilities  and  proportions 
of  cells  bearing  various  surface  markers  in  human  tonsil  and  peripheral 
blood  mononuclear  cells.  Clin.  Exp.  Immunol.  32:186-191,  1978. 

6.  Reddick,  R.  L,  Fauci,  A.  S.,  Valsamis,  N.  P.,  and  Mann,  R.  B.: 
Immunoblastic  sarcoma  of  the  central  nervous  system  in  a  patient  with 
lymphomatoid  granulomatosis.  Cancer.  42:652-659,  1978. 

7.  Blitzer,  B.  L. ,  Weiss,  G.  B.,  Osbaldiston,  G.  W. ,  Markham,  R.  B., 
Aamodt,  R. ,  Berk,  P.  D.,  Wolff,  S.  M. ,  and  Fauci,  A.  S.:   Early  idiopathic 
hemochromatosis  with  absent  stainable  bone  marrow  iron  stores. 
Gastroenterology.  In  press. 


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Project  No.  Z01  AI  00055-07  LCI 

8.  Katz,  P.,  and  Fauci,  A.  S.:   Inhibition  of  polyclonal  B  cell  activation 
by  suppressor  monocytes  in  patients  with  sarcoidosis.  Clin.  Exp.  Immunol. 
32:554-562,  1978. 

9.  Fauci,  A.  S.:   Vasculitis.  In  Clinical  Immunology,  C.  W.  Parker,  Editor, 
W.  B.  Saunders  and  Co.,  Philadelphia.  In  press. 

10.  Dimitriu,  A.,  and  Fauci,  A.  S.:   Activation  of  B  lymphocytes.  IX. 
Modulation  of  antibody  production  by  products  of  activated  macrophages. 
J.  Immunol.  120:1818-1823,  1978. 

11.  Fauci,  A.  S.:   Granulomatous  hepatitis.  In  Principles  and  Practice  of 
Infectious  Disease,  G.  L.  Mandell,  R.  G.  Douglass,  Jr.,  J.  E.  Bennett, 
Editors.  John  Wiley  and  Sons,  New  York.  In  press. 

12.  Parrillo,  J.  E.,  and  Fauci,  A.  S.:   Comparison  of  the  effector  cells  in 
human  spontaneous  cellular  cytotoxicity  and  antibody-dependent  cellular 
cytotoxicity:   Differential  sensitivity  of  effector  cells  to  in  vivo 
and  _in  vitro  corticosteroids,  Scand.  J.  Immunol.  8:99-107,  1978. 

13.  Fauci,  A.  S.:   Mechanism  of  action  of  glucocorticosteroids .  In  Annual 
Reports  in  Medicineal  Chemistry,  Vol.  13,  F.  H.  Clarke,  Editor.  Academic 
Press,  New  York,  1978,  pp.  179-183. 

14.  Gelfand,  J.  A.,  Hurley,  D.  H. ,  Fauci,  A.  S.,  and  Frank,  M.  M. :   The  role 
of  complement  in  experimental  disseminated  candidiasis.  J.  Infect. 
Pis.,  138:9-16,  1978. 

15.  Fauci,  A.  S.:   Mechanisms  of  the  immunosuppressive  and  antiinflammatory 
effects  of  glucocorticosteroids.  J.  Immunopharmacol .  1:1-25,  1978. 

16.  Fauci,  A.  S.,  Haynes ,  B.  F.,  and  Katz,  P.:   Drug-induced  T  and  B 
lymphocyte  dysfunction.  In  Infections  Complicating  the  Abnormal  Host, 
M.  H.  Grieco,  Editor.  Yorke  Medical  Books,  New  York.  In  press. 

17.  Haynes,  B.  F.,  and  Fauci,  A.  S.:   Activation  of  human  B  lymphocytes.  X. 
Heterogeneity  of  concanavalin  A  generated  suppressor  cells  of  the  pokeweed 
mitogen  induced  plaque  forming  cell  response  of  human  peripheral  blood 
lymphocytes.  J.  Immunol.  121:559-565,  1978. 

18.  Hunninghake,  G.  W. ,  and  Fauci,  A.  S.:   Suppression  of  the  generation  of 
human  Con  A-induced  cytotoxic  effector  cells  by  Con  A-activated  suppressor 
cells.  J.  Immunol.  120:1828-1831,  1979. 

19.  Fauci,  A.  S.,  Pratt,  K.  R. ,  Whalen,  G.:   Activation  of  human  B  lymphocytes. 
VIII.  Differential  radiosensitivity  of  subpopulations  of  lymphoid  cells 
involved  in  the  polyclonally-induced  PFC  responses  of  peripheral  blood 

B  lymphocytes.  Immunology. ,  35:715-720,  1978. 


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Project  No.  Z01  AI  00055-07  LCI 

20.  Doppman,  J.  L. ,  Dunnick,  N.  R. ,  Girton,  M. ,  and  Fauci,  A.  S.:   Bile  duct 
cysts  secondary  to  liver  infarcts:   Experimental  production  by  small 
vessel  hepatic  artery  occlusion  and  clinical  correlation.  Radiology. 
130:1-5,  1979. 

21.  Fauci,  A.  S.,  Steinberg,  A.  D. ,  Haynes ,  B.  F.,  and  Whalen,  G.:   Immuno- 
regulatory  aberrations  in  systemic  lupus  erythematosus.  J.  Immunol. 
121:1473-1479,  1978. 

22.  Aduan,  R.  P.,  Fauci,  A.  S.,  Dale,  D.  C,  Herzberg,  J.  H. ,  and  Wolff, 
S.  M. :   Factitious  fever  and  self- induced  infection.  A  report  of  32 
cases  and  a  review  of  the  literature.  Ann.  Intern.  Med.  90:230-242,  1979. 

23.  Editor:   Ricci,  M. ,  Fauci,  A.  S.,  Arcangeli,  P.,  and  Torzuoli,  P. 
(Eds.):   Developments  in  Clinical  Immunology.  London,  Academic  Press, 
1978. 

24.  Fauci,  A.  S.,  Haynes,  B.  F.,  Pratt,  K.  R.,  and  Whalen,  G.:   Regulation 
of  PWM-induced  plaque  forming  cell  responses  of  human  peripheral 
blood  lymphocytes.  In  Ricci,  M. ,  Fauci,  A.  S.,  Arcangeli,  P.,  and 
Torzuoli,  P.,  (Eds.):   Developments  In  Clinical  Immunology.  London, 
Academic  Press,  1978,  pp.  23-29. 

25.  Parrillo,  J.  E.,  Fauci,  A.  S.,  and  Wolff,  S.  M. :   Therapy  of  the 
hypereosinophilic  syndrome.  Ann.  Intern.  Med.  89:167-172,  1978. 

26.  Katz,  P.,  Haynes,  B.  F.,  and  Fauci,  A.  S.:  Alterations  of  T  lymphocyte 
subpopulations  in  sarcoidosis.  Clin.  Immunol.  Immunopathol.  10:350-354, 
1978. 

27.  Fauci,  A.  S.,  and  R.  E.  Ballieux  (Eds.):   Antibody  Production  in  Man: 
In  Vitro  Synthesis  and  Clinical  Implications.  Academic  Press,  Inc., 
New  York,  19  79. 

28.  Fauci,  A.  S.,  and  Haynes,  B.  F.:   Pokeweed  mitogen  induced  plaque-forming 
cell  responses  of  human  peripheral  blood  lymphocytes:   Regulation  of  B 
cell  triggering.  In  Antibody  Production  in  Man:   In  Vitro  Synthesis  and 
Clinical  Implications.  Edited  by  A.  S.  Fauci  and  R.  E.  Ballieux.  Academic 
Press,  Inc.,  New  York,  1979,  pp.  17-34. 

29.  Haynes,  B.  F.,  Katz,  P.,  and  Fauci,  A.  S.:   Effect  of  hydrocortisone  on 
the  kinetics  and  function  of  peripheral  blood  immunoregulatory  cells  in 
man.   In  Antibody  Production  in  Man:   In  Vitro  Synthesis  and  Clinical 
Implications.  Edited  by  A.  S.  Fauci  and  R.  E.  Ballieux.   Academic  Press, 
Inc.,  New  York,  1979,  pp.  291-302. 

30.  Katz,  P.,  Haynes,  B.  F.,  and  Fauci,  A.  S.:   Aberrant  regulation  of  B 
cell  function  in  immunologically  mediated  diseases:   Systemic  lupus 
erythematosus  and  sarcoidosis.   In  Antibody  Production  in  Man:   In 
Vitro  Synthesis  and  Clinical  Implications.   Edited  by  A.  S.  Fauci  and 
R.  E.  Ballieux.  Academic  Press,  Inc.,  New  York,  1978,  pp.  351-366. 

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Project  No.  Z01  AI  00055-07  LCI 

31.  Fauci,  A.  S.,  and  R.  E.  Ballieux:   Human  B  cell  function:   Recent 
advances,  unanswered  questions,  and  future  directions.   In 

Antibody  Production  in  Man:   In  Vitro  Synthesis  and  Clinical  Implications. 
Edited  by  A.  S.  Fauci  and  R.  E.  Ballieux.   Academic  Press,  Inc.,  New 
York,  1979,  pp.  393-396. 

32.  Haynes,  B.  F.,  and  Fauci,  A.  S.:   Wegener's  granulomatosis.  In  Current 
Ocular  Therapy,  F.  T.  Fraumf elder  and  F.  Hampton  Roy,  (Eds.),  W.  B. 
Saunders  Co.,  Philadelphia,  PA,  1979.   In  press. 

33.  Haynes,  B.  F. ,  and  Fauci,  A.  S.:   Cogan's  syndrome.   In  Current  Ocular 
Therapy,  F.T.  Fraumf elder  and  F.  Hampton  Roy  (Eds.),  W.  B.  Saunders 
Co.,  Philadelphia,  PA,  1979.   In  press. 

34.  Haynes,  B.  F.,  and  Fauci,  A.  S.:  Diabetes  insipidus  associated  with 
Wegener's  granulomatosis  successfully  treated  with  cyclophosphamide. 
N.  Engl.  J.  Med. ,  299:764,  1978. 

35.  Five,  M.  W. ,  G.  H.  Mundinger,  and  A.  S.  Fauci:   Diagnostic  and 
therapeutic  aspects  of  the  surgical  approach  to  Wegener's  granulomatosis. 
J.  Thor.  Cardiovasc.  Surg.,  1978.  In  press. 

36.  Kornblut,  A.  S.,  J.  E.  Gadek,  A.  S.  Fauci,  and  S.  M.  Wolff:   Head  and 
neck  manifestations  of  histocytic  medullary  reticulosis.  The  Laryngoscope, 
LXXXVIII: 1596-1602,  1978. 

37.  Katz,  P.,  and  A.  S.  Fauci:   The  effects  of  corticosteroids  on 
immuno regulation  in  sarcoidosis.   Cell.  Immunol. ,  42:308-318,  1979. 

38.  Katz,  P.,  B.  F.  Haynes,  and  A.  S.  Fauci:   Lack  of  generation  of  killer 
cells  in  the  mixed  lymphocyte  reaction  between  mitogen  stimulated  and 
unstimulated  autologous  lymphocytes.  J.  Immunol. ,  121:1998-2001,  1978. 

39.  Fauci,  A.  S.,  F.  F.  Haynes,  and  P.  Katz:   The  spectrum  of  vasculitis: 
Clinical,  pathologic,  immunologic  and  therapeutic  considerations. 
Ann.  Intern.  Med.,  89:660-676,  1978. 

40.  Dimitriu,  A.  and  A.  S.  Fauci:   Activation  of  human  B.  lymphocytes.  XI. 
Differential  effects  of  azathioprine  on  B  lymphocytes  and  lymphocyte 
subpopulations  regulating  B  cell  function.  J.  Immunol.  121:2335-2339, 
1978. 

41.  Hunninghake,  C.  W.  and  A.  S.  Fauci:  Pulmonary  manifestations  of  the 
collagen  vascular  diseases.  Am.  Rev.  Resp.  Pis . ,  119:471-503,  1979. 

42.  Gallin,  J.  I.,  R.  E.  Elin,  R.  T.  Hubert,  A.  S.  Fauci,  M.  A.  Kaliner, 
and  S.  M.  Wolff:   Efficacy  of  ascorbic  acid  in  the  Chediak-Higashi 
syndrome:   Studies  in  humans  and  mice.  Blood,  53:226-234,  1979. 

43.  Parrillo,  J.  E.  and  A.  S.  Fauci:   Mechanisms  of  glucocorticoid  action 
on  immune  processess.  Ann.  Rev.  Pharmacol.  Toxicol.,  19:179-201,  1979. 

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Project  No.  Z01  AI  00055-07  LCI 

44.  Haynes ,  B.  F.  and  Fauci,  A.  S.:   Mechanisms  of  corticosteroid  action 
on  lymphocyte  subpopulations .  IV.  Effects  of  in  vitro  hydrocortisone 
on  the  generation  and  function  of  mitogen- induced  and  naturally 
occuring  suppressor  cells  in  man.  Cell.  Immunol. ,  44:157-168,  1979. 

45.  Haynes,  B.  F.,  Katz,  P.,  and  Fauci,  A.  S.:   Mechanisms  of  corticosteroid 
action  on  lymphocyte  subpopulations.  V.  Effects  of  in  vivo  hydrocortisone 
on  the  kinetics  of  mitogen-induced  and  naturally  occuring  suppressor 
cells  in  man.  Cell.  Immunol.,  44:169-178,  1979. 

46.  Haynes,  B.  F.,  R.  T.  Schooley,  J.  E.  Grouse,  C.  R.  Payling-Wright ,  R. 
Dolin,  and  A.  S.  Fauci:   Characterization  of  thymus-derived  lymphocyte 
subsets  in  acute  Epstein-Barr  virus  induced  infectious  mononucleosis. 
J.  Immunol.,  122:699-702,  1979. 

47.  Fauci,  A.  S.:   Human  B  cell  function  in  a  polyclonally  induced  plaque 
forming  cell  system.   Cell  triggering  and  immunoregulation. 
Immunological  Rev. , .   In  press. 

48.  Clark,  R.  A.  F.,  J.  G.  Gallin,  and  A.  S.  Fauci:   Effect  of  in  vivo 
prednisone  on  ±n   vitro  eosinophil  and  neutrophil  adherence  and 
chemotaxis.  Blood.  53:633-641,  1979. 

49.  Dimitriu,  A.,  and  A.  S.  Fauci:   Differential  sensitivity  of  human 
lymphocyte  subpopulations  to  azathioprine.  Transplant.  Proc.  11:878- 
881,  1979. 

50.  Katz,  P.,  R.  A.  Goldstein  and  A.  S.  Fauci:   Immunoregulation  in 
tuberculosis:   Alteration  of  T  lymphocyte  subpopulations  and 
presence  of  suppressor  monocytes.  J.  Infect.  Pis.  In  press. 

51.  Parrillo,  J.  E.,  T.  J.  Lawley,  M.  M.  Frank,  A.  P.  Kaplan  and  A.  S.  Fauci: 
Immunologic  reactivity  in  the  hypereosinophilic  syndrome.  J.  Allergy 
Clin.  Immunol.  In  press. 

52.  Fauci,  A.  S.,  T.  Murakami,  D.  D.  Brandon,  D.  L.  Loriaux,  and  M.  B. 
Lipsett:   Mechanisms  of  corticosteroid  action  on  lymphocyte  subpopulations, 
VI.  Lack  of  correlation  between  glucocorticosteroid  receptors  and  the 
differential  effects  of  glucocorticosteroids  on  T  cell  subpopulations. 
Cell.  Immunol.  In  press. 

53.  Stevenson,  H.  D.  and  A.  S.  Fauci:   Activation  of  human  B  lymphocytes. 
XII.   Differential  effects  of  _in  vitro  cyclophosphamide  on  human 
lymphocyte  subpopulations  involved  in  B  cell  activation.  Immunology . 
In  press. 


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Project  No.  Z01  AI  00055-07  LCI 

54.  Fauci,  A.  S.:   Mechanisms  of  altered  immunoregulation  in  systemic  lupus 
erythematosus  and  Sjogren's  syndrome.  Proc.  Soc.  Exp.  Biol.  In  press. 

55.  Fauci,  A.  S.:   Wegener's  granulomatosis,  lymphomatoid  granulomatosis, 
and  related  diseases.  CME  Medicine.  1(09) :l-6,  1979. 

56.  Parrillo,  J.  E.  and  A.  S.  Fauci:   Necrotizing  vasculitis,  coronary 
angiitis,  and  the  cardiologist.  Amer.  Heart  J.  In  press. 

57.  Dimitriu,  A.,  B.  F.  Haynes ,  and  A.  S.  Fauci:  Activation  of  human  B 
lymphocytes.  XIV.  Characterization  of  the  percursor  of  the  pokeweed 
mitogen- induced  anti-sheep  red  blood  cell  plaque  forming  cell. 

J.  Immunol.  In  press. 

58.  Katz,  P.,  D.  W.  Ailing,  B.  F.  Haynes,  and  A.  S.  Fauci:   Association  of 
Wegener's  granulomatosis  with  HLA-B8.  Clin.  Immunol.  Immunopathol. 

In  press. 

59.  Kingry,  K.  R.  and  A.  S.  Fauci:   Activation  of  human  B  lymphocytes.   The 
in  vitro  polyclonal  B  cell  activator-induced  plaque-forming  cell  system. 
La  Ricerca.  In  press. 

60.  Fauci,  A.  S.:   Immunoregulation  by  human  lymphocyte  subpopulations .  In: 
Regulatory  T  Lymphocytes,  B.  Pernis  and  H.  J.  Vogel  (Eds.),  Academic 
Press,  New  York.  In  press. 

61.  Parrillo,  J.  E. ,  J.  S.  Borer,  W.  L.  Henry,  S.  M.  Wolff  and  A.  S.  Fauci: 
The  cardiovascular  manifestations  of  the  hypereosinophilic  syndrome. 
Prospective  study  of  26  patients  with  review  of  the  literature. 

Amer.  J.  Med.  In  press. 

62.  Fauci,  A.  S.,  P.  Katz,  B.  F.  Haynes,  and  S.  M.  Wolff:   Cyclophosphamide 
therapy  of  severe  systemic  necrotizing  vasculitis.  N.  Engl.  J.  Med. 

In  press. 

63.  Fauci,  A.  S.:   Assays  for  suppressor  cells.   In:   Manual  of  Clinical 
Immunology.   N.  R.  Rose  and  H.  Friedman  (Eds.),  ASM  publication. 

In  press. 

64.  Fauci,  A.  S.,  B.  F.  Haynes,  and  G.  Whalen:   Multiple  subpopulations  of 
lymphoid  cells  regulating  human  B  cell  function.  In:   Cell  Biology  and 
Immunology  of  Leukocyte  Function,  M.  Quastel  (Ed.),  Academic  Press, 
New  York,  1979,  pp.  485-488. 

65.  Haynes,  B.  F,  G.  S.  Eisenbarth,  and  A.  S.  Fauci:   Human  lymphocyte 
antigens:   Production  of  a  monoclonal  antibody  which  defines  functional 
thymus-derived  lymphocyte  subsets.   Proc.  Natl.  Acad.  Sci.  (USA).   In 
press . 


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Project  No.  Z01  AI  00055-07  LCI 

66.  Haynes,  B.  F.,  R.  T.  Schooley,  C.  R.  Payling-Wright ,  J.  E.  Grouse, 

R.  Dolin,  and  A.  S.  Fauci:   Emergence  of  suppressor  cells  of  immunoglo- 
bulin synthesis  during  acute  Epstein-Barr  virus-induced  infectious 
mononucleosis.   J.  Immunol.  In  press. 

67.  Katz,  P.,  and  A.  S.  Fauci:   Autologous  and  allogeneic  intercellular 
interactions:   Modulation  by  adherent  cells,  irradiation;  in  vitro 
and  in  vivo  corticosteroids.  J.  Immunol.  In  press. 

68.  Fauci,  A.  S.:   Vasculitis:   New  insights  amid  old  enigmas.  Amer . 
J.  Med.  In  press. 


20-59 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00056-06 


PERIOD    COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Biochemical  Pathways  of  Mediator  Release  and  Mechanism  of  Tissue  Injury 
in  Allergic  Diseases 


NAMES,    LABORATORY    AND    INSTITUTE   AFFILIATIONS,    AND    TITLES   OF    PRINCIPAL    INVESTIGATORS   AND    ALL   OTHER 
PROFESSIONAL    PERSONNEL    ENGAGED    ON   THE   PROJECT 


PI: 


OTHER: 


A. P.  Kaplan 


Head,  Allergic  Diseases  Section 


M.A. 

Kaliner 

Senior  Investigator 

J.I. 

Gallin 

Senior  Investigator 

R.J. 

Mandle,  Jr. 

Guest  Worker 

G.G. 

Miller 

Clinical  Associate 

H.L. 

Meier 

Chemist 

R.E. 

Thompson 

Chemist 

A.L. 

Weinstein 

Guest  Worker 

LCI  NIAID 

LCI  NIAID 
LCI  NIAID 
LCI  NIAID 
LCI  NIAID 
LCI  NIAID 
LCI  NIAID 
LCI  NIAID 


OPERATING    UNITS    (if    any)  _ 

Z.  Horakova  Senior  Investigator 

S.  Katz  Acting  Director,  Dermatology  Branch 

R.  Sigler  Fellow,  Walter  Reed  Army  Hospital 


NHL  I 
NCI 


LAB/BRANCH 

Laboratory   of   Clinical    Investigation 


;ection 

Allergic  Diseases  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 

5   7/12 


PROFESSIONAL: 

3   7/12 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   C  (*2)  INTERVIEWS 


G  (b)  HUMAN  TISSUES 


□   (c)    NEITHER 


SUMMARY   OF   WORK    (200   words   or   less   -   underline    keywords) 
INACTIVE   DURING   CURRENT   YEAR.      TERMINATED. 


20-60 


PHS-6040 
(Rev.    10-76) 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZQ1  AI   00057-06   LCI 


PERIOD  COVERED 


October  1.  1978  to  September  30,  1979 


TITLE  OF    PROJECT   (80  characters   or  less) 


Basic   Studies   on  Pathogenic   Fungi 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


K.J.  Kwon-Chung 


Senior  Investigator 


LCI  NIAID 


OTHER:    J.E.  Bennett         Head,  Clinical  Mycology  Sec, 
Itzhack  Polacheck    Visiting  Fellow 


COOPERATING  UNITS  (if  any) 

A.K.  Bhattacharjee  (NIAMDD) 


lab/branch 


Laboratory  of  Clinical  Investigation 


Clinical  Mycology  Section 


INSTITUTE  AND  LOCATION 

NTATT),  NTHr  Bethesda.  Maryland   20205 


TOTAL  MANYEARS: 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 

G  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


3  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  interests  and  proposed  course  of  our  unit  are  to  cover  basic  and 
applied  aspects  of  various  pathogenic  fungi  including  their  morphology,  taxon- 
omy, pathology,  epidemiology,  biochemistry  and  genetics.   Topics  of  present 
interest  include:   1)  structure  of  the  capsular  polysaccharide  of  serotype  D 
of  Cryptococcus  neoformans,  2)  metabolic  pathway  of  creatinine  in  Cryptococcus 
neof ormans  and  C^.  bacillisporus,  3)  nuclear  status  of  basidiospores  in  the 
sexual  state  of  C_.  neoformans,  and  4)  distribution  of  a_  and  a.  mating  types 
among  clinical  and  natural  isolates  of  C_.  neoformans  and  C_.    bacillisporus. 


20-61 


PHS-6040 
(Rev.    10-76) 


Project   No,      Z01     AI     00057-06     LCI 

Project  Description: 

Objectives: 

Objectives  of  the  project  cover  both  basic  and  applied  aspects  of  vari- 
ous pathogenic  fungi,  including  their  morphology,  taxonomy,  pathogenicity, 
life  cycle,  biochemistry  and  genetics.   The  topics  of  present  interest 
include: 

1)  Chemical  structure  of  capsular  polysaccharide  of  serotype  D  of 
Cryptococcus  neof ormans . 

2)  Metabolic  pathway  of  creatinine  in  C.    neoformans  and  C.    bacil- 
lisporus . 

3)  Nuclear  status  (cytogenetics)  of  basidiospores  in  the  sexual  state 
of  jC.  neoformans . 

4)  Distribution  of  a_  and  a_  mating  types  among  clinical  and  natural 
isolates  of  C^  neoformans  and  C_.  bacillisporus. 

Methods  Employed: 

1)  Growth  and  the  Preparation  of  the  Cell-free  Extracts:   Cells  of 
C.    neoformans  and  C_.  bacillisporus  were  grown  on  minimal  medium  containing 
creatinine  or  ammonium  sulfate  as  the  nitrogen  source.   The  cells  were  har- 
vested, washed  and  suspended  in  phosphate  buffer  (0.05  m) ,  and  broken  by 
using  glass  beads  and  voltex  mixture.   The  supernatant  obtained  after  10  min. 
breakage  was  used  as  cell-free  extracts. 

2)  Metabolic  Product  of  Creatinine:   The  cell-free  extract  was  incu- 
bated with  radiolabled  creatinine.   The  radiolabled  metabolites  were  de- 
tected by  thin  layer  chromatography  on  silica  gel  with  phenol-ethanol-water 
as  the  developing  solution.   The  product  was  identified  by  autoradiographic 
technique. 

3)  Enzyme  Assay:   A  very  sensitive  assay  method  was  developed  for 
creatinine  deiminase  by  using  the  reaction  mixture  containing  radiolabled 
creatinine  buffer  and  cell-free  extract.   The  enzyme  reaction  was  stopped  by 
adding  acetic  acid.   The  reaction  mixture  was  shaken  with  cation  exchanger 
Dowex-50  and  then  filtered.   Under  this  condition,  the  substrate  was  found  on 
the  filter  and  the  radioactivity  of  the  filtrate  represented  the  enzymatic 
product. 

Genetic  Analysis:   Crosses  of  isolates  with  appropriate  genetic 
markers  were  made  on  hay  infusion  agar.   The  progeny  from  such  crosses  were 
isolated  by  micromanipulation  and  the  genetic  markers  were  analyzed. 

Production  of  Capsular  Polysaccharide:   Cultures  were  grown  on 
Sabouraud  dextrose  broth,  and  cells  were  killed  by  adding  buffered  formalin 


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Project  No.   Z01  AI   00057-06  LCI 

and  removed  by  centrifugation,   The  supernatant  was  treated  with  sodium  ace- 
tate, acetic  acid  and  95%  ethanol  to  precipitate  polysaccharide.   The  poly- 
saccharide was  dissolved  in  distilled  water  and  treated  with  sodium  acetate 
and  acetic  acid.   The  solution  was  deproteinated  with  chloroform  and  butanol. 
The  aqueous  solution  was  treated  with  sodium  acetate,  acetic  acid  and  etha- 
nol.  The  precipitate  was  redissolved  in  distilled  water  and  reprecipitated 
with  95%  ethanol.   The  polysaccharide  was  chromatographed  on  a  column  of 
DEAE-cellulose  using  0.01  M  phosphate  buffer,  pH  7.3,  and  a  linear  salt 
gradient  of  0-1  M  NaCl.   The  80%  of  material  was  eluded  at  a  salt  cone.  0.2  M, 
and  this  material  was  dialized  and  freeze  dried. 

Chemical  Structure  of  the  Polysaccharide:   Gel  filtration,  ultra- 
centrifugation,  paper  chromatography,  hydrolysis  by  1  N  HC1,  methylation, 
periodate  oxidation  and  chromium  trioxide  oxidation  methods  were  used  to 
analyze  the  chemical  structure  of  the  polysaccharide. 

Major  Findings  and  Significance  to  Biomedical  Research  and  the  Program  of 
the  Institute: 

1)  The  most  significant  series  of  developments  in  this  laboratory  over 
the  past  year  with  important  implications  to  biomedical  research  have  been 
the  series  of  studies  which  have  shown  that  the  etiologic  agent  of  crypto- 
coccosis is  two  distinct  species  of  genus  Cryptococcus  rather  than  a  single 
species,  C.   neoformans,  as  has  been  believed  for  the  past  80  years.   We  have 
utilized  genetical,  biochemical,  chemical  and  epidemiological  aspects  to 
reveal  the  distinct  characteristics  of  the  two  species,  C_.  neoformans  and 

C.   bacillisporus. 

2)  Significant  Findings  in  the  Area  of  Biochemistry: 

a)   The  demonstration  of  two  different  regulatory  mechanisms  for 
the  synthesis  of  creatinine  deirainase  between  C.   neoformans  (serotype 
A-D)  and  C_.  bacillisporus  (serotype  B-C)  .   One  of  the  best  known  natural 
reservoirs  of  the  etiologic  agent  of  cryptococcosis  is  pigeon  droppings. 
The  widely  accepted  view  is  that  pigeon  droppings  contain  high  concen- 
tration of  creatinine,  and  it  serves  as  a  selective  medium  for  the 
growth  of  cryptococcal  pathogens.   We  reported  in  the  past  that  only 
two  serotypes  (A,D)  of  Cryptococcus  neoformans  were  found  in  the  pigeon 
droppings  throughout  the  world.   Also  reported  was  that  serotype  B-C 
utilized  creatinine  better  than  A-D  and  the  natural  reservoir  of  the 
B-C  remained  unknown.   We  described  the  serotype  B-C  as  a  distinct 
species,  C.    bacillisporus.   Metabolic  pathway  of  creatinine  is  known  in 
several  species  of  bacteria  but  not  in  fungi.   Our  study  demonstrated 
that  the  creatinine  metabolism  in  the  cryptococci  involved  one  step 
resulting  in  methylhydantoin  and  ammonia.   The  enzyme  responsible  for 
this  degradation  was  identified  as  creatinine  deiminase  and  was  found 
to  be  inducible  in  both  species.   However,  the  enzyme  synthesis  was 
regulated  by  the  presence  of  ammonia  in  C_.  neof ormans  but  not  in  C_. 
bacillisporus.   This  difference  may  be  due  to  their  ecological 
difference. 


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Project  No.   Z01  AI   00057-06   LCI 

b)   An  extremely  sensitive  assay  method  for  creatinine  desimidase 
was  developed  during  this  study  by  using  autoradiogram. 

3)  Findings  in  the  Area  of  Genetics:   We  have  further  extended  our 
previous  studies  on  the  cytogenetics  of  basidiospore  formation  in  Filobasi- 
diella  neoformans  (C_.  neoformans) .   Using  two  stable  genetic  markers,  nuclear 
status  of  spore  chains  was  clarified.   Nuclear  inclusion  in  the  spore  chains 
was  found  to  be  at  random.   Also  demonstrated  was  the  formation  of  diploid 
spores  which  go  through  meiosis  during  the  blastospore  formation. 

4)  Findings  in  the  Area  of  Epidemiology:   Survey  revealed  that  the 
mating  type  a_   is  predominant  among  natural  and  clinical  isolates  of  Crypto- 
coccus  neoformans  regardless  of  the  serotype.   The  ratio  of  a_   and  a.  type  was 
about  40:1  among  105  natural  isolates  and  30:1  in  233  clinical  isolates. 

5)  Findings  in  the  Area  of  Immunochemistry :   Capsular  polysaccharide 
of  Cryptococci  contains  antigenic  determinants  defining  their  serotypes.   We 
extended  the  study  on  the  chemical  structure  of  serotype  D  of  C_.  neoformans. 
The  major  sugars  were  found  to  be  similar  to  that  of  C_.    bacillisporus  but 
contained  0-acetyl  group  which  was  not  found  in  C_.  bacillisporus.   Also  found 
was  that  the  substitution  of  mannose  backbone  by  xylose  and  glucose  was  sim- 
pler than  serotype  A  of  C_.    neoformans  and  also  isolates  of  C^.  bacillisporus . 

Proposed  Course: 

The  biochemical  and  genetical  aspects  of  5FC  resistant  strains  and 
phenoloxidase  negative  strains  of  C.  neoformans  will  be  studied.   The  chemi- 
cal structure  of  capsular  polysaccharide  of  serotype  B,  C.    bacillisporus, 
will  be  studied. 

Publications: 

1.  Kwon-Chung,  K.J.  and  Bennett,  J.E.:   Distribution  of  a_  and  a_  mating  types 
of  Cryptococcus  neoformans  among  natural  and  clinical  isolates.   Am.  J_. 
Epidemiol.  108:337-341,  1978. 

2.  Kwon-Chung,  K.J.,  Bennett,  J.E.  and  Theodore,  T.S.:   Cryptococcus  bacil- 
lisporus sp .  nov. :   Serotype  B-C  of  Cryptococcus  neoformans.   Int ■  ^J. 
Syst.  Bacteriol.  28:616-620,  1978. 

3.  Young,  N.A.,  Kwon-Chung,  K.J.,  Kubota,  T.T.  and  Jennings,  A.E.:   Dissem- 
inated infection  by  Fusarium  monilif orme  during  treatment  for  malignant 
lymphoma.   J.  Clin.  Microbiol.  7:589-594,  1978. 

4.  Bhattacharjee,  A.K.,  Kwon-Chung,  K.J.  and  Glaudemans,  C.P.J. :  On  the 
structure  of  the  capsular  polysaccharide  from  Cryptococcus  neoformans 
serotype  C.   Immunochemistry  15:673-679,  1978. 

5.  Kwon-Chung,  K.J.:   Comparison  of  Sporothrix  schenckii  isolates  obtained 
from  fixed  cutaneous  lesions  with  isolates  from  other  types  of  lesions. 


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Project  No.   Z01  AI   00057-06   LCI 
J.  Infect.  Pis.  139:424-431,  1979. 

6.  Bhattacharjee,  A.K.,  Kwon-Chung,  K.J.  and  Glaudemans.  C.P.J. :  On  the 
structure  of  the  capsular  polysaccharide  from  Cryptococcus  neof ormans 
serotype  C  II.  Immunochemistry .   In  press. 

7.  West,  B.C.  and  Kwon-Chung,  K.J.:   Mycetoma  caused  by  Microsporum 
audouinii.   Am.  J_.  Clin.  Path.   In  press. 

8.  Kwon-Chung,  K.J.  and  West,  B.C.:   Mycetoma  caused  by  dermatophytes. 
First  Int.  Sym.  Mycetoma,  Venezuela,  1978.   In  press. 

9.  Kwon-Chung,  K.J.:   Serotypes,  epidemiology  and  the  sexual  life  cycle  of 
Cryptococcus  neof ormans.   British  Mycopathological  Soc .  Report,  1979.   In 
press. 

10.  Bhattacharjee,  A.K.,  Kwon-Chung,  K.J.  and  Glaudemans,  C.P.J. :  On  the 
structure  of  the  capsular  polysaccharide  from  Cryptococcus  neof ormans 
serotype  D.   Carbohydrate  Research.   In  press. 


20-65 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl   AI   00058-06  LCI 


PERIOD    COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (30  characters  or  less) 

The  Pathogenesis  and  Chemotherapy  of  Herpesvirus  Infections 
in  Man 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:      R.  Dolin        Head,  Medical  Virology  Section 
(until  Jan  1,  1979) 

S.E.  Straus     Senior  Investigator,  MVS 
(since  Jan  1,  1979) 

Other:    R.Schooley      Clinical  Associate 
R.  Berg  " 


LCI  NIAID 


COOPERATING  UNITS  (if  any) 

P.  Howley  (LP,  NCI)  and  R.  Whitley  (Cooperative  Antiviral  Study  Group) 


LAB/BRANCH 


Laboratory  of  Clinical  Investigation 


Medical  Virology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland   20205 


TOTAL  MANYEARS: 

2 


PROFESSIONAL: 

1-1/2 


1/2 


CHECK  APPROPRIATE  BOX(ES) 
5§  (a)   HUMAN  SUBJECTS 

D  (al)  MINORS   □  (a2)  INTERVIEWS 


(b)  HUMAN  TISSUES 


Q  (c)  NEITHER 


SUMMARY  OF  «I0RK  (200  words  or  less  -  underline  keywords) 

The  pathogenesis  and  natural  history  of  herpesvirus  infections  in  man  are  being 
investigated.   Patients  are  being  identified,  followed  clinically  and  diagnosed 
definitively  by  virus  isolation.   Studies  of  patients  with  infectious  mononu- 
cleosis have  documented  a  variety  of  effects  of  the  causative  agent,  the  EB 
virus,  upon  the  target  B  lymphocyte.   The  development  and  nature  of  T-cell 
mediated  immune  responses  to  the  EBV  infected  B  cells  have  been  characterized. 
The  results  of  initial  trials  of  adenine  arabinoside  therapy  of  serious  herpes- 
virus infections  have  been  substantiated.    The  tools  are  being  developed  to 
study  the  biology  and  molecular  epidemiology  of  varicella-zoster  virus 
infection. 


20-66 


PHS-6040 
(Rev.  IO-76) 


Project  No.   Z01   AI   00058-06   LCI 

Project  Description; 
Objectives: 

1)  To  determine  the  pathogenesis  and  natural  history  of  herpesvirus 
infections  in  both  normal  and  immune  compromised  humans. 

2)  To  evaluate  the  efficacy  of  antiviral  agents  in  the  treatment  of 
human  herpesvirus  infection.   These  infections  include  both  minor  and  serious 
diseases  produced  by  herpes  simplex  types  1  and  2,  varicella-zoster  and  EB 
virus. 

Methods  Employed: 

1)  Patients  with  suspected  herpesvirus  infections  are  referred  to  the 
Medical  Virology  Section  from  the  Clinical  Center,  the  National  Naval  Medical 
Center,  local  hospitals  and  practicing  physicians.   The  patients  are  evalu- 
ated and  appropriate  specimens  are  collected  for  virus  isolation  and  sero- 
logic testing. 

2)  Virus  isolation  is  performed  in  primary  or  continuous  human  or 
simian  cell  lines.   The  viruses  are  identified  by  the  appearance  of  typical 
cytopathic  changes  and  typed  by  immunof luorescent  procedures  where  required. 

3)  In  selected  cases,  the  viral  DNA  is  purified,  digested  by  restric- 
tion endonucleases  and  analyzed  by  agarose  gel  electrophoresis.   This  pro- 
vides epidemiologic  information  regarding  the  transmission  of  the  virus  and 
may  provide  structural  correlates  of  viral  pathogenicity. 

4)  The  clinical  efficacy  of  adenine  arabinoside,  adenine  arabinoside 
monophosphate,  acycloguanosine  and  ribavirin  is  being  investigated  in  the 
treatment  of  herpesvirus  infections.   This  unit  is  collaborating  in  a 
NIAID-sponsored  multicenter  trial  evaluating  adenine  arabinoside  in  the 
treatment  of  herpes  simplex  encephalitis,  herpes  zoster  infection  of  the 
immune  compromised  host,  and  chronic  mucocutaneous  herpes  simplex  infection. 

Major  Findings: 

1)   EB  Virus  Infection. 

a)   Studies  of  cell-mediated  immune  responses  in  patients  with  in- 
fectious mononucleosis  have  progressed.   T  lymphocytes  from  patients 
with  antibody  to  EBV  are  capable  of  suppressing  lymphoblastoid  trans- 
formation of  EBV  infected  autologous  lymphocytes.   The  acquisition  of 
this  suppressive  capacity  is  absent  early  during  primary  clinical  in- 
fection, develops  after  the  first  several  days  and  remains  present  for 
life.   This  T-cell  function  is  independent  of  humoral  antibody  and  is 
not  HLA  restricted. 


20-67 


Project  No.   ZQ1  AI   00058-06   LCI 

b)  EBV  has  been  found  to  be  a  potent  in  vitro  polyclonal  activator 
of  B  cell  function  as  manifested  by  induction  of  a  plaque  forming  cell 
response  and  production  of  intracytoplasmic  antibody.   The  PFC  response 
is  not  T  cell  dependent  but  may  be  modulated  by  helper  and/or  suppressor 
T  cells  when  present. 

c)  Lymphocytes  from  patients  with  infectious  mononucleosis  are 
hyporesponsive  to  polyclonal  stimulation  by  EBV. 

d)  Polyclonal  stimulation  of  B  cells  requires  successful  infection 
by  live  EB  virus.   Not  all  virally  infected  B  cells,  however,  produce 
immunoglobulin,  suggesting  that  cellular  control  mechanisms  may  play  a 
role  in  the  expression  of  EBV  induced  B  lymphocyte  activation. 

2)  Clinical  trials  of  systemically  administered  adenine  arabinoside  in 
herpesvirus  infections. 

a)  Herpes  simplex  encephalitis:   The  results  of  the  earlier  small 
collaborative  study  wherein  adenine  arabinoside  (AEA-A)  was  demonstrated 
to  substantially  reduce  mortality  in  herpes  simplex  encephalitis  have 
been  confirmed.   An  additional  78  patients  have  been  treated  with  ARA-A 
in  the  nationwide  trial,  with  a  fatality  rate  of  31%,  quite  comparable 
to  the  28%  incidence  reported  in  the  first  study.   Additional  trials 
will  be  initiated  within  the  next  several  months  to  compare  two  new 
promising  agents,  adenine  arabinoside  monophosphate  (ARA-AMP)  and 
acycloguanosine,  with  ARA-A. 

b)  Varicella-zoster  infections:   Patients  will  continue  to  be  en- 
rolled for  the  next  few  months  into  the  collaborative  trial  of  ARA-A 
for  VZ  infections  of  immunocompromised  patients.   The  earlier  prelimi- 
nary trial  suggested  that  the  drug  speeds  clearing  of  virus  from  vesi- 
cles and  healing  of  lesions.   The  present  trial  is  aimed  at  defining 
whether  treatment  initiated  within  the  first  72  hours  of  infection  is 
capable  of  preventing  dissemination  of  virus  and/or  subsequent  post- 
herpetic neuralgia. 

3)  Characterization  of  nucleic  acid  from  varicella-zoster  virus: 
VZV  DNA  purified  from  clinical  isolates  is  digested  with  restriction  endo- 
nucleases  and  analyzed  by  agarose  gel  electrophoresis.   Highly  labeled  DNA 
will  be  prepared  by  nick  translation  and  used  in  sensitive  reassociation 
kinetic  analyses  to  compare  the  DNA  from  different  isolates. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

Herpesviruses  produce  substantial  morbidity  and  mortality  each  year. 
Normal  adults  and  children  suffer  individual  or  recurrent  infections  caused 
by  members  of  the  herpesvirus  family.   These  infections  range  from  rather 
benign  afflictions  to  those  such  as  herpes  simplex  encephalitis  which  when 
untreated  causes  death  in  60-70%  of  individuals.   Immune  compromised  indi- 
viduals, of  which  there  are  an  ever  increasing  number  and  variety,  are 


20-68 


Project  No.   Z01  Al  Q0058-Q6  LCI 

especially  susceptible  to  herpes  infections.   The  above  studies  have  ex- 
panded current  knowledge  of  the  natural  history  and  pathogenesis  of  these 
infections.   The  therapeutic  trials  in  which  we  collaborate  have  been  the 
first  to  clearly  demonstrate  efficacy  of  antiviral  compounds  for  these  types 
of  infections.   These  results  have  been  encouraging  and  pave  the  way  for 
future  studies  with  even  more  potent  antiviral  drugs. 

Proposed  Course: 

With  the  recent  departure  of  the  former  principal  investigator,  the 
direction  of  this  laboratory  will  undergo  some  changes.   It  is  expected  that 
a  more  molecular  approach  will  be  applied  to  the  analysis  of  the  herpesvirus 
infection  with  particular  emphasis  on  herpes  simplex  and  varicella  zoster 
infections.   The  mechanism  of  virus  latency  and  reactivation  will  be  explored. 
Sensitive  DNA  reassociation  analyses  using  highly  labeled  VZ  DNA  probes  will 
be  performed  in  an  effort  to  detect  the  presence  of  viral  sequences  in  the 
genome  of  tissues  obtained  at  autopsy  from  patients  with  a  known  history  of 
zoster  infection. 

Clinical  trials  with  antiviral  agents  will  continue.   Within  the  next 
year,  the  NIAID  sponsored  collaborative  study  group  will  initiate  trials  of 
ARA-AMP  and  acycloguanosine.   These  agents  hold  particular  promise  because 
they  are  minimally  toxic,  are  able  to  attain  higher  tissue  levels  and  appear 
significantly  more  potent  than  ARA-A.   In  addition,  this  laboratory  will  at- 
tempt to  initiate  within  the  next  several  months  a  trial  of  topical  therapy 
of  herpes  genital  infection  with  ribavirin,  another  promising  new  agent. 
This  compound  has  been  found  in  uncontrolled  use  to  be  extremely  effective 
in  superficial  herpes  simplex  infection.   Its  true  merits  must  be  defined  by 
placebo  controlled  trials. 

Publications: 

1.  Whitley,  R.J.,  et  al:   Adenine  arabinoside  therapy  of  biopsy  proved 
herpes  simplex  encephalitis.   N^.  Engl.  J_.  Med .  297:289,  1977. 

2.  Whitley,  R.J.,  et  al:   Adenine  arabinoside  therapy  of  herpes  zoster  in 
the  immunosuppressed:   NIAID  Collaborative  Antiviral  Study.   N.  Engl.  ^J. 
Med.  294:1193,  1976. 

3.  Haynes,  B.F.,  Schooley,  R.T.,  Grouse,  J.E.,  Payling-Wright ,  C.R. ,  Dolin, 
R.  and  Fauci,  A.S.:   Characterization  of  thymus-derived  lymphocyte  sub- 
sets in  acute  Epstein-Barr  virus-induced  infectious  mononucleosis. 

J.  Immunol.  122:699-702,  1979. 

4.  Schooley,  R.T.,  Haynes,  B.F.,  Grouse,  J.E.,  Payling-Wright,  C.R.,  Fauci, 
A.S.  and  Dolin,  R.:   Quantitative  assessment  of  suppression  of  Epstein- 
Barr  virus  induced  B-lymphocyte  outgrowth.   Manuscript  in  preparation. 

5.  Schooley,  R.T.,  Haynes,  B.F.,  Grouse,  J.E.,  Payling-Wright,  C.R.,  Fauci, 
A.S.  and  Dolin,  R. :   Mechanism  of  EBV-induced  B-lymphocyte  activation. 
Manuscript  in  preparation. 

20--69- 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE      U.S.  DEPARTMENT  OF 
PROJECT  NUMBER  (Do  NOT  use  this  space)   HEALTH,  EDUCATION,  AND  WELFARE 

PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00154-04  LCI 


PERIOD    COVERED 

October  1,  1978  to  September  30. 


1979 


TITLE  OF  PROJECT  (80  characters  or  less 

Immunologic,  Neurophysiologic ,  Biochemical  and  Cellular  Events  in  Immediate 
Hypersensitivity 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


Michael  A.  Kaliner 


Senior  Investigator 


LCI  NIAID 


OTHER: 

J. 

Shelhamer 

L. 

Steel 

H. 

Oertel 

M. 

Donlon 

S. 

Wescott 

G. 

Myers 

P. 

Davis 

Clinical  Associate  LCI  NIAID 

Staff  Fellow  LCI  NIAID 

Visiting  Fellow  LCI  NIAID 

Guest  Worker  LCI  NIAID 

Bio.  Lab.  Tech.  (Biol.)  LCI  NIAID 

Bio.  Lab.  Tech.  (Biol.)  LCI  NIAID 


Clinical  Associate 


PMB  NIAMD 


COOPERATING  UNITS  (if  any) 

Walter  Reed  Army  Medical  Center  (R.  Evans,  R.  Summers,  L.  Smith  and  R.  Sigler) 


LAB/BRANCH 

Laboratory  of  Clinical  Investigation 


SECTION 

Allergic  Diseases  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  MD   20205 


TOTAL  MANYEARS: 
6  1/2 


PROFESSIONAL: 
4  1/2 


OTHER: 
2 


CHECK  APPROPRIATE  30X(ES) 
Lx(a)   HUMAN  SUBJECTS 

□  (al)  MINORS   Q  (a2)  INTERVIEWS 


H  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Human  asthma  and  rhinitis  involve  the  excessive  secretions  of  mucus. 


Cultured 


human  airways  incorporate  radiolabeled  molecules  permitting  monitoring  of 
mucous  secretion.   Human  lung  mucus  has  been  characterized  biochemically  and 
the  immunologic  as  well  as  neuropharmacologic  control  of  mucous  secretion 
defined.   The  site  and  control  of  human  lung  parenchyma  versus  airway 
prostaglandin  production  has  been  identified  and  the  factors  generated  by 
anaphylaxis  causing  prostaglandin  synthesis  isolated.   The  histologic  responses 
to  rat  mast  cell  granules  have  been  characterized  both  in  rat  and  monkey  skin 
and  the  factors  responsible  for  eliciting  the  inflammation  isolated  and 
characterized.   The  relationship  between  calcium  influx  and  rat  mast  cell 
degranulation  has  been  analyzed  as  has  the  response  of  mast  cells  to  histamine 
stimulation.   Finally,  the  neuropharmacologic  responsiveness  of  subjects  with 
asthma,  allergic  rhinitis  and  cystic  fibrosis  has  been  studied. 


20-70 


PHS-6040 
(Rev.  10-76) 


Project  No.  Z01  Al  00154-04  LCI 

Project  Description: 

Objectives: 

The  goals  of  the  Allergic  Diseases  Section  are  to  expand  our  knowledge 
of  all  aspects  of  immediate  hypersensitivity  reactions.   Our  particular  ex- 
pertise and  focus  are  defining  the  biochemical  events  which  accompany  and 
control  the  triggering  of  allergic  reactions,  the  neurophysiologic  mechanisms 
which  potentially  modulate  these  events,  the  characterization  of  the  mediators 
of  anaphylaxis  and  identification  of  the  responses  of  relevant  target  tissues 
to  these  mediators. 

We  are  concentrating  on  three  primary  areas:  1)  Employing  human  lung 
tissue,  we  study  "asthma  in  a  test  tube"  in  order  to  understand  the  immuno- 
logic triggers,  biochemical  concomitants  and  neurophysiologic  controls  of 
mediator  release;  the  identification  of  additional  mediators  of  anaphylaxis 
and  the  study  of  mucous  secretion.   2)  Rat  peritoneal  mast  cells  are  utilized 
as  a  pure  source  of  effector  cells  which  may  be  readily  studied  in  vitro 
and  may  amplify  as  well  as  initiate  new  areas  of  interest  in  relationship 
to  the  human  lung  model  and  3)  Clinical  allergic  asthma  is  being  evaluated 
in  an  extensive  protocol  designed  to  expand  our  understanding  of  the  neuro- 
physiologic mileau  of  allergic  individuals  as  compared  with  cystic  fibrosis 
and  normal  subjects. 

Methods  Employed: 

1.    Human  lung  model:   Lungs  removed,  usually  for  cancer  resection,  almost 
always  contain  large  amounts  of  normal,  functioning  lung  tissue.   We  obtain 
these  lungs  in  cooperation  with  the  surgical  and  pathology  departments  of 
all  the  major  hospitals  in  the  Bethesda  area.   The  lung  tissue  is  washed, 
fragmented  and  replicated  into  200  mg  samples  and  subsequently  incubated  in 
dilutions  of  allergic  serum  for  2  hours  at  37  °C  or  18  hours  at  25 °C.   After  this 
"sensitization"  period,  the  serum  is  removed  and  the  fragments  challenged 
with  allergen.   The  interaction  of  the  allergen  with  tissue  (mast  cell) 
bound  IgE  induces  the  release  of  histamine,  slow-reacting  substance  of  ana- 
phylaxis (SRS-A),  eosinophil  chemotactic  factor  of  anaphylaxis  (ECF-A) ,  and 
prostaglandin  E   and  F    (PGE  and  PGF    ) .   These  mediators  may  be  assayed 
on  the  isolated  terminaiportion  of  the  guinea  pig's  ileum  (histamine,  SRS- 
A) ,  chemotactic  chambers  (ECF-A)  or  by  radioimmunoassay  (PGE  ,  PGF2a  ). 

We  have  been  determining  alterations  in  the  tissue  levels  of  cyclic 
AMP  and  cyclic  GMP  after  treating  lung  with  various  agonists  in  order  to 
define  the  role  these  nucleotides  play  in  modulating  the  release  of  mediators. 
Cyclic  AMP  may  be  measured  by  either  a  protein  kinase  binding  assay  or  radio- 
immunoassay while  cyclic  GMP  is  determined  by  radioimmunoassay  after  acety- 
lation  or  succinylation. 

3 
Mucous  glycoproteins  are  monitored  by  incorporating  H-glucosamine  in- 
to the  mucous  glands  during  an  18  hour  incubation  period.   Secretions  are 
dialyzed  against  6M  urea,  filtered  over  sepharose  2B,  and  analyzed  by  ion 

20-71 


Project  No.  Z01  AI  00154-04  LCI 

exchange  chromatography.   Mucous  glycoproteins  may  also  be  hydrolyzed  by 
NaBH,  or  be  focused  on  a  sucrose  gradiant  in  the  presence  of  ampholytes. 

2.  Rat  mast  cells:   Rat  peritoneal  and  pleural  mast  cells  are  obtained  from 
freshly  sacrificed  male  Sprague-Dawley  rats  by  lavage  of  these  cavities.   The 
peritoneal  cells  include  5%  mast  cells  (or  approximately  500,000  to  1  million/ 
animal) .   The  cells  are  used  either  in  mixed  cell  suspensions  or  after  purifi- 
cation by  centrifugation  into  albumin  cushions.   Mast  cells  granules  are 
isolated  with  intact  perigranular  membranes  by  sonication  and  centrifugation 
through  sucrose  cushions.   Granules  free  of  membranes  are  obtained  by  osmotic 
lysis  of  mast  cells. 

Calcium  studies  with  rat  mast  cells  involve  incubation  of  cells  with 
4o 

Ca,  followed  by  rapid  centrifugation  through  silicone  oil  into  water.   Hista- 
mine released. into  the  upper  layer  is  assayed  by  an  automated  fluorometric 
assay  while   Ca  associated  with  the  cells  spun  into  the  bottom  layer  is 
quantitated  by  scintillation. 

3.  Clinical  asthma  study:   The  methods  employed  in  this  study  are  covered 
in  detail  in  the  clinical  protocol  and  include:  a)  measurement  of  cutaneous 
blood  flow  by    xenon  disappearance;  b)  pupillary  responses  as  measured  by 
a  binocular  pupillometer ;  c)  airway  obstruction  as  measured  by  flow-volume 
curves  with  and  without  helium  inhalation;  d)  serum  cyclic  nucleotide  re- 
sponses as  measured  after  intravenous  isoproterenol  administration. 

Major  Findings: 

1)    Human  Lung 

a)  The  response  of  human  and  guinea  pig  peripheral  lung  preparations  to 
histamine  stimulation  was  compared  with  airway  smooth  muscle  preparation  from 
the  same  species.   Histamine  induced  both  PGE  and  PGF?    from  both  parenchymal 
preparations,  only  PGE  from  human  airways  and  both  PGE  and  PGF.    from  guinea 
pig  airways.   In  order  to  analyze  the  mechanism  of  histamine- induced  prosta- 
glandin synthesis,  the  responses  to  KCL  at  membrane  depolarizing  concentrations 
and  the  muscle  stimulant  carbachol  were  studied.   Both  guinea  pig  and  human 
airway  preparations  were  equivalently  stimulated  with  KCL  or  carbachol  while 
neither  of  these  agents  stimulated  lung  parenchyma.   Therefore,  histamine, 
through  H-l  receptor  stimulation,  generates  prostaglandin  synthesis  from  lung 
by  direct  stimulation  of  parenchymal  cells  and  by  causing  muscle  contraction 

in  the  airways. 

b)  Human  airways  can  be  maintained  in  organ  culture  for  at  least  96  hours. 
The  mucous  secreting  cells  take  up  radiolabeled  sugars,  amino  sugars,  amino 
acids  and  sulfate  and  incorporate  these  labels  into  newly  synthesized  glyco- 
proteins.  The  glycoproteins  produced  can  be  differentiated  by  size 
(fraction  A  is  >  7,000,000  daltons  while  fraction  B  is  400,000  daltons)  on 

gel  filtration  and  carbohydrate:  protein  ratios  (fraction  A=70:30;  fraction 
B=20:80;  wt:wt).  However,  both  molecules  elute  from  anion  exchange  columns  at 
the  same  salt  concentration,  each  has  an  identical  isoelectic  focusing  point, 
each  has  an  identical  constituent  sugar  composition,  each  has  similar  amino 
acids  constituting  the  protein  core  and  the  size  of  the  protein  core 
of  each  molecule  is  the  same.   Thus,  human  airway  submucosal  glands 
synthesize  two  similar  but  distinct  glycoproteins. 

20-72 


Project  No.  Z01  AI  00154-04  LCI 

Employing  quantitation  of  non-dialyzable  glycoprotein  radiolabeled  with 
3H-glucosamine  to  monitor  mucous  secretion,  the  influence  of  immunologic 
challenge  as  well  as  several  neurohornomal  agonists  on  mucous  secretion  was 
analyzed.   Reversed  and  direct  anaphylaxis  of  airways  increased  mucous 
secretion.   As  supernatants  rich  in  the  various  mediators  released  during 
anaphylaxis  were  also  effective  in  increasing  mucous  release,  we  determined 
which  mediator  was  responsible.   Histamine  acting  through  H-2  receptor  stimu- 
lation was  found  to  cause  mucous  secretion,  as  did  PGF    ,  PGF   ,  PGD?  and 
PGA?.   PGE„  and  thromboxane  B?  were  ineffective.   Muscarinic  stxmulation  of 
mucous  glands  by  methacholine~augmented  mucous  release  as  did  alpha  adrenergic 
stimulation.   Beta  adrenergic  stimulation  was  ineffective.   Therefore,  several 
mediators  of  anaphylaxis  (including  histamine  and  several  prostaglandins) 
and  several  neurophormones  (cholinergic  and  alpha  adrenergic  agonists)  are 
capable  of  increasing  mucous  secretion  from  human  airways. 

c)   Prostaglandin  formation  by  human  lung  after  anaphylaxis  may  in 
part  be  attributed  to  histamine.   However,  about  50%  of  the  prostaglandin 
formation  is  independent  of  histamine.   Several  factors  released  from 
human  lung  during  anaphylaxis  are  able  to  cause  guinea  pig  lung  parenchymal 
preparations  to  produce  thromboxane  B„  ,  PGF,,    and  PGE.   We  have  characterized 
these  factors  and  now  recognize  that  they  consist  of  two  major  components. 
The  larger  is  retained  on  a  DM5  filter  (>  5000  MW) ,  and  separates  on  a 
Sephadex  G-150  column  into  two  factors  with  apparant  molecular  weights 
of  >200,000  and  55000  daltons.   The  second  component  filters  through  DM5 
but  is  retained  by  UM05  filters  (MW  =  <5000  but  >5000),  filters  on  Sephadex 
G25  with  an  apparant  molecular  weight  of  1500,  adheres  to  DE52  in  0.001 
of  NH.HC0  ,  pH7.8,  and  elutes  in  one  major  peak  at  0.1M  NH  HCO  .   Thus, 
human  lung  undergoing  anaphylaxis  generates  at  least  three  molecules  in  addi- 
tion to  histamine  which  are  capable  of  causing  prostaglandin  synthesis.   Fur- 
ther purification  and  biologic  characterization  of  these  molecules  are 
underway. 

2)   Rat  Mast  Cells 

Mast  Cell  granules  injected  into  the  skin  of  rats  and  monkeys  were 
found  to  cause  significant  inflammatory  reactions.   Granules  isolated  with 
intact  perigranular  membranes  caused  a  polymorphonuclear-rich  infiltrate  apparent 
in  2  hours  which  peaked  by  8  hours.   Subsequently,  an  intense  mononuclear 
infiltrate  replaced  the  PMN's  and  peaked  at  24-48  hours.   Granules  isolated 
free  of  membranes  and  washed  free  of  elutable  mediators  injected  into  skin 
caused  a  somewhat  less  pronounced  polymorphonuclear  infiltrate  and  a  somewhat 
greater  mononuclear  infiltrate.   The  responsible  factors  were  found  primarily 
in  close  association  with  the  granule  matrix.   In  fact,  granule  preparations 
washed  free  of  elutable  mediators  were  found  to  have  three  separable  factors 
capable  of  inducing  these  infiltrates.   These  factors  may  be  separated  on 
filters  -  the  two  larger  factors  are  retained  on  a  UM10  filter  while  the  small 
factor  filters  through  UM10  but  is  retained  by  a  UM05  filter.   The 
larger  molecules  have  apparent  molecular  weights  of  200,000  and  70000  daltons 
respectively  on  gel  filtration  through  Sephadex  G-200 .   The  smaller  molecule 
chromatographs  on  Sephadex  G-25  with  a  apparent  MW  of  1250.   The  smaller 
molecule  binds  to  both  anion  and  cation  exchange  columns  and  elutes  as 
one  major  peak  in  a  salt  gradient.   The  larger  molecules  may  be  precipitated 

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Project  No.  Z01  AI  00154-04  LCI 

by  10%  ethyl  alcohol  but  not  by  (NH.)-SO,.   Therefore,  there  are  three 
factors  constituent  within  the  mast  cell  granule  which  elicit  delayed 
inflammatory  responses  in  rat  skin.  Further  characterization  of  the 
structure  and  biologic  activity  of  these  molecules  is  underway. 

b)  Initiation  of  rat  mast  cell  secretion  involves  transmembrane  move- 
ment of  extracellular  calcium.   The  equilibration  with  extracellular  calcium 
in  resting  mast  cells  is  extremely  rapid,  being  completed  within  60  sec 

at  37 °C  in  the  presence  of  0.8  mM  calcium.   Analysis  of  calcium  movement 
into  the  rat  mast  cell  undergoing  a  secretory  event  reveals  that  uptake 
is  increased  within  5-10  sec,  only  50%  is  removed  by  subsequent  exposure 
to  EGTA  and  the  time  course  of  calcium  uptake  continues  after  granule 
release  is  completed.   Calcium  uptake  was  initiated  by  phosphatidyl 
serine  (PS)  added  to  the  bathing  medium  in  the  absence  of  histamine 
release.   As  PS  augments  antigen- induced  histamine  release,  the  calcium 
translocation  observed  may  relate  to  this  property.   ATP  (10  mM) 
inhibits  mast  cell  degranulation  and  chelates  calcium;  at  ImM,  ATP 
induces  release  and  stimulates  calcium  uptake.   ATP  does  not  require 
hydrolysis  to  ADP  or  AMP  for  this  response  and  there  is  insufficient  ATP 
released  by  the  mast  cell  to  suggest  that  secreted  ATP  has  any  influence 
on  the  release  reaction.   Further  characterization  of  the  role  which 
calcium  plays  in  degranulation  by  mast  cells  is  ongoing. 

c)  Rat  mast  cell  cyclic  AMP  and  cyclic  GMP  levels  are  important  in  so 
far  as  these  levels  determine  the  capacity  of  mast  cells  to  undergo  degranu- 
lation.  We  are  completing  an  analysis  of  factors  responsible  for  changes 

in  mast  cell  cyclic  nucleotides  and  have  found  that  histamine  fails  to 
change  the  mast  cell  levels.   This  observation  suggests  that  the  mast  cell 
which  is  a  major  synthesis  and  storage  site  for  histamine  is  either  desensi- 
tized to  histamine  or  has  no  functional  histamine  receptor  sites. 

3)    Clinical  Studies 

The  autonomic  responses  of  asthmatic  subjects  have  been  compared  with 
those  of  subjects  with  allergic  rhinitis  and  normal  controls.   Alpha  adrenergic 
function  was  assessed  by  pupillometry  and  cutaneous  vascular  responses.   In 
both  instances,  the  subjects  with  asthma  were  significantly  more  sensitive 
than  both  allergic  rhinitis  and  normal  control  subjects.   Beta  adrenergic 
function  has  been  analyzed  by  the  cardiovascular  and  cyclic  AMP  responses  to 
intravenous  isoproterenol  in  these  subjects.   Patients  with  either  asthma  or 
allergic  rhinitis  have  significant  impairment  of  both  beta  adrenergic  responses 
as  compared  with  controls.   Parasympathetic  function  has  been  analyzed  by 
pupillometry,  sweat  responses  and  bronchial  challenge.   Again,  patients  with 
either  allergic  rhinitis  or  asthma  demonstrate  the  same  degree  of  exaggerated 
responsiveness  in  comparison  with  normal  controls.   Therefore,  asthmatics  are 
selectively  more  responsive  to  alpha  adrenergic  stimulation  while  both  asthmatics 
and  subjects  with  allergic  rhinitis  have  increased  cholinergic  and  dimished 
beta  adrenergic  responses. 


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Project  No.  Z01  AI  00154-04  LCI 

Subjects  manifesting  reproducibly  positive  allergic  skin  tests  but  who 
are  asymptomatic  have  also  been  examined.   These  subjects  who  are  "pre-allergic" 
have  reduced  beta  adrenergic,  hyper-responsive  cholinergic  and  normal  alpha 
adrenergic  responses  much  as  the  subjects  with  allergic  rhinitis.   Taken 
together  these  data  indicate  that  reduced  beta  adrenergic  responsivity  is 
associated  with  the  atopic  state  as  is  excessive  cholinergic  responsiveness. 
However,  excessive  alpha  adrenergic  responses  are  seen  only  in  asthmatics. 

More  recently,  we  have  completed  an  examination  of  11  young  adult 
subjects  with  cystic  fibrosis  and  9  parents  (  obligate  heterozygotes)  of  cystic 
fibrosis  children.   The  CF  subjects  were  significantly  more  sensitive  than 
asthmatics  in  regards  to  both  alpha  adrenergic  and  cholinergic  responsive- 
ness and  equally  abnormal  in  regards  to  depressed  beta  adrenergic  responses. 
Only  3  of  the  CF's  were  concomitantly  allergic  and  these  3  were  relatively 
less  abnormal  than  the  other  8.   Therefore,  the  autonomic  abnormalities  un- 
covered may  be  found  in  diseases  other  than  allergy  and  may  relate  to  glandular 
(mucus)  secretion  rather  than  muscle  spasm. 

The  obligate  heterozygotes  were,  as  a  group,  abnormal  in  both  alpha 
adrenergic  and  cholinergic  responses  although  much  less  so  than  the  affected 
children.   This  observation  suggests  that  the  hyper-responsiveness  is  inherited 
as  a  primary  defect  rather  than  appearing  as  a  secondary  phenomena. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute 

Increased  understanding  of  the  mechanisms  of  immediate  hypersensitivity 
and  its  controls  may  eventually  allow  improved  therapy  of  the  44  million 
Americans  with  allergic  rhinitis,  the  9  million  with  asthma  and  the  22 
million  with  urticaria.   The  model  systems  employed  permit  sophisticated 
analysis  of  many  of  the  aspects  involved  _in  vivo.   Unquestionably,  our 
analysis  of  mucous  secretion  will  enable  us  to  approach  clinical  problems 
of  bronchorrhea  (asthma,  cystic  fibrosis,  chronic  bronchitis)  with  a 
greater  understanding  as  well  as  permit  _in  vitro  analysis  of  new  modalities 
of  therapy. 

The  observation  that  mast  cell  granules  may  elicit  delayed  inflammatory 
responses  may  provide  the  mechanisms  for  explaining  certain  long- 
observed  but  hitherto  poorly-understood  clinical  problems  such  as:  the 
epithelial  destruction  accompanying  asthma,  the  chronic  inflammation 
associated  with  perennial  rhinitis,  the  hyper-reactive  airways  disease  occur- 
ing  in  asthma  and  delayed  responses  seen  with  allergy  skin  tests.   Isolation 
of  a  low  molecular  weight  molecule  eliciting  this  late  phase  response  implies 
potent  chemotactic  properties  and  should  allow  studies  of  the  cellular  mechanisms 
of  chemotaxis  employing  a  biologically-relevant  molecule. 

Characterization  of  prostaglandin  generating  factors  elaborated  by 
human  tissues  undergoing  biologic  processes  (allergic  responses) 
will  permit  identification  of  the  role  these  factors  play  in  a  variety 
of  diseases  including  asthma.   Further,  purification  of  these  factors 
will  permit  a  detailed  analysis  of  the  mechanisms  of  prostaglandin 
synthesis. 

20-75 


Project  No.  Z01  AI  00154-04  LCI 

Finally,  analysis  of  the  autonomic  responsiveness  of  allergic  and 
cystic  fibrosis  patients  enables  us  to  approach  these  diseases  with  a 
unique  appreciation  of  the  capacity  of  neurophysiologic  processes  to 
modulate  and  complicate  these  diseases.   Clearly  the  abnormalities 
uncovered  have  therapeutic  implications  in  both  the  CF  and  asthma  populations. 

In  summary,  the  Allergic  Diseases  Section  is  approaching  immediate 
hypersensitivity  at  several  levels  with  interests  in  both  clinical  and  basic 
areas.   This  approach  is  providing  a  mileau  in  which  results  are  rapidly 
transmitted  from  the  laboratory  bench  to  the  clinic  and  from  which  a 
finer  appreciation  of  disease  processes  is  evolving.   The  direction  in 
which  the  lab  is  headed  should  continue  to  translate  basic  observations 
to  enhanced  clinical  practice. 

PUBLICATIONS 

1.  Raphael,  G.D.,  Henderson,  W.R. ,  and  Kaliner,  M. :  Isolation  of 
membrane  bound  rat  mast  cells.  Exp.  Cell  Res.  115:428-431,  1978. 

2.  Kaliner,  M. :   Human  lung  tissue  and  anaphylaxis:   The  effect  of 
histamine  upon  the  immunologic  release  of  mediatos,  Am.  Rev.  Resp . 
Pis.   118:  1015-1022,  1978. 

3.  Gallin,  J. I.,  Elin,  R.J.,  Hubert  R.T.,  Fauci,  A.S.,  Kaliner,  M.A. , 
and  Wolff,  S.M. :  Efficacy  of  ascorbic  acid  in  the  Chediak-Higashi 
Syndrome:   Studies  in  humans  and  mice.   Blood.  53:  226-234,  1979. 

4.  Platshon,  L.,  and  Kaliner,  M. :   The  effect  of  the  immunologic 
release  of  histamine  upon  human  lung  cyclic  nucleotide  levels  and 
prostaglandin  generation.  J  Clin.  Invest.   62:1113-1121,  1978. 

5.  Henderson,  W.R.,  and  Kaliner,  M. :   Mast  cell  granule  peroxidase: 
Location,  secretion  and  SRS-A  inactivation.  J_.    Immunol.   122:1322-1328, 
1979. 

6.  Henderson,  W.R. ,  Shelhamer,  J.E. ,  Reingold,  D.B.,  Smith,  L.J.,  Evans, 
R. ,  and  Kaliner,  M. :   Alpha  adrenergic  hyperresponsiveness  in 
asthma  -  analysis  of  vascular  and  pupillary  responses.   N.  Engl.  J. 
Med.,  300:  642-647,  1979. 

7.  Metcalfe,  D.D.,  Corash,  L.M.  and  Kaliner,  M. :  Type  II  arylsul- 
fatases  of  human  platelets:  identification  and  characterization. 
Immunology,  in  press. 

8.  Kaliner,  M. :  Human  Lung  tissue  and  anaphylaxis:  Cyclic  nucleotide 
response  and  prostaglandin  synthesis.   12th  Symposium  of  Collegium 
Internationale  Allergologicium.   Karger  Press.  In  Press. 


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Project  No.  Z.01  AI  00154-0.4  LCI 

9.  Steel,  L. ,  Platshon,  L. ,  and  Kaliner,  M. :  Prostaglandin  generation 
by  human  and  guinea  pig  lung  tissue.  J_;_  Allergy,  Clin.  Immunol. , 
in  press. 

10.  Kaliner,  M. :  Prostaglandins  and  allergic  inflammation.   Elsevier  Press. 
In  press. 

11.  Kaliner,  M. :  Mast  cell  derived  mediators  and  bronchial  asthma,  in  press. 


20-77 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  Al  00155-04  LCI 


PERIOD  COVERED 

October  1,  1977  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 


Phagocyte  Cell  Function 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS, 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:       John  I.  Gall in 

Other:     Steven  Whited 
John  Davis 
Anthony  S.  Fauci 
Charles  H.  Kirkpatrick 

Bruce  Seligmann 
Deborah  H.  Cotton 
Mark  Fletcher 
Haig  Donabedian 
Michael  M.  Frank 


AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 


Head,  Bacterial  Disease  Section 

Clinical  Associate 

Guest  Worker 

Head,  Clinical  Physiology  Section 

Head,  Clinical  Allergy  and 

Hypersensitivity  Section 
Staff  Fellow 
Clinical  Associate 
Clinical  Associate 
Clinical  Associate 
Chief,  Laboratory  of  Clinical 

Investigation 


LCI 

NIAID 

LCI 

NIAID 

LCI 

NIAID 

LCI 

NIAID 

LCI 

NIAID 

LCI 

NIAID 

LCI 

NIAID 

LCI 

NIAID 

LCI 

NIAID 

LCI 

NIAID 

cooperating  UNITS  (if  anykr.    David   Ailing    (NIAID,    NIH)  ,    E.    K.    Gall  in    (Div.    Exp.    Hem., 
Armed   Forces   Radio.    Biol.    Res.    Inst.),    E.    B.    Cramer    (Dept.    Anat.,    Downstate  Med. 
Ctr.),    M.    Klempner   and   B.    Babior    (Dept.    Med.,    Tufts-New  Engl.    Med.    Ctr.),    E. 
Schiffmann    (NIDR,    NIH) 


LAB/BRANCH 

Laboratory  of  Clinical  Investigation 


bacterial  Disease  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland 


TOTAL  MANYEARS: 

4   1/12 


PROFESSIONAL: 

4   1/12 


OTHER: 
0 


CHECK  APPROPRIATE  BOX(ES) 
g  (a)   HUMAN  SUBJECTS 


S  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


i2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords)  ,  ,  . 

The  mechanism  of  leukocyte  activation  by  chemotactic  factors  has  been  studied 
using  electrophysiology,  fluorescent  probe,  surface  charge  and  ultrastructural 
techniques. 

Studies  assessing  the  mechanism  of  modulating  leukocyte  locomotion  indicate 
that  limited  secretion  of  specific  granules,  which  accompanies  chemotaxis,  is 
associated  with  increased  cell  adhesiveness  and  increased  availability  of  chemo- 
attractant  receptors.   Vigorous  exocytosis  is  associated  with  depressed  chemo- 


taxis, decreased  availability  of  chemoattractant  receptors,  chemoattractant 
hydrolysis  by  secreted  products  and  markedly  increased  cell  adherence  and  aggre- 
gation.  Human  pyrogen  has  been  shown  to  be  a  potent  stimulator  of  neutrophil 
exocytosis  and  activation  of  the  hexose  monophosphate  shunt. 

Studies  of  the  two  populations  of  neutrophils  we  had  identified  previously  in- 
dicate that  during  the  neutropenia  that  follows  in  vivo  endotoxin  or  hemodialy- 
sis, a  subpopulation  of  neutrophils  with  poorly  demonstrable  Fc  receptors  is  the 
predominant  neutrophil  left  in  the  circulation.   Clinical  studies  assessing  the 
effect  of  pharmacologic  agents  on  the  neutrophil  subpopulations  in  normal  sub- 
jects,  patients  with  recurrent  infection  and  host  defense  defects  are  underway. 


PHS-6040 
(Rev.  10-76) 


20-78 


Project  No.  Z01  Al  00155-04  LCI 
Project  Description 
Objectives: 

1)  Study  the  mechanism  of  leukocyte  chemotaxis. 

2)  Study  the  phenomenon  of  deactivation  of  leukocyte  chemotaxis  and  to  relate 
this  to  the  pathophysiology  of  leukocyte  dysfunction  syndromes. 

3)  Develop  new  techniques  to  quantitate  the  chemotaxis  and  the  spreading  of 
living  phagocytes. 

4)  Study  the  role  of  cations  in  leukocyte  activation  with  particular  emphasis 
on   the  utilization  of  indirect  probes  of  membrane  potential  to  study  the 
relationship  between  changes  in  membrane  potential  and  initiation  of  neutro- 
phil motility,  phagocytosis,  degranulation  and  superoxide  generation. 

5)  Study  neutrophil  subpopulations  in  normal  subjects  and  in  patients  with 
neutrophil  dysfunction. 

6)  Study  the  mechanism  of  neutrophil  margination  and  aggregation  at  capillary 
beds  prior  to  cell  migration  into  tissues. 

7)  Study  the  mechanism  of  mobilization  and  secretion  of  neutrophil  lysosomal 
granules,  study  the  relationship  of  the  secretory  process  to  neutrophil 
function  and  dysfunction  and  to  study  how  neutrophil  secretory  products  may 
influence  homeostasis. 

8)  Characterize  and  define  the  mechanism  of  abnormal  neutrophil  chemotaxis 
following  thermal  injury. 

9)  Study  the  role  of  fibroblast  secretory  products  in  the  inflammatory 
response. 

10)  Study  the  effects  of  pharmacologic  agents  on  leukocyte  function  in  vitro 
and  in  vivo. 

11)  Assess  the  chemotactic  activity  of  histamine  for  eosinophils  in  vivo. 

Methods  Employed 

Peripheral  blood  leukocytes  were  separated  from  heparinized  whole  blood  by 
dextran  sedimentation  and  Hypaque-Ficoll  separation.   Chemotaxis  was  evaluated 
using  a  radioassay  employing   Cr  labeled  leukocytes  and  a  double  micropore 
filter  system  or  a  single  filter  measuring  the  distance  migrated  into  the  filter 
by  the  population  of  responding  cells.   Cell  adherence  was  measured  by  quanti- 
tating  the  number  of  leukocytes  adhering  to  plastic  surfaces  or  to  nylon  wool 
columns.   Phagocytosis,  bactericidal  capacity,  nitroblue  tetrazolium  dye  re- 
duction, hexose  monophosphate  shunt  activity  and  superoxide  generation  were 

20-79 


Project  No.  Z01  Al  00155-04  LCI 
measured  using  standard  techniques.   PMN  receptors  for  the  chemoattractant 
f-met-leu-phe  were  quantitated  as  published  previously. 

The  electrophysiology  of  cultivated  human  macrophages  was  studied  using 
standard  intracellular  recording  techniques.  The  fluorescent  probe  dipentylo- 
xacarbocyanine  and  the  radioisotope  trimethylphosphonium  were  used  as  indirect 
probes  of  membrane  potential  changes  in  neutrophils.  Leukocyte  surface  charge 
was  measured  with  a  Zeiss  cytopherometer .  Intracellular  calcium  was  localized 
in  human  neutrophils  by  the  intracellular  precipitation  of  pyroantimonate  anion 
followed  by  microprobe  analysis  and  studies  with  the  metal  chelators  (EDTA  and 
EGTA) . 

The  cytoskeleton  of  polymorphonuclear  leukocytes  was  studied  during 
conditions  of  chemotaxis  and  chemokinesis  using  0.45  ym  micropore  filters. 
These  small  filters  impede  leukocyte  migration  but  permit  pseudopod  penetration 
and  a  fixed  orientation  of  leukocytes  is  thereby  established.   Orientation  of 
living  cells  was  monitored  by  well  established  techniques .   Secretion  of  leuko- 
cyte granule  enzymes  were  monitored  by  standard  spectrophotometric  assays. 
Polymorphonuclear  leukocyte  granules  were  separated  and  fractionated  using 
sucrose  gradient  techniques  and  various  enzyme  markers  were  utilized  to  identify 
granule  types. 

Neutrophils  were  fractionated  into  subpopulations  based  on  their  ability 
to  rosette  IgG  coated  erythrocytes.   In  some  studies,  in  normal  human  volunteers, 
the  effects  of  intravenous  endotoxin  on  neutrophil  subpopulations  was  examined. 

Major  Findings 

1.  Technologic  advances  have  been  made  for  quantitating  leukocyte  chemo- 
taxis, and  bactericidal  activity.   Using  an  Optomax  image  analyzer  the  amount 
of  time  required  to  quantitate  the  number  of  cells  migrating  a  distance  into  a 
micropore  filter  or  the  number  of  bacteria  on  a  pour  plate  has  been  reduced  by 
90%.   In  addition,  the  image  analyzer  has  made  possible  a  quantitative  assay 
of  leukocyte  spreading.   Preliminary  studies  indicate  the  analyzer  will  make 
possible  the  tracking  of  moving  cells  thereby  providing  quantitative  information 
on  initial  rates  of  locomotion  (Ailing) . 

2.  During  chemotaxis  in  vitro  and  exudation  in  vivo  neutrophils  secrete 
their  granule  contents  and  this  secretion  is  preferential  for  specific  granules. 
It  has  been  shown  that  this  secretory  event  modulates  chemotactic  responsiveness. 
With  limited  exocytosis  there  is  increased  chemoattractant  binding  to  the  cell 
membrane  and  this  increased  binding  of  chemoattractants  increases  availability 

of  chemoattractant  receptors  without  altered  affinity  of  binding.   Preliminary 
data  indicates  the  increased  receptor  availability  is  related  to  translocation 
of  specific  granule  membrane,  which  contains  the  chemoattractant  receptors,  to 
the  cytoplasmic  membrane.   This  may  provide  the  mechanism  for  membrane 
turnover  during  sustained  locomotion.   Following  vigorous  exocytosis  both  chemo- 
taxis and  cell  orientation  in  a  gradient  of  chemoattractant  are  inhibited. 
This  inhibition  is  related  to  decreased  binding  of  a  chemotactic  factor  to  the 
cell.   The  latter  has  been  associated  with  decreased  peptide  affinity  and 
increased  hydrolysis  of  chemoattractant  by  granule  products  secreted  extracellu- 

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Project  No.  Z01  Al  00155-04  LCI 
larly  (Fletcher,  Schif fmann) . 

3.  Exocytosis  of  neutrophil  granules  in  vitro  increases  neutrophil 
adhesiveness  and  aggregation.   This  has  been  related  to  neutralization 

of  the  net  negative  surface  charge  of  the  cell  membrane  and  facilitation  of 
cell-cell  and  cell-surface  adhesion.   Treatment  of  neutrophils  with  neuraminidase 
or  poly-1-lysine  also  increases  their  adhesiveness  without  causing  degranulation. 
In  addition,  submembranous  deposition  of  cations  (calcium)  at  the  leading  edge 
of  cells  in  a  gradient  of  chemoattractant  was  observed  and  speculated  to  facil- 
itate fusion  of  intracellular  granules  with  the  cytoplasmic  membrane.   In  this 
regard,  it  is  of  interest  that  the  specific  granules,  which  are  most  accessible 
to  extracellular  release,  are  more  negatively  charged  than  the  azurophil  granules 
(Gallin,  Cramer) . 

4.  Using  fluorescent  cyanine  dyes  and  tritiated  trimethyl  phosphonium 
ions  chemoattractants  and  degranulating  stimuli  were  shown  to  stimulate  membrane 
potential  changes  in  neutrophils.   The  potential  changes  in  neutrophils  are 
similar  to  those  observed  in  macrophages  using  direct  intracellular  recording 
techniques.   The  data  using  degranulating  stimuli  indicate  low  concentrations 

of  degranulating  stimuli  increase  f-met-leu-phe  receptor  efficacy  for  eliciting 
membrane  potential  changes  as  monitored  with  a  fluorescent  dye.   Vigorous  secre- 
tion inhibits  receptor  efficacy.   These  latter  observations  support  and  extend 
the  data  on  the  effect  of  degranulating  stimuli  on  chemoattractant  receptor 
availability  and  on  chemotactic  responsiveness.   In  addition,  the  data  suggest 
that  alteration  of  neutrophil  plasma  membrane  ion  permeability  or  the  concen- 
tration of  intracellular  ions  by  degranulating  stimuli  modulates  the  locomotory 
responsiveness  of  the  cell. 

5.  Assessment  of  chemoattractant  elicited  membrane  potential  changes  using 
indirect  probes  indicates  that  neutrophils  from  patients  with  chronic  granulo- 
matous disease  have  a  major  abnormality.   Neutrophils  from  other  patients  with 
chemotactic  defects  did  not  show  any  such  abnormality  (Seligmann) . 

6.  Highly  purified  leukocyte  pyrogen  causes  selective  secretion  of  human 
neutrophil  specific  granules  in  vitro  and  rabbit  neutrophils  in  vivo .   In 
addition,  pyrogen  stimulates  neutrophil  nitroblue  tetrazolium  dye  reduction, 
hexosemonophosphate  shunt  activation  and  superoxide  generation  (Klempner) . 

7 .  The  acquired  defect  of  neutrophil  locomotion  seen  following  thermal 
injury  precedes  sepsis  and  was  shown  to  be  a  function  of  burn  wound  area  and 
to  be  linearly  related  to  the  degree  of  lysosome  lost  from  the  neutrophil.   A 
rabbit  model  for  thermal  injury  has  been  developed  to  enable  further  study  of 
this  acquired  chemotactic  defect  (Davis) . 

8.  Twenty  minutes  following  intravenous  administration  of  E.  coli  endo- 
toxin to  normal  volunteers,  during  the  period  of  neutropenia,  the  predominant 
circulating  neutrophil  is  a  "subpopulation"  without  readily  demonstrable  IgG 
(Fc)  receptors.   Plasma  obtained  at  the  time  of  neutropenia  increases  neutro- 
phil adhesiveness.   In  related  studies,  twenty  minutes  after  initiating  hemo-    , 
dialysis  in  patients  with  chronic  Schizophrenia,  also  during  a  period  of  neutro- 


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Project  No.  Z01  Al  00155-04  LCI 

penia,  the  remaining  circulating  neutrophils  are  significantly  enriched  with  a 
subpopulation  of  neutrophils  with  poorly  demonstable  Fc  receptors.   Plasma 
obtained  at  this  time  during  hemodialysis  increased  the  adhesiveness  and 
aggregation  of  control  leukocytes  (Klempner,  Cotton). 

9.  Hydrocortisone  sodium  succinate  reversibly  inhibits  adherence  of  IgG 
sensitized  erythrocytes  to  human  peripheral  blood  neutrophil  monolayers  suggest- 
ing hydrocortisone  interferes  with  the  availability  of  the  neutrophil  Fc  recep- 
tor for  binding  (Klempner) . 

10.  Normal  human  neutrophils  were  separated  into  two  populations  based 
on  their  ability  to  rosette  human  IgG  coated  erythrocytes  and  tested  for  their 
to  rosette  complement-coated  erythrocytes.   Both  neutrophil  populations  formed 
rosettes  with  complement-coated  erythrocytes  equally  well.   Moreover,  neither 
population  of  cells  displayed  those  complement  receptors  felt  to  be  markers  of 
immature  granulocytes  (Whited,  Frank) . 

11.  Administration  of  prednisone  to  normal  human  subjects  inhibits  in 
vitro  neutrophil  and  eosinophil  adherence  to  nylon  wool.   In  vivo  prednisone 
also  inhibits  eosinophil  but  not  neutrophil  chemo taxis  (Fauci) . 

12.  Collection  of  human  neutrophils  by  filtration  leukapheresis  for  sub- 
sequent intravenous  administration  results  in  functional  changes  of  the  neutro- 
phils attributable  to  degranulation  and  secretion  of  granule  contents.   Col- 
chicine pretreatment  of  filtration  leukapheresis  donors  significantly  reduces 
these  adherence  induced  changes  (Wright) . 

13.  Human  fibroblasts  cultured  in  vitro  secrete  at  least  two  chemoattrac- 
tants  which,  based  on  their  elution  from  G-75  Sephadex  chromatography  columns, 
have  molecular  weights  of  greater  than  150,000  and  less  than  5,000  daltons . 
These  attractants  appear  distinct  from  collagen  and  other  previsouly  described 
chemotactic  factors.   They  are  protein  in  nature  and  attract  both  polymorpho- 
nuclear leukocytes  and  monocytes.   Elaboration  of  this  material  in  vitro  is 
inhibited  by  colchicine  (10  M)  and  hydrocortisone  sodium  succinate  (1  mM) 
(Gallin) . 

14.  In  studies  of  patients  with  recurrent  pyrogenic  infections,  a  patient 
with  abnormal  neutrophil  chemotaxis  whose  neutrophils  are  missing  a  membrane 
glycoprotein  and  impaired  neutrophil  spreading  has  been  identified  (Babior) . 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute 

The  accumulation  of  leukocytes  at  inflammatory,  immune  and  allergic  sites 
is  critical  for  appropriate  responses.   Understanding  the  physiologic  basis 
for  events  regulating  these  processes  will  provide  the  basis  for  therapeutic 
manipulation. 

One  of  the  first  steps  in  leukocyte  mobilization  from  the  blood  stream  is 
increased  cell  adhesiveness  to  the  endothelium.   This  is  followed  by  local 


20-8^ 


Project  No.  Z01  Al  00155-04  LCI 

leukocyte  aggregation  and  then  diapedesis.   Our  finding  that  limited  degranu- 
lation  and  exocytosis  of  intracellular  granules  markedly  enhances  these  events, 
together  with  the  observation  that  human  pyrogen  initiates  these  processes, 
provides  a  clue  as  to  what  controls  margination  and  then  migration  of  leuko- 
cytes from  the  blood  stream.   The  related  observation  that  vigorous  degranu- 
lation  in  vitro  inhibits  chemotaxis  suggested  that  some  acquired  chemotactic 
defects  relate  to  excessive  degranulation  in_  vivo.   In  support  of  this  is  our 
finding  that  following  thermal  injury  the  severity  of  the  acquired  chemotactic 
defect  is  linearly  related  to  the  amount  of  intracellular  lysozyme  released. 
Clinically,  it  is  of  interest  that  the  degranulation  and  defective  chemotaxis 
precedes  the  severe  and  often  lethal  pyrogenic  infections  that  follow  thermal 
injury. 

In  related  studies  we  confirmed  that  neutrophil  specific  granules  contain 
lactoferrin  and  showed  this  is  secreted  when  cells  are  incubated  with  pyrogen. 
Lactoferrin  irreversibly  binds  iron  which  is  then  sequestered  in  the  reticu- 
loendothelial system.   Another  specific  granule  component,  B  „  binding  protein, 
may  effect  B  „  related  events  when  secreted  from  the  neutrophil.   Thus,  the 
potential  regulatory  role  of  neutrophil  products  on  a  number  of  systems  is 
interesting  and  currently  under  investigation. 

Two  observations  from  our  studies  of  the  mechanism  of  the  phenomena  of 
degranulation,  cell  adhesiveness  and  chemotaxis  may  be  particularly  important 
to  the  cell  biology  of  chemotaxis  and  perhaps  relevant  to  clinical  studies  as 
well.   We  have  shown  that  leukocyte  adherence,  aggregation  and  secretion  may 
be  under  the  modulating  influence  of  electrostatic  forces.   Development  of 
techniques  for  controlling  these  forces  in  vivo  may  have  potential  clinical 
use.   Our  data  that  neutrophil  specific  granules  are  a  potential  source  of  new 
cytoplasmic  membrane  and  chemoattractant  receptors  is  intriguing  in  terms  of 
understanding  the  basis  for  membrane  turnover  during  chemotaxis.   These  studies 
need  to  be  extended  and  explored  further. 

The  studies  of  the  effect  of  steroids  on  PMNs  are  interesting  and  have 
shown  that  prednisone  iri  vivo  inhibits  both  neutrophil  and  eosinophil  adherence 
and  eosinophil  chemotaxis.   Hydrocortisone  sodium  succinate  also  blocks  the 
expressability  of  neutrophil  Fc  receptors  and  inhibits  chemotactic  factor  induced 
changes  of  neutrophil  and  macrophage  membrane  potential.   These  data  contribute 
to  our  understanding  of  the  physiologic  basis  for  steroid  action  on  these  cells. 

The  use  of  fluorescent  carbocyanine  dyes  to  measure  membrane  potential  is 
emerging  as  a  rapid  test  of  cell  reponsiveness  that  appears  to  be  as  reliable 
and  easier  to  use  than  other  indirect  probes  of  membrane  potential.   The  demon- 
stration of  a  severe  abnormality  of  chronic  granulomatous  disease  neutrophils 
using  this  test  may  provide  a  new  simple  rapid  diagnostic  test  for  this  disease. 
In  addition,  the  implications  that  the  observed  abnormality  in  chronic  granu- 
lomatous disease  reflects  abnormal  ion  flux  studies,  perhaps  related  to  abnormal 
activation  mechanisms  provides  new  insights  into  the  nature  of  the  defect. 

The  continuation  of  our  studies  of  neutrophil  subpopulations  has  indicated 
that  one  population  of  cell  (PMNs  with  readily  demonstrable  Fc  receptors)  is 

20-83 


Project  No.  Z01  Al  00155-04  LCI 

particularly  available  for  margination.   We  are  presently  extending  these  studies 
of  neutrophil  subpopulations  to  explore  their  kinetics  of  circulation,  and 
their  functional  role.   In  addition,  the  effect  of  various  disease  states  and 
pharmcologic  agents  on  these  two  neutrophil  populations  in  man  and  in  animals 
is  under  study. 

Prosposed  Course 

We  plan  to  continue  some  of  the  studies  outlined  above. 

Publications 

1.  Wright,  D.  G.,  Kauffmann,  J.  C,  Terpstra,  G.  K.,  Graw,  R.  G., 
Deisseroth,  A.  B.,  and  Gallin,  J.  I.   Mobilization  and  exocytosis  of 
specific  (secondary)  granules  by  human  neutrophils  during  adherence  to 
nylon  wool  in  filtration  leukapheresis .   Blood.   52:770-782,  1978. 

2.  Wright,  D.  G.,  Ungerleider,  R.  S.,  Gallin,  J.  I.,  and  Deisseroth,  A.  B. 
Pretreatment  of  leukaphersis  donors  with  colchicine.   Blood .   52:783- 
792,  1978. 

3.  Gallin,  J.  I.,  Wright,  D.  G.,  and  Schiffmann,  E.  Role  of  secretory 
events  in  modulating  human  neutrophil  chemotaxis.  J.  Clin.  Invest. 
62:1364-1374,  1978.  "~ " 

4.  Klempner,  M.  S.  and  Gallin,  J.  I.   Inhibition  of  neutrophil  Fc 
receptor  function  by  corticosteroids.   CI in .  Exp .  Immunol .   34:137- 
143,  1978. 

5.  Gallin,  J.  I.,  Elin,  R.  J.,  Hubert,  R.  T.,  Fauci,  A.  S.,  Kaliner,  M.  A., 
and  Wolff,  S.  M. :  Efficacy  of  ascorbic  acid  in  the  Chediak-Higashi  syn- 
drome:  Studies  in  humans  and  mice.   Blood.   53:226-234,  1979. 

6.  Clark,  R.  A.  F.,  Gallin,  J.  I.  and  Fauci,  A.  S.   Effects  of  in  vivo 
prednisone  on  in  vitro  eosinophil  and  neutrophil  adherence  and  chemo- 
taxis.  Blood.   53:633-641,  1979. 

7.  Sobel,  J.  D.  and  Gallin,  J.  I.:   Polymorphonuclear  leukocyte  and 
monocyte  chemoattractants  produced  by  human  fibroblasts.   J.  Clin. 
Invest.   63:609-618,  1979. 

8.  Gallin,  J.  I.   The  Compromised  Host.   In  P.  B.  Beeson  and  W.  McDermott 
(Eds.):   Textbook  of  Medicine,  15th  Edition,  pp.  145-153,  1979. 

9.  Wright,  D.  G.  and  Gallin,  J.  I.   Secretory  responses  of  human  neutro- 
phils: Exocytosis  of  specific  (secondary)  granules  by  human  neutrophils 
during  adherence  in  vitro  and  during  exudation  in  vivo .   J .  Immunol . 
123:285-294,  1979.  , 


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Project  No.  Z01  Al  00155-04  LCI 

10.  Cramer,  E.  B.  and  Gallin,  J.  I.:   Localization  of  submembranous 
cations  to  the  leading  end  of  human  neutrophils  during  chemotaxis. 
J.  Cell  Biol.   In  Press. 

11.  Gallin,  J.  I.,  Gallin,  E.  K.,  and  Schiffmann,  E.   The  Mechanism  of 
Leukocyte  Chemotaxis.   In  Proceedings  of  the  International  Congress  of 
Inflammation.   Raven  Press,   In  Press. 

12.  Gallin,  E.  K.,  Seligmann,  B.  and  Gallin,  J.  I.  Alteration  of  macrophage 
and  monocyte  membrane  potential  by  chemotactic  factors.  In  R.  Van  Furth 
(Ed.):   Mononuclear  Phagocytes.   Martinus  Nijhoff  B.  V.,  The  Hague. 

In  Press. 

13.  Seligmann,  B.  and  Gallin,  J.  I.   Secretagogue  modulation  of  the  response 

of  human  neutrophils  to  chemoattractants :  studies  with  a  membrane  potential 
sensitive  cyanine  dye.   Molecular  Immunology.   In  Press. 

14.  Whited,  S.  C.  and  Gallin,  J.  I.   Neutrophil  chemotaxis.   Interna. 
J.  Dermatol.   In  Press. 

15.  Davis,  J.  M.  and  Gallin,  J.  I.   The  Neutrophil.   The  Cell  Biology  of 
Immunity  and  Inflammation.   Ed.  by  Oppenheim,  J.  J.,  Rosenstreich,  D.  L. 
and  Potter,  M.   Elsevier  North-Holland.   In  Press. 

16.  Klempner,  M.  S.,  Dinarello,  C.  A.,  Henderson,  W.  R.  and  Gallin,  J.  I. 
Stimulation  of  neutrophil  oxygen-dependent  metabolism  by  human  leuko- 
cytic pyrogen.   J.  Clin.  Invest.   In  Press. 

17.  Gallin,  J.  I.   The  Cell  Biology  of  Leukocyte  Chemotaxis.   In. 
G.  Weissmann  Ed.   Handbook  of  Inflammation.   New  York,  Elsevier 
North  Holland.   In  Press. 

18.  Schiffmann,  E.  and  Gallin,  J.  I.   Biochemistry  of  Phagocyte  Chemotaxis. 
In  E.  R.  Stadtman  Ed.  Cellular  Regulation.   New  York,  Academic  Press. 
In  Press. 


20-85 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  AI   00188-01   LCI 


PERIOD    COVERED 


October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT 


characters  or  less 


Rapid  Diagnosis  of  Infections  by  Enzyme-Linked  Immunoadsorbent  Assays 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:       Stephen  E.  Straus 
Other:    Richard  A.  Berg 


Senior  Investigator 
Medical  Virology  Section 

Clinical  Associate 


LCI  NIAID 


COOPERATING  UNITS  (if  any) 

J.E.  Bennett  (LCI,  NIAID),  R.  Brown  (LCI,  NIAID),  P.  Pizzo  (LPO,  NCI), 
B.  Murphy  (LID,  NIAID),  and  S.  Rennard  (LDBA,  NIDR) 


LAB/BRANCH 


Laboratory  of  Clinical  Investigation 


Medical  Virology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland   20205 


TOTAL  MANYEARSi 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 

C  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Enzyme-linked  immunoadsorbent  assays  have  been  developed  to  detect  influenza  A 
hemagglutinin,  Candida  cell  wall  mannan,  human  adenoviruses,  herpes  simplex 
virus,  and  antibodies  to  pneumococci.   Current  efforts  are  designed  to  augment 
the  sensitivity  of  the  assays  and  establish  their  utility  in  clinical  and 
research  applications. 


20-86 


PHS-6040 
(Rev.  10-76) 


Project  No.    Z01  AI   00188-01  LCI 
Project  Description: 
Objectives: 

1)  To  develop  an  assay  which  will  rapidly,  simply  and  reliably  detect 
Candida  antigenemia,  with  the  aim  of  identifying  those  patients  who  may 
benefit  from  early  therapy  with  amphotericin  B. 

2)  To  develop  a  rapid  assay  for  influenza  infection  in  order  to  iden- 
tify individuals  and  patient  groups  which  may  benefit  from  amantadine 
chemoprophylaxis . 

3)  To  develop  rapid,  more  sensitive  assays  to  detect  adenoviruses  and 
herpes  viruses  in  body  fluids  and  in  research  specimens. 

4)  To  develop  a  convenient  assay  for  the  development  of  antibodies 
directed  against  specific  strains  of  pneumococci. 

5)  To  detect  group  A  streptococcal  polysaccharide  in  throat  swabs  of 
patients  with  pharyngitis.   The  rapidity  of  the  test  would  eliminate  the 
1-2  day  wait  for  identification  by  routine  microbiologic  methods. 

Methods  Employed: 

Either  direct  or  competitive  assays  are  employed.  Briefly,  the  desired 
antigen  is  adsorbed  to  the  surface  of  plastic  microtiter  wells.  Specific 
antisera  is  added  and  binds  to  the  antigen  where  present.  Development  of 
the  antigen-antibody  reaction  is  detected  by  addition  of  a  second  enzyme- 
linked  antibody  directed  at  the  first  antibody.  The  reaction  is  detected 
colorimetrically  by  addition  of  a  chromogenic  substrate  specific  for  the 
enzyme. 

Major  Findings: 

1)  Candida:   An  ELISA  has  been  developed  which  detects  mannan  at  the 
nanogram  level.   A  limited  retrospective  clinical  trial  has  been  completed. 
Briefly,  sera  of  patients  with  proven  disseminated  candidiasis  and  those 
without  candidiasis  were  tested  in  a  blinded  fashion.   The  assay  discerned 
significant  differences  in  level  of  Candida  mannan  between  the  groups. 

2)  An  ELISA  capable  of  detecting  less  than  1  hemagglutinin  unit  of 
influenza  A  virus  has  been  developed.   The  assay  is  currently  being  tested 
in  experimental  infections  in  ferrets. 

3)  Adenoviruses:   An  ELISA  has  been  developed  to  reliably  detect  all 
of  26  types  of  human  adenoviruses  tested  thus  far.   Modifications  of  the 
assay  are  being  attempted  which  would  permit  virus  typing. 

4)  Herpesviruses:   ELISA  tests  for  both  types  of  herpes  simplex  are 
being  developed  using  monoclonal  type  specific  antisera. 


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Project  No.    Z01  AI   00188-01   LCI 

5)  Pneumococci:   A  successful  assay  is  currently  being  tried  in 
experimental  pneumococcal  infection  of  guinea  pigs.   A  pilot  study  has  been 
completed  in  which  the  ELISA  was  used  to  quantitate  the  antibody  response  to 
pneumococcal  polyvalent  vaccine  in  six  volunteers.   The  assay  results  cor- 
related very  highly  with  results  obtained  by  standard  radioimmunoassay. 

6)  Streptococci:   An  assay  using  monoclonal  specific  antisera  is  being 
developed.   The  test  thus  far  is  insufficiently  sensitive. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

Rapid  assay  for  a  variety  of  antigens  and  antibodies  has  broad  appli- 
cability to  both  clinical  and  research  areas.   The  tests  currently  being 
developed  will  speed  up,  simplify,  and  reduce  the  cost  of  detecting  infec- 
tions.  At  the  same  time,  they  will  permit  clinical  decisions  regarding 
therapy  to  be  soundly  based  at  an  earlier  time  in  the  course  of  an  illness 
than  is  currently  practiced. 

Proposed  Course: 

Once  the  ELISA  tests  are  fully  developed  and  their  sensitivity  and 
specificity  defined,  they  will  be  examined  prospectively  with  patient  ma- 
terial.  Their  ability  to  correctly  identify  infectious  agents  will  be 
tested  by  comparison  with  the  results  of  traditional  methods.   Assays  for 
viral  agents  will  also  be  applied  to  routine  testing  of  research  materials 
generated  in  the  study  of  virus  pathogenesis  and  treatment. 

Publications: 

1.  Weiner,  M.H.,  Yount,  W.J.:   Mannan  antigenemia  in  the  diagnosis  of 
invasive  Candida  infections.   J_.  Clin.  Invest .  58:1045,  1976. 

2.  Voller,  A.,  et  al:   Microplate  enzyme  immunoassays  for  the  immuno- 
diagnosis  of  virus  infections.   In  N.R.  Rose  and  H.  Friedman  (eds.) 
Manual  of  Clinical  Immunology.   ASM,  Washington,  D.C.,  1976,  p  506. 

3.  Yolken,  R.H.,  et  al:  Measurement  of  rotavirus  antibody  by  an  enzyme- 
linked  immunoadsorbent  assay  blocking  assay.  ^J.  Clin.  Microb .  8:283, 
1978. 

4.  Segal,  E.,  Berg,  R.A.,  et  al:   Detection  of  Candida  antigens  in  sera  of 
patients  with  candidiasis  by  an  ELISA  test.   J_.  Clin-  Microb.   In  press. 

5.  Berg,  R.A. ,  et  al:   Type-specific  pneumococcal  antibody  measurement  with 
enzyme-linked  immunoadsorbent  assay.   In  preparation. 


10-88 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANC 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI   00189-01  LCI 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Clinical  and  Biochemical  Studies  of  Human  Enteral  Adenovirus 
Infections 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


S.E.  Straus 


Senior  Investigator 
Medical  Virology  Section 


LCI  NIAID 


COOPERATING  UNITS  (if  any) 

A.Z.  Kapikian  (LID,  NIAID) 

H.S.  Ginsberg  (Columbia  University) 


LAB/BRANCH 

Laboratory  of  Clinical  Investigation 


Medical  Virology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland   20205 


TOTAL  MANYEARS: 


PROFESSIONAL: 
1 


CHECK  APPROPRIATE  BOX(ES) 
8  (a)   HUMAN  SUBJECTS 


S  (b)  HUMAN  TISSUES 


(c)  NEITHER 


□  (a1  )  MINORS 


a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Enteral  adenoviruses  are  a  recently  defined  group  of  agents  which  cause  gastro- 
enteritis in  children.   They  differ  from  other  known  human  adenoviruses  in  their 
inability  to  be  propagated  in  tissue  culture.   Adenoviruses  recovered  from  pa- 
tients will  be  examined  for  their  relation  to  known  respiratory  adenoviruses  by 
serologic,  DNA  hybridization,  restriction  endonuclease  and  protein  electrophor- 
esis studies.   The  mechanism  of  restriction  of  growth  of  these  agents  will  be 
examined  by  nucleic  acid  hybridization,  immuno fluorescent  microscopy  and  AAV 
helper  studies.   Attempts  will  be  made  to  grow  the  viruses  in  tissue  culture 
using  adenovirus  transformed  cell  lines  and  a  series  of  early  and  late  tempera- 
ture sensitive  mutants  of  adenovirus  5  as  helpers.   Enteral  adenovirus-induced 
diarrheal  illness  will  be  studied  in  normal  volunteers.   The  features  of  the 
illness,  virus  shedding  and  immune  resolution  will  be  examined. 


20-89 


PHS-6040 
(Rev.  10-76) 


Project   No.         Z01      AI      00189-01     LCI 
Project  Description: 

Objectives: 

1)  To  define  the  clinical  spectrum  of  illness  induced  by  enteral  adeno- 
viruses in  both  naturally  occurring  and  experimental  infections. 

2)  To  define  the  biochemical  properties  of  these  adenoviruses  and  their 
relation  to  other  respiratory  adenoviruses. 

3)  To  develop  methods  of  promoting  the  growth  of  these  agents  in  tissue 
culture  so  that  further  studies  will  be  simplified. 

Methods  Employed: 

1)  Source  of  viral  agents:   Enteral  adenoviruses  will  be  obtained  from 
specimens  collected  during  natural  infections  at  Children's  Hospital  and 
identified  as  enteral  adenoviruses  by  their  EM  appearance,  reactivity  in 
ELISA  tests,  and  their  fastidious  behavior  in  standard  tissue  culture  lines. 

2)  In  vitro  culture  system:   Standard  primary  and  continuous  human  and 
simian,  adult  and  fetal  tissues  will  be  employed  for  attempted  virus  isola- 
tion.  A  variety  of  methods  will  be  employed  to  promote  the  efficient  repli- 
cation of  these  viruses  in  tissue  culture.   Stool  specimens  will  be  inocu- 
lated into  an  adenovirus  transformed  cell  line  (293)  with  the  hope  that  the 
integrated  viral  sequences  will  complement  the  presumably  restricted  func- 
tions of  the  enteral  viruses.   Similar  complementation  studies  will  be  at- 
tempted using  early  and  late  temperature-sensitive  mutants  of  adenovirus 
type  5  which  have  been  developed  at  Columbia  University.   Simian  cells  will 
be  coinfected  with  SV40  and  enteral  adenoviruses  to  determine  whether  the 
helper  function  that  SV40  provides  for  respiratory  adenoviruses  in  monkey 
cells  extends  to  these  agents  as  well. 

3)  Biochemical  Studies:   The  mechanism  of  restricted  growth  in  tissue 
culture  will  be  examined  by  performing  DNA-DNA  and  DNA-RNA  hybridizations  on 
infected  cell  extracts  using  in  vitro  labeled  DNA  probes.   Immunofluorescence 
microscopy  will  indicate  whether  adenovirus  T  antigen  or  late  proteins  are 
being  synthesized  in  the  restricted  system.   The  expression  of  early  adeno- 
virus function  will  be  assessed  by  testing  for  helper  activity  for  adeno- 
associated  viruses. 

4)  Clinical  Studies:  After  rigorous  safety  testing,  enteral  adenovirus- 
containing  stool  filtrates  will  be  innoculated  into  normal  adult  volunteers. 
The  ability  of  these  agents  to  reproduce  the  typically  mild  diarrheal  illness 
will  be  explored.   The  illness  will  be  characterized  by  close  clinical  obser- 
vation, virologic  and  serologic  studies  of  blood,  stool  and  respiratory 
secretions,  and  electron  microscopic  examination  of  stools. 


20-90 


Project  No.    Z01  AI   00189-01   LCI 
Major  Findings: 

Our  laboratory  has  to  date  developed  the  tools  necessary  to  perform 
these  studies.   We  are  now  proficient  with  the  maintenance  of  the  required 
cell  lines,  the  growth  and  replication  of  respiratory  adenoviruses,  labeling 
and  extraction  of  viral  nucleic  acids  and  proteins,  nucleic  acid  hybridiza- 
tion, restriction  endonuclease  analysis,  SDS-PAGE,  immunof luorescent  micros- 
copy, HA,  HI,  neutralization  and  ELISA  tests.   We  are  currently  awaiting  our 
first  stool  specimen. 

Proposed  Course: 

Detailed  investigation  of  the  viral  pathogenesis  and  host  defense 
mechanisms  will  depend  upon  our  ability  to  generate  substantial  quantities 
of  virus  and  reproduce  the  clinical  illness  in  volunteers.   Since  the  other 
major  viral  diarrhea  agents  (rotaviruses,  parvovirus-like  agents)  can  not  be 
grown  in  the  laboratory,  our  success  with  these  enteral  adenoviruses  may 
provide  the  model  for  the  study  of  human  viral  gastroenteritis. 

Publications: 

1.  Flewett,  T.H.,  et  al.:  Epidemic  viral  enteritis  in  a  long-stay 
children's  ward.   Lancet  1:4,  1975. 

2.  Richmond,  S.J.,  et  al.:   An  outbreak  of  gastroenteritis  in  young 
children  caused  by  adenoviruses.   Lancet  1:1178,  1979. 

3.  Dolin,  R. :   Viral  infections  of  the  gastrointestinal  tract.   In 
G.J.  Galasso,  et  al.  (eds.)  Antiviral  Agents  and  Viral  Diseases. 
Raven  Press,  New  York,  N.  Y. ,  1979,  p.  289. 

4.  Wadell,  G. :   Classification  of  human  adenoviruses  by  SDS-polyacrylamide 
gel  electrophoresis  of  structural  polypeptides.   Intervirology  11:47, 
1979.  ' 


20-91 


LABORATORY  OF  IMMUNOGENETIGS 

1979  Annual  Report 

Table  of  Contents 


Z01-AI 


Project  Number  Page 

Summary  Report  21-1 

00166-02       Chemical  Characterization  of  Rabbit  21-5 

Histocompatibility  Antigens 

0016  7-02       Mass  Spectrometry  and  Sequence  Analysis  21-8 

of  Polypeptides 

00168-02       Structure  of  Rabbit  Immunoglobulin  and  21-11 

Antibody  Heavy  and  Light  Chains 

00169-02       Primary  Structural  Analysis  of  Murine  21-13 

Transplantation  Antigens 

00170-02       Structural  Studies  on  Precursors  to  21-17 

Immunoglobulin  Molecules 

00171-02       Genetic  Studies  on  Rabbit  Immunoglobulins         21-19 
and  Other  Serum  Proteins 

00172-02       Carbohydrate  and  Glycoprotein  Antigens  of         21-22 
Microbial  Cell  Walls 

00173-02       Nonallelic  Expression  of  Genes  Encoding  Rabbit    21-24 
Immunoglobulins 

00180-01       Monoclonal  Antibodies  Directed  Against  Cell       21-27 
Surface  Proteins 

00191-01      Immunoregulation  of  T  Lymphocytes  -  The  Role     21-29 
of  Anti-idiotype  and  Immune  Complexes 


Annual  Report  of  the  Laboratory  of  mmunogenetics,  National  Institute  of 
Allergy  and  Infectious  Diseases,  NIH,  October  1,  1978  -  September  30,  1979 

Summary  Report 

The  Laboratory  of  Immunogenetics  continues  to  conduct  research  aimed 
at  elucidation  of  the  genetic  basis  of  immunologic  function.   Studies  on 
genetic  markers  of  immunoglobulins  have  concerned  the  expression  of  latent 
allotypes.   In  the  past  year  progress  has  been  made  on  structural 
determination  of  molecules  bearing  latent  allotypes  and  more  recently, 
interspecies  cell  hybrids  (mouse-rabbit)  are  being  utilized  as  a  source  of 
mRNA  which  will  be  used  to  initiate  molecular  biological  studies  which 
hope  to  elucidate  the  genetic  basis  for  latent  allotypes.   Studies  on  the 
structure  of  histocompatibility  molecules  continue;  the  280  residue  papain 
fragment  of  the  murine  K  molecule  is  near  completion  and  intense 
structural  analysis  of  molecules  from  this  and  other  mouse  MHC  haplotypes 
have  begun.   In  additon,  progress  has  been  made  on  studies  of  rabbit 
histocompatibility  antigens  this  year.   Other  areas  of  active  interest 
include  studies  of  cell  surface  molecules  using  hybridoma  reagents  which 
have  been  applied  with  some  effectiveness  in  our  H-2  studies.   Initially 
the  hybridoma  antisera  will  be  directed  against  T-cells  from  the  rabbit. 
Work  on  the  control  of  the  immune  response  by  T-cell  components  has 
recently  been  initiated  and  this  will  involve  a  chemical  and  biological 
approach  to  this  problem. 

Recent  additions  to  the  LIG  staff  include  Dr.  Lee  Maloy  a  Biochemist 
from  Case  Western  Reserve  University  and  Dr.  Thomas  Folks  who  joins  us 
from  the  Naval  Medical  Research  Unit  in  Bethesda.   Dr.  John  Sogn  has  been 
given  the  position  of  Chemist,  GS-13  after  serving  a  year  and  one-half  as 
Senior  Staff  Fellow  in  the  Laboratory.   Dr.  Blair  Fraser,  a  Staff  Fellow 
left  to  accept  a  permanent  position  with  the  Bureau  of  Biologies.   In 
addition  to  the  permanent  personnel  our  program  has  been  greatly  enriched 
by  participation  of  students  from  various  part-time  and  summer  programs. 

Research  Accomplishments 

Rabbit  Histocompatibility  Antigens.   Products  of  the  rabbit 
histocompatibility  genes  were  isolated  from  a  rabbit  lymphoid  tumor  cell 
line  (RL-5)  following  growth  in  culture  with  radioactive  amino  acids. 
Molecules  were  obtained  from  detergent  lysates  and  glycoprotein  fractions 
were  isolated  by  affinity  chromatography  using  immobilized  lentil  lectin. 
Chromatography  on  purified  sheep  anti-rabbit  beta-2-microglobulin  yielded 
a  43,000  dalton  fraction  which  was  noncovalently  associated  with 
beta-2-microglobulin.   Amino  terminal  sequence  analysis  allowed  assignment 
of  28  of  the  amino  terminal  35  residues.   These  data  revealed  89%  homology 
between  this  RLA-11  product  and  the  human  HLA-b7  molecule.   Similar 
comparison  with  the  mouse  H-2K  histocompatibility  antigen  yielded  a 
homology  of  82%.   Although,  beta-2-microglobulin  is  associated  with 
histocompatibility  antigens  of  several  types  (K,D,  and  L  in  the  mouse)  in 
other  species  only  a  single  molecular  species  could  be  detected  in  our 
studies  even  though  beta-2-microglobulin  was  the  basis  upon  which  our 
molecule  was  isolated.   The  extent  of  similarity  between  the  rabbit  and 
other  histocompatibility  cmplexes  is  currently  under  investigation. 

21-1 


Mass  Spectrometric  Analysis  of  Peptides.   Immunogenetic  studies  require 
fast,  reproducible  and  sensitive  methods  for  amino  acid  sequence  analysis 
of  proteins  which  are  not  intrinsically  radiolabelled.   One  possibility  to 
attain  this  goal  is  mass  spectrometric-gas  chromatograhic  (MS-GC) 
techniques.   Work  in  the  past  year  has  included  studies  on  a  method  which 
involves  digestion  of  polypeptides  into  dipeptides  from  the  amino  terminus 
and  identification  of  these  dipeptides  by  computer  aided  MS  methodology. 
More  recently,  the  use  of  an  enzyme  dipeptidyl  carboypeptidase  (DCP) ,  has 
been  investigated  for  use  with  these  techniques.   This  enzyme  cleaves 
dipeptides  from  the  carboxy  terminus.   It  is  anticipated  that  this  enzyme 
will  be  extremely  useful  either  as  an  adjunct  to  the  DAP  digestions,  or  as 
a  method  to  complement  normal  Edman  degradation.   Model  compounds  under 
study  at  present  include  rabbit  and  mouse  Ig  heavy  chains  as  well  as  rat 
beta-2-microglobulin. 

Murine  Transplantation  Antigens.   Gene  products  from  murine  major 
histocompatibility  locus  are  involved  in  diverse  functions  which  are 
associated  with  immune  recognition  and  reaction.   Our  structural  studies 
thus  far,  have  dealt  with  classical  transplantation  antigens  encoded  at 
the  K  and  D  loci.   Studies  using  radiochemical  methodology  have  allowed 
assignment  of  nearly  280  residues  to  the  K  glycoprotein  molecule.   Data 
indicate  a  high  degree  of  homology  to  human  histocompatibility  antigens 
and  furthermore  show  that  the  differences  between  these  molecules  are  not 
spread  over  the  molecule  but  rather  cluster  in  discrete  areas.   In 
addition  to  the  K  molecule,  primary  structural  analysis  is  being  carried 
out  on  the  D  ,  D   and  L  molecules.   In  addition,  the  determination  of  the 
primary  structure  of  murine  beta-2-microglobulin,  a  molecule  found  in 
association  with  major  histocompatibility  antigens,  is  near  completion. 
It  is  anticipated  that  knowledge  of  the  primary  structure  of  these 
molecules  will  aid  in  an  understanding  of  their  function  and  mechanism  of 
action.   The  major  histocompatibility  antigens  are  deeply  involved  in 
allograft  rejection  and  in  the  response  of  T  lymphocytes  to  altered  cell 
surface  antigens. 

Precursors  of  Immunoglobulin  Molecules.   When  certain  protein  products  are 
formed  by  _in  vitro  translation  using  mRNA  the  products  yielded  are  larger 
than  those  found  for  the  normal  secreted  products.   The  amino  terminus  of 
certain  of  these  proteins  may  be  extended  by  as  many  as  30  amino  acids. 
These  precursor  sequences  have  been  reported  for  immunoglobulin  light  and 
heavy  chains  as  well  as  a  number  of  hormones  and  secreted  enzymes.   The 
present  studies  are  directed  toward  a  study  of  the  immunoglobulin 
precursor  molecules  of  the  rabbit.   This  study  will  attempt  to  correlate 
the  variable  region  extension  with  the  constant  region  allotypes  of  the 
rabbit  L  chains.   The  source  of  mRNA,  which  in  the  past  has  proved 
troublesome,  will  be  hybrid  cell  lines  which  secrete  a  single  heavy  or 
light  chain  of  the  rabbit. 

Rabbit  Latent  Allotypes.   Rabbit  immunoglobulin  allotypes  are  antigenic 
determinants  which  were  thought  to  be  inherited  as  autosomal  codominant 
allele.   This  notion  has  been  challenged  by  observations  of  low 
concentrations  of  allotypes  which  were  not  detected  by  qualitative  tests 


21-2 


nor  predicted  by  parental  genotypes.   Detailed  structural  analyses  have 
shown  that  latent  allotypes  isolated  from  the  sera  of  pedigreed  rabbits 
are  indistinguishable  from  the  normal  allotypes.   Current  research  focuses 
on  hybridoma  cells  maintained  in  culture  that  are  able  to  synthesize 
chains  with  rabbit  allotypes.   RNA  which  encodes  the  immunoglobulin  chains 
secreted  by  these  cells  will  be  isolated  and  cDNA  probes  will  be  prepared 
in  order  to  carry  out  molecular  biological  studies  aimed  at  elucidation  of 
the  basis  for  inheritance  of  latent  allotypes.   Other  studies  in  this  area 
have  included  measurement  of  the  clearance  of  normal  and  latent  allotypes 
in  animals  using  doubly  radiolabelled  material.   Other  genetic  studies  in 
this  area  have  concentrated  on  the  linked  expression  of  V  markers  of 
immunoglobulin  expressing  latent  allotypes. 


21-3 


Honors  and  Awards 

Dr.  Kindt  serves  on  the  editorial  boards  of  the  Journal  of 
Immunology,  Proceedings  of  the  Society  for  Experimental  Biology  and 
Medicine  the  Zeitschrift  fur  Immunotatsforschung,  the  Journal  of 
Experimental  Medicine  and  Molecular  Immunology.   He  serves  as  a  member  of 
the  program  committee  of  the  American  Association  of  Immunologists  and  was 
elected  this  year  to  the  American  Society  for  Biological  Chemists.   Dr. 
Kindt  was  invited  to  speak  at  the  Josiah  Macy  Foundation  Conference  on 
membranes  and  human  disease  which  was  held  in  New  Orleans  and  the 
Institute  Pasteur  Conference  on  allotypes  and  idiotypes  which  was  held  in 
Paris.   Seminars  of  laboratory  work  were  presented  by  Dr.  Kindt  at  the 
University  of  North  Carolina  at  Chapel  Hill,  at  New  York  University 
Medical  School  in  New  York,  at  Scripps  Clinic  and  Research  Foundation  in 
La  Jolla,  at  the  Molecular  Immunology  Institute  at  the  University  of 
Marseille,  at  the  Institute  for  Genetics  at  the  University  of  Koln  and  at 
the  German  Cancer  Research  Institute  in  Heidelberg. 

Dr.  Coligan  presented  his  recent  data  on  the  structure  of  mouse 
histocompatibility  antigens  in  seminars  at  the  Mayo  Clinic  in  Rochester, 
Minnesota  and  at  Harvard  University.   He  also  presented  data  at  a 
symposium  on  T  and  B  lymphocytes  held  at  Keystone,  Colorado  and  at  the 
International  Congress  of  Biochemistry  in  Toronto,  Canada. 

Martin  Yarmush  received  his  Ph.D.  degree  from  Rockefeller  University 
this  Spring  and  was  accepted  at  a  number  of  medical  schools.   He  will 
attend  Yale  University  Medical  School  in  the  Fall. 


21-4 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl   AI   00166-02  LIG 


PHbMVeErRHl,    1978    to    September   30,    1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Chemical  Characterization  of  Rabbit  Histocompatibility  Antigens 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:      Dr.  Thomas  J  Kindt 

Others:   Dr.  Edward  S.  Kimball 

Dr.  John  E.  Coligan 

Dr.  Frederick.  T.  Gates 


Chief,  LIG  LIG  NIAID 

Staff  Fellow  LIG  NIAID 

Research  Microbiologist  LIG  NIAID 

Staff  Fellow  LIG  NIAID 


COOPERATING  UNITS  (if  any) 

Dept.  of  Genetics,  University  of  Illinois  at  Chicago  (Dr.  R.  Tissot) 


lab/branch 

Laboratory  of  Immunogenetics 


SECTION 

Immunogenetics  Research  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 
1.5 


PROFESSIONAL: 

0.9 


0.6 


CHECK  APPROPRIATE  BOX(ES) 
Q  (a)   HUMAN  SUBJECTS 

□  (a1  )  MINORS  Q   (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


XX(=)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

This  laboratory  has  been  engaged  in  the  isolation  and  purification  of 
histocompatibility  antigens  of  the  rabbit.   There  are  approximately  12 
serologically  defined  haplotypes  in  the  domestic  rabbit  and  our  study  has 
concentrated  on  the  RLA  11  haplotype.   The  major  source  of  antigen  has 
been  a  tumor  cell  line  derived  from  the  Bar  Harbor  B  strain  rabbit. 
Antigen  isolation  was  carried  out  by  detergent  solubilization  of  cells 
labelled  with  tritiated  amino  acids.   Partial  amino  acids  sequence 
analysis  was  carried  out  on  the  histocompatibility  antigen  isolated  and  it 
revealed  a  high  degree  of  homology  especially  to  histocompatiblity 
antigens  isolated  from  humans.   Beta-2  microglobulin,   a  low  molecular 
weight  protein  that  is  associated  with  histocompatibility  antigens,  was 
subjected  to  total  amino  acid  sequence  analysis.   Although  this  protein  is 
homologous  to  analogs  from  other  species  some  significant  differences  were 
observed  in  its  structure. 


21-5 


PHS-6040 
(Rev.  10-76) 


Z01  AI  00166-02  LIG 

Chemical  Characterization  of  Rabbit  Histocompatibility  Antigens 

Graft  rejection  and  control  of  the  ability  to  respond  to  specific 
antigens  are  directed  by  products  of  genes  residing  in  the  major 
histocompatibility  complex.   Little  is  known  about  the  structure  and 
function  of  the  major  histocompatibility  gene  products  although 
preliminary  work,  has  been  reported  for  proteins  from  the  mouse  and  human. 
It  is  our  intention  to  study  the  rabbit  as  a  third  species  in  order  to 
compare  MHC  gene  products  structures.   There  are  about  twelve 
serologically  defined  MHC  haplotypes  in  the  domestic  rabbit. 

Current  work  has  concentrated  on  the  RLA-11  haplotype.  The  major 
source  of  antigen  has  been  a  tumor  cell  line  derived  from  the  Bar  Harbor  B 
strain  of  rabbits.   Antigen  isolation  has  been  carried  out  by  detergent 
solubilization  of  cell  membranes  that  were  intrinsically  labeled  with 
tritiated  amino  acids.   A  glycoprotein  fraction  was  isolated  by  lentil 
lectin  chromatography  and  the  histocompatibility  antigen  isolated  from 
this  by  affinity  chromatograhy  on  a  column  of  highly  purified  antibodies 
directed  against  beta-2  microglobulins.   Amino  terminal  amino  acid 
sequence  analysis  using  radiochemical  methods  allowed  the  assignment  of  28 
of  the  amino  terminal  35  residues.   The  data  obtained  revealed  89% 
homology  of  the  RLA-11  protein  with  the  human  HLA  B7  and  82%  with  the 
mouse  H-2K  .   Comparison  of  the  RLA-11  to  sequence  data  obtained  from 
major  histocompatibility  antigens  of  other  species  revealed  no 
substitutions  unique  to  the  rabbit  antigen. 

The  complete  amino  acid  sequence  of  rabbit  beta-2  microglobulin  has 
been  determined  and  compared  to  the  sequence  reported  for  human 
beta-2  microglobulin.   This  comparison  showed  a  homology  of  71%  with  a 
minimum  of  13%  difference  in  nucleotide  sequences  of  genes  encoding  the 
two  proteins.   However,  a  single  insertion  must  be  introduced  before 
position  68  of  the  rabbit  protein  to  maintain  this  maximum  homology. 
Amino  acid  substitutions  are  distributed  throughout  the  molecule. 
Although  the  majority  of  those  requiring  multiple  base  changes  are 
restricted  to  the  carboxy-terminal  third  of  the  molecule.   Although  the 
rabbit  protein  analyzed  in  this  study  was  isolated  from  the  pooled  urine 
of  15  rabbits  no  heterogeneity  in  amino  acid  sequence  was  observed. 

Publications 

Kimball,  E.S.,  Coligan,  J.E.,  and  Kindt,  T.J.:   Structural 
characterization  of  antigens  encoded  by  rabbit  RLA-11  histocompatibility 
genes.   Immunogenetics  8:   201-211,  1979. 

Kindt,  T.J.,  Coligan,  J.E.,  Kimball,  E.S.,  Ewenstein,  B.,  Uehara,  H. , 
Martinko,  J.,  and  Nathenson,  S.G. :   Use  of  radiochemical  techniques  for 
primary  structural  analysis  of  mouse  and  rabbit  histocompatibility 
antigens.   Proceedings  of  the  Josiah  Macy  Foundation,  in  press,  1979. 


21-6 


Z01  AI  00166-02  LIG 

Gates,  III,  F.T.,  Coligan,  J.E.,  and  Kindt,  T.J.:   The  complete  amino  acid 
sequence  of  rabbit  beta-2-microglobulin.   Biochemistry  18:   2267-2272 
1979.  


21-7 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION.  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00167-02  LIG 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 


Mass  Spectrometry  and  Sequence  Analysis  of  Polypeptides 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 

Others: 


Dr.  Henry  C.  Krutzsch 
Dr.  Thomas  J.  Kindt 


Sr.  Staff  Fellow 
Chief,  LIG 


LIG   NIAID 
LIG  NIAID 


COOPERATING  UNITE 

None 


lab/branch 
Laboratory    of    Immunogenetics 


SECTION 

Immunogenetics  Research  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

1.9 


PROFESSIONAL: 

0.9 


1.0 


CHECK  APPROPRIATE  BOX(ES) 
G  (a)   HUMAN  SUBJECTS 


□  (b)  HUMAN  TISSUES 


□ 


MINORS   Q  (a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

A  major  need  in  immunogenetic  studies  are  fast,  reproducible  and 
sensitive  methods  for  amino  acid  sequence  analysis  of  proteins. 
Mass  spectrometric  (MS)  techniques  provide  a  promising  means  of  meeting 
tnese  goals.   Methods  under  study  involve  enzymatic  digestion  of 
polypeptides  into  dipeptides  using  either  dipeptidyl  aminopeptidase  or 
dipeptidyl  carboxypeptidase  (DAP/DCP  method) and  use  of  MS  for  rapid 
identification  of  dipeptides  produced.   Another  employs  direct  introduction 
of  larger  derivatized  peptides  into  the  MS  for  analysis  (DI  method) . 
Computer  assisted  data  analysis  programs  for  both  MS  methods  are  under 
development . 

The  DAP/DCP  and  DI  methods  are  being  tested  using  rabbit  and  mouse 
heavy  chains  as  model  proteins  because  these  proteins  are  not  readily 
amenable  to  routine  sequence  analysis  techniques.   The  structure  of 
rat  g-2  microglobulin  is  also  under  study  using  this  method. 

21=3 


PHS-6040 
(Rev.  10-76) 


Z01  AI  00167-02  LIG 

Mass  Spectrometry  and  Sequence  Analysis  of  Polypeptides 

It  is  the  aim  of  this  program  to  develop  rapid  and  sensitive  methods 
for  polypeptide  sequence  analysis  and  to  apply  these  methods  to  molecules 
of  immunogenetic  interest.   Development  of  this  method  will  include 
devising  strategies  for  isolaton  and  purification  of  peptides  in  amounts 
sufficient  for  characterization.   These  techniques  will  facilitate 
polyeptide  sequence  analysis,  helping  to  determine  more  rapidly  the 
primary  structures  of  immunogenetically  important  molecules  such  as 
antibodies  and  histocompatibility  antigens. 

An  LKB  Model  2091  EI-CI  gas  chromatograph-mass  spectrometer  (GC-MS) 
was  used  to  identify  trimethylsilylated  (TMS)  dipeptides  from  dipeptidyl 
peptidase  (DP)  digestion  of  polypeptides.   Two  MS  ions  are  necessary  for 
identification;  the  sequence-determining  ion  resulting  from  loss  of  the 
C-terminal  residue  plus  the  N-terminal  residue's  carbonyl  group,  and  the 
molecular  weight-determining  ion.   Alignment  of  the  dipeptides  to  give  the 
polypeptide  sequence  is  done  by  using  the  dipeptides  from  a  second  DP 
digestion  of  the  polypeptide  after  addition  of  another  amino  acid  residue, 
or  by  homology  to  a  similar  peptide  of  known  structure. 

Some  more  areas  of  the  DP/GC-MS  method  have  been  investigated.   The 
utilization  of  dipeptidyl  carbonsypeptidase  (DCP)  ,  in  this  case 
Angiotensin-I  converting  enzyme,  in  place  of  DAP  for  sequence  analysis 
from  the  C-terminus  (instead  of  the  N-terminus)  of  the  polypeptide  has 
been  one  of  these.   Use  of  this  type  of  enzyme  system  will  provide 
sequence  information  that  may  not  be  available  from  Edman  chemistry  (or 
DAP  digestion)  due  to  a  blocked  N-terminus,  or  to  inability  to  go 
completely  to  the  C-terminus  in  peptide  with  an  unblocked  N-terminus.   At 
the  present  time,  no  totally  reliable  technology  exists  for  such  an 
operation. 

In  this  area,  three  sections  were  investigated;  enzymology,  scope  of 
digestion,  including  available  sensitivity  and  associated  chemistry.   The 
enzymology  was  studied  to  determine  what  enzyme  concentration,  buffer 
conditions  and  associated  miscellaneous  conditions,  such  as  digestion 
times,  which  gave  maximum  dipeptide  yields.   The  enzyme  system  was  also 
checked  for  the  presence  of  materials  that  would  interfere  with  the 
subsequent  GC  MS  analysis,  such  as  contaminating  proteases  or  components 
that  would  create  spurious  GC  peaks.   The  scope  of  the  digestion  was 
studied  to  determine  what  types  and  lengths  of  polypeptides  could  be 
digested,  as  well  as  the  amount,  in  nmoles,  of  polypeptide  that  could  be 
determined.   The  chemical  studies  were  mainly  concerned  with  determining 
and  optimizing  (and  carrying  out)  the  best  method  for  obtaining  the 
"overlapping"  peptide  to  provide  the  necessary  alignment  information  for 
the  dipeptides  from  DCP  digestion  of  the  native  polypeptide. 

The  results  obtained  up  to  the  present  indicate  that  the  method  has 
broad  applicability.   Thus,  this  system  has  been  applied  to  peptides 
containing  all  types  of  amino  acids,  with  chain  lengths  of  up  to  greater 
than  50  residues,  and  at  levels  less  than  5  nmoles. 

21-9 


Z01  AI  00167-02  LIG 

Four  computer  programs,  written  in  Fortran- IV,  have  been  almost 
completely  developed  to  allow  rapid  manipulation  of  the  GC-MS  DP  digestion 
data.   Two  of  these  programs  are  for  dipeptide  identification,  one  a 
manual  system,  one  an  automatic  system.   The  other  two  are  for  dipeptide 
alignment,  either  from  the  N-terminus  (DAP  digestion)  or  from  the 
C-terminus  (DCP  digestion) . 

Two  proteins  are  currently  under  study  with  these  enzyme  systems;  the 
N-terminal  225  residue  fragment  resulting  from  CNBr  cleavage  of  a  rabbit 
(??4135,  a3  allotype)  homogeneous  antibody  heavy  chain,  and  rat  B-2 
microglobulin.   All  of  the  work,  up  to  the  present  time  has  been  with  the 
DAP  system,  using  as  substrates  peptides  from  both  restricted  (Arg 
cleavage  only)  and  total  tryptic  cleavage  of  the  rabbit  H-chain  CNBr 
fragment.   Several  partial  or  total  sequences  have  been  obtained  for  these 
peptides.   Because  several  of  these  tryptic  fragments  are  blocked  at  the 
N-terminus  due  to  glutamine-pyroglutamate  coversion,  the  DCP  enzyme  will 
be  a  useful  addition  to  the  present  technology. 

Publications 

Krutzsch,  H.C.  and  Pisano,  J.   Preparation  of  dipeptidyl  aminopeptidase  IV 
for  polypeptide  sequencing.   Biochem.  Biophys.  Acta.  576:   280-289,  1979. 

Krutzsch,  H.C.  and  Kindt,  T.J.   The  identification  of  trimethylsilylated 
dipeptides  with  chemical  ionization  mass  spectrometry.   Analytical 
Biochemistry  92:   525-531,  1979. 

Rao,  D.N.,  Rudikoff,  S. ,  Krutzsch,  H. ,  and  Potter,  M.   Structural  evidence 
for  independent  joining  region  gene  in  immunoglobulin  heavy  chains  from 
anti-galactan  myeloma  proteins  and  its  potential  role  in  generating 
diversity  in  complementary-determining  regions.   Proc.  Nat.  Acad.  Sci.  76: 
2890-2894,  1979. 

Keeton,  T.K.,  Krutzsch,  H.C,  and  Lovenberg,  W.   A  specific 
radioimmuneassay  for  3-methoxy-4-hydroxyphenyl-ethylene  glycol  (mopeg) . 
Proc.  4th  International  Catecholamine  Symposium.,  Pergamon  Press,  1978. 

Yarmush,  M.L.,  Krutzsch,  H.C,  and  Kindt,  T.J.   Amino  acid  sequence 
analysis  of  immunoglobulin  light  cains  by  gas  chromatographic-mass 
spectrometric  techniques:   structural  identity  of  latent  b9  and  nominal  b9 
molecules.   Molecular  Immunology,  in  press,  1979. 


11-10 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Oo  NOT  use  this  sDace) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl   AI   00168-02   LIG 


PTOoTOECL,    1978    to    September   30,    1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Structure  of  Rabbit  Immunoglobulin  and  Antibody  Heavy  and  Light  Chains 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  ANO  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:       Dr.  Thomas  J.  Kindt 
Others:   Dr.  John  A.  Sogn 

Dr.  Henry  Krutzsch 


Chief 

Chemist 

Sr.  Staff  Fellow 


LIG  NIAID 
LIG  NIAID 
LIG  NIAID 


COOPERATING  UNITS  (if  any) 

None 


LAB/BRANCH 

Laboratory  of  Immunogenetics 


SECTION 

Immunogenetics  Research  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland   20205 


TOTAL  MANYEARS: 

1.0 


PROFESSIONAL: 

0.3 


0.7 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


XX(c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Homogeneous  rabbit  antibodies  to  streptococcal  carbohydrates,  are 
used  to  investigate  structural  aspects  of  the  allotypic  and 
idiotypic  markers  of  immunoglobulins.   The  partial  amino  acid  sequence  was 
determined  for  a  rabbit  light  chain  bearing  latent  b9  allotype.   In 
addition,  structural  work  on  a  rabbit  heavy  chain  bearing  the  a3  allotype 
is  presently  under  way. 

Studies  such  as  these  that  combine  serological  and  structural 
investigations  of  antibodies  have  the  potential  to  define  genetic 
mechanisms  involved  in  antibody  synthesis. 


21-11 


PHS-6040 
(Rev.  IO-76) 


Z01  AI  0U168-02  LIG 

Structure  of  Rabbit  Immunoglobulin  and  Antibody  Heavy  and  Light  Chains 

The  structural  aspects  of  serologically  detected  polymorphic  forms  of 
rabbit  immunoglobulins  will  aid  in  understanding  the  genetic  mechanisms 
involved  in  antibody  synthesis.   Study  of  the  covalent  structure  of 
immunoglobulins  involves  both  separation  of  the  protein  into  component 
peptides  and  amino  acid  sequence  analysis  of  these  materials.   Separation 
of  heavy  and  light  chais  from  rabbit  IgG  is  a  standard  procedure,  but 
structural  analysis  of  the  components  must  be  treated  in  a  unique  manner 
depending  on  the  chain  and  its  allotypic  specificities.   During  the  past 
year  we  have  been  determining  structural  features  of  light  and  heavy 
chains  representing  various  allotypes. 

In  the  area  of  light  chain  structure,  the  amino  acid  sequence  for  a 
latent  b9  allotype  light  chain  was  determined,  using  the  DAP/GC-MS  method 
for  residues  110-118,  following  mild  acid  cleavage  and  full  reduction  and 
alkylation  of  the  whole  isolated  light  chain.   Comparison  with  similarly 
treated  and  DAP/GC-MS  analyzed  nominal  b9  allotype  isolated  light  chain 
showed  the  same  sets  of  dipeptides,  indicating  that  they  are  structurally, 
(latent  and  nominal  b9  allotype)  as  well  as  serologically,  identical. 

In  the  area  of  heavy  chain  structure,  work,  has  been  concentrated  on 
determining  the  amino  acid  sequence  for  the  N-terminal  225  residue  piece 
from  CNBr  cleavage  of  heavy  chain  of  allotype  a3,  isolated  from 
homogeneous  rabbit  anti-streptococcal  antibody.   This  fragment  has  been 
fully  reduced  and  alkylated  and  subjected  to  partial  and  full  tryptic 
cleavage.   The  amino  acid  sequence  determination  of  these  tryptic  peptides 
is  currently  under  way;  both  the  conventinal  Edman  method  and  the 
DAP-DCP/GC-MS  method  are  being  employed.   The  structural  studies  on  this 
CNBr  fragment  will  serve  as  a  prototype  for  future  structural  work  on 
otner  proteins  and  peptides.   New  methods  of  peptide  isolation  and 
detection  have  been  developed  and  are  currently  being  applied  to  obtain 
peptides  from  chains  such  as  this  to  allow  complete  structural  analysis. 

Publications 

Yarmush,  M.L. ,  Krutzsch,  H.C. ,  and  Kindt,  T.J.:   Amino  acid  sequence 
analysis  of  immunoglobulin  light  chains  by  gas  chromatographic-mass 
spectrometric  techniques:   Structural  identity  of  nominal  and  latent  b9 
molecules.   Molecular  Immunology,  in  press,  1979. 

Fraser,  B.A. ,  Thunberg,  A.L. ,  and  Kindt,  T.J.:   Variable  region  correlates 
of  group  b  allotypes:   amino  acid  sequence  studies  of  b9  L  chains  from 
homogeneous  antibodies.   Eur.  J.  Immunol.  8:   380-385,  1978. 

Kindt,  T.J.   Antibody  (Immunoglobulin)  Structure.   In  Baron,  S.  (Ed.): 
Medical  Microbology:   Principles  and  Concepts,  Addison-Wesley  Publishing 
Company,  Inc.,  California,  1979,  in  press. 


21-12 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00169-02  LIG 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 


Primary  Structural  Analysis  of  Murine  Transplantation  Antigens 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


Research  Microbiologist  LIG  NIAID 

Chief,  LIG  LIG  NIAID 

Staff  Fellow  LIG  NIAID 

Staff  Fellow  LIG  NIAID 

Staff  Fellow  LIG  NIAID 


PI: 

Dr. 

John  E.  Coligan 

Others: 

Dr. 

Thomas  J.  Kindt 

Dr. 

Frederick.  T.  Gates 

Dr. 

W.  Lee  Maloy 

Dr. 

Edward  S.  Kimball 

COOPERATING  UNITS  (if  any) 

Dept.  of  Microbiology,  Albert  Einstein  School  of  Medicine  (Dr.  S.  Nathenson) 
National  Cancer  Institute,  National  Institutes  of  Health  (Dr.  David  Sachs 
and  Dr  .  Ted  Hansen) 


lab/branch 
Laboratory  of  Immunogenetics ,  NIAID 


SECTION 

Immunogenetics  Research  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 
4.5 


PROFESSIONAL: 

2.7 


1.8 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   j_  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□__?_)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  genes  in  the  mouse  H-2  major  histocompatibility  complex  determine 
a  diversity  of  functions  associated  with  immune  recognition  and  reaction. 
Three  of  these  genes,  H-2K,  H-2D  and  H-2L  encode  the  major  antigens 
involved  in  allograft  rejection;  a  feature  undoubtedly  related  to  the 
genetic  polymorphism  of  these  H-2  molecules.   In  addition,  these  molecules 
play  a  role  in  T-lymphocyte  responses  to  altered  cell-surface  antigens. 
These  H-2  molecules  are  integral  membrane  glycoproteins  and  are  associated 
on  the  cell  surface  with  Bn -microglobulin.   The  purpose  of  this  work  is 
to  determine  the  primary  structure  of  these  molecules  in  order  that  we 
may  better  understand  their  function  and  mechanism  of  action.   Toward 
this  goal,  we  are  determining  the  primary  structure  of  each  of  these 
molecules  from  mice  of  the  b  and  d  haplotypes  (i-£.  H-2K  ,  H-2D  ,  H-2D  , 
H-2L  ) .   The  amino  acid  sequence  analysis  of  H-2K   is  near  completion  and 
limited  amounts  of  sequence  data  are  available  for  the  other  molecules. 
In  addition,  the  determination  of  tne  primary  structure  of  murine 
g9-microglobulin  is  near  completion.   21-13 

PHS-6040 
(Rev.  10-76) 


Z01  AI  00169-02  LIG 

Primary  Structural  Analysis  of  Murine  Transplantation  Antigens 

In  order  to  relate  structural  differences  among  mouse  H-2 
glycoproteins  to  changes  in  their  biological  activity  and  to  examine 
various  genetic  and  evolutionary  relationships  among  the  genes  of  the 
major  histocompatibility  complex,  the  complete  amino  acid  sequences  of 
the  H-2K  and  H-2D  glycoproteins  are  being  determined. 

Because  of  the  limited  amounts  of  these  materials  available, 
radio-chemical  methodology  has  been  developed  in  order  to  pursue  these 
studies.  H-2  molecules  are  intrinsically  radiolabeled  by  growing 
appropriate  tumor  cell  lines  in  the  presence  of  radioactive  amino  acids. 
Initial  studies  have  utilized  the  EL4.BU  (H-2  )  and  C14  (H-2  ) 
lymphoblastoid  cell  lines.   Following  detergent  extraction,  the  H-2 
glycoproteins  are  isolated  by  immunoprecipitation  with  alloantiserum. 
Peptide  fragments  are  produced  by  CNBr  cleavage  at  methionine  residues 
and  are  isolated  by  gel  filtration  and  ion  exchange  chromatographic 
procedures. 

Microsequencing  analyses  utilizing  radiochemical  methodology  have 
made  possible  the  complete  primary  structural  determination  of  the  H-2K 
(H-2. 33)  molecule.   Approximately  95%  of  the  amino  acid  sequence  of  the 
NH-terminal  270  residues  have  been  assigned.   This  portion  of  the 
molecule  is  roughly  equivalent  to  the  papain  fragment  and  accounts  for 
the  extra  cellular  portion  of  the  molecule.   Amino  acid  sequence 
determinations  of  the  transmembrane  (30  residues)  and  the  intracellular 
(40  residues)  portions  of  the  molecule  are  approximately  50%  complete. 

Comparison  of  the  amino  acid  sequences  for  the  H-2K  molecule  and 
the  human  HLA-B7  molecule  in  regions  currently  available  for  comparision 
indicate  an  overall  homology  of  70%.   Greater  than  90%  of  the  amino  acid 
interchanges  require  only  one  nucleotide  base  change  at  the  DNA  level. 
To  date,  comparision  of  the  molecules  has  shown  two  regions 
(positions  61-82  and  87-105J  of  less  than  50%  homology  and  in  which  many 
of  the  amino  acids  require  multiple  base  changes  for  interconversion. 
These  regions  may  be  involved  in  alloantigenic  specificity. 

Comparison  of  the  H-2K   amino  acid  sequence  to  the  primary  structure 
of  other  proteins  reveals  that  an  internal  region  of  the  molecule 
encompassing  the  second  disulfide  is  homologous  to  the  constant  region 
domains  of  immunoglobulin  heavy  and  light  chains  as  well  as 
3  -microglobulin.   This  indicates  that  all  these  molecules  of  the  immune 
system  evolve  from  the  same  ancestral  gene. 

Studies  on  the  H-2Db  (H-2. 2)  and  H-2D   (H-2. 4)  molecules  have 
progressed  to  the  point  where  the  CNBr  peptides  of  the  H-2  molecules  have 
been  isolated  and  aligned  by  homology  to  H-2K  .   NH  -terminal  sequence 
analyses  of  each  of  these  peptides  has  allowed  the  assignment  of  residues 


21-1L 


Z01  AI  00169-02  LIG 

which  account  for  approximately  40%  of  the  total  sequence.   The  homology 
of  these  molecules  to  the  H-2K  molecule  is  approximately  85%  for  the 
positions  available  for  comparision. 

Similar  studies  on  the  H-2K  and  H-2L  molecules  have  been 
initiated.   Because  of  their  proximity  in  the  genome,  it  has  been 
difficult  to  differentiate  the  H-2D  and  H-2L  molecules.   NH  -terminal 
sequence  analysis  of  the  H-2L   indicated  that  this  molecule  can  be 
distinguished  from  H-2D  by  its  lack  of  a  valine  and  phenylalanine  at 
positions  9  and  17,  respectively,  and  by  the  substitution  of  an 
isoleucine  for  a  methionine  at  position  23. 

The  determination  of  the  amino  acid  sequence  of  murine  g  - 
microglobulin  is  95%  complete  and,  as  expected,  it's  primary  structure  is 
highly  homologous  (~80%)  to  that  of  6?M  from  other  species. 

A  major  goal  of  our  structural  studies  on  the  H-2  glycoproteins  is 
to  locate  positions  of  structural  changes  in  the  H-2  molecules  isolated 
from  the  series  of  H-2K  and  H-2D  utants  of  the  b  and  d  haplotypes. 
Strains  of  each  K  and  D  mutant  series  show  strong  histogenic  reactivity 
in  vivo  and  MLR  and  CML  in  vitro  with  the  parents.   In  five  of  the  H-2K 
mutants  that  have  been  examined,  there  appear  to  be  only  one  or  two  amino 
acid  interchanges  between  the  H-2K  molecules,  produced  by  the  parent  and 
mutant  strain.   Further  studies  on  the  precise  nature  of  the  amino  acid 
interchanges  in  these  and  other  H-2K  molecules  of  this  mutant  series  will 
serve  to  localize  the  region  of  the  molecule  in  which  polymorphism  can 
serve  as  a  specific  and  effective  signal  for  recognition  by  the  immune 
system. 

An  added  impetus  to  the  study  of  these  molecules  is  given  by  the 
relationships  in  humans  between  the  homologous  loci  (HLA  A  and  B)  and 
predisposition  to  certain  diseases.   Furthermore,  molecules  coded  for  by 
the  K  and  D  loci  of  the  major  histocompatibility  complex  appear  to  play  a 
major  role  in  the  stimulation  and  specificity  of  T-lymphocyte  response  to 
virally  induced  and  other  cell  surface  antigens. 

Publications 


Coligan,  J.E.,  Kindt,  T.J.,  Ewenstein,  B.M.  ,  Uehara,  H. ,  Nisizawa,  T. , 
and  Nathenson,  S.G.   Primary  structure  of  murine  MHC  alloantigens:   II. 
Aminp  acid  sequence  studies  of  the  cyanogen  bromide  fragments  of  the 
H-2K  glycoprotein.   Proc.  Natl.  Acad,  of  Sciences,  USA  75:   3390-3394, 
1978. 

Coligan,  J.E.,  Kindt,  T.J.,  Ewenstein,  B.M. ,  Uehara,  H. ,  Martinko,  J.M. 
and  Nathenson,  S.G.  Further  analysis  of  the  murine  H-2K  glycoprotein 
using  radiochemical  methodology.   Mol.  Immunol.  16:   3-8,  1979. 


21-15 


Z01  AI  00169-02  LIG 

Kindt,  T.J.,  Coligan,  J.E.,  Kimball,  E.S,  Ewenstein,  B. ,  Uehara,  H. , 
Martinko,  J.,  and  Nathenson,  S.G.   Use  of  radiochemical  techniques  for 
primary  structural  analysis  of  mouse  and  rabbit  histocompatibility 
antigens.   Proceedings  of  the  Josiah  Macy  Foundation,  in  press,  1979. 

Nathenson,  S.G.,  Ewenstein,  B.M.,  Uehara,  H. ,  Martinko,  J.M.,  Coligan, 
J.E.,  and  Kindt,  T.J. :   Recent  Studies  on  the  Structure  of  the  H-2K 
Glycoprotein  and  on  the  H-2K  MHC  Mutants.   In  Ferrone,  S.  and  Resfeld, 
R.A.  (Eds.):   Current  Trends  in  Histocompatibility.   Plenum  Press,  1979, 
in  press. 

Uehara,  H. ,  Ewenstein,  B.,  Martinko,  J.M. ,  Nathenson,  S.G. ,  Coligan, 
J.E.,  and  Kindt,  T.J.   Primary  structure  of  murine  MHC  alloantigens: 
amino  acid  sequence  of  the  amino  terminal  173  residues  of  the  K 
glycoprotein.   Biochemistry,  in  press,  1979. 


il-16 


SMITHSONIAN  SCIENCE  INFORMATION  cXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00170-02  LIG 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Structural  Studies  on  Precursors  to  Immunoglobulin  Molecules 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:       Dr.  Thomas  J.  Kindt         Chief,  LIG  LIG  NIAID 

Others:   Dr.  Frederick  T.  Gates       Staff  Fellow         LIG  NIAID 


COOPERATING  UNITS  (if  any) 

Institute  of  Biochemistry,  University  of  Glasgow,  Scotland  (Dr.  A.  Williamson, 
H.  Singer) 


lab/branch 
Laboratory  of  Immunogenetics 


SECTION 

Immunogenetics  Research  Section 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS  □  (b)  HUMAN  TISSUES  &(<=)  NE!THER 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

0.8 


PROFESSIONAL:  OTHER: 

0.5 I     0.3 


□  (al)  MINORS   C  (a2)  INTERVIEWS 


SUMMARY  OF  ilORK    (200  words  or  less  -  underline  keywords) 

The  in  vitro  translation  of  the  mRNA  for  some  proteins  yields 
products  which  are  larger  than  those  found  in  vivo.   The  amino  termini  of 
these  precursor  proteins  are  extended  by  up  to  thirty  amino  acids  when 
compared  to  their  mature  counterparts.   Precursor  sequences  have  been 
reported  for  murine  (plasmacytoma)  immunoglobulin  light  and  heavy  chains, 
with  the  conclusion  that  these  sequences  represent  extensions  of  the 
variable  region  genes.   In  order  to  better  understand  the  multi-gene 
nature  of  immunoglobulin  genes,  the  purpose  of  this  work,  is  to  sequence 
other  mouse  immunoglobulin  precursor  molecules  for  comparison  and  to 
establish  the  nature  of  such  sequences  in  the  rabbit.   The  latter  goal 
requires  the  construction  of  hybrid  cell  lines  capable  of  secreting  rabbit 
immunoglobulin  chains  as  sources  for  the  isolation  of  rabbit 
immunoglobulin  mRNA.   This  mRNA  must  then  be  isolated  and  translated 
in  vitro,  and  the  translation  products  must  be  analyzed  using 
radiosequence  techniques. 

21-17 


PHS-6040 
(Rev.  10-76) 


Z01  AI  00170-02  LIG 

Structural  Studies  on  Precursors  to  Immunoglobulin  Molecules 

Peptides  of  as  many  as  thirty  amino  acids  may  be  present  at  the  amino 
terminus  of  a  protein  when  it  is  synthesized  and  these  peptides  may  then 
be  cleaved  from  the  rotein,  presumably  durng  the  secretion  process.   These 
precursor  peptides,  which  are  hydrophobic,  have  been  identified  for 
hormones,  digestive  track  enzymes,  and  serum  proteins.   It  is  postulated 
that  these  peptides  direct  the  passage  of  the  nascent  polypeptide  chain 
through  the  membrane  of  the  endoplasmic  reticulum. 

Precursor  peptides  for  several  BALB/c  mouse  immunoglobulin  light 
chains  and  a  single  heavy  chain  have  been  previously  reported.   The  amino 
acid  sequence  data  have  indicated  that  light  chan  precursors  appear  to  be 
somewhat  homologous  within  and  between  light  chain  subgroups,  while  much 
less  homology  exists  between  precursor  peptides  of  Kappa  and  lambda  light 
chains.   In  collaboration  with  A.  Williamson  and  H.  Singer  (University  of 
Glasgow)  we  have  determined  a  partial  amino  acid  sequence  for  the 
precursor  peptides  of  a  gamma  heavy  chain  and  a  Kappa  light  chain  from  a 
C3H  mouse  myeloma.   These  sequences  are  not  homologous  to  those  reported 
for  BALB/c  mice,  indicating  a  broader  diversity  of  precursor  peptide 
sequence  than  was  previously  assumed. 

In  order  to  study  the  structure  of  precursor  peptides  of 
immunoglobulins  from  another  species,  we  are  constructing  mouse-rabbit 
hybrid  cell  lines  (hybridomas)  which  secrete  rabbit  immunoglobulin  chans. 
With  these  cell  lines  as  sources  of  mRNA  for  in  vitro  protein  synthesis, 
we  are  immunoprecipitating  the  subsequent  rabbit  immunoglobulin  products 
prior  to  radiosequence  analysis.   The  multiplicity  of  kappa  constant 
region  genes  in  the  rabbit  allows  a  study  of  gene  rearrangement  and 
regulation  of  gene  expression  which  is  not  possible  in  the  mouse  system. 
This  analysis  will  help  to  define  the  structure-function  relationship  of 
immunoglobulin  precursor  peptides  and  the  genes  which  encode  them. 

Publications 

None 


21-18 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00171-02  LIG 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 


Genetic  Studies  on  Rabbit  Immunoglobulins  and  Other  Serum  Proteins 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:       Dr.  John  A.  Sogn 

Others:   Dr.  Thomas  J.  Kindt 

Dr.  Martin  Yarmush 

Mr.  Mark  Simpson 


Chemist 
Chief,  LIG 
Chemist 
Veterinary-Costep 


LIG  NIAID 

LIG  NIAID 

LIG  NIAID 

LIG  NIAID 


COOPERATING  UNITS  (if  any) 

Dr.  Ben  Wolf,  Univ.  of  Penn. ,  School  of  Vet.  Medicine,  Philadelphia,  Pa. 
Dr.  John  Coe,  Rocky  Mountain  Laboratory,  NIAID 


LAB/BRANCH 

Laboratory  of  Immunoaenetics 


SECTION 

Immunogenetics  Research  Section 


INSTITUTE  AND  LOCATION 

NIAID.  NIM.  Bethesda. 


Maryland  20205 


TOTAL  MANYEARS: 

1.35 


PROFESSIONAL: 

0.5 


0.85 


CHECK  APPROPRIATE  BOX(ES) 
G  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   3  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


D^c)  NE 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  serologic  markers  of  rabbit  immunoglobulins-allotypes  and 
idiotypes ,  are  under  study  because  they  can  provide  information  concerning 
the  number  and  arrangement  of  genes  encoding  the  immunoglobulins.   A  major 
effort  has  been  made  to  characterize  allotype  b4  ,  a  new  allele  of  the 
group  b  allotypes  of  the  C   region.   This  allotype,  first  discovered  in 
this  laboratory  during  amino  acid  sequence  studies,  has  now  been  shown  by 
genetic  analysis  to  be  an  allele  of  the  other  group  b  allotypes  and  has 
been  characterized  serologically.   The  utility  of  chain  specific  idiotypes 
for  genetic  studies  in  the  rabbit  has  been  confirmed,  and  the  initial 
study  of  an  L  chain  specific  idiotype  has  been  extended  with  an  H 
chain-specific  idiotype.   It  was  possible  to  show  linkage  of  this  idiotype 
to  V  and  C   allotypes  within  a  single  allogroup,  and  to  demonstrate 
latent  expression  of  an  allogroup.   Attempts  to  improve  breeding 
efficiency  of  inbred  rabbits  are  underway  at  present.   Idiotypy  studies 
will  be  extended  to  these  animals  when  sufficient  numbers  are  available. 

21-19 

' PHS-6040 
(Rev.  10-76) 


Z01  AI  00171-02  LIG 

Genetic  Studies  on  Rabbit  Immunoglobulins  and  Other  Serum  Proteins 

The  various  serologic  markers  of  rabbit  immunoglobulins  have  been 
valuable  probes  for  the  study  of  genetic  interactions  involved  in  the 
humoral  immune  response.   The  rabbit  has  a  uniquely  diverse  repertoire  of 
allotypes  (intra-species  antigenic  variants),  encompassing  most  regions  of 
the  antibody  molecule.   The  structural  correlates  for  some  allotypic 
markers  are  simple,  while  for  others  they  are  either  complex  or  unknown. 
Studies  within  this  project  are  designed  to  expand  the  allotypic 
repertoire,  to  increase  its  structural  definition,  and  to  use  these 
serologic  markers  to  examine  questions  about  the  number  of  genes  involved 
in  immunoglobulin  synthesis  and  the  mechanisms  controlling  interactions 
among  these  genes.   The  studies  based  on  allotypes  have  been  complemented 
and  extended  by  use  of  idiotypes  as  markers  for  the  antibody  combining 
site.   The  obvious  complexity  of  idiotypes  as  genetic  markers  has  been 
lessened  somewhat  by  increasing  the  specificity  of  idiotypic  antisera, 
either  by  fractionation  based  on  antigen  competition  or  by  limiting 
idiotypic  recognition  to  determinants  on  one  of  the  antibody  chains. 

Serologic  characterization  is  now  complete  for  a  new  C,  allotype, 
b4V,  which  has  been  under  study  for  two  years.   While  conventional 
antiallotypic  reagents  do  not  distinguish  b4  from  b4  ,  three  methods  of 
serologic  distinction  have  been  found.   First,  as  reported  previously,  one 
anti-b4  serum,  upon  adsorption  on  a  column  containing  bound  cottontail 
rabbit  IgG,  yielded  an  antibody  fraction  which  reacted  well  with  b4  IgG 
but  poorly  with  b4v  IgG.   An  inhibition  of  binding  radioimmunoassay  could 
then  be  used  to  differentiate  b4   from  b4.   Second,  in  collaborative 
studies  with  Dr.  John  Coe,  several  bull-frog  antisera  raised  against  b4 
IgG  or  crosslinked  b4v  L  chains  reacted  specifically  with  b4   IgG  after 
adsorption  with  b4  IgG.   The  reaction  could  be  detected  either  by 
radiobinding  or  precipitin  assays.   Although  only  low  levels  of  activity 
were  found,  the  result  established  that  b4   IgG  does  possess  antigenic 
determinants  not  found  on  b4  IgG.   Third,  collaborative  studies  with  Dr. 
Jan  Naessens  have  proven  that  b4   and  an  allotpe  described  in  Belgium, 
b4.2,  are  serologically  and  structurally  identical.   This  finding  has  made 
tne  serologic  detection  of  b4   in  serum  much  easier  because  an  antiserum 
exists  (anti-MS7)  which  reacts  only  with  IgM  containing  L  chains  of 
allotype  b4.2  (or  b4v) .   The  reaction  can  be  seen  readily  by  precipitin 
reaction.   Another  serum  of  the  MS  series,  anti-MS3,  reacts  in  a  similarly 
specific  way  with  IgM  containing  b4.1  (b4)  L  chains.   Structural  identity 
of  b4V  with  b4.2  was  shown  by  sequence  analysis  of  succinylated,  acid 
cleaved  L  chains. 

Further  genetic  analysis  of  the  rabbit  immune  response  will  be 
difficult  without  rabbit  of  more  defined  genetic  composition.   To  take 
advantage  of  available  in  vitro  techniques  for  dissecting  the  cellular 
components  of  the  unimmune  response  fully  inbred  rabbits  would  be 
particularly  valuable.   Some  attempts  to  incorporate  existing  inbred 
rabbits  into  studies  in  ths  laboratory  have  been  made  in  the  past  year. 
The  B/J  strain  has  been  examined  to  determine  optimal  parameters  for 


il-20 


Z01  AI  00171-02  LIG 

immunization  with  antigens  inducing  monoclonal  responses.   Idiotypic 
analysis  will  be  done  to  examine  idiotypic  diversity  in  an  inbred 
population.   Large  scale  studies  will  require  numbers  of  B/J  rabbits  not 
available  with  current  breeding  techniques.   Methods  for  enhancing 
fertility  and  reproductive  rate  are  being  examined  in  cooperation  with  the 
Veterinary  Resources  Branch  and  Dr.  Ben  Wolf. 

Limited  structural  studies  are  underway  in  collaboration  with  Dr. 
John  Goe  on  a  protein  isolated  by  Dr.  Coe  from  the  serum  of  female 
hamsters.   The  protein  is  present  only  in  trace  amounts  in  nude  hamsters. 
Amino  acid  composition  and  sequence  analysis  are  being  used  to  determine 
whether  the  female  protein  exhibits  structural  homology  to  biological 
active  proteins  of  several  types  already  sequenced  in  other  species. 

Publication 


Sogn,  J. A.  and  Kindt,  T.J..   Genetic  characterization  of  a  new  allele  of 

the  rabbit  group  b  C  allotypes.   Immunogenetics  7:   141-147,  1978. 
K  

Kindt,  T.J.  and  Capra,  D.   Gene-insertion  theories  of  antibody  diversity: 
A  re-evaluation.   Immunogenetics  6:   309-321,  1978. 

Smith,  L.J.,  Sogn,  J. A.,  Kindt,  T.J.,  and  Mandy,  W.J.   Serologic 
distinction  between  the  rabbit  kappa  L  chain  allotype  b4  and  an  allele 
b4  .   European  Journal  of  Immunology  9:   27-31,  1979. 

Yarmush,  M.L.  and  Kindt,  T.J.   Idiotypes  of  Rabbit  Antistreptococcal 
Antibodies:   Probes  for  Inheritance  and  Immune  Regulation.   In  Rudbach  and 
Baker  (Eds.):   Immunology  of  Bacterial  Polysaccharides,  Elsevier  North 
Holland,  Inc.,  1979,  pp.  41-65. 

Capra,  J.D.  and  Kindt,  T.J.  One  From  Many:   Immunoglobulin  V  Regions  are 
the  Products  of  Interacting  Genes.   In  Cunningham  and  Sercarz  (Eds.): 
The  Strategy  of  Immune  Regulation,  Academic  Press,  New  York,  1979,  in 
press. 


21-21 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  Al  00172-02  LIG 


PERIOO  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 


Carbohydrate  and  Glycoprotein  Antigens  of  Microbial  Cell  Walls 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


Dr.  John  Coligan 


Research  Microbiologist 


LIG  NIAID 


COOPERATING  UNITS  (if  any) 

Dept.  of  Micro.  Univ.  of  Alabama  at  Birmingham  (Dr.  David   Pritchard) 


LAB/BRANCH 

Laboratory  of  Immunogenetics 


SECTION 

Immunogenetics  Research  Section 


INSTITUTE  AND  LOCATION 

NIAID.  NIH.  Bethesda.  Maryland  202U5 


TOTAL  MANYEARS: 

0,2 


PROFESSIONAL: 


0.1 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (a!)  MINORS   C  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


sxf 


c   NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Previous  compositional  and  immunological  studies  have  defined  the 
chemical  and  antigenic  structure  of  the  group-specific  carbohydrates  for 
Groups  A,  A  variant  and  C  streptococci.   These  polysaccharides  were  shown 
to  have  a  common  core  structure.   Work  is  in  progress  to  determine  the 
structure  of  the  group  specific  carbohydrate  of  the  Group  B  streptococcus 
in  order  to  chemically  define  its  antigenic  determinants  and  to  determine 
its  chemical  relationship  to  the  other  streptococcal  group  specific 
carbohydrates . 


21-22 


PHS-6040 
(Rev.  10-76) 


Z01  AI  00172-02  LIG 

Publications 

Coligan,  J.E.  and  Slayter,  H.   Physical,  chemical  and  immunological 
characterization  of  saline-extracted,  concanavalin  A  purified 
carcinoembryonic  antigen.   Mol.  Immunol.  16:   129-135,  1979. 

Coligan,  J.E.  and  Kindt,  T.J.   Use  of  Structurally  Defined  Streptococcal 
Carbohydrate  Antigens  in  Studies  of  Rabbit  Antibody  Idiotypes.   In  Read, 
S.  and  Zabriskie,  J.  (Eds.):   Streptococcal  Disease  and  the  Immune 
Response.   Academic  Press,  New  York,  in  press,  1979. 

Coligan,  J.E.,  Kindt,  T.J.,  and  Krause,  R.M.   Structure  of  the 
streptococcal  groups  A,  A-variant  and  C  carbohydrates. 
Immunochemis  try  15:   755-760,  1978. 

Coligan,  J.E.  and  Krause,  R.M.   Antibodies  to  streptococcal  carbohydrate, 
substitutes  for  the  myeloma  proteins.   J.  Infect.  Pis. ,  in  press,  1979. 


21-23 


SMITHSONIAN    SCIENCE    INFORMATION    EXCHANGE 
PROJECT   NUMBER    (Do    NOT    use    this    space) 


U.S.    DEPARTMENT   OF  PROJECT   NUMBER 

HEALTH,    EDUCATION,    AND    WELFARE    ! 
PUBLIC   HEALTH   SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH   PROJECT  ZQ]_    ^    qQ173_q2    l1q 


PERIOD    COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (30  characters  or  less) 


Nonallelic  Expression  of  Genes  Encoding  Rabbit  Immunoglobulins 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:       Dr.  T.J.  Kindt  Chief,  LIG 

Others:   Dr.  Martin  Yarmush  Chemist 

Dr.  John  A.  Sogn  Chemist 

Dr.  F.T.  Gates,  III  Staff  Fellow 


LIG  NIAID 

LIG  NIAID 

LIG  NIAID 

LIG  NIAID 


COOPERATING  UNITS  (if  any) 

Dr.  William  J.  Mandy,  University  of  Texas,  Austin,  Texas 

Dr.  Ben  Wolf,  University  of  Pennsylvania  School  of  Veterinary  Med. 

Philadelphia,  Pa. 


lab/branch 

Laboratory  of  Immunogenetics 


SECTION 

Immunogenetics  Research  Section 


INSTITUTE    AND    LOCATION 

NTATD,     NTH,     RpfhPsHa,,   Maryland     2Q205 


TOTAL  MANYEARS: 

2.3 


PROFESSIONAL: 

1.3 


CHECK  APPROPRIATE  BOX(ES) 
Z   (a)   HUMAN  SUBJECTS 

G  (al)  MINORS   Q  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


GX&)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Rabbit  immunoglobulin  allotypes  are  antigenic  determinants  thought  to 
be  inherited  as  autosomal  co-dominant  alleles .   This  notion  has  been 
challenged  by  observations  of  low  concentrations  of  allotypes  not  detected 
by  qualitative  tests  or  predicted  by  parental  genotypes.   In  the  past 
year,  L  chains  with  "latent"  allotypes  were  isolated  from  the  sera  of 
pedigreed  rabbits  ana  shown  by  amino  acid  sequence  analysis  to  be 
indistinguishable  from  the  normal  allotypes.   Current  research  focus  on 
hybridoma  cells  maintained  in  culture  that  synthesized  rabbit 
immunoglobulin  RNA  will  be  isolated  from  these  cell  lines  and  cDNA  probes 
will  be  prepared  to  carry  out  molecular  biological  studies  in  order  to 
determine  the  basis  for  inheritance  of  latent  allotypes.   Other  studies  in 
this  area  have  included  clearance  of  normal  versus  latent  allotypes  in 
animals  using  doubly  labelled  material  and  studies  on  the  linked 
expression  of  V^  markers  on  immunoglobulins  expressing  latent  allotypes. 


21-; 


PHS-6040 
(Rev.  10-76) 


Z01  AI  00173-02  LIG 

Nonallelic  Expression  of  Genes  Encoding  Rabbit  Immunoglobulins 

In  the  past  year,  studies  on  latent  allotypes  of  rabbit 
immunoglobulins  have  included  complete  serologic  characterization  of 
allotypic  markers  of  both  the  constant  and  variable  regions  of  the  rabbit 
molecules.   It  has  been  shown  by  structural  studies  that  the  molecules 
expressing  latent  allotypes  have  markers  that  are  indistinguishable  from 
those  of  the  allotypes  normally  expressed.   Latent  Group  b  markers  of  the 
constant  region  of  the  rabbit  antibody  light  chain  were  previously 
detected  in  sera,  in  IgG  preparatins,  and  on  isolated  L  chains  from 
rabbits  bred  for  homozygosity  at  the  b  locus.   Serologic  analysis  of  sera 
from  an  extended  family  of  homozygous  b4  rabbits  revealed  the  presence  of 
latent  b  allotypes  in  5  of  37  sera  tested.  Latent  b5  and  b9  markers  were 
identified.   None  of  the  sera  tested  contained  latent  b6.   In  two 
instances,  the  level  of  latent  b9  allotype  was  sufficiently  high  to  permit 
isolation  and  detailed  characterization  of  the  immunoglobulin  population 
bearing  this  latent  allotype.   These  studies  which  are  completely 
described  in  another  section  indicated  that  the  nominal  and  latent 
allotype  light  chains  were  identical  in  sequence. 

Studies  concerning  the  linked  expression  of  C  and  V  allotype  genes 
have  involved  isolation  of  molecules  on  the  basis  of  a  latent  group  a 
allotype.   This  is  carried  out  by  use  of  immunoadsorbent  chromatography 
employing  specific  antibodies  directed  against  the  allotype  specificities. 
The  isolated  molecules  were  then  typed  for  their  group  d  markers  (dll,  dl2) 
by  serologic  and  by  chemical  means.   Approximately  half  of  the  molecules 
isolated  were  shown  to  carry  latent  group  d  markers  in  addition  to  the 
latent  group  a  allotype.   However,  in  no  case  was  found  a  molecule  with 
combinations  of  group  a  and  group  d  markers  that  are  not  normally 
expressed  in  the  rabbit  populations  studied.   This  finding  indicates  that 
the  mechanism  for  synthesis  of  latent  allotypes  are  similar  to  those  for 
nominal  allotypes  and  do  not  represent  products  of  events  such  as 
misspairing  of  multiple  genes  involved  in  H  chain  synthesis. 

A  promising  new  tool  for  the  study  of  latent  allotypes  has  been 
introduced  by  the  use  of  interspecies  (mouse-rabbit)  hybridoma  cells. 
Cells  have  been  prepared  that  will  secrete  rabbit  heavy  or  light  chains  in 
culture  and  in  ascites  fluid  of  nude  mice.   These  cells  are  being  prepared 
in  bulk  culture  and  RNA  encoding  the  rabbit  immunoglobulin  chains  will  be 
isolated  and  used  to  prepare  cDNA  which  can  be  used  as  a  probe  to  study 
the  occurrence  of  latent  allotype  genes  in  various  rabbits. 

IgG  clearance  rates  were  measured  by  intravenous  injection  of  rabbits 
with  differentially  labelled  (    I  and    I)  allotype  matched  and 
nonmatched  IgG  samples.   In  no  instance  was  the  allotype  matched  IgG 
cleared  faster  than  nonmatched  although  the  converse  was  true  in  several 
instances.   These  data  suggest  that  there  is  an  in  vivo  recognition  of 
allotypes  which  is  a  possible  regulatory  mechanism.   Many  questions  remain 
to  be  answered  concerning  mechanisms  for  the  regulation  of  synthesis  and 
the  selection  of  genes  involved  in  the  preparation  of  normal  and  latent 
allotypes. 

21-25 


Z01  AI  00173-02  LIG 

Publications 

Yarmush,  M.L.  and  Kindt,  T.J.   Isolation  and  characterization  of  IgG 
molecules  expressing  latent  group  b  allotypes  from  pedigreed  b  b   rabbits. 
J.  Exp.  Med.  148:  522-533,  1978. 

Mudgett-Hunter,  M.  ,  Yarmush,  M.L. ,  Fraser,  B.A. ,  and  Kindt,  T.J.   Rabbit 
latent  group  a  allotypes:   Characterization  and  relationship  to  nominal 
group  a  allotypic  specificities.   J.  Immunology  121:   1132-1138,  1978. 

Yarmush,  M.L.,  Sogn,  J. A.,  and  Kindt,  T.J.:   Latent  allotypes:   A  window 
to  a  genetic  enigma.   Ann.  Immunol.  (Inst.  Pasteur)  130C:   143-156,  1979. 

Yarmush,  M.L.,  Krutzsch,  H. ,  and  Kindt,  T.J.:   Amino  acid  sequence 
analysis  of  immunoglobulin  light  chains  by  gas  chromatographic  -  mass 
spectrometric  techniques:   structural  identity  of  latent  b9  and  nominal  b9 
molecules.   Molecular  Immunol.,  in  press,  1979. 


Wolf,  B.,  Urban,  R. ,  Miller,  A.B.,  Kimball,  E.S.,  Mudgett,  M. ,  Catty,  D. 
and  Danemann,  J.:   Nonallelii 
Cell  surface  and  serum  studit 
J .  Immunol . ,  in  press,  1979. 


and  Danemann,  J.:   Nonallelic  inheritance  of  V   region  a  group  allotypes. 
Cell  surface  and  serum  studies  in  double  and  triple  expressing  rabbits. 


21-26 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl    AI   00180-01   LIG 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 


Monoclonal  Antibodies  Directed  Against  Cell  Surface  Proteins 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


LIG  NIAID 

LIG  NIAID 

LIG  NIAID 

LIG  NIAID 


PI: 

Dr. 

John  A 

Sogn 

Chemist 

Others: 

Dr. 

Thomas 

J.  Kindt 

Chief,  LIG 

Dr. 

Edward 

S.  Kimball 

Staff  Fellow 

Dr. 

Thomas 

Folks 

Staff  Fellow 

COOPERATING  UNITS  (if  any) 

None 


LAB/BRANCH 

Laboratory  of  Immunogenetics 


SECTION 

Immunogenetics  Research  Section 


NSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland 


20205 


TOTAL  MANYEARS: 

2^2 


PROFESSIONAL: 
i 1.2 


±^L 


CHECK  APPROPRIATE  SOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


&fC 


SUMMARY    OF    WORK    (200   words    or   less   -   underline    keywords) 

Preliminary  studies  are  under  way  on  antibodies  to  cell 
surface  proteins  produced  by  hybridoma  techniques.   The  initial  targets 
for  this  investigation  is  cell  line  RL-5,  a  rabbit  T-cell  tumor.   The  cell 
line  has  been  characterized  by  conventional  techniques  and  several 
mouse-mouse  hybridomas  with  activity  against  this  cell  have  been  produced, 
cloned  and  propagated  _in  vivo  and  _in  vitro.   Biochemical  characterization 
of  the  molecular  specificity  of  these  antibodies  is  under  investigation. 


21-27 


PHS-6040 
(Rev.   IO-76) 


Z01  AI  00180-01  LIG 

The  ability  to  produce  monoclonal  hybridoma  antibodies,  in  unlimited 
amounts,  directed  against  any  antigenic  determinant  of  interest,  no  matter 
how  impure,  has  revolutionized  the  study  of  the  biologically  important 
proteins  of  the  cell  surface.   These  proteins  are  difficult  or  impossible 
to  purify  by  conventional  techniques  because  they  are  present  in 
vanishingly  small  amounts  and  because  their  isolation  is  complicated  by 
the  hydrophobic  nature  of  these  integral  membrane  constituents. 
Characterization  of  such  proteins  has  been  feasible  only  by  indirect 
methods  using  genetically  defined  organisms,  where  specific  antibodies  may 
be  produced  by  cross-immunization  of  antimals  differing  at  a  limited 
number  of  genetic  loci.   Structural  studies  in  outbred  species  such  as 
rabbit  and  man  have,  therefore,  been  especially  limited.   Hybridoma 
technology  removes  these  restrictions.   The  research  interests  of  several 
groups  in  the  Laboratory  of  Immunogenetics  currently  involve  molecules  of 
immunologic  importance  found  on  cell  surfaces.   The  goal  of  this  project 
is  to  raise  hybridoma  antisera  against  these  molecules  so  that  structural 
studies  can  proceed  rapidly. 

The  principal  current  interest  is  the  rabbit  cell  line  RL-5 .   This 
virus- induced  lymphoma  has  been  characterized  as  a  member  of  the  T-cell 
lineage  on  the  basis  of  reactivity  with  two  specific  anti-rabbit  thymocyte 
sera,  the  absence  of  cytoplasmic  and  surface  immunoglobulin,  and  the 
absence  of  receptors  for  Fc   or  C3.   Mice  have  been  immunized  with  RL-5 
and  spleen  cells  from  the  immunized  mice  have  been  fused  with  appropriate 
myeloma  cells  to  obtain  hybrid  antibodies  against  RL-5.   Antibody  activity 
is  measured  in  an  ELISA  assay.   The  membrane  components  against  which  the 
antibodies  react  are  currently  being  identified  by  immunoprecipitation  and 
electrophoretic  techniques. 

Publication 

None 


21-21 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01-A1-00191-01    LIG 


PERIOD  COVERED 

New  -  October  1  ,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Immunoregulation  of  T  Lymphocytes  -   The  Role  of  Anti-idiotype  and 
Immune  Complexes 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:     Kenneth  W.   Sell 
Other:     Tom  Folks 


Head,    Immunobiology  Section         LIG,   NIAID 
Staff  Fellow  LIG,   NIAID 


COOPERATING  UNITS 


Dr.  Aftab  Ahmed  (Merck  Corporation,  Rahway,  N.J.),  Dr.  James  Woody  (NMRI) 


LAB/BRANCH 

Laboratory  of  Immunogenetics 


SECTION 

Immunobiology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

3-  1/2 


PROFESSIONAL: 

1-  1/2 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


$  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Investigation  of  mechanisms   by  which  immunoregulatory  signals   are  transmitted  to 
T-lymphocytes   to  cause  activation  or  suppression  of  their  activity.      Project 
attempts  to  examine  both  antigen  and  allogeneic  receptors  on  T-lymphocytes  and 
the  means  by  which  they  may  be  activated,  blocked  or  stimulated  to  active  sup- 
pression of  T-cell    functions     In  particular,   the   role  of  immune  complexes  and 
the   related  role  of  anti-idiotype  antibodies   in   the  regulatory  process  will    be 
examined.     The  interaction  between  antigen,   antibody,   complement,  anti-idiotype 
immunoconglutinjnand  rheumatoid  factor  will    be  examined  in  the  make-up  of  immune 
complexes   to  determine  the  role  of  each,   individually  or  acting  in   concert,   to 
produce  effects  on  T-cell   activation  or  effector  function.      In  addition,   attempts 
will    be  made  to  identify  immune  complexes   in  disease  states   such  as   juvenile 
diabetes   and  hepatitis   to  determine  what  role  they  may  play  i_n  vivo  in  the 
suppression  of  cell-mediated  immunity  in  these  diseases  which  are  related  to 
latent  or  persistent  viral    infections. 


'21-29' 


PHS-6040 
(Rev.    IO-76) 


Z01-A1-00191-01    LIG 
Project  Description 

Objectives 

The  specific  suppression  or  inactivation  of  those  clones  of  antigen- 
reactive  cells   directed  against  foreign   antigens  or  donor  histocompatibility 
antigens  has   been  the  object  of  intensive   research.     A  recent  finding  has 
been  the  disclosure  that  F,    hybrids   between  two  inbred  strains  of  mice  will 
synthesize  anti-allo-anti bodies   if  injected  with  either  parental    lymphoid 
cells  or  in  al lo-antiserum  produced  in   the  same  parental   strain  against  the 
allo-antigens  of  the  other  parental   strain.     These  observations  have  led  to 
the  concept  that  the  recognition  of  allo-antigen  by  the  T-lymphocyte   is   the 
primary  step  towards  allograft  rejection  and  initiation  of  antigen   responsive- 
ness.    The  exact  nature  of  the  T-lymphocyte   recognition  structure   is   still    an 
unresolved  question.      Binz  and  Wigzell    have  obtained  evidence  that  suggest 
that  the   recognition  structure  is  similar, if  not  identical,   to  the  combining 
site  of  allo-antibody   (idio-type).      Isolated  T-cell    receptors  have  been  shown 
to  have  a  single  chain  structure  with   a  molecular  weight  of  70,000  daltons. 
It  has  been  suggested  that  both  T  and  B  cells   employ  a  common   pool   of  heavy 
chain  V  genes   in  synthesis  of  the  antigen  binding  site  on  the  respective  an- 
tigen receptors.     The  use  of  the  term  "idiotype"   for  the  recognition  structure 
on  T-cells   is   based  on  the  evidence  for  shared  idiotypic  determi nance  on  both 
T  and  B  cells.      Both   normal    T-cells  as  well    as   T-lymphoblasts  which   have  been 
specifically  activated  by  antigen  or  the  mixed  lymphocyte  reaction   both 
express   idiotypic  determinants  against  the  relevant  histocompatibility  or 
foreign  antigen.     Although  these  i diotype-posi ti ve  cells   specific  for  a 
given  set  of  major  histocompatibility  complex  antigens  are  found  with   very 
low  frequency,   less   than  3  percent,   in   the  normal    resting  population  of 
peripheral    blood  lymphocytes,   they  are  greatly  enriched  during  the  in  vitro 
proliferation   in  an  MLC  response  against  the  relative  MHC. 

Auto  and  anti-idiotype   (anti-receptor)  antibodies   can  be  produced  by  immuni- 
zation with   lymphoblasts.      These   can   deplete  the   responder  T-lymphocyte  popu- 
lation apparently  through  cytotoxic   killing   in   the   presence  of  complement. 
They  are  not  capable  of  blocking  MLC  reactions   in   the  absence  of  complement 
as   they  apparently  compete   ineffectively  for  binding  with   the   receptor  site 
on  T-cells   responsible  for  the  MLC  reaction. 

Inhibitors  of  T-cell   mediated  immunity  have  been  identified  in   certain  clini- 
cal   diseases.      In  subacute  sclerosing  panencephalitis    (SSPE)   and  in   CMV  virus 
disease,   immune  complexes   have  been   identified  which  apparently  are  capable 
of  specifically  inhibiting  T-cell    responses   to  the  virus   and  specifically 
blocking  effector  functions   such  as   the   release  of  macrophage   inhibition 
factor.     We  hypothesize  that  these   immune  complexes  may  exert  their  immuno- 
suppressive activity  on  T-lymphocytes   because  of  the  presence  of  antiidio- 
types.     It  is   possible  that  the   immune  complex  serves   as   a   focusing  mechanism 
or  perhaps   a  stabilizing  mechanism  allowing  the  more  effective   interaction  of 
anti-idio-type  with  T-cell    receptors,   thus   providing  for  blocking  or  initia- 
tion of  inactivation  events   in   the  T  lymphocyte. 

We,   therefore,   propose  to  study  the   role  of  anti-idiotype  and  immune  complexes 
as   they  interact  with   receptors   on  T-lymphocytes   in  order  to  elucidate  the 

21-30 


Z01-A1-00191-01    LIG 
mechanisms  of  immunoregulation  of  cell -mediated  immune  events. 

Methods   Employed: 

1.  Attempts  will    be  made  to  construct  model   immune  complexes   in  order  to 
evaluate  their  interaction  with  T-cell    receptors  and  subsequent  T-cell   acti- 
vity.    Specific  antibody  and  anti-idiotype  as  well   as   immunoconglutinin  and 
rheumatoid  factor  will    be  prepared  using  hybridomas.     Balb/C  mice  will    be 
immunized  with  specific  carrier  proteins  such  as   BSA,   OVA,   BGG,   KLH,   etc., 
and  their  spleen  cells  fused  with  appropriate  mouse  myeloma  cells   in  order  to 
establish  specific  clones  of  antibody  producing  cells.     The  Elisa  technique 
will   be  utilized  to  assay  for  the  presence  of  specific  antibodies.     It  is  an- 
ticipated that  spleens  of  immunized  animals  will    provide  cells   producing,   not 
only  specific  antibody  against  the  relevant  antigen  but  also  cells  expressing 
anti-idiotype  against  antibody.      In  addition,  autoantibodies  against  C-3 
(immunoconcluti nin)  and  an  auto-antibody  against  IgG   (rheumatoid  factor)  will 
be  anticipated  to  occur  in  the  immunized  mice.     Each  of  these  antibodies  then 
can  be  used  along  with  purified  complement   (C3)   to  construct  immune  complexes 
which  can  be  tested  in  cell-mediated  immune  tests   both  for  recognition   (trans- 
formation) or  for  effector  function   (MIF  assay)  or  killer  cell   assays. 

2.  Lymphocytes  and  serum  will    be  collected  from  chimpanzees  which  have  been 
infected  with  hepatitis  virus.     The  lymphocytes  will    be  assayed  for  cell- 
mediated  immune  activity  including  transformation  and  MIF  production.     Serum 
will   be  assayed  for  the  presence  of  inhibitors  of  cell-mediated  immunity.      If 
inhibitors  are  discovered,  specificity  will    be  determined  and  nature  of  the 
inhibitor  will   be  examined  with   particular  emphasis  on  techniques   to  identify 
immune  complexes.     Assays  will    be  made  regularly  during  the  infective  stage 
of  the  disease  in  the  chimpanzee  to  determine  the  sequential   appearance  of 
cell-mediated  immunity  and  inhibition. 

3.  Patients  with  juvenile  diabetes  as  well    as   control   patients  will    be  tested 
for  the  presence  of  humoral   and  cell-mediated  immunity  against  viruses  which 
have  been  suspected  in   the  etiology  of  diabetes.      In  particular,   coxsachie  B4 
will   be  evaluated  and  cell -mediated  immunity   (transformation  and  MIF)  will    be 
studied  and  compared  with  a  control    group  of  sex  and  age  matched  normal   child- 
ren.    Serum  of  diabetic  patients  will   be  examined  for  inhibitors  of  eel  1  - 
mediated  immunity  against  candidate  viruses  with  immunochemical    studies   to 
determine  the  presence  of  immune  complexes. 


21-31 


LABORATORY  OF  IMMUNOLOGY 

1979  Annual  Report 

Table  of  Contents 


Z01-AI 
Project  Number 

Summary 

00030-11 

00148-04 

00035-0.6 


Antigen  Recognition  and  Activation 
of  Immunocompetent  Cells. — Paul 

Lymphocyte  Interactions,  Receptors 
and  Functions. — Green 

Specificity  in  Immune  Responses. — 
Inman 


Page 
22-1 
22-13 

22-19 

22-25 


00036-14 


00037-12 


00038-07 


00040-05 


Immunoglobulin  Genetics:   Regulation       22-30 
of  Gene  Expression  and  Lymphoid 
Differentiation. — Mage 

Immuno gene tics  of  Mouse  Immunoglobulin     22-38 
and  Genetic  Control  of  Antibody 
Response . — Lieberman 

Structure  and  Activity  Studies  on         22-43 
Immunologically  Important  Cells  and 
Proteins . — Waxdal 


Genetic  Control  of  Immunocompetent 
Cell  Interactions. — Shevach 


22-48 


00147-04 


The  Mechanism  of  Activation  of 
Thymus -Derived  Lymphocytes . — 
Schwartz 


22-56 


PHS-NIH 
Summary  Statement 
Office  of  the  Chief 
Laboratory  of  Immunology 
October  1,  1978  through  September  30,  1979 

Recombination  between  Genes  for  u  and  <S  Immunoglobulin  Heavy  Chains 

The  genes  controlling  the  constant  regions  of  heavy  (H)  immunoglobulin 
(Ig)  chains  of  various  classes  exist  as  a  genetic  linkage  group  termed  the 
IgC  complex  in  the  mouse.   The  genes  specifying  IgA  and  the  various  IgG 
Hcnains  are  very  tightly  linked.   Indeed,  in  the  examination  of  several 
thousand  individual  mice  and  large  numbers  of  humans,  no  instance  of  a 
genetic  recombination  separating  these  genes  had  been  observed.   It  is  known 
that  genes  for  the  \i   and  6  H  chains  are  also  found  in  the  IgC   complex  but 
the  degree  of  linkage  has  not  been  extensively  studied  because  allotypic 
markers  for  the  p  and  6  genes  have  only  recently  been  described.   In  the 
course  of  experiments  aimed  at  studying  the  linkage  of  Lyb7  and  IgD, 
scientists  in  the  Laboratory  of  Immunology  and  at  the  Naval  Medical  Research 
Institute,  have  observed  two  instances  of  recombination  between  the  IgG 
and  IgD  genes.   The  initial  observation  was  that  two  progeny  (numbers  72 
and  74)  of  a  cross  between  (C57BL/6  x  DBA/2)  and  C57BL/6  possessed  the 
"DBA/ 2"  allele  of  IgD  [IgDa]  but  lacked  the  "DBA/ 2"  allele  of  IgG    [IgG   C]. 
This  phenotype  was  transmitted  to  progeny  on  subsequent  crossings  of 
individuals  72  and  74  to  C57BL/6  to  create  families  72  and  74.   Proof  that 

Q 

the  failure  of  these  mice  to  express  IgG    was  due  to  its  replacement  by 
_Ig_G„    [the  allele  possessed  by  C57BL/6]  was  obtained  by  crossing  members  of 
families  72  and  74  to  A  mice  and  demonstrating  that  progeny  that  inherited 
IgD  ,  which  is  distinguishable  from  the  IgD   of  A,  also  inherited  IgG?   .   It 
has  also  been  possible  to  show  that  in  both  recombinants  the  genes  for  IgG..  , 
IgA,  IgM  and  prealbumin  segregated  with  the  IgG?  gene.   These  recombinant 
families  represent  the  first  documented  examples  of  intra-IgC  genetic 
recombination.   Studies  of  segregation  of  IgV  markers  in  these  mice,  which 
are  now  in  progress,  will  allow  construction  of  more  detailed  genetic  maps 
of  this  genetic  region,  which  is  of  key  importance  in  the  specification  of 
antibody  molecules.   In  turn,  this  will  prove  of  considerable  utility  in 
understanding  the  genetic  basis  of  the  immune  response  (Subbarao,  Lieberman 
and  Paul,  LI/NIAID;  Ahmed  and  Scher,  Naval  Medical  Research  Institute). 

Genetic  Regulation  of  the  Anti-f ructosan  Antibody  Response 

Bacterial  levan  (BL)  is  a  g  (2-»-6)  linked  polyfructosan  with  g  (2-KL)  branch 
points.  Anti-BL  antibodies  produced  in  BALB/c  mice  consist  of  two  families 
of  molecules.   One  of  these  bind  BL  but  not  inulin,  a  g  (2-K1)  polyfructosan, 
and  thus  appear  to  be  specific  for  g  (2-*-6)  fructosans.   The  other  set  of  anti- 
bodies bind  inulin  and  BL,  are  principally  specific  for  g  (2-KL)  linkages  and 
express  one  or  more  of  a  family  of  cross  reactive  idiotypes  (IdX)  found  on 
inulin-binding  myeloma  proteins  of  BALB/c  mice.   It  has  previously  been  shown 
that  the  capacity  to  produce  both  anti-inulin  antibody  and  IdX-bearing  mole- 
cules in  response  to  immunization  with  BL  requires  the  presence  of  genes 
linked  to  the  IgC   gene  complex.   During  the  past  year,  Laboratory  of  Immunol- 
ogy scientists  have  demonstrated  that  non-IgC   genes  exert  major  effects  on  the 

22-1 


amount  and  clonal  diversity  of  the  anti-inulin  antibodies  produced  in  response 
to  BL  immunization.   Thus,  BALB/c  mice  immunized  with  BL  produce  inulin- 
binding  antibodies  which,  when  analyzed  by  isoelectric  focusing  (IEF) ,  consist 
of  characteristic  "triplet"  of  bands.   C57BL/6  mice,  or  C.B20,  which  are 
BALB/c  congenic  mice  expressing  IgC  genes  of  C57BL/6,  make  no  anti-inulin 
antibodies  after  BL  immunization"!   Nonetheless,  F-  hybrids  between  C57BL/6 
and  BALB/c  make  a  more  vigorous  and  more  diverse  anti-inulin  response  to 
BL,  as  judged  by  IEF,  than  do  BALB/c.   The  genetic  influence  contributed  by 
the  C57BL/6  depends  on  genes  outside  the  Ig  H  chain  genetic  region  since 
B.C8  mice,  which  are  congenic  to  C57BL  put  possess  BALB/c  IgC   genes,  make 
a  response  similar  to  the  F..  in  its  amount  and  diversity.   Through  the  analysis 
of  amount  and  diversity  of  the  anti-inulin  response  to  BL  of  recombinant 
inbred  lines  between  C57BL/6  and  BALB/c,  it  has  been  tentatively  concluded 
that  two  non-IgC   genes  regulate  the  clonal  diversity  and  amount  of  the  anti- 
inulin  response.   These  studies  should  shed  considerable  light  on  the 
genetic  factors  which  regulate  immune  responses  to  simple  antigens,  parti- 
cularly those  which  are  excellent  models  for  polysaccharide  antigens  used 
in  bacterial  vaccines  (Stein,  Bona,  Lieberman  and  Paul,  LI/NIAID) . 

A  Specialized  Subset  of  B  Lymphocytes  is  Stimulated  By  Anti-Immunoglobulin 
Antibodies 

One  of  the  most  direct  and  potentially  informative  approaches  to  the 
study  of  the  requirements  for  B  lymphocyte  activation  is  the  use  of  anti- 
bodies to  B  lymphocyte  membrane  receptors  to  stimulate  these  cells. 
Laboratory  of  Immunology  scientists  have  established  that  specifically 
purified  antibodies  to  u  H  chains  and  to  k  L  chains  cause  marked  stimula- 
tion of  proliferative  responses  by  B  lymphocytes.   The  response  is 
independent  of  any  requirement  for  T  lymphocytes  or  macrophages.   However, 
anti-Ig  antibodies  do  not  stimulate  the  synthesis  of  Ig  by  B  lymphocytes; 
indeed,  these  antibodies  are  powerful  inhibitors  of  stimulation  of  Ig 
synthesis  by  both  antigens  and  polyclonal  B  lymphocyte  activators. 
During  the  past  year,  scientists  in  the  Laboratory  of  Immunology  and  the 
Naval  Medical  Research  Institute  have  shown  that  the  capacity  of  lympho- 
cytes to  respond  to  anti-u  and  anti-K  is  a  property  of  a  specialized 
subset  of  B  lymphocytes  which  has  a  characteristic  density  of  membrane  (m) 
Ig  and  expression  of  differentiation  antigens.   To  show  this,  B  lymphocytes 
were  incubated  with  fluorescein  (Fl)  conjugated  anti-Ig,  anti-u  or  anti-6 
reagents  and  separated  into  populations  with  characteristic  densities  of 
mlg  using  the  fluorescence  activated  cell  sorter  (FACS) .   Cells  with  a  high 
density  of  total  Ig,  a  relatively  low  density  of  IgM,  and  a  moderate  to 
high  density  of  IgD  were  most  responsive  to  anti-Ig.   By  contrast,  cells 
with  a  low  density  of  total  Ig ,  a  high  density  of  IgM,  and  a  low  density 
of  IgD  were  poorly  responsive.   The  results  also  suggest  that  the  bulk 
of  cells  responsive  to  anti-Ig  are  Lyb5  .   These  results  establish  that 
responsiveness  even  to  simple  stimulants  such  as  anti-u   is  a  property  of 
a  discrete  B  lymphocyte  subpopulation.   Further  progress  in  delineating  the 
biochemical  events  in  B  lymphocyte  activation  by  specific  stimuli  will  require 
techniques  to  purify  and/or  clone  specific  populations  of  B  lymphocytes. 
Sieckmann  and  Paul,  LI;  Scher,  Naval  Medical  Research  Institute). 


22-2 


Failure  to  Detect  V  Framework  Determinants  on  T  Lymphocytes 

The  delineation  of  the  chemical  nature  of  the  antigen-binding  re- 
ceptors of  T  lymphocytes  has  been  one  of  the  most  challenging  and  critical 
problems  of  modern  immunology.   There  is  an  increasing  body  of  functional 
and  genetic  data  which  indicates  that  some  idiotypic  determinants,  pre- 
sumably markers  of  hypervariable  and  J  segments  of  immunoglobulin  H  chains, 
are  expressed  on  T  lymphocyte  receptors  as  well  as  on  antibodies  and  B 
lymphocyte  receptors.   These  findings  provide  strong  evidence  that 
structural  genes  in  the  IgV  genetic  region  code  for  at  least  part  of  the 
T  lymphocyte  receptor.   Laboratory  of  Immunology  scientists  have  sought  to 
determine  whether  antigenic  determinants  associated  with  framework  areas  of 
Ig  H  chain  variable  (V  )  regions  were  also  expressed  on  T  lymphocytes.   In 
order  to  examine  this,  anti  V  framework  antibodies  against  allotypic  and 
species-specific  determinants  have  been  prepared.   The  former  are  the  well 
known  antibodies  to  ^  locus  allotypic  determinants;  the  latter  are  anti- 
sera  to  rabbit  V  determinants  prepared  in  goats.   Such  reagents  have  been 
fluoresceinated  and  their  ability  to  stain  B  and  T  lymphocytes  determined 
by  direct  examination  and  by  use  of  the  fluorescence  activated  cell  sorter 
(FACS).   Thus  far,  no  evidence  for  T  lymphocytes  expressing  V  determinants 
has  yet  been  obtained.   Since  T  lymphocytes  have  been  shown  to  lack 
classical  light  (L)  chains,  efforts  have  been  made  to  chemically  identify 
V  bearing  molecules  which  lack  L  chains.   Again,  no  such  molecules  have 
yet  been  found.   This  data  is  consistent  with  the  failure  of  V  framework 
determinants  to  be  expressed  on  T  lymphocytes  at  all,  even  on  T  lymphocytes 
which  appear  to  express  idiotypic  determinants.   It  is  also  consistent  with 
the  possibilities  that  only  a  very  small  fraction  of  T  cells  possess  V  - 
bearing  receptors  or  that  each  T  cell  expresses  very  small  amounts  of  such 
receptors.   These  studies  are  being  pursued  both  with  an  effort  to  in- 
crease their  sensitivity  and  to  obtain  data  on  the  effects  of  anti-V 
sera  on  T  lymphocyte  functions.   They  promise  to  offer  important  insights 
into  the  nature  of  antigen-recognition  by  T  lymphocytes  (Wilder  and 
Mage,  LI/NIAID;  Scher,  Naval  Medical  Research  Inst.). 

Phospholipid  Methylation  is  an  Early  Event  in  T  Lymphocyte  Activation 

T  lymphocytes  can  be  stimulated  to  proliferate  by  exposure  to  a  wide 
variety  of  agents  of  which  the  plant  lectin  concanavalin  A  (Con  A)  is  a 
prototype.   It  has  long  been  known  that  many  intracellular  events  are 
associated  with  stimulation  of  DNA  synthesis  by  Con  A  but  no  clear  under- 
standing of  the  critical  initial  events  which  lead  to  the  proliferative 
response  has  yet  been  developed.   Recently,  scientists  in  the  Laboratory  of 
Immunology  and  in  the  National  Institute  of  Mental  Health  have  shown  very 
rapid  changes  in  membrane  phospholipid  methylation  following  exposure  of  T 
lymphocytes  to  Con  A.   Thus,  there  is  a  maximum  incorporation  of  H-methyl 
groups  into  membrane  phospholipids  within  10  minutes  after  the  addition  of 
Con  A  followed  by  a  somewhat  slower  calcium  dependent  degradation  so  that 
normal  levels  of  methylation  are  found  at  40  minutes.   Increases  in  phos- 
pholipid methylation  are  closely  correlated  with  subsequent  proliferative 
events.   Thus,  the  dose-response  curve  for  increased  phospholipid  methylation 
and  for  stimulation  of  DNA  synthesis  are  similar;  only  cell  types  which 
' proliferate  in  response  to  Con  A  exhibit  increased  methylation;  and,  only 

2  2-3 


those  lectins  which  are  mitogenic  induced  methylation.   Furthermore, 
inhibition  of  methylation  by  addition  of  a  methylation- inhibitor  prevents 
subsequent  DNA  synthesis  but  only  if  the  inhibitor  is  added  before  the 
increase  in  methylation  caused  by  Con  A.   These  results  suggest  that 
membrane  transmethylases  are  activated  by  binding  of  Con  A  to  the  cell, 
that  this  leads  to  an  increase  in  several  methylated  phospholipids  in- 
cluding monomethylethanolomine.   Such  an  increase  may  lead  to  decreased 
membrane  viscosity  and  the  entry  of  calcium  into  the  cell.   Calcium  is 
apparently  associated  with  the  accelerated  degradation  of  methylated  phos- 
pholipids and  release  of  a  series  of  compounds,  such  as  thromboxane, 
prostaglandins,  and  lysolecithin  which  may  be  of  considerable  importance 
in  subsequent  events  of  lymphocyte  activation.   These  early  biochemical 
events  appear  to  be  excellent  candidates  to  be  critical  steps  in  the 
cellular  processes  which  determine  the  capacity  of  physiologic  stimulants, 
such  as  antigens,  to  activate  lymphocytes  (Toyoshima  and  Waxdal,  LI/NIAID; 
Hirata  and  Axelrod,  NIMH). 

Antigen  Presentation  by  Macrophages:  both  Antigen  and  la  Gene  Products  must 
be  Expressed  on  the  Surface  of  the  Antigen-presenting  Cell 

The  activation  of  proliferative  responses  and  of  lymphokine  production 
by  specific  T  lymphocytes  depends  on  presentation  of  antigen  by  a  special- 
ized macrophage- like  cell.   Antigen-recognition  by  T  lymphocytes  has  been 
shown  by  Laboratory  of  Immunology  and  Laboratory  of  Clinical  Investigation 
scientists  to  involve  recogntion  of  both  I-region  associated  (la)  major 
histocompatibility  complex  antigens  and  the  conventional  antigen  to  which 
the  donor  of  the  T  lymphocytes  had  been  primed.   The  role  of  la  antigen 
recognition  in  this  process  has  been  strongly  supported  by  the  finding  that 
anti-la  antisera  are  potent  inhibitors  of  T  cell  activation.   Paradoxically, 
antisera  to  the  "conventional"  antigen  have  been  uniformly  ineffective  in 
the  blocking  of  T  lymphocyte  activation  by  antigen-pulsed  macrophages. 
Although  this  failure  of  antibody  to  cause  inhibition  has  certain  physiologic 
advantages,  it  also  raises  the  possibility  that  the  stimulatory  antigen 
may  not  be  on  the  surface  of  the  antigen-presenting  cell.   To  further  study 
this  critical  point,  Laboratory  of  Immunology  scientists  have  developed 
a  T  lymphocyte  activation  system  in  which  the  antigen-presenting  cell  is 
covalently  derivatized  with  trinitrophenyl  (TNP)  groups  and  responding  T 
cells  are  primed  to  TNP-macrophages  _in  vivo  or  in  vitro.   The  antigen- 
presenting  retains  its  capacity  to  stimulate  responses  by  T  lymphocytes 
even  if  it  is  incubated  for  24  hours  after  derivatization  before  addition 
to  T  cells.   Despite  the  capacity  of  these  "aged"  TNP-macrophages  to 
stimulate  a  TNP-specific  response,  anti-TNP  antibody  has  no  inhibitory, 
effect  on  the  activation  process.   However,  if  the  macrophage  is  subject 
to  a  second  round  of  derivatization  with  either  TNP  or  DNP ,  just  prior  to 
being  added  to  the  T  cells,  its  stimulatory  activity  is  now  sensitive 
to  inhibition  by  anti-TNP  antibody.   This  is  particularly  striking  for  the 
case  in  which  DNP  derivatization  is  used  because  DNP-derivatized  macro- 
phages do  not  stimulate  responses  by  T  cells  primed  to  TNP-macrophages, 
although,  anti-TNP  antibody  does  bind  DNP.   This  result  provides  strong 
evidence  that  the  stimulatory  antigen  is  on  the  surface  of  the  antigen- 
presenting  cell.      it  appears  the  antigen  is  present  at  a  density  too  low 


22-4 


to  be  cross-linked  and  cleared  from  the  surface  by  anti-TNP  antibody.   The 
second  round  of  derivatization  increases  the  antigen  density  and  thus  leads  to 
the  result  that  the  cell  is  now  susceptible  to  blockade.   However,  these 
results  still  lead  to  the  conclusion  that  the  antigen  on  the  macrophage, 
surface  can  stimulate  T  cell  responses  even  in  the  presence  of  saturating 
amounts  of  antibody.   These  results  have  considerable  significance  in 
the  understanding  of  the  regulation  of  T  lymphocyte  immunity  by  antibody 
and  in  the  elucidation  of  the  molecular  events  in  antigen  recognition. 
(Thomas  and  Shevach;  LI/NIAID) . 

Cellular  Expression  of  Responder  Phenotype  in  Complementing  Immune  Response 
Gene  Systems 

Specific  immune  response  (Ir)  genes  of  the  major  histocompatibility 
complex  control  the  ability  of  T  lymphocytes  to  be  activated  by  specific 
antigens.   In  certain  systems,  such  as  responses  to  poly (Glu,Lys,Phe) 
[GL$]  and  to  pigeon  cytochrome  C,  responsiveness  depends  upon  the  possession 
of  responder  alleles  of  Tr  genes  in  both  the  I -A  and  I-E/C  subregions  of 
the  mouse  MHC.   It  had  previously  been  shown  in  these  and  related  systems 
that  antigen-specific  T  lymphocyte  activation  by  antigen-presenting  cells 
(APC)  required  that  the  donor  of  the  APC  be  a  responder  to  the  antigen 
under  study.   However,  whether  the  responding  T  cell  had  to  be  derived 
from  a  responder  donor  was  uncertain.   Laboratory  of  Immunology  scientists 
have  studied  this  problem  through  the  creation  of  bone  marrow  chimeras. 
Thus,  F..  hybrids  prepared  by  crossing  complementing  non-responder  parents 
(i.e.  Ir-GL$a  3   and  Ir-GL$oi  8  mice)  are  capable  of  responding  to  GL$. 
If  these  mice  are  lethally  irradiated  and  reconstituted  with  bone  marrow 
from  both  parents,  the  resulting  mice  are  unresponsive  to  immunization 
with  GL$,  indicated  that  some  cell  must  possess  both  complementing  Ir 
genes  in  order  for  a  response  to  ensue.   However,  if  T  lymphocytes  are 
transferred  from  such  a  reconstituted  mouse  to  a  lethally  irradiated  F.. 
recepient  together  with  a  source  of  F,  APC  and  immunized  immediately 
with  GL$,  they  respond   to  GL$.   This  indicates  that  the  inability  of 
non-responder  T  lymphocytes  to  respond  is  not  a  genotypic  character  of 
the  T  cells,  but  depends,  at  least  in  part,  upon  cells  in  their  sensitizing 
microenvironment.   On  the  other  hand,  when  bone  marrow  cells  from  F 
(responder)  donors  were  used  to  reconstitute  lethally  irradiated  mice  of 
either  of  the  non-responder  parental  types  or  of  the  responder  F,  type, 
only  the  F-i+F-,  chimera  could  subsequently  respond  to  antigen.   This 
indicates  that  the  developmental,  as  well  as  the  sensitizing,  microenviron- 
ment is  critical  for  the  acquisition  of  the  responder  phenotype  by  the 
T  lymphocytes.   These  results  provide  further  insight  into  the  nature  of 
the  reagulation  of  immune  responses  by  MHC  I_r  genes  and  should  allow  a 
better  understanding  of  the  linkage  of  disease  susceptibility  and  resistance 
to  MHC  genes.   (Longo  and  Schwartz,  LI/NIAID). 

Three  Cells  Participate  in  the  In  Vitro  T  Lymphocyte  Proliferative  Response 
to  Antigen 

It  is  now  well  recognized  that  antigen-specific  activation  of  T  lympho- 
cytes for  proliferative,  lymphokine  producing,  or  helper  responses  can  be 


22-5 


effeciently  achieved  by  the  presentation  of  antigen  to  the  T  cells  in 
association  with  an  la  macrophage-like  cell.   The  analysis  of  this  antigen- 
presentation  system  has  led  to  the  demonstration  that  T  lymphocytes  recog- 
nize both  antigen  and  I_-region  gene  products  expressed  on  the  surface  of 
the  antigen-presenting  cell.   Nonetheless,  this  analysis  only  demonstrates 
that  cellular  interactions  are  important  in  the  artificial  condition  of 
addition  of  antigen-pulsed  cells  to  a  primed  T  lymphocyte  population.   It 
does  not  give  a  picture  of  the  relative  importance  of  such  interactions  in 
the  normal  in  vitro  response  of  unseparated  lymphoid  cells. 

In  order  to  examine  cellular  interactions  in  unseparated  cell  popula- 
tions, Laboratory  of  Immunology  scientists  have  studied  the  relation  between 
numbers  of  cells  cultured  and  response  to  added  antigens.   In  an  ideal 
situation,  the  diminution  of  response  upon  diluting  a  cell  population  by  a 
factor  of  two,  should  be  two-fold  if  a  single  cell  type  limits  the  response, 
four-fold  if  there  are  two  interacting  cell  types  both  of  which  are  limiting, 
and  eight-fold,  if  there  are  three  such  limiting  cell  types.   A  plot  of  the 
log  of  number  of  added  cells  against  log  of  the  response  will  yield  a 
slope  of  1  for  one  limiting  cell  type,  2  for  two  limiting  cell  types,  3  for 
three  limiting  cell  types,  etc.   In  a  large  series  of  experiments,  we  found 
that  the  relationship  between  the  log  of  number  of  primed  nylon  wool  passed 
lymph  node  cells  and  the  log  of  thymidine  uptake  in  response  to  the  immuniz- 
ing antigen  was  3.   This  suggests  that  the  _in  vitro  response  normally  in- 
volves the  participation  of  three  cell  types.   The  nature  of  these  inter- 
acting cells  can  be  determined  by  adding  a  constant  (and  excess)  number  of 
a  given  cell  type  to  all  dilutions  of  the  primed  lymph  node  cells  and 
measuring  its  effect  on  the  slope  of  the  resultant  response.   Using  this 
analysis,  it  was  shown  that  one  of  the  cells  was  a  radio- resistant  cell 
present  in  spleens  of  non-primed  mice;  this  cell  is  very  likely  an  antigen- 
presenting  cell.   A  second  cell  was  a  radio-sensitive  T  lymphocyte  found 
in  lymph  nodes  of  primed  and  unprimed  donors.   This  cell  appears  to 
represent  a  cell  which  is  non-specif ically  "recruited"  to  proliferate.   The 
third  cell  type  appears  to  be  the  antigen-specific  T  lymphocyte.   Analysis 
of  histocompatibility  restrictions  and  expression  of  immune  response  gene 
function  in  these  systems  are  now  in  progress.   These  experiments  provide 
an  approach  to  determine  the  extent  to  which  rules  derived  from  "synthetic" 
systems  apply  to  cellular  interactions  in  unseparated  lymphoid  cell  popula- 
tions and  will  thus  give  a  more  complete  understanding  of  the  relative  role 
of  various  cellular  interactions  in  intact  systems.   (Tse,  Schwartz  and 
Paul;  LI/NIAID). 

Patients  with  Systemic  Lupus  Erythematosus  have  a  Defect  in  Suppressor 
Cell  Function  Associated  with  a  Serum  Autoantibody 

Systemic  lupus  erythematosus  (SLE)  is  complex  disorder  associated 
with  derangements  in  the  regulation  of  immune  responses  and  with  the 
presence  of  antibodies  to  various  autoantigens.   Scientists  in  the 
Laboratory  of  Immunology  and  in  the  Arthritis  and  Rheumatism  Branch  of 
NIAMDD  have  shown  that  blood  lymphocytes  of  SLE  patients  have  a  markedly 
diminished  capacity  to  develop  into  suppressor  cells  as  a  result  of 
stimulation  with  concanavalin  a  (Con  A) .   Con  A  induced  suppressor  cells 


22-6 


from  normal  individuals  will  suppress  in  vitro  proliferative  responses  to 
Both  pokeweed  mitogen  and  autologous  lymphoid  cells  (i.e.  mixed  lymphocyte 
responses  [MLR]).   In  many  patients  with  SLE,  Con  A  fails  to  induce  cells 
capable  of  suppressing  either  the  pokeweed  mitogen  response  or  the  MLR, 
whereas  as  other  patients  are  defecient  in  only  one  of  these  suppressor 
functions .   Sera  from  SLE  patients  with  defects  in  capacity  to  develop 
suppressor  cells  contain  cytotoxic  antisera  capable  of  causing  complement 
mediated  lysis  of  suppressor  cell  precursors  in  normal  lymphocyte  populations. 
Moreover,  serum  from  patients  with  selective  defects  caused  the  same 
selective  defect  in  normal  lymphocyte  populations  treated  with  that  serum  and 
complement.   These  results  suggest  that  separate  T  lymphocyte  populations 
suppress  the  pokeweed  mitogen  response  and  the  MLR.   Fractionation  of  T  lympho- 
cytes into  those  possessing  Fc  receptors  for  IgG  (T  cells)  and  those  lacking 
such  receptors  indicates  that  T  cells  preferentially  suppress  the  MLR. 
These  studies  provide  important  insights  into  the  pathophysiology  of  the 
immune  response  in  SLE  patients  and  help  to  expand  our  understanding  of 
the  normal  regulation  of  the  human  immune  response.   (Sakane  and  Green, 
LI/NIAID;  Steinberg,  Arthritis  and  Rheumatism  Branch,  NIAMDD) . 

Lack  of  Role  for  the  Fourth  Component  of  Complement  (C4)  in  In  Vitro  T 
Lymphocyte  Activation 

Other  investigators  have  reported  that  antisera  to  human  C4  inhibit  the 
in  vitro  mixed  lymphocyte  response  (MLR) .   This  implies  that  C4,  perhaps  as  a 
membrane  molecule,  may  have  a  critical  role  in  the  response  of  T  lymphocytes, 
either  by  acting  on  the  stimulatory  or  responding  cell.   Because  of  the 
potential  importance  of  this  finding,  Laboratory  of  Immunology  scientists 
have  carefully  examined  this  issue  in  guinea  pig  systems.   Antisera  to  C4, 
raised  in  guinea  pigs,  rabbits  and  goats,  were  found  to  have  no  effect  on 
in  vitro  proliferative  responses  to  antigens  and  mitogens  and  did  not 
block  the  MLR.   Furthermore,  T  lymphocytes  from  guinea  pigs  with  a  genetic 
defeciency  in  C4  (C4D)  responded  normally  to  each  of  these  stimulants  and 
cells  of  C4D  guinea  pigs  had  normal  capacity  to  present  antigens  and  to 
stimulate  mixed  lymphocyte  responses.   These  results  suggest  that  if  C4 
has  an  important  role  in  the  regulation  of  T  lymphocyte  activation,  that 
role  cannot  be  a  general  one  involving  all  (or  even  most)  types  of  such 
activation  (Burger  and  Shevach,  LI/NIAID) . 


22-7 


22-8 


Administrative,  Organization  and  Other  Changes 

The  Laboratory  of  Immunology  plays  an  important  role  in  the  training  of 
young  scientists.   During  the  past  year,  Drs.  Theo  Kirland,  Virgil  Woods,  Jr., 
Loran  Clement  and  Ronald  Wilder  completed  their  appointments  as  research 
associates.   Dr.  Constantin  Bona,  who  was  a  Visiting  Scientist;  Drs.  Bondada 
Subbarao,  Tsuyoshi  Sakane  and  Franc  Meloni,  who  were  Visitng  Fellows;  and 
Drs.  Herbert  Herscowitz,  Ole  Werdelin,  Kim  Bottomly  and  Harley  Tse,  who  were 
Guest  Workers,  completed  research  and  training  programs  in  the  Laboratory  of 
Immunology.   Margaret  Simons  received  her  Ph.D.  in  genetics  for  research 
carried  out  in  the  Laboratory  under  the  supervision  of  Dr.  Rose  Mage.   Each 
of  these  scientists  made  substantial  research  contributions  during  their  ten- 
ure in  the  Laboratory  of  Immunology. 

Drs.  Laurie  Glimcher,  Dan  Hansburg,  John  Schmidt  and  David  Cohen  entered 
the  Laboratory  as  research  associates  while  Dr.  Dan  Longo  was  converted  from 
a  Guest  Worker  to  a  Research  Associate.   Drs.  Louis  Matis  and  Robert  Clark, 
clinical  associates  in  the  Laboratory  of  Clinical  Investigation,  were  assigned 
to  the  Laboratory  of  Immunology.   Drs.  John  Kung,  Joe  Chiba,  Benjamin  Sredni, 
and  Shunichi  Kumagai  were  appointed  as  Visiting  Fellows  and  Dr.  Leona 
Fitzmaurice  entered  the  Laboratory  as  an  Expert  Consultant. 

Finally,  one  of  the  senior  staff  of  the  Laboratory,  Dr.  Donald  Mosier, 
resigned  his  position  in  order  to  accept  a  major  appointment  at  the 
Institute  for  Cancer  Research,  Fox  Chase,  Pennsylvania.   Dr.  Mosier  had 
established  an  internationally  recognized  research  program  in  cellular 
immunology.   His  loss  as  a  contributor  to  the  Laboratory  program  will  be 
deeply  felt. 


22-9 


22-10 


Honors,  Awards,  and  Scientific  Recognition 

Laboratory  scientists  play  important  roles  in  the  national  and  inter- 
national scientific  community.   Many  serve  on  editorial  boards  of  important 
journals.   Dr.  Paul  was  appointed  to  the  editorial  board  of  the  Journal  of 
Immunology;  Drs.  Schwartz  and  Shevach  are  associate  editors  of  this  journal. 
Dr.  Green  and  Dr.  Shevach  are  referee  editors  of  the  Proceedings  of  the 
Society  for  Experimental  Biology  and  Medicine.   Dr.  Mage  is  chairman  of  the 
editorial  board  of  Federation  Proceedings  and  is  a  member  of  the  editorial 
board  of  the  Journal  of  Immunologic  Methods.   In  addition,  Dr.  Paul  is  an 
advisory  editor  of  the  Journal  of  Experimental  Medicine  and  an  associate 
editor  of  Immunologic  Reviews,  The  Scandinavian  Journal  of  Immunology, 
Immunologic  Communications,  CRC  Critical  Reviews  in  Immunology,  and 
Human  Immunology.   Dr.  Green  is  an  associate  editor  of  Clinical  Immunology 
and  Immunopathology,  and  Dr.  Inman  is  an  advisory  editor  of  Molecular 
Immunology.   Ms.  Lieberman  completed  a  term  as  an  advisory  editor  of  that 
journal.   Dr.  Shevach  is  a  member  of  the  editorial  advisory  board  of  the  FASEB 
Handbook  on  Inbred  Strains  of  Laboratory  Animals. 

Dr.  Paul  presented  invited  lectures  at  the  Southeastern  Immunology 
Workshop,  the  Armand  Hammer  Conference  on  the  Regulation  of  the  Immune 
Response,  the  Midwest  Student  Medical  Research  Forum,  the  Scottsdale 
Conference  on  B  Lymphocytes  in  the  Immune  Response,  the  Jane  Coffin  Childs 
Symposium,  the  Fogarty  Center  Workshop  on  Mediation  of  Effector 
Functions  by  Antibodies  and  the  Symposium  on  Carcinogeneses  of  the  Given 
Institute.   He  served  as  summarizing  speaker  at  the  National  Institute  on 
Aging  Conference  on  Immunology  and  at  the  International  Symposium  on  Aging 
and  Immunity  and  was  a  session  chariman  at  the  Brook  Lodge  Conference  on 
Mononuclear  Phagocytes  and  the  Scottsdale  Conference  on  B  Lymphocytes  in  the 
Immune  Response.   In  addition  Dr.  Paul  was  elected  President-elect  of  the 
American  Society  for  Clinical  Investigation  and  was  appointed  chairman  of 
the  American  Association  of  Immunologists  Program  Committee.   He  is  a  member 
of  The  American  Cancer  Society  Committee  on  Personnel  for  Research  and  the 
Arthritis  Foundation  Fellowships  sub-committee  and  is  co-organizer  of  the 
Cold  Spring  Harbor  Immunogenetics  Course. 

Dr.  Green  was  an  invited  lecturer  at  the  Intenational  Congress  on 
Systemic  Lupus  Erythematosus,  held  in  Kyoto;  at  the  International  Congress 
on  Vasculitis  in  Innsbruck;  and  at  the  Summer  Course  in  Methods  of 
Immunological  Research  and  Diagnosis.   He  is  co-chairmen  of  the  American 
Association  of  Immunologists  Program  Committee  and  is  a  member  of  the 
Transplantation  Immunology  Committee  of  NIAID. 

Dr.  Shevach  gave  major  lectures  at  the  ICN-UCLA  meeting  on  T  and  B 
Lymphocytes,  at  the  Annual  Meeting  of  the  New  England  Society  for  Allergy 
and  at  the  Brook  Lodge  Conference  on  Mononuclear  Phagocytes.   He  is  a 
member  of  the  Allergy  and  Immunology  Study  Section  of  the  Division  of 
Research  Grants,  NIH. 


22-11 


Dr.  Schwartz  gave  invited  lectures  at  the.  ICN-UCLA  Meeting  on  T  and  B 
Lymphocytes  and  at  the  Brook  Lodge  Conference  on  Mononuclear  Phagocytes.   He 
was  co-chairmen  of  a  minisymposium  at  the  annual  meeting  of  the  American 
Association  of  Immunologists. 

Dr.  Mage,  Ms.  Lieberman  and  Dr.  Bona  were  invited  lecturers  at  the 
International  Colloquium  in  honor  of  Jacques  Oudin  at  the  Institut  Pasteur, 
in  Paris.   Dr.  Mage  is  Vice-President  of  the  D.C.  Chapter  of  the  Society  of 
the  Sigma  Xi.   Ms.  Lieberman  is  a  member  of  the  American  Association  of 
Immunologists  Membership  Committee. 

Dr.  Inman  was  an  invited  speaker  at  the  European  Molecular  Biology 
Organization  Workshop  on  Accuracy  in  Biology,  at  the  Netherlands  Red  Cross 
Transfusion  Service  and  at  the  Central  Laboritorium  TNO  Ryswegh,  Netherlands. 
Dr.  Waxdal  was  a  major  speaker  at  the  Colloquium  on  Membrane  Glycoconjugates, 
in  Seillac,  France,  and  at  the  Colloquium  on  Protides  of  the  Biological 
Fluids,  in  Brussels.   He  was  an  invited  discussion  leader  at  the  Symposium 
on  Biomedical  Applications  of  the  Horseshoe  Crab  at  Woodshole  MA.  and  was 
chairman  of  the  session  on  Biochemical  Consequences  of  Mitogen  Activation 
at  the  annual  meeting  of  the  American  Association  of  Immunologists. 

In  addition,  laboratory  scientists  presented  research  seminars  at  major 
universities  and  research  institutes  in  the  United  States  and  abroad. 


22-12 


SMITHSONIAN  SCIENCE 

PROJECT  NUMBER  (Do  NOT  use  th 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT  .  .        .    ^ 

Principal  Investigator:  William  E.  Paul,  Chief,  LI/NIAID 

Rose  Lieberman,  Sr.  Investigator,  LI/NIAID 

Donald  E.  Mosier,  Inst,  for  Cancer 


NFORMATION  EXCHANGE 
space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00030-11  LI 


PERIOD  COVERED 


ctober  1,  1978  -  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Antigen  Recognition  and  Activation  of  Immunocompetent  Cells 


Other  Investigators: 


Research,  Phila. ,   PA. 
Bondada  Subbarao ,  Inst,  for  Cancer 
Research,  Phila.,  PA. 


Donna  G.  Sieckmann,  Staff  Fellow,  LI/NIAID 

Kathryn  Stein,  Staff  Fellow,  LI/NIAID 

Toby  Hecht,  Sr.  Staff  Fellow,  LI/NIAID 

Constant in  Bona,  Visiting  Scientist,  LI/NIAID 

Patricia  Mongini,  Guest  Worker,  LI/NIAID 

John  Kung,  Visiting  Fellow,  LI/NIAID 

Ellen  Heber-Katz,  Staff  Fellow,  LI/NIAID 

James  Mond,  Uniformed  Services  University  of  the  Health  Sciences 

Irwin  Scher,  Naval  Med.  Res.  Inst. 

Aftab  Ahmed,  Naval  Med.  Res.  Inst. 

Steven  Kessler,  Naval  Med.  Research  Institute  


COOPERATING  UNITS  (if  any) 

LMI/NIAID;  Naval  Medical  Research  Institute;  Institute  for  Cancer  Research, 

Philadelphia,  PA 


LAB/BRANCH 

Laboratory   of   Immunology 


institute  and  locat I ONNat ional  Institute  of  Allergy  and  Infectious  Diseases. 
National  Institutes  of  Health,    Bethesda,   Maryland  20014 


TOTAL  MANYEARS: 
9 


PROFESSIONAL: 


CHECK   APPROPRIATE   BOX(ES) 
G  (a)      HUMAN   SUBJECTS 

□  (a1 )    MINORS       □  (a2)    INTERVIEWS 


□  (b)    HUMAN    TISSUES 


^  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  general  goal  of  this  project  is  to  delineate  the  mechanisms  involved 
in  antigen  recognition  by  and  activation  of  thymus-independent  (B)  and  thymus - 
dependent  (T)  lymphocytes.   During  the  past  year  our  efforts  have  concentrated 
on:  1)  the  development  of  new  genetic  models  of  _B  lymphocyte  deficiency; 
2)  the  identification  and  genetic  mapping  of  B  lymphocyte  differentiation 
antigens;  3)  the  study  of  genetics  of  IgD  and  of  recombination  within  the  IgC^ 


gene  complex;  4)  the  genetic  regulation  and  chemical  characteristics  of  anti- 
bodies produced  in  response  to  polysaccharide  haptens;  5)  the  regulation  of 
antibodies  and  T_  lymphocytes  capable  of  interacting  with  membrane  immunoglobulin, 


In  addition,  studies  of  the  regulation  of  growth  of  vesicular  stomatitis 
yirus  in  spleen  cells,  both  _in  vivo  and  _in  vitro  are  underway. 


22-13 


PHS-6040 
(Rev.    10-76) 


Z01  AI  00030-11  LI 

Genetic  models  for  the  study  of  B  lymphocyte  development 

Over  the  past  several  years  we  have  investigated  the  developmental  and 
functional  heterogeneity  of  thymus-independent  (B)  lymphocytes  through  the 
use  of  a  mutant  mouse  strain.   Mice  of  this  strain,  CBA/N,  are  unresponsive 
to  thymus-independent  type  2  (TI-2)  antigens  and  lack  a  subset  of  B  lympho- 
cytes which  express  the  differentiation  antigens  Lyb  3,5,  and  7.   Recently, 
we  have  observed  that  breeding  the  mutant  CBA/N  gene,  xid,  on  to  a  C3H  back- 
ground leads  to  a  much  more  profound  defect  due  to  a  genetic  synergy  between 
the  xid  gene  and  one  or  more  C3H  genes.   The  "synergistic  defect"  (SD)  is 
characterized  by  _in  vitro  unresponsiveness  to  TI-1  as  well  as  TI-2  antigens 
and  by  a  failure  of  B  lymphocytes  to  proliferate  in  response  to  a  series  of 
mitogens  including  lipopolysaccharide,  bacterial  lipoprotein,  and  Nocardia 
water  soluble  mitogen.   Mice  with  the  SD  phenotype  have  diminished  numbers  of 
B  lymphocytes  and  the  B  lymphocytes  they  possess   express  very  low  ratios  of 
membrane  (m)  IgD:mIgM.   We  are  currently  in  the  process  of  preparing  inbred 
and  congenic  mouse  strains  expressing  the  SD  phenotype  in  order  to  make  these 
mice  available  for  the  detailed  study  of  B  lymphocyte  function  and  development. 

A  concurrent  program  is  the  analysis  of  B  lymphocyte  subsets  through 
the  development  of  antisera  to  differentiation  antigens  expressed  by  these 
subpopulations.   In  last  year's  report,  we  described  the  identification  and 
mapping  of  Lyb  5  and  Lyb  7.   Both  markers  are  found  on  that  subpopulation 
of  B  lymphocytes  which  is  lacking  in  mice  with  the  CBA/N  immune  defect. 
Lyb  7  is  particularly  interesting  because  antisera  to  it  block  B  lymphocyte 
activation  by  TI-2,  but  not  TI-1,  antigens  and  because  Lyb  7  is  genetically 
linked  to  genes  specifying  immunoglobulin  heavy  chain  constant  regions 
(IgC  genes) . 

We  are  attempting  to  expand  our  "library"  of  antibodies  to  B  lymphocyte 
differentiation  antigens  by  the  preparation  of  rat  anti-mouse  B  lymphocyte 
hybridomas.   A  series  of  such  hybrid  tumors  have  been  produced,  and  pre- 
liminary analysis  of  their  secreted  products  is  in  progress.   Several  of  these 
reagents  appear  to  identify  B  lymphocyte  subpopulations  and  will  be  of 
considerable  interest  in  our  continued  study  of  B  lymphocyte  function. 

Linkage  and  recombination  of  immunoglobulin  structural  genes. 

Studies  of  the  genetic  regulation  of  mlg  expression  are  also  in  progress. 
We  have  available  a  series  of  monoclonal,  alio-  and  hetero-  anti-6  anti- 
bodies and  have  recently  completed  an  extensive  strain  survey  allowing  us  to 
more  completely  make  IgC   haplotype  assignments  among  common  inbred 
laboratory  strains.   An  interesting  result  of  this  is  the  observation  that 
the  d  and  e  haplotype  groups  need  to  be  further  subdivided  because  strains 
previously  assigned  to  each  of  these  groups  differ  in  IgD  allotype  from  other 
strains  within  the  same  group.   On  this  basis,  we  have  defined  the  n  and 
o  IgC  haplotypes.   Furthermore,  one  inbred  strain,  AL/N, appears  to  be  a 
natural  IgC  recombinant  between  the  A  and  AKR  strains,  possessing  the  IgD 
alleles  of  A1  and  the  IgG  alleles  of  AKR.   This  observation  is  supported  by 
our  identification  of  two  actual  intra-IgC   recombinant  mice,  which  arose 

22T-14 


Z01  AI  00030-11  LI 

during  backcrossing  of  (C57BL/6  x  DBA/2)F..  hybrids  to  C57BL/6.   These  mice, 
individuals  72  and  74,  express  mlgD  and  serum  IgD  of  the  DBA/2  type  but  lack 
DBA/ 2  type  IgM,  IgG  ,  and  IgG   .   This  phenotype  is  stable  on  subsequent 
breeding  of  72  and  74  to  C57BE/6.   Furthermore,  when  mice  of  the  72  and  74 
families  are  crossed  to  strain  A  mice,  individuals  which  inherit  DBA/2  IgD 
also  inherit  C57BL/6  IgG.  ,  proving  that  these  allelic  genes  are  on  the  same 
chromosome  and  establishing  that  a  recombination  has  actually  occurred.   The 
availability  of  these  IgM-IgD  recombinant  strains  should  make  possible  the 
precise  mapping  of  genes  for  V  region  markers  with  reference  to  these  two 
critical  constant  region  genes. 

Genetics  of  immune  responses 

In  association  with  these  studies  of  genetics  of  immunoglobulin 
structural  genes,  studies  of  the  genetic  basis  of  antibody  responses  to 
highly  defined  polysaccharide  antigens  are  in  progress.   Lieberman  and  her 
colleagues  have  previously  established  that  the  capacity  to  make  antibodies 
to  6(2-^1)  fructosan  determinants,  such  as  those  in  inulin,  upon  immunization 
with  bacterial  levan,  a  g  (2->6)  polyfructosan  with  0  (2-*-l)  branch  points,  is 
determined  by  genes  linked  to  IgC  genes.   Our  current  studies  indicate  that 
non-IgC  genes  play  a  critical  role  in  the  magnitude  and  clonal  diversity  of 
the  response.   Most  strikingly,  BALB/c  mice  immunized  with  bacterial  levan 
make  a  highly  restricted  anti-inulin  response,  dominated  by  an  idiotype- 
positive  antibody  appearing  as  a  characteristic  triplet  of  bands  on  iso- 
electric focusing.   Although  C57BL/6  mice  lack  the  structural  genes  for  the 
triplet  and  related  anti-inulin  antibodies,  they  possess  non- immunoglobulin 
genes  which  increase  and  diversify  the  response.   This  is  shown  by  an 
analysis  of  responses  of  (BALB/c  x  C57BL/6)F..  hybrids,  allotype  congenic 
B.C8  mice,  and  recombinant  inbred  CXB  mice.   Our  current  analyses  suggests 
that  two  distinct  genes  are  involved;  we  are  in  the  process  of  verifying 
this  and  mapping  those  genes.   In  addition,  we  are  studying  the  genetic 
regulation  of  the  response  to  isomaltotriose  and  isomaltohexose  derivatives  of 
proteins  in  normal  mice  and  in  mice  with  the  CBA/N  immune  defect. 

Regulation  of  B  lymphocyte  activation  through  mlg 

Over  the  last  several  years,  we  have  developed  persuasive  evidence 
that  purified  antibodies  to  IgM  and  to  L  chains  can  stimulate  substantial 
proliferative  responses  on  the  part  of  B  lymphocytes  while,  at  the  same 
time,  inhibiting  the  secretion  of  immunoglobulin  in  response  to  other 
stimulants.   Our  data  has  indicated  that  this  proliferative  response  does 
not  require  the  presence  of  either  T  lymphocytes  or  macrophages  but  that  it 
is  a  function  of  a  late  appearing  subset  of  B  lymphocytes  which  is  absent  in 
mice  with  the  CBA/N  immune  defect.   During  the  past  year,  a  detailed  study 
of  the  phenotype  of  the  responding  cells  has  been  carried  out.   Particular 
emphasis  has  been  placed  on  the  characterization  of  these  cells  in  terms  of 
the  density  of  mlg  of  various  classes  expressed  on  their  membranes.   To 
carry  out  these  studies,  B  lymphocytes  have  been  prepared  and  separated  into 
subpopulations  with  distinct  amounts  of  Ig  on  their  surface  through  the  use 
of  the  fluorescence  activated  cell  sorter.   Through  this  approach,  we  have 

22-15 


Z01  AI  00030-11  LI 

shown  that  cells  which  proliferate  in  response  to  both  anti-p  and  anti-K 
express  a  high  density  of  total  Ig,  a  low  density  of  IgM,  and  an  intermediate 
to  high  density  of  IgD.   Preliminary  studies  indicate  these  cells  also  express 
Lyb  5.   We  are  now  in  the  process  of  preparing  hybridoma  rat  anti-mouse  u 
antibodies  inorder  to  more  decisively  explore  the  recognition  events  involved 
in  B  cell  activation. 

In  a  closely  related  set  of  studies,  we  have  shown  that  T  lymphocytes 
with  receptors  specific  for  idiotypic  determinants  found  on  the  TNP-binding 
myeloma  protein  MOPC460  suppress  the  production  of  anti-TNP  antibodies  which 
express  this  idiotype.   Furthermore,  we  have  evidence  that  such  cells  not 
only  exist  but  that  they  play  an  important  normal  role  in  regulating  the 
clonal  nature  of  the  anti-TNP  response  to  TNP  antigens.   These  studies  are 
particularly  interesting  because  they  provide  one  of  the  first  examples  of 
direct  action  of  suppressor  T  cells  on  B  cells  and  because  they  comprise  one 
of  the  first  instances  of  the  physiologic  activity  of  the  idiotype-anti- 
idiotype  network. 

Regulation  of  infection  of  spleen  cells  with  vesicular  stomatitis  virus  (VSV) 

VSV  is  a  membrane  budding  virus  which  fails  to  grow  in  resting  lympho- 
cytes and  which  has  been  reported  to  contain  host  cell  membrane  proteins,  such 
as  H-2  antigens,  in  its  envelope.   We  are  in  the  process  of  testing  the  thesis 
that  H-2  antigens  associated  with  VSV  virions  may  provide  a  means  of  attack  of 
the  host  immune  system  upon  virus  and  that  H-2  antigens  associated  with  virus 
play  a  strong  evolutionary  role  in  promoting  H-2  diversity  and  the  high  level 
of  "spontaneous"  immunity  to  H-2.   To  study  this,  we  have  developed  procedures 
for  the  m.  vivo  and  in  vitro  infection  of  mouse  spleen  cells  with  VSV  obtained 
from  various  cell  lines  and  have  shown  that  immunity  to  the  cell  line  in  which 
VSV  is  grown  profoundly  effects  the  capacity  of  the  virus  to  infect  spleen 
cells  in  vivo  or  in  vitro.   Studies  are  in  progress  to  obtain  more  reproducible 
conditions  for  inducing  in  vivo  and  in  vitro  infection  of  lymphocytes  by  VSV, 
for  more  precisely  evaluating  the  immunologic  mechanisms  underlying  the 
distinctions  made  between  VSVs  obtained  from  cell  lines  of  distinct  H-2  type, 
and  to  directly  determine  what  host  component  is,  in  fact,  responsible  for  the 
capacity  of  the  immune  system  to  determine  the  "lineage"  of  the  virus. 


22-16 


Z01  AI  00030-11  LI 

PUBLICATIONS 

Paul,  W.E.,  Scher,  I.,  Mond,  J.J.,  Ahmed,  A.,  Subbarao,  B.  and  Mosier,  D.E.: 
B  lymphocyte  development,  heterogeneity  and  function.  Arthritis  and 
Rheumatism21:  5175,  1978. 

Mond,  J.J.,  Scher,  I.,  Mosier,  D.E.,  Blaese,  M.  and  Paul,  W.E.:   T  independent 
responses  in  B  cell  defective  CBA/N  mice  to  Brucella  abortus  and  to  TNP 
conjugates  of  Brucella  abortus.   Eur .  J .  Immunol .  8:  459,  1978. 

Sieckmann,  D.G.,  Scher,  I.,  Asofsky,  R.,  Mosier,  D.E.  and  Paul,  W.E.: 
Activation  of  mouse  lymphocytes  By  ant i- immunoglobulin.   II.   Requirement 
for  a  mature  subset  of  B  lymphocytes.   J.  Exp.  Med.  148:  1628-1643,  1978. 

Mosier,  D.E.,  Zitron,  I.M. ,  Mond,  J.J.  and  Paul,  W.E.:   Requirements  for 
induction  of  antibody  formation  in  the  newborn  mouse.   Strasburger 
Symposium  on  Developmental  Immunobiology  (Eds.  G.W.  Siskind  and  M.  Weksler) 
Grune  and  Stratton,  New  York.   In  press. 

Paul,  W.E.,  Subbarao,  B. ,  Mond,  J.J.,  Sieckmann,  D.G.,  Zitron,  I.M., 
Ahmed,  A.,  Mosier,  D.E.  and  Scher,  I.   B  lymphocyte  development  and 
activation:   Analysis  with  a  mutant  mouse  strain.   Cells  of  Immunoglobulin 
Synthesis  (Eds.  B.  Pernis  and  H.J.  Vogel) .   In  press. 

Paul,  W.E.:   Genetic  regulation  of  the  immune  response.   Immunopa t ho lo gy 
(Eds.  J.  Mohn  and  F.  Milgrom)  S.  Karger  AG,  Basel,  Switzerland.   In  press. 

Ahmed,  A.,  Subbarao,  B.,  Scher,  I.,  Ryan,  J.J.,  Paul,  W.E.,  Mosier,  D.E.  and 
Sell,  K.W. :   Preliminary  evidence  for  a  new  non-H-2  locus  which  codes  for 
lymphocyte  activating  determinants  distinct  from  Mis.   Transplant.  Proc. 
In  press. 

Paul,  W.E.:   Lymphocyte  biology.   Clinical  Immunology  (Ed.  C.W,  Parker) 
Saunders,  New  York.   In  press. 

Bona,  C.  and  Paul,  W.E.:   Cellular  basis  of  regulation  of  expression  of 
idiotypes.   I.  Suppressor  cells  specific  for  MOPC460  idiotype  regulate  the 
expression  of  cells  secreting  anti-TNP  antibodies  bearing  460  idiotype. 
J.  Exp.  Med.  149:  592-600,  1972. 

Bona,  C,  Mond,  J.J. ,  Kessler,  S.,  Scher,  I.  and  Paul,  W.E.:   "Synergistic" 
immune  defect  in  backcross  male  mice  from  matings  of  CBA/N  and  C3H/HeJ  lines. 
In  B-Cell  Differentiation  (Edited  by  I.  Scher).   Academic  Press.   In  press. 

Bona,  C. ,  Hooghe,  R. ,  Cazenave,  P. A.,  Leguern,  C.  and  Paul,  W.E.:   Cellular 
basis  of  regulation  of  expression  of  idiotype.   II.  Enhancement  of  MOPC460 
idiotype  component  of  anti-TNP  antibodies  by  antibody  against  anti-MOPC460 
Id  antibodies.   J.  Exp.  Med  149:  815-823,  1979. 


22-17 


Z01  AI  00030-11  LI 

Bona,  C. ,  Cazenave,  P. A.  and  Paul,  W.E.:   REgulation  of  anti-TNP  response  by 
antiidiotypic  and  anti-(antiidiotypic)  antibodies.   Ann.  Immunol.  (Inst. 
Pasteur)  130C:  303,  1979. 

Paul,  W.E.:   Genetic  control  of  the  immune  response.   In  Textbook  of 
Rheumatology. 

Sieckmann,  D.G.,  Scher,  I.  and  Paul,  W.E.:   B  lymphocyte  activation  by 
anti- immunoglobulin  antibodies.   In  Physical-Chemical  Aspects  of  Cell  Surface 
Events  in  Cellular  Regulation.   (edited  by  R.  Blumenthal  and  C.  DeLisi) 
Academic  Press.   In  press. 

Subbarao,  B. ,  Hosier,  D.E.,  Ahmed,  A.,  Mond,  J.J.,  Scher,  I.  and  Paul,  W.E.: 
Role  of  a  non-Ig  cell  surface  determinant  in  the  activation  of  B  lymphocytes 
by  thymus- independent  antigens.   J .  Exp .  Med .  149:  495-506,  1979. 

Subbarao,  B. ,  Ahmed,  A.,  Paul,  W.E.,  Scher,  I.,  Lieberman,  R.  and  Mosier,  D.E. 
Lyb  7,  A  B  cell  alloantigen  controlled  by  genes  linked  to  the  IgC,,  locus. 
J.  Immunol.   In  press. 

Bona,  C.  and  Paul,  W.E.:   Cellular  basis  of  the  regulation  of  production  of 
anti-TNP  antibodies  carrying  MOPC460  idiotype.   1979  ICN-UCLA  Symposia, 
Keystone,  Colorado. 

Mond,  J.J.,  Stein,  K.  and  Paul,  W.E.:   Analysis  of  B  cell  activation  require- 
ment using  the  conjugated  polyacrylamide  beads.   J .  Immuno 1 .   In  press. 

Perlmutter,  R.M. ,  Nahm,  M. ,  Stein,  K.E. ,  Slack,  J.,  Zitron,  I.,  Paul,  W.E. 
and  Davie>  J-M..:   Immunoglobulin  subclass-specif ic  immunodeficiency  in  mice 
with  an  X-linked  B-lymphocyte  defect.   J.  Exp.  Med.  149:  993-998,  1979. 

Stein,  K.E.,  Mond,  J.J.,  Brennan,  C,  MatikelH,  0.  and  Paul,  W.E.:   Antibody 
affinity  in  CBA/N  mice.   ICN-UCLA  Symposia,  Keystone,  Colorado. 

Mond,  J.J.,  Sehgal,  E.,  Sachs,  D.H.  and  Paul,  W.E.:  Expression  of  la  antigen 
on  adult  and  neonatal  B  lymphocytes  responsive  to  thymus  independent  antigens. 
J.  Immunol.   In  press. 


22-18 


ITHSONIAN  SCIENCE  INFORMATION  EXCHAN 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01-AI-00148-04  LI 


PERIOD  COVERED 

October  1,  1979  -  September  30,  1980 


TITLE  OF  PROJECT  (80  characters  or  less) 

Lymphocyte  Interactions,  Receptors  and  Functions 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Principal  Investigator:   Ira  Green,  M.D.,  Senior  Investigator,  LI/NIAID 


Other  Investigators: 


Tsuyoshi  Sakane,  LI/NIAID 

Myron  Waxdal,  LI/NIAID 

Ethan  Shevach,  LI/NIAID 

Alfred  Steinberg,  Arthritis  &  Rheumatism  Branch, 

NIAMDD/NIH 
Herbert  Herscowitz,  Dept.  of  Microbiology,  Georgetown 

University  School  of  Medicine 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Immunology 


institute  and  location    National  Institute  of  Allergy  and  Infectious  Diseases,   National 
nrgs    of    HPalfh.    RpfhPsria,    Maryland    2  0? 05 


Tnsti  ti 


TOTAL  MANYEARS: 


PROFESSIONAL: 
2_ 


CHECK  APPROPRIATE  BOX(ES) 
Q  (a)   HUMAN  SUBJECTS 

□   (a1  )    MINORS       □  (a2)    It 


EJj]  (b)    HUMAN   TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

A  B  cell  leukemia  (L  C)  of  inbred  strain  2  guinea  pigs  and  a  new  myelogenous 
leukemia  (G-13)  of  inbred  strain  13  guinea  pigs  are  being  investigated  as  models 
of  human  leukemia.   The  chemical  nature  of  the  tumor  specific  transplantation 
antigen  (TSTA)  of  the  L„C  leukemia  is  being  investigated  by  immunization  protec- 
tion tests  in  syngeneic  animals.   The  TSTA  of  the  L  C  leukemia  is  unusual  in 
that  it  is  of  low  molecular  weight  and  is  extremely  resistant  to  heating.   The 
G-13  leukemia  is  being  examined  for  the  presence  of  a  TSTA  and  the  presence  of 
cell  surface  markers.   As  in  the  human,  the  myeloblasts  of  this  leukemia  are 
la  .   The  immune  function  of  these  cells  are  also  being  investigated. 

Patients  with  systemic  lupus  erythematosus  (SLE)  have  a  defect  in  the 
development  of  Con  A  induced  suppressor  cells.   Moreover,  the  sera  from  lupus 
patients  (more  particularly  the  IgM  fraction)  can  induce  suppressor  cell  defects 
in  populations  of  normal  lymphocytes.   T   and  T     cells  mediate  different  type^ 
of  suppression;  certain  lupus  sera  are  specifically  cytotoxic  for  T  cells. 
Finally  T  cells  able  to  proliferate  in  response  to  autologous  non  T  cells  act  as 
precursors  of  Con  A  induced  supppressor  cells. 


PHS-6040 
(Rev.  IO-76) 


-±9- 


Z01-AI-00148-04  LI 

I.   Studies  of  the  Guinea  Pig  L  C  Leukemia  and  a  New  Myelogenous  Leukemia 

The  L  C  lymphatic  leukemia  of  inbred  strain  2  guinea  pigs  arose  about  25 
years  ago  in  an  old  female  strain  2  guinea  pig.   Studies  in  our  laboratory 
starting  8  years  ago  have  shown  that  this  leukemia  cell  is  of  B  cell  origin 
and  has  surface  IgM  and  C3  receptors.   There  are  several  variants  of  this 
leukemia;  one  variant  exists  which  lacks  la  antigens  (an  antigen  of  the  major 
histocompatibility  complex  of  the  guinea  pig) .   Our  laboratory  has  demonstra- 
ted that,  as  measured  by  immunization  protection  tests  in  inbred  strain  2 
guinea  pigs,  the  L  C  cell  has  a  powerful  tumor  specific  transplantation 
antigen  (TSTA) .   However,  in  the  la  negative  variant  of  the  L  C  cell,  this 
TSTA  is  not  immunogenic.   Our  previous  studies  suggested  that  the  TSTA  is 
not  related  to  the  surface  IgM  molecule. 

The  main  goal  of  the  present  study  is  to  identify  the  chemical  nature  of 
the  TSTA  of  the  L„C  leukemia  cell.   A  3M  KC1  extract  of  the  L9C  cell  contains 
the  TSTA  as  measured  by  immunization  protection  tests  in  syngeneic  strain  2 
animals.   Immunization  with  as  little  as  100  ug  of  this  material  per  animal 
confers  protection  against  a  lethal  challenge  of  L9C  cells.   Fractionation 
of  the  KC1  extract  on  Sephadex  G-200,  DEAE  cellulose  and  CM  cellulose 
indicate  that  the  immunogenic  molecule  has  a  M.W.  of  less  than  20,000  and  is 
basic.   The  material  is  very  resistant  to  heat  and  extremes  of  pH  but  is 
destroyed  by  treatment  with  trypsin,  neuramnidase  and  periodate.   These 
properties  suggest  that  the  TSTA  is  a  low  m.w.  glycoprotein.   Attempts  to 
further  fractionate  the  material  by  polyacrylamide  gel  electrophoresis  were 
unsuccessful,  none  of  the  fractions  eluted  from  these  gels  were  immunogenic. 

Most  recent  studies  have  used  CM  cellulose  followed  by  G-50  chromato- 
graphy.  Several  immunogenic  regions  from  G-50  chromatography  have  been 
obtained  and  are  now  being  currently  investigated.   Progress  in  the  chemical 
identification  of  the  TSTA  of  the  L0C  cell  has  been  slow  because  each 
individual  assay  takes  7-8  weeks  to~complete  (in  vivo  protection  being  the 
final  end  point) . 

In  addition  to  the  studies  of  the  L„C  leukemia,  we  have  been  studying 
a  new  myelogenous  leukemia  of  strain  13  guinea  pigs  induced  with  the  carcino- 
gen N-nitroso-N-butylurea  (Evans  et  al. ,  Cancer  Research,  Vol.  38:  130,  1978). 
This  leukemia  is  serially  transplantable  in  inbred  strain  13  guinea  pigs 
using  intradermal  or  intraperitoneal  injection  of  whole  cells.   Two  to  three 
weeks  after  injection  of  the  cells  there  is  a  rise  in  polymorphonuclear 
leukocytes;  this  is  soon  followed  by  an  increased  number  of  myeloblasts.   The 
WBC  finally  rises  to  >  100,000  cmm  and  the  animal  succumbs.   Paralysis  of  the 
hind  legs  is  often  observed  as  a  terminal  event. 

We  have  characterized  the  surface  markers  on  these  myeloblasts;  these 
cells  lack  surface  Ig  and  do  not  have  C3  or  Fc  receptors.   These  cells  do 
have  la  antigens  on  their  surface  as  determined  by  immunof luorescent 
techniques;  biosynthetic  studies  indicate  that  the  la  antigens  found  on  these 
cells  are  actually  synthesized  by  the  cells. 


22-20 


Z01-AI-00148-04  LI 

To  determine  whether  la  antigens  were  also  found  on  more  mature  cells 
of  the  myelocytic  series  the  cells  were  first  stained  in  suspension  in  the 
living  state  for  the  presence  of  surface  la  antigens.   The  cells  were  then 
smeared  on  gridded  slides  (each  grid  having  a  number)  and  the  position  of  the 
la  positive  (stained)  cells  was  noted.   The  smear  was  then  stained  by  Giemsa 
stain,  the  same  cells  were  again  located  and  their  morphology  noted.   The 
results  of  such  experiments  demonstrated  that  most  myeloblasts  were  la  where- 
as most  bands  and  polymorphonuclear  leukocytes  were  negative. 

The  la  positive  myeloblasts  were  then  studied  for  two  immunological 
functions.   First  the  myeloblasts  were  tested  for  their  ability  to  act  as 
stimulator  cells  for  the  MLR.   Second  the  myeloblasts  were  tested  for  their 
ability  to  act  as  antigen  presenting  cells  for  the  T  cell  proliferative 
response  to  antigen.   They  failed  in  both  of  these  functional  tests.   This 
observation  strongly  suggests  that  the  presence  of  surface  la  antigens  is 
necessary  but  not  sufficient  to  allow  a  cell  to  perform  such  immunological 
functions. 

Preliminary  immunization  protection  studies  to  determine  whether  these 
cells  possess  a  tumor  specific  transplantation  antigen  (TSTA)  indicate  that 
the  cells  do  have  a  TSTA.   In  immunized  animals,  large  f ungating  tumor  masses 
were  observed  to  regress. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute 

The  properties  of  the  TSTA  of  the  L?C  leukemia  cell  are  highly  unusual 
and  differ  from  the  properties  of  most  other  TSTA  described  by  other  investi- 
gators.  Most  other  TSTA  have  a  m.w.  greater  than  40,000  daltons  and  are 
sensitive  to  heat. 

The  fact  that  this  TSTA  is  on  a  malignant  B  lymphocyte  is  of  particular 
interest;  once  the  L„C  TSTA  is  more  completely  identified  one  could  look  for 
a  similar  TSTA  in  several  different  human  B  cell  leukemias.   Another  possi- 
bility is  that  this  TSTA  could  represent  a  marker  for  a  particular  stage  of  B 
cell  differentiation  present  on  only  a  very  small  number  of  normal  B  cells. 

Studies  of  this  myelogenous  leukemia  are  of  potentially  great  interest 
since  in  some  cases  of  human  myeloid  leukemia  the  myeloblasts  are  also  la  pos- 
itive.  The  availability  of  large  numbers  of  la  myeloblasts  may  make  possible 
further  chemical  studies  of  these  la  antigens  and  to  allow  comparison  of  these 
la  antigens  with  those  on  lymphoid  cells.   Further  functional  studies  using 
these  la  myeloblasts  may  provide  a  clue  as  to  why  la  antigens  are  present  on 
myeloblasts.   This  leukemia  may  also  serve  as  a  model  for  chemo- immunotherapy 
of  myelogenous  leukemia. 

Human  Suppressor  Cells  in  Health  and  Disease 

The  second  aspect  of  our  studies  concerns  suppressor  cells  in  patient 
with  systemic  lupus  erythematosus  (SLE) .   The  system  we  are  using  is  adapted 
'from  Shou  _et  al. ,  J.  Exp.  Med.  143,  1100,  1976.   The  use  of  this  system  to 

22-21 


Z01-AI-00148-04  LI 

describe  concanavalin  induced  suppressor  cells  in  normal  individuals  has 
already  been  published  by  us  in  the  J.  of  Immunology  119,  1169,  1977. 

A  two  stage  assay  system  is  employed  in  which  T  lymphocytes  are  exposed 
to  concanavalin  A  (Con  A)  for  3  days  to  induce  suppressor  function  and  then 
these  cells,  after  mitomycin  C  treatment,  are  added  to  an  assay  system  con- 
sisting of  lymphoid  cells  obtained  from  the  same  donor  3  days  later.   As  a 
control,  another  aliquot  of  cells  incubated  without  Con  A  is  used.   These 
assay  cultures  are  then  stimulated  by  either  mitogen  or  allogeneic  cells, 
cells  from  the  first  culture  are  added  and  the  degree  of  proliferation  is 
measured  by  H-thymidine  incorporation. 

We  have  recently  demonstrated  that  aliquots  of  these  same  Con  A  activated 
T  cells  can  also  suppress  a  pokeweed  mitogen  (PWM)  induced  polyclonal  B  cell 
response. 

Our  major  effort  has  been  to  analyze  suppressor  cell  defects  in  patients 
with  SLE.   We  first  demonstrated  that  patients  with  SLE  have  a  relative  lack 
of  T  cells  capable  of  being  induced  to  form  suppressor  cells.   Furthermore 
when  certain  SLE  sera  are  added  to  cultures  of  normal  lymphocytes  during  the 
Con  A  mediated  induction  of  suppressor  cells,  no  suppressor  cells  develop. 
We  had  previously  shown  that  the  IgM  fraction  of  SLE  sera  was  responsible  for 
this  effect  and  that  complement  is  necessary.   Our  most  recent  studies  have 
revealed  that  the  SLE  sera  is  only  effective  when  added  at  the  beginning  of 
the  Con  A  inducing  culture  but  not  at  the  end.   We  also  observed  that  certain 
patients  with  SLE  had  a  selective  loss  of  only  one  type  of  suppressor  cell. 
That  is,  the  patients  lymphocytes  could  suppress  the  PWM  proliferative  re- 
sponse but  not  the  MLR.   The  sera  from  these  same  patients  could  induce  in  an 
in  vitro  culture  exactly  this  same  pattern  of  defects  in  the  lymphocytes  from 
normal  individuals.   The  fine  cytotoxic  specificity  of  the  sera  from  these 
selected  patients  was  then  tested  on  different  classes  of  T  cells.   T  cells 
can  be  separated  into  two  classes,  one  class  having  a  surface  receptor  for  the 
Fc  portion  of  IgG.   (T^  cells)  and  another  class  lacking  this  receptor  (T 
cell).   These  sera  were  selectively  cytotoxic  for  T  cells.   We  then  investi- 
gated whether  different  T  cell  classes  could  differentially  suppress  either 
the  PWM  proliferative  response  or  the  mixed  lymphocyte  response  (MLR) .   We 
observed  that  T  cells  could  preferentially  suppress  the  MLR.   Thus  the  ability 
of  certain  SLE  sera  to  eliminate  suppressor  cells  for  the  MLR  can  be  ascribed 
to  their  preferential  cytotoxicity  for  T  cells. 

We  have  also  performed  two  other  related  studies.   First  we  have  serially 
studied  patients  with  SLE  during  the  active  and  inactive  phases  of  their 
disease  for  both  defects  in  suppressor  cells  development  for  defects  in  the 
autologous  MLR  (discussed  in  last  year's  annual  report). 

In  each  case  these  _in  vitro  tests  were  abnormal  during  the  active  phase 
of  disease  and  returned  to  normal  when  the  disease  activity  decreased.   These 
results  strongly  suggest  that  the  abnormalities  of  suppressor  cell  generation 
and  the  autologous  MLR  observed  in  lymphocytes  from  patients  with  SLE  are  not 
"intrinsic  to  the  lymphocyte  or  genetically  determined. 

22-22 


Z01-AI-00148-04  LI 

In  the  second  study  we  wished  to  determine  whether  cells  proliferating 
in  the  autologous  MLR  were  particularly  able  to  develop  Con  A  induced  supp- 
ressor function  as  compared  to  cells  proliferating  in  the  usual  cells  MLR. 
Therefore  cells  undergoing  either  auto  MLR  or  allogeneic  MLR  were  treated  or 
not  treated  with  BUdR  and  light. 

The  T  cells  were  then  harvested  and  tested  for  their  ability  to  generate 
suppressor  function.   The  cells  undergoing  an  autologous  MLR  in  the  presence 
of  BUdR  and  light  failed  to  subsequently  develop  suppressor  function  whereas 
the  cells  undergoing  an  allogeneic  MLR  were  able  to  develop  suppressor  func- 
tion. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute 

The  defect  in  the  development  of  suppressor  cells  in  patients  with  SLE 
may  be  one  of  the  causes  of  increased  auto  antibody  production  in  this 
disease.   Furthermore,  sera  from  patients  with  SLE  can  induce  suppressor  cell 
abnormalities  in  normal  T  lymphocyte.   Strategies  to  increase  suppressor 
function  in  patients  with  SLE  might  have  therapeutic  potential.   Selective 
removal  of  antibodies  to  suppressor  cells  might  be  a  step  in  this  direction. 

PUBLICATIONS : 


Sakane,  T. ,  Steinberg,  A.D.  and  Green,  I.:  Failure  of  suppressor  T  cell 
activity  in  patients  with  systemic  lupus  erythematosus  (SLE) .  Arthritis 
and  Rheumatism.  21:  657,  1978. 

Gelfand,  M.C.,  Frank,  M.M. ,  Green,  I.  and  Shin,  M.L.:   Binding  sites  for 
immune- complexes  containing  IgG  in  the  renal  interstitum  of  man  and 
other  species.   Clinical  Immunology  and  Immunopathology  13:  19,  1979. 

Stingl,  G. ,  Katz,  S.I.,  Clement,  L.,  Green,  I.  and  Shevach,  E.M. ;   Immuno- 
logical functions  of  la-bearing  epidermal  Langerhans  cells.   J.  Immunol. 
121:  2005,  1978. 

Carter,  R. ,  Gwadz,  R.W.  and  Green,  I.:   Plasmodium  gallinaceum:  Transmission 
Blocking  Immunity  in  the  Chicken  with  Aedes  algypti  as  the  Mosquito  Vector. 
II.  The  Effect  of  Anti-Gamete  Antibodies  _In  Vitro  and  InVivo  and  their 
Elaboration  During  Infection.   Experimental  Parasitology.   In  press. 

Nilsson,  S.F.,  Edelson,  R. ,  Mann,  D. ,  Green,  I.  and  Waxdal,  M.J.:   Concanavalin 
A  binding  proteins  on  the  surface  of  human  malignant  and  normal  lymphocytes. 
Scand.  J.  Immunol.   In  press,  1979. 

Bona,  C,  Lieberman,  R.,  House,  S.,  Green,  I.  and  Paul,  W.E.  :   Immune 
Response  to  Levan.   II.  T-Independence  of  Suppression  of  cross-Reactive 
Idiotypes  by  Anti-Idiotype  Antibodies.   J.  Immunol.  122:  1614,  1979. 


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Z01-AI-00148-04  LI 

Sakane,  T.,  Steinberg,  A.D.,  Reeves,  J. P.  and  Green,  I.:   Studies  of 
Immune  Functions  of  Patients  with  Systemic  Lupus  Erythematosus.   Complement 
Dependent  IgM  anti-T  Cell  Antibodies  Preferentially  Inactivate  Suppressor 
Cells.   J.  Clin.  Investigation.  63:  954,  1979. 

Sakane,  T.,  Steinberg,  A.D.,  Arnett,  F.C.,  reinertsen,  J.L.  and  Green,  I.: 
Studies  of  immune  function  of  patients  with  systemic  lupus  erythematosus. 
III.   Characterization  of  the  lymphocyte  subpopulations  responsible  for 
the  defective  autologous  MLR.   Arthritis  and  Rheumatism.   In  press. 

Sakane,  T.  and  Green,  I.:   Specificity  and  suppressor  function  of  human 
T  cells  responsive  to  autologous  non-T  cells.   J.  Immunol.   In  press. 

Sakane,  T.,  Steinberg,  A.D.,  Reeves,  J. P.  and  Green,  I.:   Studies  of 
immune  functions  of  patients  with  systemic  lupus  erythematosus.   T  cell 
subsets  and  antibodies  to  T  cell  subsets.   J.  Clin.  Investigations.   In 
press. 

Sakane,  T.,  Steinberg,  A.D.  and  Green,  I.:   Studies  of  immune  functions  of 
patients  with  systemic  lupus  erythematosus.   V.  T-cell  suppressor  function 
and  autologous  MLR  during  active  and  inactive  phases  of  disease.   Arthritis 
and  Rheumatism.   In  press. 


22-24 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  AI   00035-06    LI 


PERIOD  COVERED 

October  1,  1978  -  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Specificity  in  Immune  Responses 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


Principal  Investigator: 
Other  Investigators: 


John  K.  Inman,  Senior  Investigator,  LI/NIAID 

Dr.  Ettore  Appella,  LCB/NCI 
Dr.  Garrett  C.  DuBois,  LCB/NCI 
Dr.  Bruce  Merchant,  BOB/FDA 


COOPERATING  UNITS  (if 

Laboratory  of  Cell  Biology,  National  Cancer  Institute;  Immunology  Branch, 
National  Cancer  Institute;   Bureau  of  Biologies,  FDA. 


iny) 

:el] 


lab/branch 

Laboratory  of   Immunology 


INSTITUTE  AND  LOCATION 


National  Institute  of  Allergy  and  Infectious  Diseases, 
National  Institutes  of  Health,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

2 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 
G  (a)   HUMAN  SUBJECTS 

Q  (al)  MINORS   G  (a2)  INTERVIEWS 


G  (b)  HUMAN  TISSUES 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  long  range  objective  of  this  project  is  to  carefully  re-examine  the 
nature  of  binding  specificity  at  antibody  combining  regions  and  other  types  of 
receptor  sites.   The  effects  of  polyvalent  binding  on  binding  energy  and  spec- 
ificity will  be  considered,  and  together  these  properties  will  be  related  to 
the  behavior  of  immune  systems.   In  particular,  multispecif ic  binding  of  disp- 
arate structures  by  individual  antibody  species  is  being  studied  through 
screening  of  specifically  purified,  radiolabeled  antibodies  with  affinity 
chromatography  columns.   The  ligands  are  bound  to  these  columns  using  large 
haptenic  reagents  synthesized  specially  for  this  purpose  using  methods  of 
peptide  synthesis.   Multispecif ic  binding  will  be  used  for  producing  and 
isolating  homogeneous  antibody  by  cross-stimulation  and  immunoadsorption .   A 
number  of  chemical  techniques  and  products  that  have  been  developed  from  the 
above  work  are  being  used  in  collaborative  studies  on  molecular  interpretation 
of  cellular  mechanisms  in  immune  responses.   Materials  employed  include  syn- 
thetic thymus- independent  antigens. 
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PHS-6040 
(Rev.  10-76) 


Z01  AI  00035-0  5  LI 

Project  Description 

Objectives 

The  overall  objective  of  the  project  is  to  gain  more  complete  under- 
standing of  the  molecular  basis  for  specificity  in  immune  responses.   The 
binding  properties  of  antibodies  and  other  receptor-bearing  molecules  are 
to  be  studied  and  related  to  the  behavior  of  immune  systems.   Specific 
objectives  are  the  following: 

1.  To  assess  the  generality  and  frequency  of  multispecif ic  binding  by 
individual  antibody  combining  regions  by  means  of  screening  experiments 
employing  many  diverse,  structurally  unrelated  haptens. 

2.  To  develop  methodology  required  for  such  screening  tests  including  the 
synthesis  of  numerous,  large  haptenic  reagents. 

3.  To  employ  multispecif ic  binding  and  cross-stimulation  in  raising  high 
titers  of  highly  restricted  antibodies  for  fine  specificity  studies  and  for 
use  as  highly  specific  reagents  in  immunological  studies. 

4.  To  use  the  large  hapten  screen  to  find  disparate  structure  cross-reactions 
useful  in  identifying  antibody-secreting  cell  clones  in  studies  assessing 
antibody  diversity. 

5.  To  use  the  screen  in  studying  the  multispecif ic  character  of  other  types 
of  receptors  and  to  employ  observed  cross-reactions  in  a  general  approach 

to  affinity  separations  of  receptors. 

6.  To  employ  methods  and  reagents  developed  in  this  project  in  collaborative 
studies  dealing  with  specificity  in  immune  responses. 

Major  Findings 

The  main  emphasis  during  the  past  year  has  been  placed  on  exploring 
strategies  for  the  efficient  synthesis  of  highly  varied  specificity  probes. 
These  probes  are  reagents  for  introducing  large  haptenic  groups  onto 
macromolecular  carriers,  and  they  play  a  key  role  in  fulfilling  the  above 
research  objectives.   With  the  collaboration  of  a  postdoctoral  fellow, 
Dr.  Suresh  Shukla,  and  my  assistant,  Miss  Barbara  Duntley,  I  have  evaluated 
a  large  number  of  synthetic  intermediates,  pathways,  tactics  and  strategies 
leading  to  branched  peptide  derivatives  bearing  a  variety  of  substituent 
structures  and  attachment  functions.   Our  findings  are  as  follows:   the  use 
of  glutamic  acid  as  a  trivalent  hub  structure  sometimes  led  to  undesired 
by-products  when  the  last  substituent  was  introducted.   This  result  seemed 
to  preclude,  for  the  time  being,  the  glutamic  diamide  approach  as  being 
sufficiently  general  and  facile  to  meet  the  demands  of  our  study.   Instead, 
we  turned  our  attention  mainly  to  another  trivalent  amino  acid,  L-lysine. 
We  have  successfully  synthesized  large  haptenic  reagents  having  attachment 
functions  and  spacing  structures  connected  with  either  the  alpha-carboxyl  or 

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Z01  AI  00035-06  LI 

epsilon-amino  group.   About  20  of  the  latter  type  of  reagent  have  been  pre- 
pared in  final  form  and  are  at  present  undergoing  purification.   Techniques 
for  achieving  final  purity  are  being  studied.   We  have  explored  adipic  acid 
as  a  spacer  for  the  attachment  function  on  lysine  and  have  concluded  that  the 
latter  should  be  a  protected  hydrazide  introduced  at  an  early  stage  of 
synthesis.   In  the  course  of  this  work  we  introduced  a  new  protecting  group 
for  acyl  hydrazides,  the  2-(methylsulf onyl)ethoxycarbonyl  (Msc)  function 
which  is  stable  during  the  removal  of  other  blocking  groups.   It  can  be  re- 
moved under  special,  mild  conditions  just  prior  to  activation  and  coupling 
of  the  hapten  to  a  carrier  (immunogen  or  adsorbent  matrix) .   The  Msc  group 
has  not,  to  our  knowledge,  been  used  for  hydrazide  protection.   This  finding 
should  have  general  application  in  peptide  synthesis:   Along  with  our 
experience  in  haptenic  reagent  synthesis,  it  was  presented  in  a  poster  paper 
at  the  Sixth  American  Peptide  Symposium  on  June  21,  1979.   Currently  we  are 
evaluating  haptens  based  on  L-ornithine  as  a  central  structure.   A  number  of 
substituent  structures  have  been  selected  or  prepared  for  the  next  series  of 
probes.   Enough  haptenic  reagents  are  on  hand  to  begin  setting  up  a  pre- 
liminary screen  for  multispecif ic  binding;  preparations  are  underway. 

Work  on  sequencing  chemistry  has  largely  been  concluded.   I  am  pre- 
sently writing  a  chapter  (with  Dr.  Appella)  summarizing  our  thoughts  and  ex- 
periences over  the  past  few  years  on  the  topic  of  newer  approaches  to  solid- 
and  liquid-phase  amino  acid  sequence  analysis. 

In  collaboration  with  Dr.  Bruce  Merchant  (BOB/FDA)  and  Dr.  Harm  Snippe 
(State  University  at  Utrecht,  The  Netherlands)  several  lines  of  investigation 
were  continued,  and  results  are  currently  being  submitted  for  publication. 
It  was  found  earlier  that  immunization  of  mice  with  various  large  haptens, 
conjugated  to  bovine  serum  albumin  and  mixed  with  the  cationic,  surface-active 
lipid,  dimethyl  dioctadecyl  ammonium  bromide  (DDA) ,  generated  delayed  type 
hypersensitivity  (DA)  without  detectable  levels  of  circulating  specific  anti- 
body.  DDA  promoted  strong  cross-reactivity  with  other  similar,  and  not  so 
similar,  haptens  on  the  same  carrier.   Both  direct  and  indirect  plaque- 
forming  cells  (PFC)  were  detected,  however,  in  peripheral  lymph  nodes  and 
spleens  4  days  after  a  challenge  injection.   The  specificity  of  the  PFC 
was  distinctly  greater  than  that  exhibited  by  DH.  Another  area  of 
collaborative  work  with  Drs.  Merchant  and  Snippe  concerns  the  nature  of  the 
X-linked,  B-cell  defect  of  CBA/N  mice.   We  had  earlier  reported  that  the 
essentially  normal  immune  response  of  these  animals  to  thymic  dependent, 
hap ten- carrier  conjugates  can  be  blocked  by  very  small  amounts  of  the  same 
hapten  bound  to  a  thymic  independent  carrier  such  as  Ficoll  or  SIII  poly- 
saccharide.  The  blockade  was  presumed  to  be  mediated  in  part  through 
efficient  binding  of  the  blockading  antigen  by  Ig  receptors  on  the  de- 
fective B  cells.   A  subsequent  study  supported  this  view:   normal  CBA/N  x 
C3H/HeN  F1  female  spleen  cells  when  exposed  to  DNP-AGG-Ficoll  in  vitro 
could  establish  a  specific  PFC  response  after  transfer  into  irradiated,  male, 
hybrid  recipients.   Although  defective  male,  hybrid  spleen  cells  could  not 
mount  this  rescue  function,  they  could  convey  immunogenic  quantities  of  DNP- 
AGG-Ficoll,  previously  bound  to  their  surfaces,  to  female  cells  in  vitro 
This  conveyor  function  was  impeded  by  pretreating  the  male  cells  with  goat 

22-27 


Z01  AI  00035-06  LI 

anti-mouse  u  serum.   The  defective  cells  thus  appear  to  bind  thymus-independ- 
ent  antigens  but  not  to  be  triggered  by  the  binding  event.   Additional  ex- 
periments with  cell  transfer  systems  using  CBA/N  x  C3H/He  F..  hybrid  spleen 
cells  and  studying  the  effects  of  the  blockading  antigen,  DNP-AGG-Ficoll, 
showed  a  different  pattern  of  blockade  than  was  observed  in  in  vivo  trials: 
Direct  and  indirect  PFC  responses,  upon  secondary  immunization  in  irradiated 
recipients,  were  subject  to  hap ten- specific  blockade;  under  some  cell  transfer 
conditions  normal  CBA  x  C3H/HeN  F   female  spleen  cells  were  just  as  suscepti- 
ble to  blockade  as  the  defective  male  hybrid  cells. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

An  essential  characteristic  of  many  immune  mechanisms  is  the  specificity 
of  action  which  is  directed  by  definitive  chemical  structures.   An  under- 
standing of  specificity  in  immune  systems  rests  first  upon  an  adequate  know- 
ledge of  the  scope  and  nature  of  specificity  at  the  level  of  single  re- 
ceptor-determinant interactions.   Therefore,  a  principal  aim  of  this  project 
is  to  carry  out  the  first  sytematic  and  general  exploration  of  the  phenomen- 
on of  multispecif ic  binding.   Because  of  the  diversity  and  availability  of 
antibodies,  these  receptor-bearing  molecules  provide  ideal  models  for  such 
studies  in  addition  to  their  being  interesting  as  important  components  of 
many  types  of  immune  reactions. 

Cooperative  studies  have  proven  the  value  of  large  hatpenic  reagents 
and  other  synthetic  or  semi-synthetic  antigenic  components  in  exploring 
specificity  as  it  is  manifested  in  more  complex,  multi-receptor-determinant 
interactions.   Reagents  or  reactions  developed  for  the  explicit  requirements 
of  this  project  can  have  interesting  applications  in  many  basic  studies  in 
immunology  such  as  those  aimed  at  elucidating  the  mechanisms  of  B  and  T  cell 
specific  activation. 

Future  Course: 


The  screening  for  multispecif ic  binding  will  commence  in  the  next  month. 
Early  studies  will  involve  some  monoclonal  antibodies  (myeloma  and  hybridoma 
proteins)  and  some  selected  antisera.   Synthesis  of  large  haptenic  reagents 
will  continue  on  a  more-or-less  routine  basis  in  order  to  enlarge  the 
repertoire  of  our  screen.   A  large  number  of  hybridoma  antibodies  will  be 
sought  (as  gifts  from  other  laboratories)  and  used  for  study.   Animals 
will  soon  be  immunized  against  our  large  haptens  for  production  of  normal 
heterogeneous  antibodies  and  possibly  for  production  of  hybridomas  (as 
a  collaboration  within  LI/NIAID) .   Collaborative  work  with  Dr.  Merchant  will 
continue. 

PUBLICATIONS: 

Snippe,  H. ,  Merchant,  B.,  Johannesen,  L.  and  Inman,  J.K. :   Effects  of  cyclo- 
phosphamide on  the  _in  vivo  response  of  outbred  athymic  (nude)  mice  to  a 
thymus-independent  antigen  (DNP-AGG-Ficoll).   Immunol.  35:  1009-1015,  1978. 


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Z01  AI  00035-96  LI 

JBrnvall,  H. ,  Inman,  J.K.  and  Appella,  E.:   Aminolysis  of  thiazolinones  in 
manual  amino  acid  sequence  analysis.   Direct  detection  of  carboxyl,  amide 
and  tryptophan  residues  in  conjunction  with  the  dansyl-Edman  method.   Anal. 
Biochem.  90:  651-661,  1978. 

Schroer,  J. A.,  Inman,  J.K. ,  Thomas,  J.W.  and  Rosenthal,  A.S.:   H-2-linked 
Ir  gene  control  of  antibody  responses  to  insulin.   I.  Anti-insulin  plaque- 
forming  cell  primary  responses.   J.  Immunol.   In  press. 

Inman,  J.K.:   Peptide  synthesis  with  minimal  protection  of  side-chain 
functions.   In  Gross,  E.  and  Meienhofer,  J.   (Eds):   The  Peptides-Analysis, 
Synthesis  and  Biology,  Vol.  3,  N.Y.,  Academic  Press,  Inc.,  in  press. 

HONORS : 

Appointed  as  Advisory  Editor  to  the  Journal,  Molecular  Immunology. 

Invited  speaker  at  The  European  Molecular  Biology  Organization,  Workshop 
on  Accuracy.   Grignon,  France,  September  1978. 

Invited  lecturer  at  the  State  University  at  Utrecht,  The  Netherlands, 
September,  1978. 


22-29 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  AI-00036-14  LI 


PERIOD  COVERED 

October  1,  1978  -  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Immunoglobulin  Genetics:   Regulation  of  Gene  Expression  and  Lymphoid 
Differentiation. 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Principal  Investigator:   Rose  G.  Mage,  Ph.D.,  Senior  Investigator,  LI/NIAID 

Other  Investigators:   G.O.  Young-Cooper,  LI/NIAID 
C.B.  Alexander,  LI/NIAID 
R.  Wilder,  Research  Associate,  LI/NIAID 
H.  Abdi,  Visiting  Fellow,  LI/NIAID 


cooperating  units  (if  any)ji.D.  Cooper  and  A.  R.  Lawton,  Univ.  Alabama,  Birmingham,  AL: 
J.  Karsh,  A.R./NIAMDD;  I.  Scher,  NMRI;  A.  Chersi,  Istituto  Regina  Elena,  Rome, 
Italy;  E.  Appella,  LCB/NCI;  R.  Asofsky,  LMI/NIAID.  In  addition,  we  cooperate  with 
a  large  number  of  investigators  at  NIH  and  elsewhere  by  supplying  research  re- 


LAB/BRanch  agents  and  allotype-def ined  rabbits  and  by  providing  typing  information 
Laboratory  of  Immunology about  rabbit  sera  sent  to  us. 


NSTITUTE  AND  location  National  Institute  of  Allergy  and  Infectious  Diseases. 
Mal-innal  Tnsfihitps  of  Health,  Bethesda,  MP  20205. 


TOTAL  MANYEARS: 

-5_3_ 


PROFESSIONAL: 


2.6 


CHECK  APPROPRIATE  BOX(ES) 
G  (a)   HUMAN  SUBJECTS 


3c(b)  HUMAN  TISSUES 


□  (c)  NEITHER 


□  (a1 )  MINORS 


(a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Overall  goals  of  this  project  are  to  define  the  nature  of  genes  which  code 
for,  or  otherwise  affect  the  structure  of  immunoglobulins (Igs)  and  to  understand 
the  processes  which  regulate  Ig-gene  expression  and  which  lead  to  synthesis  of 
highly  specific  antibody  molecules.   We  prepare  immunological  reagents  which  de- 
tect structural  differences  between  genetically  controlled  polymorphic  forms  of 
rabbit  Igs  (Ig  allotypes).   We  use  immunological,  chemical  and  molecular  genetic 
approaches  to  define  the  Ig-structural  differences  and  to  learn  more  about  the 
chromosomal  organization  of  Ig  genes,  and  the  regulated  expression  of  antibodies 
and  antigen-specific  surface  receptors  on  lymphocytes.   Inheritance  patterns  and 
the  levels  of  regulation  of  phenotypic  expression  of  alleles  and  chain  types  are 
investigated  by  breeding  experiments,  by  studies  of  ontogeny  and  differentiation 


of  cells  of  the  rabbit  immune  system  and  by  direct  molecular  genetic  analyses 
Two  major  new  efforts  which  are  planned  will  involve  the  use  of  recombinant  DNA 
molecules  to:  1)  examine  genome  organization  by  isolation  of  rabbit  Ig-genes  and 


analysis  of  variable  and  constant  region  DNA  sequences  as  well  as  adjacent  se- 
quences; and  2)  identify  nuclear  RNA  precursors  and  their  processing  steps  in  the 
orndnrtinn  nf  immunoglobulin -mRNA.    Z2-JU 


-hS-6040 

10-76) 


ZHI  AI  00036-14  LI 


Objectives 

The  objectives  of  this  research  program  are  to  define  in  molecular  terms, 
the  organization  and  regulated  expression  of  rabbit  immunoglobulin  genes,  the 
levels  of  regulation  of  lymphoid  cell  differentiation,  and  the  mechanisms 
which  lead  to  the  synthesis  of  highly  specific  antibody  molecules  and  antigen 
specific  lymphocyte  cell  surface  receptors. 

Methods  Employed 

For  the  studies,  we  prepare  and  characterize  antisera  which  detect 
structural  differences  between  genetically  controlled  polymorphic  forms  of 
rabbit  Igs  (Ig  allotypes)  and  between  classes  and  subclasses,  types  and  sub- 
types of  their  polypeptide  chains.   We  use  immunological  and  chemical 
approaches  to  define  structural  differences  and  conduct  classical  and  molecu- 
lar genetic  experiments  to  learn  more  about  the  chromosomal  organization  of 
genetic  information  related  to  Ig  molecules.   Problems  which  until  recently 
have  been  approached  at  the  levels  of  serology  and  classical  Mendelian  genet- 
ics are  now  amenable  to  direct  molecular  genetic  analysis.  We  are  initiating 
studies  which  will  utilize  characterized  clones  containing  mouse  immunoglobu- 
lin heavy  and  light  chain  sequences  as  well  as  efforts  to  clone  rabbit  genomic 
DNA  and  complementary  DNA  copies  of  mRNA  so  that  we  can  examine  the  organiza- 
tion of  rabbit  immunoglobulin  genes  and  identify  nuclear  RNA  precursors  and 
the  processing  steps  in  production  of  immunoglobulin  mRNA. 

Major  Findings 

Ontogeny,  lymphoid  cell  differentiation,  and  the  regulation  of  Ig-gene 

expression. 

Detection  of  V  and  studies  of  allelic-exclusion  in  pre-B  cells. 

In  previous  studies,  we  described  a  primitive  lymphoid  cell  found  in  fetal 
liver  and  in  the  bone  marrow  of  older  rabbits  which  contained  cytoplasmic  IgM 
but  lacked  surface  IgM  detectable  by  immunofluorescence  (Hayward  et  al. ,1978) . 
In  heterozygous  b  b  rabbits,  the  pre-B  cells  in  which  we  could  detect  these 
kappa  chain  allotypes  appeared  to  exhibit  allelic  exclusion.   Comparable  stud- 
ies have  now  been  done  (Gathings,  Cooper,  Lawton,  Young-Cooper  and  Mage — work 
in  progress)  with  the  allotypes  associated  with  the  variable  regions  of 
immunoglobulin  heavy  chains.   Affinity-purified  and  cross-absorbed  al  anti-a2 
and  al  anti-a3  reagents  were  used  to  identify  V  -bearing  immunoglobulin  within 
pre-B  cells  of  a2,  a3  and  heterozygous  a  a  rabbits.   We  found  that  essen- 
tially all  pre-B  cells  from  adult  and  newborn  heterozygous  animals  which  are 
detectable  with  our  fluorescent  reagents  exhibit  allelic  exclusion  of  the  a_ 
allotypes.   The  majority  of  cells  with  cytoplasmic  IgM  have  detectable  V  a 
allotypes  (56-100%).   We  have  also  investigated  B  cells  with  the  reagents 
described  above  (Gathings  et^  al . )  as  well  as  with  a2  anti-a3  and  a3  anti-a2 
reagents  (Wilder,  Abdi,  Scher  and  Mage,  in  progress).   In  the  latter  studies, 
analytical  flow  microfluorometry  (FACS  analysis)  was  performed  on  fluorescein 
labeled  cells  rather  than  doubly  stained  cells.   Doubly  heterozygous  rabbits 


22-31 


ZO1-AI-00036-14  LI 

were  used  and  single  populations  were  scored  for  proportions  of  total  Ig-, 
b4-,  b5-,  a2-,  and  a3-positive  cells.   The  proportions  of  Ig-positive  were 
compared  to  the  sums  of  a2  +  a3  positive  and  b4  +  b5  positive  cells.   No 
evidence  for  B  cells  expressing  two  allelic  types  was  obtained  by  either  the 
double  staining  or  the  FACS  technique.   Cells  tentatively  identified  as  mye- 
loid or  pre-myeloid  cells  were  found  in  adults  which  appear  to  bind  Ig  of  both 
allotypes  in  heterozygotes,  presumably  through  high  avidity  Fc-receptors  (the 
cells  still  stain  for  both  allotypes  after  overnight  incubation  at  37°C) 
(Gathings  et   al) .   Some  reports  of  "allelic  inclusion"  from  other  laboratories 
may  be  explained  by  scoring  of  such  cells.   We  are  currently  comparing  the 
results  of  analysis  of  surface  Ig  allotypes  on  peripheral  blood  lymphocytes 
detectable  by  FACS  analysis  and  by  a  specific  resetting  assay  because  some  of 
the  reports  of  "doubles"  utilized  rosetting  assays  (Abdi,  Scher  and  Mage,  in 
progress).   Reagents  have  been  prepared  and  their  specificity  demonstrated  so 
that  additional  studies  (in  progress)  can  determine  whether  imbalances  in 
allelic  allotype  expression  which  are  quite  marked  at  the  B  cell  level  in 
b  b  and  b  b   rabbits  are  also  detected  at  the  pre-B  cell  level.   Also  in  pro- 
gress are  searches  for  kappa  chain  allotypes  in  pre-B  cells  of  mutant  rabbits 
of  the  BASILEA  strain  which  are  defective  in  expression  of  Ig  with  kappa  type 
light  chains. 

In  studies  of  the  effects  of  allotype-suppression  and  its  neutralization 
on  the  expression,.of  b4  and  b5  allotypes  by  B  and  pre-B  cells  from  spleens 
and  marrow  of  b  b  rabbits,  we  found  that  pre-B  cells  of  the  suppressed  allo- 
type persist  when  B  cells  of  that  allotype  are  absent.   The  persistence  of 
pre-B  cells  of  the  suppressed  type  supports  the  view  that  pre-B  cells  differ 
in  their  responsiveness  to  external  influences  such  as  anti-Ig  compared  to  B 
lymphocytes.   Surviving  pre-B  cells  are  a  likely  source  of  the  B  cells  which 
appear  within  24  hr  of  administration  of  "neutralizing"  antigen  to  14-23  day 
old  suppressed  heterozygotes  as  well  as  for  B  cells  of  suppressed  allotype 
which  appear  during  the  recovery  phase  of  allotype-suppression  (Simons  et  al^. , 
1979) .   We  have  found  that  cells  bearing  slg  of  the  suppressed  type  appear 
and  increase  toward  normal  proportions  even  when  serum  Ig  levels  of  the 
suppressed  type  are  markedly  depressed  (Simons,  M. ,  Ph.D.  thesis,  1979  and 
Abdi,  Scher  and  Mage,  in  progress). 

Experiments  are  in  progress  to  test  the  working  hypothesis  that  Rheuma- 
toid Factors  produced  by  some  individuals  are  reflections  of  exaggerated 
production  of  normal  regulatory  anti-globulins  analogous  to  the  regulatory 
idiotypic  network  described  by  Jerne.   The  specificity  of  such  factors  may 
not,  however,  be  for  idiotypes,  but  for  allotypes  or  other  characteristic 
antigenic  determinants  on  immunoglobulins.   We  are  using  allotype-def ined 
rabbits  and  human  sera  containing  rheumatoid  factors  to  investigate  these 
questions.   In  rheumatoid  patients,  there  clearly  appear  to  be  complexes  of 
rheumatoid  factors  and  circulating  immunoglobulins  of  the  patient.   Isolated 
human  rheumatoid  factor  does  not  seem  to  distinguish  between  specific  rabbit 
CII  al^otyPes  °|  IgG.   Rheumatoid-factor-producing  rabbits  are  now  being 
generated  to  test  the  specificity  of  the  analogous  rheumatoid  factors  pro- 
duced in  the  rabbit  model  (DeRito,  Wilder,  Karsh  and  Mage,  in  progress). 


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Z01-AI-00036-14  LI 

Antigen-specific  surface  receptors  of  B  and  T  cells. 

Rabbit  and  mouse  splenic  lymphocytes  were  radiolabeled  by  the  lactoperox- 
idase  technique,  extracted  with  non- ionic  detergent,  immunoprecipitated  with 
high-titered  rabbit  anti-kappa  antisera  and  compared  by  SDS-PAGE.  Mouse  slg 
peaks  were  reproducibly  larger  in  size  than  rabbit  slg  peaks  (often  greater 
than  ten  times) .   Neither  differences  in  incorporation  of  label  into  the  rabbit 
cell  surface  nor  differences  in  average  slg  density  explain  this  result.   We 
conclude  that  incorporated  radioactivity  may  not  reflect  relative  cell  surface 
receptor  density  (Wilder  et.  al. ,  1979a)  .   A  rabbit  cell  surface  Ig  that  bears 
light  chain  and  V  but  lacks  y,  y-   and  a-allotypes  was  demonstrated.  The 
molecule  appears  to  be  proteolytically  labile  but  has  an  undegraded  heavy 
chain  that  co-electrophoreses  with  p  chains  on  reduced  SDS-PAGE.   The  similar- 
ities to  published  data  on  human  and  rat  lymphocyte  IgD  strongly  suggest  but 
do  not  formally  prove  that  we  have  identified  the  rabbit  homologue  of  IgD 
(Wilder  _et  a_l. ,  1979b) .   Staphylococcal  protein  A  and  several  different 
immunoglobulins  were  radiolabeled  to  high  specific  activities  (10  cpm/yg)  by 
reductive  methylation  with  tritiated  sodium  borohydride.   The  proteins  retain- 
ed excellent  functional  and  antigenic  properties.   The  reagents  were  used  in  a 
variety  of  assays  for  cell  surface  antigens  as  well  as  solution  immunoassays. 
This  radiolabeling  procedure  has  become  the  method  of  choice  for  many  cell 
surface  and  solution  immunoassays  currently  being  performed  in  the  Laboratory 
of  Immunology  (Wilder  et_   al. ,  1979c) .   T  and  B  lymphocytes  appear,  at  least 
partially  to  share  idiotypic  determinants.   Sharing  of  V  framework  determin- 
ants is  more  controversial.   In  order  to  examine  this  question,  we  have  pre- 
pared heterologous  anti-rabbit  and  anti-mouse  Vn  antisera.   Considerable 
characterization  has  been  done  of  these  reagents  (Wilder  et  al. ,  1979d,  and 
work  in  progress)  and  the  sera  are  being  used  to  further  examine  the  T  cell 
receptor  question  (Wilder  _et  a^. ,  1979e,  and  in  progress) .   In  addition  we  have 
supplied  anti-rabbit  V   to  a  number  of  investigators  for  further  testing 
immunochemically  and  in  cell  functional  assays) .   We  have  studied  the  extent 
to  which  V  framework  determinants  are  represented  on  T  cells  in  the  rabbit 
using  anti-a  (V  framework)  allotype  sera  and  anti-rabbit  V  sera  made  in  goat 
(Wilder  jet  al.  I979d,e)  and  chicken  (work  in  progress).   Flow  microf luorometric 
analyses  of  splenic  lymphocytes  from  hyperimmune  rabbits  demonstrated  closely 
similar  frequencies  of  cells  stained  with  the  anti-V   framework  antisera 
compared  to  anti-light  chain  antisera.   To  search  for  potentially  hidden  mole- 
cules, additional  studies  were  performed  with  radioiodinated,  detergent 
solubilized  plasma  membranes  derived  from  immune  spleen  and  lymph  nodes.   No 
evidence  for  a  population  of  cells  or  a  class  of  molecule(s)  bearing  V  in 
the  absence  of  light  chain  constant  region  determinants  was  obtained  (Wilder 
et  al. ,  1979e) .   One  possible  interpretation  of  our  data  is  that  only  a  small 
T  cell  subpopulation  expresses  V  determinants.   Conceivably,  different 
immunization  protocols  might  have  expanded  the  proportion  of  such  T  cells. 
We  are  currently  investigating  other  immunization  protocols  and  have  enriched 
for  antigen  binding  T  cell  subpopulations  to  pursue  this  question. 

Structure  of  immunoglobulins  from  allotype-def ined  rabbits. 

The  complete  sequence  of  the  variable  region  of  a  light  chain  from  a  rab- 


22-33 


Z01-AI-00036-14  LI 

bit  anti-type  III  polysaccharide  antibody  of  b4  allotype  was  determined.   Com- 
parisons with  other  sequenced  rabbit  kappa  chains  showed  striking  similarity 
to  light  chains  with  the  rare  amino  terminal  Ile-Val-Met  sequence  from  anti- 
bodies elicited  against  Streptococcal  A-variant  and  Micrococcus  lysodeikticus 
carbohydrates.   Residues  28-33  within  the  first  complementarity-determining 
region  (CDR)  were  identical  in  sequence  to  anti-A  variant  carbohydrate  anti- 
body light  chains  but  the  third  CDR  differed  considerably.   An  unusual  in- 
sertion of  three  amino  acids  following  homology  position  58  was  found  which 
has  not  been  previously  observed  in  rabbit  kappa  chains  (Chersi  e_t  al. ,  1979a) . 
The  partial  sequence  (positions  29-71)  of  the  variable  region  of  light  chains 
of  predominately  b5  allotype  from  the  IgG  of  a  single  allotype-suppressed 
rabbit  was  obtained  by  traditional  sequencing  methods  on  isolated  tryptic  and 
chymotryptic  peptides.   The  peptides  from  this  region  were  isolated  in  rela- 
tively high  yields  and  probably  represent  a  dominant  sequence.   The  framework 
sequence  between  positions  35  and  49  (FR2)  is  identical  to  an  FR2  sequence 
commonly  found  in  antibodies  produced  by  b4  rabbits  as  well  as  in  murine  and 
human  myeloma  light  chains,  with  the  exception  of  an  interchange  of  threonine 
for  proline  at  position  43  or  44.  This  may  be  b5  allotype-related  since  to 
date  all  b4  light  chains  have  had  proline  and  a  b9  light  chain  was  found  with 
arg  at  position  43.   The  fact  that  a  dominant  sequence  could  also  be  found  for 
positions  corresponding  to  the  second  CDR  (50-56)  in  other  species,  confirms 
previous  observations  that  this  portion  of  the  light  chain  is  not  extremely 
variable  in  the  rabbit  (Chersi  e_t  al. ,  1979b). 

Breeding  Experiments  and  Investigations  of  Chromosomal  Organization. 

To  date,  only  one  recombinant  has  been  documented  among  746  offspring  of 
informative  matings  for  the  detection  of  V  C  recombinant  phenotypes.   490  of 
these  progeny  were  informative  for  both  hinge  region  (dll-dl2)  and  C„2  (el4- 
el5)  allotypes.   The  analysis  of  the  linked  IgM  and  IgA  allotypes  in  this  re- 
combinant as  well  as  those  of  Kindt  and  Mandy  and  of  Hamers  et_  a_l.  continues. 
A  reinterpretation  of  the  IgM  phenotype  of  our  recombinant  was  proposed  based 
on  reports  of  V  -dependent  conformational  determinants  of  rabbit  IgM  (Mage, 
1979) .   New  data  on  the  structure  of  Ig  genes  at  the  DNA  level  stimulated 
a  re-evaluation  of  a  number  of  questions  concerning  the  arrangement  and  re- 
arrangement of  genes  for  rabbit  Ig-heavy  chains.   A  tabulation  of  the  postu- 
lated heavy  chain  J-coding  sequences  from  IgGs  of  known  a-allotypes  showed  no 
apparent  correlates  of  sequence  and  a.-allotype  for  this  portion  of  the  V 
domain  (Mage,  1979).   Another  general  question  raised  was  whether  the  apparent 
recombinations  observed  by  ourselves  and  others  within  the  heavy  chain  link- 
age group  were  intra-V   between  V  and  J,  intra-J ,  between  J  and  C   or  indeed 
several  of  these.   Some  recombinations  in  the  H-chain  genetic  region  may  be 
undetectable  or  poorly  detectable  because  the  recombinations  result  in  dis- 
ruption of  mechanisms  for  efficient  gene  reorganization.   Occasional,  al- 
though infrequent,  expression  of  certain  genes  after  such  recombinations  might 
then  lead  to  observation  of  "hidden  or  latent  allotype  or  idiotype"  pro- 
duction (Mage,  1979). 


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Z01-AI-00036-14  LI 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute. 

Immunogenetics  continues  to  be  at  the  forefront  of  our  understanding  of 
the  nature  of  structural  and  regulatory  genes  in  complex  mammalian  systems.   As 
we  increase  our  knowledge  of  the  genetics  and  evolution  of  Ig  genes  and  of  the 
chemistry  of  the  Ig  proteins  and  of  the  DNAs  which  code  for  and  regulate  their 
production,  the  Ig  system  becomes  one  of  the  most  valuable  models  for  investi- 
gation and  understanding  of  the  functioning  of  complex  genetic  regions  on 
chromosomes,  and  the  molecular  basis  for  cellular  differentiation  in  higher 
organisms.   The  genetics,  evolution  and  chemistry  of  cell  surface  isoantigens 
in  the  major  histocompatibility  locus  are  analogous  in  many  respects  to  the  Ig 
systems.   Increased  knowledge  of  Ig  genetics  has  also  provided  models  of  direct 
relevance  to  the  evolution  and  immunogenetics  of  cell  surface  antigenic  sys- 
tems.  Understanding  of  the  chemistry,  genetics  and  evolution  of  Igs,  the 
factors  which  control  their  genetic  expression  and  the  differentiation  path- 
ways of  the  cells  which  produce  them  is  thus  of  broad  fundamental  interest. 

Proposed  Course  of  the  Project. 

Most  of  the  projects  described  are  in  progress  and  continuing.  Much  of 
the  evidence  of  linked  inheritance  and  cis  expression  of  V  and  C  genetic 
information,  allelic  exclusion,  imbalances  in  quantitative  expression  of 
"allelic"  genes  and  "latent  allotypes,  or  hidden  genes"  has  been  developed  in 
the  rabbit  model.   The  phenomena  continue  to  be  studied  intensively.   In  addi- 
tion to  our  studies  at  the  immunochemical,  T  cell,  pre-B  and  B-cell  levels,  we 
are  initiating  experiments  (Dr.  L.  Fitzmaurice,  Research  Expert),  which  will 
utilize  cloned  recombinant  DNA  molecules  to  investigate  the  above  phenomena 
and  obtain  molecular  descriptions  of  the  events  at  the  DNA  and  mRNA  levels 
which  contribute  to  these  phenomena.   The  studies  of  specificity  characteris- 
tics of  Rheumatoid  Factors  in  man  and  in  animal  models  of  Rheumatoid  Disease 
will  continue  as  a  collaboration  between  this  Laboratory  and  Drs.  R.  Wilder 
(Clinical  Associate  A  &  R/NIAMDD  as  of  7/1/79  and  J.  Karsh  A  &  R/NIAMDD) . 

Appendix 

Contract  number  N01  AI  82565-Maintenance  and  Breeding  of  Rabbits  of 
Known  Genotype  and  Their  Use  of  Immunological  Studies. 

The  objectives  and  methods  are  related  to  those  outlined  above.  The  con- 
tract has  continued  to  provide  outstanding  support  for  production  and  mainten- 
ance of  alio type-defined  rabbits,  immunizations,  bleedings,  and  various  short- 
term  and  long-term  projects  involving  breeding,  immunizations  and  suppression. 
Rabbits  and  reagents  produced  and  supplied  through  this  contract  were  made 
available  to  numerous  other  investigators  at  the  NIH  and  were  shipped  to  labor- 
atories throughout  the  US  and  around  the  world. 

Publications. 

Ansari,  A. A.  and  Mage,  R.:   Immunochemical  Studies  of  the  a  Allotypes  of  Rabbit 


22-35 


Z01-AI-00036-14  LI 

Heavy  Chain  Variable  Regions-  I.   Comparisons  of  a3  Allotypic  Determinants  on 
Normal  IgG  and  IgG  of  Limited  Heterogeneity  by  Radioimmunoassays  with  Purified 
Labeled  Anti-Allotype  Antibodies.   Immunochem.  15:  561-568,  1978. 

Ansari,  A. A. ,  Mage,  R.G. ,  Carta-Sorcini,  M. ,  Carta,  S.  and  Appella,  E.: 
Immunochemical  Studies  of  Rabbit  Heavy  Chain  Variable  Regions  -  II.   Immunol- 
ogical Properties  of  Peptides  From  Variable  Regions  of  Heavy  Chains  of  Limited 
Heterogeneity  and  a3  Allotype.   Immunochem.  15:  569-575,  1978. 

Hayward,  A.R.,  Simons,  M.A.,  Lawton,  A.R. ,  Mage,  R.G.  and  Cooper,  M.D. :   Pre-B 
and  B  cells  in  rabbits:   Ontogeny  and  allelic  exclusion  of  kappa  light  chain 
genes.   J.  Exp.  Med.  148:   1367-1377,  1978. 

Wilder,  R.L. ,  Yuen,  C.C.  and  Mage,  R.G.:   Lactoperoxidase  catalyzed  radioiodin- 
ation  of  cell  surface  immunoglobulin:   Incorporated  radioactivity  may  not  re- 
flect relative  cell  surface  Ig  density.   J.  Immunol.  122:   459-463.  1979a. 

Wilder,  R.L.  ,  Yuen,  C.C,  Coyle,  S.A.  and  Mage,  R.G.:   Demonstration  of  a 
rabbit  cell  surface  Ig  which  bears  light  chain  and  V  but  lacks  u,  a  and  y 
allotypes  -  rabbit  IgD?  J.  Immunol.  122:   464-468,  1979b. 

Chersi,  A.,  Appella,  E.,  Carta,  S.  and  Mage,  R.:   The  amino  acid  sequence  of 
a  variable  region  of  rabbit  b4  light  chain  from  an  anti-SIII  antibody:  compar- 
ison with  light  chains  of  the  same  subgroup  from  anti-A-variant  carbohydrate 
antibodies.   Mo lee.  Immunol.   In  press,  1979a. 

Mage,  R.G. :   A  new  look  at  the  biological  and  genetic  significance  of  rabbit 
heavy  chain  allotypes.   Ann.  Immunol.  (Inst.  Pasteur)  130C:   105-114,  1979. 

Wilder,  R.L.,  Yuen,  C.C,  Subbarao,  B.,  Woods,  V.L.  ,  Alexander,  CB.  and  Mage, 
R.G.:   Tritium  (  H)  radiolabeling  of  protein  A  and  antibody  to  high  specific 
activity:   application  to  cell  surface  antigen  radioimmunoassays.   J.  Immunol. 
Methods,  in  press,  1979c. 

Wilder,  R.L.,  Yuen,  C.C  and  Mage,  R.G.:   Preparation  and  characterization  of 

a  heterologous  anti-rabbit  V  antiserum.   Mo  lee.  Immunol.,  in  press,  1979d. 

H 

Wilder,  R.L.,  Yuen,  C.C,  Scher,  I.  and  Mage,  R.G.:   Are  V   framework  anti- 
genic determinants  expressed  on  both  rabbit  B  and  T  lymphocytes?   Eur .  J . 
Immunol. ,  in  press,  1979e. 

Simons,  M.A. ,  Hayward,  A.R. ,  Gathings,  W.E. ,  Lawton,  A.R. ,  Young-Cooper,  CO., 
Cooper,  M.D.  and  Mage,  R.G. :   Expression  of  b4  and  b5  kappa  light  chain  allo- 
types by  B  and  pre-B  cells  in  allotype-suppressed  and  neutralized  b  b 
rabbits.   Eur.  J.  Immunol. ,  in  press,  1979. 

Chersi,  A.,  Mage,  R.G.  and  Alexander,  CB.  :  Partial  sequence  of  the  variable 
region  of  IgG  light  chains  of  b5  allotype  from  b  b  rabbit.  Molec.  Immunol., 
in  press,  1979b. 


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Z01-AI-00036-14  LI 

Honors 

Chairman,  Editorial  Board,  Federation  Proceedings 

Editorial  Board,  J.  Immunological  Methods 

Vice  President  D.C.  Chapter,  Sigma  Xi 

Invited  Lecturer,  International  Colloquium,  Institute  Pasteur,  Jan.  4-5,  1979. 


22-37 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  CERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl-AI  00037-12  LI 


PERIOD  COVERED 

October  1,  1978 


September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Immunogenetics  of  Mouse  Immunoglobulin  and  Genetxc  Control  of  Antibody 
Response 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Principal  Investigator:   Rose  Lieberman,  Research  Microbiologist,  LI/NIAID 


Other  Investigators: 


Constantin  Bona,  LI/NIAID 

Subbarao  Bondada,  Philadelphia  Cancer  Research  Institute 

Philadelphia,  PA 
Aftab  Ahmed,  Naval  Research  Institute 
Herbert  Morse.  LMI/NIAID 
Franco  Meloni,  LI/NIAID 
Stuart  Rudikoff,  LCB/NCI 

Goran  MBller,  Karolinska  Institute,  Stockholm 
Kathryn  Stein,  LI/NIAID 

Edward  A.  Boyse,  Sloan  Kettering  Institute 
F.  W.  Shen,  Sloan  Kettering  Institute 
W.  E.  Paul,  LI/NIAID 


COOPERATING  UNIT: 


Laboratory  of  Immunology 


INSTITUTE  and  location  National  Institute  oi  Allergy  and  Infectious  Disease,  NaLiuual 
Institutes  of  Health,  Bethesda,  Maryland  20014 


TOTAL  MANYEARS: 


PROFESSIONAL: 
1 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

Q  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


§  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

This  laboratory  is  mainly  concerned  with  the  genetic  and  cellular  regula- 
tion of  the  expression  of  variable  and  constant  region  antigenic  determinants 
present  on  light  and  heavy  chains  of  immunoglobulin  molecules  before  and  after 
immunization  with  specific  haptens.   Idiotypes  or  V-region  structural  antigenic 
determinants  associated  with  specific  antibodies  (e.g.  anti  polyf ructosan, 
anti  phosphorylcholine,  anti  a  1-6  dextran)  have  proven  to  be  very  useful 
as  genetic  markers  or  phenotypes  in  studies  of  genetic  and  cellular  regulation 
of  immunoglobulin  expression.   The  preparation  of  monoclonal  antibody  (hybridomas 
carrying  specific  idiotypes  provide  sources  of  large  amounts  of  monospecific 
antisera  that  are  invaluable  in  identification  of  specific  markers  in  studies 
of  interaction  of  cells  in  immunoglobulin  expression. 


22-38 


PHS-6040 
(Rev.  10-76) 


Z01-AI  00037-12  LI 


A.   Immune  Response  to  Levan 

I.  Capacity  to  produce  anti-inulin  antibodies  and  cross  reactive  idiotypes 
appears  late  in  ontongeny. 

Adult  BALB/c  mice  that  have  been  immunized  with  bacteral  levan  (BL) 
produce  anti-inulin  and  anti  BL  antibodies  which  exhibit  cross-reactive 
idiotypes  (IdX-G,  IdX-A  and  IdX-B)  found  on  inulin  binding  myeloma  proteins. 
These  mice  also  produce  anti  BL  antibodies  which  do  not  bind  inulin  nor 
express  IdX's.   Young  BALB/c  mice  immunized  with  BL  or  a  variety  of  inulin 
conjugates  do  not  produce  anti-inulin  antibodies  but  do  produce  anti  BL 
antibodies.   The  late  appearance  of  anti-inulin  IdX  antibodies  appears  to  be 
due  to  a  late  maturation  of  precursor  cells  and  not  to  suppressor  cells  in 
the  neonate.   This  has  been  shown  by  the  fact     that  C.B20  mice  that  have 
received  spleen  cells  from  BALB/c  neonates  do  not  produce  anti-inulin  anti- 
bodies and  that  T  cells  from  BALB/c  neonates  do  not  suppress  the  anti-inulin 
response  of  adult  BALB/c.   Furthermore,  adult  spleen  cells  transferred  into 
neonates  induce  an  anti-inulin  response. 

Although  neonates  do  not  produce  IdX  anti-inulin  antibody  in  response 
to  immunization  with  levan,  their  serum  contains  IdXA  molecules  which  lack 
IdX-G  and  IdX-B  and  which  do  not  bind  inulin  as  BL.   These  IdXA  antibodies 
may  possibly  represent  products  of  a  "protoclone"  which  may  subsequently 
develop  into  IdX-G  .  A  and  B  anti-inulin  antibody  forming  cells. 

II.  Suppression  of  antibody  response  by  pretreatment  with  antibodies  to 
cross-reactive  idiotypes. 

Pretreatment  of  BALB/c  mice  with  antibodies  directed  at  cross-reactive 
idiotypes  of  anti-inulin  antibodies  profoundly  suppresses  the  inulin-anti- 
body  response  to  bacterial  levan.   This  is  shown  by  the  failure  of  such 
pretreated  mice  to  develop  anti-inulin  hemagglutinins  or  to  develop  anti- 
bodies detectable  on  isoelectric  focusing.   By  contrast,  B.C8  mice,  which 
are  congenic  to  C57BL  mice  but  which  possess  the  IgC   haplotype  fail  to 
be  suppressed  by  pretreatment  with  anti-idiotype  antibody.   This  suggests 
that  capacity  to  be  suppressed  by  anti-idiotype  pretreatment  is  determined 
by  non-IgC  genes. C57BL  mice  possess  genes  determining  resistance  to 
suppression  and  BALB/c  mice  possess  genes  determining  sensitivity  to 
suppression.   As  noted  in  Z01-AI-0030-11  LI,  a  similar  situation  pertains 
for  the  determination  of  clonal  diversity  and  magnitude  of  anti-inulin 
response  to  BL  immunization  as  assessed  by  isoelectric  focusing.   C57BL  genes 
determined  an  increased  response  and  a  greater  diversity.   This  is  parti- 
cularly striking  since  C57BL  mice  appear  to  lack  the  structural  genes 
coding  for  the  anti-inulin  antibodies  produced  in  response  to  BL.   Efforts 
are  in  progress  to  determine  whether  the  genes  and  those  regulating  the 
magnitude  and  diversity  of  the  anti-inulin  response  are  the  same  and  to 
map  these  genes. 


22-39 


Z01-AI  00037-12  LI 

III.   Anti  bacterial  Levari  Monoclonal  Antibodies. 

Hybridomas  were  produced  by  fusion  of  spleen  cells  from  BALB/c  mice 
immunized  with  BL  with  the  P31x63  cell  line  grown  from  a  BALB/c  plasmacytoma. 
P31x63  cell  cultures  produce  and  secrete  IgG.   Approximately  4000  clones 
were  obtained  and  these  were  examined  for  anti  BL  and  anti-inulin  activity. 
3/4000  clones  showed  anti  BL  antibodies  and  two  of  these  also  expressed 
idiotypes  (IdX)  that  have  been  identified  on  normal  BALB/c  anti-inulin  anti- 
bodies and  also  on  myeloma  proteins  that  bind  inulin  and  BL.   One  of  the 
hybridomas  also  expressed  an  idiotype  A48  that  has  been  found  only  on  a 
myeloma  protein  that  binds  levan  (£2^-6)  and  not  inulin  (£2->-l)  polyf ructosans. 
Monoclonal-ant i  BL  antibodies  from  ascites  fluid  from  BALB/c  mice  injected 
with  these  two  hybridomas  are  being  collected  and  purified. 

B.   Allotype  Related  Research. 

I.  al-6  Dextran  Response  Linked  to  the  IgCH  Locus. 

Responsiveness  to  a.   1-6  dextran  is  independent  of  H-2  but  is  determined 
by  the  IgCH  allotype  of  the  strain.   Mice  of  the  IgCH  and  IgCH"1  allotypes, 
such  as  the  C57BL  and  CBA/J  strains,  are  high  responders  while  strains  of 
other  allotypes  are  low  responders.   Backcross  analysis  of  low  and  high  re- 
sponders indicate  that  the  gene  controlling  responsiveness  is  linked  to  the 
IgCH  gene  complex.   F..  hybrids  of  low  and  high  responder  strains  indicate 
that  responsiveness  was  inherited  as  a  codominant  trait. 

II.  Determination  of  the  IgCH  Domains  that  Express  IgCH  allotypes. 

Cell  lines  of  MPC11,  an  IgG2b  plasmacytomas  of  BALB/c  origin  that  had 
been  treated  with  mutagens  were  found  to  have  switched  to  producing  IgG2a 
class  of  immunoglobulin  identified  by  allotype  analysis.   Antisera  prepared 
to  the  wild  type  identified  MPC11  idiotype  and  IgG2b  allotypes.   Antisera 
prepared  to  the  supernatant  Ig  secreted  by  the  mutagen  treated  cell  lines 
of  MPC11  identified  the  MPC11  idiotype  which  cross  reacted  to  MPC11  idiotype 
of  wild  type  but  also  identified  the  IgG2a  allotype.   CH  fragments  were  pre- 
pared from  the  MPC  IgG2a  protein  by  trypsin  treatment  of  CH  fragments  H2L2  but 
lacking  the  CH3  domain,  did  not  express  IgG2a  or  IgG2b  allotypes.   Fc  fragment 
consisting  of  the  CH3  domain  expressed  the  IgG2a  but  not  the  IgG2b  allotype. 

III.  Studies  of  possible  Linkage  of  A  Allotype  with  A    and  A^ 
Regulatory  and  other  Genetic  Markers 

Allotypic  A  markers,  X  and  A  ~  ,  have  been  identified  hy  allo- 

antisera  prepared  against  BALB/c  A  myeloma  proteins  in  SJL  mice.   In 
addition,  an  antiserum  has  been  prepared  in  a  rabbit  against  the  A^  Bence- 
Jones  protein  RPC  20.   Using  this  heteroantiserum,  it  can  be  shown  that  some 
mouse  strains  have  high  A.,  levels  and  others  have  low  A.,  levels.   Genetic 
studies  of  crosses  of  strains  with  low  levels  of  A^  with  strains  of  high  A^ 
levels  show  that  the  expression  of  level  of  A1  is  controlled  by  regulatory 


22-40 


Z01  AI  00037-12  LI 

genes  and.  the  X-         is  linked  to  the  A..   .   In  addition  we  examined  A.  allotype 
genes  A    in  relation  to  other  markers  including  IgCH  Ly  1.1,  Ly  2.1,  Ly  5.1, 
H-2  C,  Hbb^and  Gpi.   No  evidence  of  linkage  of  any  of  these  markers  to 
A,    or  A    has  been  found  except  for  the  ly  1.1  markers  where  in  the 
appropriate  progeny  crosses,  there  was  some  evidence  of  loose  linkage  C75%) . 

IV.   p  -  S    Heavy  Chain  Recombinants. 

44  backcross  progeny  of  (C57BL/6  x  DBA/2)  x  C57BL/6  were  prepared  for  the 
genetic  studies  of  the  linkage  of  Lyb7.1  genes  to  the  IgCH  genes;  these  mice 
were  also  examined  for  serum  and  membrane  S    allotype  and  for  y  allotypes. 
Two  progeny  from  the  same  litter  were  found  to  express  serum  and  membrane  S 
allotype  of  the  DBA/2  strain  and  \i,    y0    ,  y       and  a  allotypes  of  the  C57BL. 
These  two  putative  recombinants  have  now  been  proven  to  be  true  recombinants 
by  appropriate  mating  tests. 

C.   Polymorphism  of  V-Region  Markers  in  the  Mouse 

I.   Phosphorylcholine  antibodies  of  BALB/c  and  most  mice  of  IgCH  haplotypes 
exhibit  an  idiotype  designated  T15  that  is  also  present  on  a  BALB/c 
phosphorylcholine  binding  myeloma  proteins.   Binding  of  antiidiotype  antibody 
to  the  The  T15  idiotype  is  not  inhibitible  by  phosphorylcholine  and,  thus, 
is  not  in  the  combining  site.   Genetic  studies  of  the  T15  idiotype  show  that 
it  is  controlled  by  a  gene  linked  to  the  IgC  locus.   Recently  we  prepared 
alloantisera  to  CBPC3,  a  phosphorylcholine  binding  myeloma  protein  from  the 
C.B20  mouse  which  carried  the  IgA   allotype  marker  of  the  IgCH  haplotype. 
Anti  CBPC3  antisera  produced  in  BALB/c,  AL/N  and  A/He  mice  were  made  specific 
for  CBPC3  idiotype  by  absorption  with  CBPC  105  (a  CB20IgA  myeloma  protein)  to 
remove  anti-allotype  antibodies.   AL  anti  CBPC3  idiotype  antibodies  cross 
reacted  with  T15  myeloma  protein.   This  cross  reaction  extended  to  anti- 
phosphorylcholine  antibodies  of  normal  C57BL  and  BALB/c.   In  addition  absorpt- 
ion of  this  antisera  with  T15  removed  the  cross  reacting  idiotype  (IdX)  anti- 
bodies.  Thereafter,  the  antisera  was  reactive  with  anti  phosphorylcholine 
antibodies  of  C57BL  but  not  BALB/c  origin.   BALB/c  anti  CBPC3  idiotype  anti- 
bodies only  reacted  to  CBPC3  myeloma  protein  and  not  with  T15.   This  anti- 
body combined  with  C57BL  anti  phosphorylcholine  antibodies.   These  findings 
suggest  polymorphism  of  V  region  markers  coding  for  H  chains  of  anti-phos- 
phorylcholine  antibodies.   One  marker  is  shared  by  BALB/c  and  C57BL  mice  while 
the  other  is  present  only  in  C57BL.   The  heavy  chains  of  both  the  CBPC-3  and 
T15  myeloma  proteins  have  been  completely  sequenced  and  their  homology  is  very 
striking.   They  differ     at  positions  14,  16,  40  and  120.   The  latter  is 
within  the  newly  recognized  J  segment. 

PUBLICATIONS : 


Lieberman,  R._:   Genetics  of  IgC„  (allotype)  locus  in  the  mouse.   Springer 
Seminars  in  Immunopathology  1:  7-30,  1978. 


22-41 


ZQ1  AI  00037-12  LI 

Chien,  C.C.,  Lieberman,  R.  and  Inman,  J.K.:   Preparation  of  functionalized 
derivatives  of  inulin:   Conjugation  of  erythrocytes  for  hemagglutination 
and  plaque-forming  cell  assays.   J.  Immunol.  Methods,  26:  38,  1979. 

Lieberman,  R.  ,  Bona,  C,  Chien,  C.C.,  Stein,  K.E.  and  Paul,  W.E.: 
Genetic  and  cellular  regulation  of  the  expression  of  specific  antibody 
idiotypes  in  the  anti-polyf ructosan  response.   Ann.  Immunol.  (Inst.  Pasteur^ 
130:  247,  1979. 

Lieberman,  R. :   Immunoglobulin  allotypes  of  inbred  and  genetically  define 
strains  of  laboratory  animals.   FASEB  -  Office  of  Biological  Handbooks 
III:  107,  1979. 

Bona,  C. ,  Lieberman,  R. ,  Green,  I.  and  Paul,  W.E.:   Immune  response  to 
levan.   II.  T- independence  of  suppression  of  cross  reactive  idiotypes  by 
anti-idiotypic  antibodies.   J.  Immunol.  122:  1614,  1979. 

Bona,  C,  Stein,  K.E.,  Lieberman,  R.  and  Paul,  W.E.:   Direct  and 
indirect  suppression  induced  by  anti-idiotype  antibody  in  the  inulin- 
bacterial  levan  antigenic  system.   Molec.  Immunol. ,  1979. 

Fernandez,  C. ,  Lieberman,  R.  and  Moller,  G.:   The  immune  response 

to  alpha  1-6  epitope  of  dextran  is  determined  by  a  gene  linked  to  the 

IgCH  locus.   Scand.  J.  Immunol.,  1979.   In  press. 

Bona,  C. ,  Mond,  J.J.,  Stein,  K.E.,  House,  S.,  Lieberman,  R.,  and 
Paul,  W.E.:   Immune  Response  to  Levan.   III.   The  capacity  to  produce 
anti-inulin  antibodies  and  cross-reactive  idiotypes  appears  late  in 
ontogeny.   J .  Immuno 1 .   In  press. 


22-42 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01-AI-00038-07  LI 


PERIOD  COVERED 

October  1,  1978  -  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less 

Structure  and  Activity  Studies  on  Immunologically  Important  Cells  and  Proteins 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT  . 

Principal  Investigator:   Myron  J.  Waxdal,  Ph.D.,  Research  Chemist,  LI/NIAID 


Other  Investigators: 


Dr.  S.  Toyoshima,  LI/NIAID 

Dr.  B.  Tyran,  LI/NIAID 

Ms.  T.  Basham,  LI/NIAID 

Mr.  D.  Skiados,  LI/NIAID 

Dr.  B.  Mathieson,  LMI/NIAID 

Ms.  B.  Fowlk.es,  LMI/NIAID 

Ms.  S.  Sharrow,  I/NCI 

Dr.  L.  Guidice,  CE/NIAMDD 

Dr.  B.  Weintraub,  CE/NIAMDD 

Dr.  F.  Finkelman,  USUHS 

Dr.  J.  Axelrod,  LCM/NIMH 


Dr.  F.  Hirata,  LCM/NIMH 


cooperating  units  (if  any)  Unif Q.r.med„Services  University  of  Health  Sciences, 
Bethesda,  MD  20014 


lab/branch 
Laboratory  of  Immunology 


institute  and  location  National  Institute  of  Allergy  and  Infectious  Diseases, 
National  Institutes  of  Health,  Bethesda,  MP  20205 


TOTAL  MANYEARS: 

6.5 


PROFESSIONAL: 


2.5 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 

D  (a!)  MINORS   Q  (a2)  INTERVIEWS 


S  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Pa-2  stimulation  of  murine  lymphocytes  causes  the  production  of  soluble 
lymphocyte  stimulating  factors  (LSF) .   These  LSF  are  produced  by  splenic  T  cells 
and  thymus  cells.   Fractionation  of  thymocytes  by  peanut  agglutinin  into 
non-agglutinable  cells  (about  10%)  and  agglutinable  cells  (90%)  followed  by 
removal  of  the  PNA  and  stimulation  with  Pa-2,  showed  that  the  non-agglutinable 
thymocytes  were  responsible  for  the  production  of  LSF.   These  helper  factors 
have  been  separated  into  6  major  pools  by  affinity  chromatography.  While  each 
stimulates  DNA  synthesis  and  immunoglobulin  production  by  spleen  cells,  five  are 
mitogenic  for  thymocytes  or  T  cells  and  the  sixth  is  B  cell  specific. 

One  of  the  earliest  biochemical  events  following  mitogenic  lectin  stimula- 
tion of  lymphocytes  is  the  activation  of  a  membrane  enzyme  system  which  causes 
the  methylation  of  phosphotidyl  ethanolamine.   A  phosphocholine  degrative  path- 
way must  also  be  activated  in  order  for  the  cells  to  synthesize  DNA  or  to  pro- 
duce soluble  factors  (LSF) . 

The  fluorescence  activated  cell  surface  has  been  used  to  identify  lectins 
which  differentially  bind  to  subsets  of  lymphocytes.   Several  such  lectins  have 
been  identified  and  are  being  Used  tu  fractionate  lymphocyte  subsets. 


PHS-6040 
(Rev.  10-7 


22=43 


Z01-AI-00038-07  LI 

I.  Stimulation  of  Lymphocytes  -  Soluble  Factors 

We  have  previously  shown  that  the  T  lymphocyte  specific  mitogen,  Pa-2, 
stimulates  soluble  helper  factor  production  by  BALB/c  thymocytes  and  splenic 
T  cells.   This  mixture  of  lymphocyte  stimulating  factors  (LSF,  formerly 
called  BSF)  contained  several  components  which  varied  in  their  biological 
activities.   LSF  has  now  been  produced  by  several  strains  of  mice  and  proven 
to  stimulate  lymphocytes  across  H-2  barriers. 

Our  recent  experiments  indicate  that  all  of  the  LSF  produced  by  BALB/c 
thymocytes  comes  from  a  small  subset  of  these  cells.   By  the  use  of  differen- 
tial agglutination  with  a  lectin  isolated  from  peanuts  (PNA) ,  murine  thymus 
can  be  separated  into  two  fractions;  those  that  do  not  agglutinate  (PNA  , 
ca  10%)  and  those  that  are  agglutinated  (PNA  ,  ca  90%).  After  removal  of  the 
PNA  with  specific  inhibitors  and  recovery  of  single  cells,  both  fractions 
were  stimulated  with  Pa-2.   Only  the  PNA  -  fraction  produced  LSF  or  synthesized 
DNA. 

Preparations  of  LSF  were  known  to  contain  several  biologically  active 
components.  Affinity  chromatography  on  columns  of  insolubilized  Concanavalin 
A  fractionates  LSF  into  6  active  pools  (I-VI),  which  appear  to  be  glycopro- 
teins.  Pools  I  to  V  stimulate  DNA  synthesis  and  immunoglobulin  production 
(measured  by  the  reverse  plaque  technique)  in  spleen  cells  from  normal  mice 
but  not  from  athymic  nude  mice.   They  also  stimulate  DNA  synthesis  in  T  cells 
and_ thymocytes.   Furthermore,  when  tested  on  PNA  fractionated  thymocytes,  the 
PHA  fraction  responded  100-200  fold  better  than  the  PNA  fraction. 

These  results  indicate  that  the  LSF  in  pools  I-V,  which  appear  to  be 
some  of  the  molecules  involved  in  normal  cellular  control  of  immune  responses, 
are  produced  by  a  small  set  of  thymocytes  contained  in  the  PNA   fraction  and 
cause  cell  division  only  in  thymocytes  with  same  PNA  fraction.   The  popula- 
tions of  cells  in  this  fraction  appear  to  largely  overlap  with  cortisone 
resistant  thymocytes. 

On  the  other  hand,  the  material  in  pool  VI  shows  little  or  no  mitogenic 
stimulation  of  thymocytes,  but  is  the  only  LSF  to  stimulate  DNA  synthesis  or 
increase  the  number  of  IgM  secreting  cells  in  the  athymic  nude  mouse  spleen. 
It  is  unclear  whether  this  LSF  acts  directly  on  the  B  lymphocyte,  or  requires 
the  presence  of  an  auxiliary  cell. 

We  are  presently  investigating  the  biochemical  properties  of  these  LSF 
and  attempting  to  further  delineate  the  cells  which  produce  them  and  their 
target  cells. 

II.  Stimulation  of  Lymphocytes  -  Early  Biochemical  Events 

Our  studies  in  collaboration  with  LCM/NIMH  have  shown  that  Concanavalin  A, 
a  mitogen  that  stimulates  DNA  synthesis  in  T  cells,  induces  a  transient  activa- 
tion of  membrane  phospholipid  methylation  followed  by  an  increase  in  degrada- 
tion of  methylated  phosolipids.   This  results  in  a  maximum  incorporation  of 

22-44 


Z01-AI-00038-07  LI 

3 
H-methyl  groups  into  membrane  phosolipids  at  10  minutes  followed  by  a  return 

to  baseline  by  40  minutes.   The  dose  response  curves  for  Con  A  on  phospholipid 

methylation  (measured  at  10  minutes)  and  DNA  synthesis  (measured  at  48  hr) 

were  nearly  identical. 

To  further  correlate  phospholipid  methylation  with  later  cell  division, 
analyses  were  performed  using  lymphocytes  from  athymic  nude  mouse  spleen, 
thymus,  and  splenic  B  and  T  cells  separated  by  adherence  to  anti- immunoglobu- 
lin coated  (Mage)  plates.   In  each  case,  Con  A  activated  the  membrane  phospho- 
lipid methylation  system  only  with  cells  that  also  responded  with  DNA 
synthesis  (T  or  thymus  cells).   Additional  experiments  using  several  mitogenic 
and  non-mitogenic  lectins  showed  a  complete  correlation  between  activation  of 
the  methylation  enzymes  and  subsequent  DNA  synthesis. 

Inhibition  of  phospholipid  methylation  with  S-isobutyrylthio-3-diaza- 
adinosine  was  effective  only  if  the  inhibitor  was  added  prior  to  Con  A 
stimulation.   After  this  initial  step,  the  inhibitor  could  be  washed  away 
without  loss  of  inhibition.   Both  the  methylation  (10  min)  and  DNA  synthesis 
(48  hr)  were  equally  inhibited.   If  the  inhibitor  was  added  at  the  same  time 
as  Con  A  and  left  for  the  duration  of  culture,  no  effect  was  seen  either  on 
phospholipid  methylation  or  DNA  synthesis. 

It  was  further  found  that  calcium  influx  into  the  cell  was  dependent 
upon  the  activation  of  phospholipid  methylation.   However,  the  degradation 
of  methylated  phospholipids  did  not  occur  in  the  absence  of  calcium.   In 
summary,  the  membrane  transmethylases  are  activated  by  the  binding  of  mito- 
genic lectins  to  the  cell  surface,  causing  a  transient  increase  in  mono- 
methyl  ethanolamine,  dimethylethanolamine,  and  phosphatidyl  choline.   The 
accumulation  of  monomethyl  ethanolomine  apparently  causes  a  decrease  in  mem- 
brane microviscosity.   At  this  point  calcium  appears  to  enter  the  cell  and 
an  increased  breakdown  of  methylated  phospholipids  occurs.   The  products  are 
arachodonic  acid,  which  may  be  further  metabolized  into  thromboxane  and 
prostaglandins,  and  lysolecithin  which  may  activate  guanidylate  cyclase.. 

The  activation  of  the  membrane  transmethylases  to  synthesize  methylated 
phospholipids,  and  the  subsequent  activation  of  degrative  enzymes  (probably 
phospholipase  A2)  appear  to  be  integral  events  in  the  stimulation  of  mitosis 
by  lectins.   We  are  currently  testing  other  lymphocyte  activators,  such  as 
lymphokines,  to  determine  whether  these  enzymes  are  in  a  common  pathway  of 
lymphocyte  activation. 

III.  Lectin  Fractionation  of  Lymphocytes 

In  collaboration  with  LMI/NIAID  and  I/NCI  we  have  surveyed  approximately 
3  dozen  lectins  for  preferential  binding  to  different  subsets  of  murine  lympho- 
cytes.  The  contribution  of  this  laboratory  has  been  the  preparation  and 
fluorescence  labeling  of  lectins.   Various  lymphocyte  populations  were  stained 
with  the  fluorescent  lectins  and  analyzed  by  flow  microfluorometry.   We  have 
^identified  several  lectins  which  bind  differentially  to  subsets  of  lymphocytes. 


22-45 


Z01-AI-00038-07  LI 

These  lectins  have  nominal  specificities  for  galactose/N-acetylgalactosamine, 
N-acetyl  glucosamine,  or  sialic  acids.   Lectins  with  the  same  nominal  specifi- 
city do  not  usually  show  the  same  cell  staining  patterns.   The  lectins  we  have 
identified  will  be  used  to  develop  techniques  for  the  separation  of  lympho- 
cyte subpopulations.   Further  experiments  will  characterize  the  cells  in  each 
subpopulation  recognized. 

IV.   Microsequencing  of  Pituitary  Glycoprotein  Hormone  Pre-a  Subunits 

The  isolation  of  poly-A  containing  RNA  from  bovine  and  mouse  pituitaries, 
from  a  murine  pituitary  thyrotropic  tumor  and  their  translation  in  a  cell  free 
wheat  germ  biosynthetic  system  containing  H-labeled  amino  acids  was 
accomplished  by  the  collaborating  unit  (CE/NIAMDD) .   The  precursors  of  the  a 
subunit  (pre-a)  of  the  glycoprotein  hormones  were  isolated  by  immunoprecipitat- 
ion  and  SDS-gel  electrophoresis.   This  laboratory  carried  out  microsequencing 
analyses  of  the  radioactively  labeled  chains.   Partial  amino  acid  sequences 
were  determined  for  the  N-terminal  22  residues  of  these  three  pre-a  subunits. 
The  data  indicate  that  both  murine  pituitary  and  pituitary  tumor  a  subunits 
are  synthesized  with  a  "signal"  prepiece  and  that  this  polypeptide  is  at  least 
22  residues  in  length.   These  pre-a  subunits  appear  identical.   The  data  also 
show  that  leucine  is  common  to  4  positions  (12,15,19  and  22)  in  murine  and 
bovine  pituitary  pre-a  as  well  as  in  human  placental  choriogonadotropin  pre-a 
(other  workers)  subunits. 

This  extends  the  homology  among  a  subunits  from  various  species  and 
organs  into  their  prepieces,  furthering  the  suggestion  that  the  a  subunits 
from  pituitary  and  placenta  are  products  of  the  same  gene.   There  are  no 
immediate  plans  to  continue  this  project. 

PUBLICATIONS : 

Bridgen,  J.  and  Waxdal,  M.J.   Development  of  a  quantitative  solid-phase 
microsequencing  procedure.   In  Solid  Phase  Methods  in  Protein  Sequence 
Analysis.   Ed.  A.  Previero  and  M.-A.  Coletti-Previero.   Elsevier/North 
Holland,  1978,  p.  153-162. 

Marchalonis,  J.J.,  Warr,  G.W. ,  Decker,  J.M.  and  Waxdal,  M.J.   Glycoproteins 
as  biologically  important  molecules  of  the  lymphocyte  membrane.   In  Biological 
Markers  of  Neoplasia:  Basic  and  Applied  Aspects.   Ed.  R.  Ruddon.   Elsevier/ 
North  Holland. 

Nilson,  S.,  Edelson,  R. ,  Mann,  D. ,  Green,  I.  and  Waxdal,  M.J.   Con  A  binding 
proteins  on  the  surface  of  human  malignant  and  normal  lymphocytes.   Scand.  J. 
Immunol.  9_:    483,  1979. 

Marchalonis,  J.J.  and  Waxdal,  M.J.  Limulus  agglutinins:  Past,  present  and 
future.  In  Biomedical  Applications  of  the  Horseshoe  Crab  (Limulidae) .  Ed. 
E.B.  Cohen,  Alan  R.  Liss,  1979.   In  press.   " 


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Z01-AI-00038-07  LI 

Waxdal,  M. J. ,  Fowlkes,  B.J.,  Sharrow,  S.O.  and  Mathieson,  B.J.   The  Use  of 
Lectins  for  the  Fractionation  of  Lymphocytes.   In  27th  Collaquium  on  Peptides 
of  the  Biological  Fluids.   Ed.  H.  Peeters.   In  press. 

Guidice,  L.C.,  Waxdal,  M.J.  and  Weintraub,  B.D.  Comparison  of  Bovine  and 
Mouse  Pituitary  Glycoprotein  Hormone  Pre-a  Subunits  Synthesized  In  Vitro. 
PNAS.   In  press. 

Hirata,  F. ,  Axelrod,  J.,  Toyoshima,  S.  and  Waxdal,  M.J.  Phospholipid 
methylation:  A  biochemical  signal  modulating  lymphocyte  mitogenesis. 
Nature.   In  press. 

Waxdal,  M.J.,  Basham,  T.Y. ,  Clement,  L.,  Shevach,  e.M.  and  Schwartz,  B. 
Amino  terminal  sequence  studies  on  the  la  antigens  of  the  guinea  pig. 
J.  Molecular  Immunology  16:  61,  1979. 


22-47 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION.  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01-AI-00040-05  LI 


PERIOD    COVERED 
October   1,    1978    to    September    ^0,    1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Genetic  Control  of  Immunocompetent  Cell  Interactions 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Principal  Investigator:   Ethan  M.  Shevach,  Senior  Investigator,  LI/NIAID 

Other  Investigators:   L.  Clement,  Research  Associate,  LI/NIAID 

E.  Heber-Katz,  Staff  Fellow,  LI/NIAID 

R.  Burger,  Guest  Worker,  LI/NIAID 

0.  Werdelin,  Guest  Worker,  LI/NIAID 

R.  Clark,  Clinical  Associate,  LCI/NIAID 


cooperating  units  (if  any)  Dr _  Hinrich  Bitter-Suermann,  Dept.  of  Pathology,  Georgetc 
University  Medical  School 


lab/branch 
Laboratory  of  Immunology 


section  National  Institute  of  Allergy  and  Infectious  Diseases,  National 
Institutes  of  Health,  Bethesda,  MD 


INSTITUTE  and  location 
As    above 


TOTAL  MANYEARS: 

6.5 


PROFESSIONAL: 

4 


2.5 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  ^ords  or  less  -  underline  keywords) 

The  long  range  goal  of  this  laboratory  is  to  determine  the  function  of 
histocompatibility-linked  immune  response  (Ir)  genes  in  the  regulation  of  the 
immune  response  and  in  the  control  of  immunocompetent  cell  interactions.   At 
present,  the  research  effort  is  directed  in  two  main  areas:   1)  the  role  of  the 
I-region  associated  (la)  antigens  and  Ir  gene  products  in  macrophage-T  lymphocyte! 
interaction,  macrophage  processing  and  presentation  of  antigen,  and  T  lymphocyte 
antigen  recognition.   2)  Genetic,  serological,  physicochemical,  and  functional 
studies  of  the  antigens  of  the  guinea  pig  major  histocompatibility  complex 
(the  GPLA  complex) . 


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?HS-b040 
(Rev.    10-76) 


Z01-AI-00040-05  LI 

I.  Role  of  the  I-region  in  the  Regulation  of  Immunocompetent  Cell  Interactions 

a.   Macrophage  Processing  and  Presentation  of  Antigen 

We  have  postulated  that  the  site  of  expression  of  the  histocompatibility 
linked  Ir  genes  may  be  at  the  level  of  the  antigen  presenting  cell  or  macro- 
phage rather  than  in  the  antigen-responsive  T  lymphocyte.   Thus,  macrophages 
from  a  non-responder  animal  would  lack  the  I-region  gene  product  necessary  to 
process  or  present  the  relevant  genetically  controlled  antigen.   In  order  to 
test  this  hypothesis  we  have  begun  a  detailed  study  of  the  biochemical  basis 
of  macrophage-lymphocyte  interaction  and  are  attempting  to  gain  an  understand- 
ing at  the  molecular  level  of  macrophage  processing  and  presentation  of  antigen. 
We  have  used  as  a  model  antigen  the  simple  trinitrophenyl  hapten  (TNP)  which 
can  easily  be  covalently  coupled  to  the  macrophage  surface.   We  initially 
examined  the  effects  of  anti-TNP  antibody  on  the  ability  of  TNP-modified 
macrophages  to  stimulate  TNP-specif ic  T  cell  proliferative  responses  in  vitro. 
The  addition  of  anti-TNP  to  TNP-modified  macrophages  immediately  following 
conjugation  inhibited  their  ability  to  stimulate  TNP-specific  T  cell  prolifer- 
ation. However,  anti-TNP  had  no  effect  on  the  TNP  specific  response  to  TNP- 
modified  macrophages  that  had  been  cultured  overnight  before  addition  to 
primed  T  cells.   This  result  initially  suggested  that  macrophage  presentation 
of  the  TNP  determinant  is  not  simply  a  surface  display  phenomenon  and  that 
the  macrophage  must  process  membrane  conjugated  TNP  in  a  manner  so  that  it  is 
inaccessible  to  anti-TNP  antibody  to  create  the  relevant  immunogen  recognized 
by  T  cells.   However,  recently  we  have  been  able  to  demonstrate  that  anti-TNP 
antibody  can  inhibit  the  proliferative  response  induced  by  cultured  TNP  modi- 
field  macrophages  provided  they  have  been  freshly  modified  with  the  non-cross 
reactive  dinitrophenyl  (DNP)  hapten.   We  could  then  study  the  effects  of  anti- 
hapten  antibody  on  macrophages  which  simultaneously  bore  low  concentrations 
of  one  hapten  (aged)  but  high  concentrations  of  the  other  (freshly  modified) . 
In  these  experiments  we  could  demonstrate  that  the  response  to  TNP-modified- 
aged-macrophages  which  were  modified  with  DNP  immediately  prior  to  addition  to 
culture  was  markedly  inhibited  with  anti-TNP  antibody.   These  experiments 
suggest  that  antigenic  determinants  are  displayed  on  the  macrophage  surface, 
but  under  normal  conditions  a  too  low  a  density  to  allow  efficient  binding  of 
anti-antigen  antibody.   At  the  present  time  we  are  conducting  studies  on  the 
mechanism  and  requirements  for  anti-antigen  induced  inhibition  of  T  cell 
proliferation  .   We  are  also  extending  this  approach  to  examine  the  effects  of 
antibody  to  soluble  protein  antigens  on  macrophage  handling  of  such  antigens. 
Hopefully,  these  studies  will  lend  insights  into  the  mechanisms  whereby  the 
products  of  the  I-region  play  a  role  in  macrophage  presentation  and  processing 
of  antigen. 

We  have  previously  demonstrated  that  treatment  of  macrophages  with  anti- 
la  sera  at  any  time  following  TNP  conjugation  resulted  in  a  reduced  ability  of 
TNP-conjugated  macrophages  to  stimulate  a  TNP-specific  T  cell  proliferative 
response.   These  results  suggested  that  anti-la  sera  interfere  with  the 
development  of  a  TNP-specific  immunogen  on  the  macrophage  and  strongly  suggest 
that  an  association  exists  between  TNP-modified  membrane  proteins  and  la 


22-49 


Z01-AI-00040-05  LI 

antigens.   We  have  therefore  performed  a  detailed  biochemical  analysis  of  the 
relationship  between  the  TNP  moiety  and  the  products  of  the  major  histocom- 
patibility complex  on  the  macrophage  cell  surface.   Macrophages  were  TNP- 
modified,  radio-iodinated,  and  lysed  in  detergent.   When  TNP-derivatized  pro- 
teins were  isolated  using  an  anti-TNP  immunoabsorbant  and  the  presence  of  TNP- 
derivatized  antigens  in  the  eluted  proteins  determined  by  immunoprecipitation 
techniques  no  hapten  modified  la  antigens  were  detected.   Furthermore,  when 
la  antigens  from  TNP-modified  cells  were  eluted  from  an  anti-la  immunoabsorb- 
ant, no  proteins  other  than  la  antigens  were  detectable.   It  is  thus  highly 
unlikely  that  la  antigens  are  strongly  bound  to  a  TNP-modified  protein  in  a 
complex  which  withstands  solubilization  of  the  membrane.   However,  a  complex 
maintained  by  the  integrity  of  the  intact  cell  membrane  might  very  well  exist. 

The  demonstration  that  la  antigens  are  not  derivatized  on  TNP-modified 
cells  strongly  suggests  that  the  antigenic  determinant  recognized  by  guinea 
pig  T  cells  does  not  consist  of  covalently  trinitrophenylated  la  antigens. 
Further  support  for  this  data  was  obtained  from  studies  where  membrane 
preparations  from  TNP-modified  macrophages  were  used  in  both  primary  and 
secondary  cultures  in  place  of  TNP-modified  macrophages.   Surprisingly,  mem- 
branes were  effective  stimulators  of  TNP-specific  T  cell  proliferation  in  both 
primary  and  secondary  cultures.   However,  effective  stimulation  was  only 
observed  in  the  presence  of  unmodified  macrophages.   A  detailed  study  of  the 
genetic  requirements  of  this  response  demonstrated  that  the  la  antigens  of  the 
unmodified  macrophages  rather  than  the  la  antigens  borne  on  the  subcellular 
membrane  fractions  determine  the  histocompatibility  restrictions  on  macro- 
phage-! cell  interaction.   These  studies  effectively  rule  out  the  possibility 
that  the  major  immunogen  recognized  by  the  T  cell  is  the  TNP  determinant  co- 
valently coupled  to  macrophage  la.   Furthermore,  these  experiments  suggest 
that  la-positive  macrophages  can  process  and  present  antigens  bound  to  mem- 
brane fragments. 

b.   Studies  on  the  Physical  Interaction  Between  Macrophages  and  T  Cells. 

Previous  studies  have  suggested  that  physical  contact  between  macrophages 
and  T  lymphocyte  is  an  obligatory  step  in  this  cell-cell  interaction.   In 
order  to  examine  the  basis  for  this  physical  interaction  between  macrophage 
and  T  cell  we  have  used  monolayers  of  macrophages  pulsed  with  soluble  protein 
antigens  as  immunosorbents  for  T  lymphocytes  from  guinea  pigs  primed  to  solu- 
ble protein  antigens.   When  T  lymphocytes  were  cultured  for  three  successive 
4  hour  periods  on  such  monolayers,  they  were  selectively  deprived  of  cells 
responding  in  an  assay  for  antigen  dependent  proliferation  against  the 
antigen  used  for  pulsing  the  absorbing  monolayer,  but  maintained  their  re- 
sponse to  other  antigens;  the  lymphocytes  adhering  to  the  M$  of  the  absorbing 
monolayer  were  enriched  in  responding  cells.   The  proliferative  response  of  F^ 
T  lymphocytes  to  antigen  in  association  with  M$  of  either  parental  strain 
could  be  absorbed  leaving  the  response  to  antigen  in  association  with  M<3>  of 
the  other  parental  strain.   The  absorption  of  the  proliferative  response  could 
not  be  inhibited  by  the  addition  of  a  large  excess  of  soluble  antigen  to  the 
medium  of  the  absorption  culture.   These  studies  are  in  support  of  the  view 
that  T  lymphocytes  recognize  and  bind  specifically  to  a  complex  of  la  antigen 

22-50 


Z01-AI-00040-05  LI 

and  protein  antigen  at  the  surface  of  the  M$. 

c.   Studies  on  the  Genetic  Regulation  of  the  Autologous  Mixed  Leukocyte 
Reaction  and  on  the  Response  to  Aldehydes  on  Cell  Surfaces . 

It  has  been  demonstrated  that  human  T  lymphocytes  can  be  specifically 
sensitized  to  antigens  present  on  autologous  non-T  cells.   We  have  extended 
this  experimental  observation  to  the  guinea  pig  system  and  demonstrated  that 
guinea  pig  T  cells  will  specifically  proliferate  when  stimulated  with  syngen- 
eic la  positive  macrophages .  We  have  also  shown  that  the  syngeneic  MLR  ex- 
hibits both  memory  and  specificity.   Thus,  T  cells  positively  selected  with 
syngeneic  macrophages  respond  specifically  in  a  second  culture  when  stimulated 
with  syngeneic  rather  than  allogeneic  macrophages.   Two  populations  of  F  T 
cells  could  be  demonstrated,  each  responsive  to  unmodified  macrophages  or 
either  parent.   Furthermore,  the  response  could  then  be  specifically  blocked 
by  anti-la  sera  directed  toward  the  stimulatory  parental  haplotype.   The 
significance  of  these  auto-reactive  cells  is  still  unclear.   One  intriguing 
possibility  is  that  they  represent  a  true  reaction  to  self  la  antigens  media- 
ted by  means  of  a  low  affinity  receptor  to  self.   Experiments  are  now  in 
progress  to  test  this  hypothesis. 

A  second  area  of  investigation  related  to  the  autologous  MLR  is  the 
induction  of  T  cell  proliferation  by  macrophages  which  have  been  treated  with 
neuraminidase  and  galactose  oxidase  (NAGO) .   In  this  experimental  system,  the 
macrophages  may  be  either  syngeneic  or  allogeneic  to  the  responding  T  cell, 
but  the  proliferation  can  be  specifically  blocked  by  anti-la  sera  directed  to 
the  stimulatory  macrophage.   To  further  probe  the  genetic  regulation  of  this 
response,  we  have  examined  whether  2  clones  of  F  T  cells  exist  which  respond 
specifically  to  aldehyde  bearing  macrophages  of  one  parent,  but  not  the  other. 
Using  the  technique  where  proliferating  cells  can  be  selectively  killed  by 
incorporation  of  bromodeoxyuridine  (BUdR)  followed  by  exposure  to  light,  we 
have  shown  that  the  F  T  cells  responding  to  modified  macrophages  of  one 
parent  are  eliminatedTiy  exposure  to  aldehyde  bearing  macrophages  of  either 
parent.   However,  the  stimulation  produced  by  parental  macrophages  is  still 
blocked  by  anti-la  sera  directed  against  the  stimulatory  parent.   Thus, 
stimulation  produced  by  NAGO  treated  macrophages  is  an  I-region  mediated 
event  that  is  not  genetically  restricted.   These  studies  suggest  that  the  la 
molecule  may  subserve  a  function  other  than  a  role  in  antigen  processing,  or 
as  a  target  for  a  part  of  the  T  cell  receptor,  but  perhaps  may  function  as  a 
trigger  or  second  signal  in  T  cell  activation. 

II.  Genetic,  Serologic,  Physicochemical,  and  Functional  Studies  of  the  Guinea 
Pig  Major  Histocompatibility  Complex. 

Although  the  major  interest  of  the  laboratory  has  shifted  over  the  past 
two  years  to  an  analysis  of  the  products  of  the  MHC  in  the  regulation  of 
immunocompetent  cell  interactions,  these  functional  studies  have  required  a 
continued  interest  both  in  the  serologic  makeup  of  the  GPLA  complex  and  in  the 
production  of  well  characterized,  potent  antisera  to  GPLA  antigens.   Indeed, 
wherever  possible  we  have  attempted  to  correlate  structural  and  physicochemi- 

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Z01-AI-00040-05  LI 

cal  studies  with  functional  studies.   Several  different  areas  have  been 
studied  during  the  past  year: 

a)  Structural  and  Functional  Studies  of  B^-microglobulin  -  We  have 
demonstrated  two  previously  undescribed  guinea  pig  molecules  reactive  with 
anti-guinea  pig  3^-m.   The  first  molecule  was  composed  of  a  36,000  dalton 
glycoprotein  associated  with  3~-m  and  was  found  on  guinea  pig  thymocytes,  but 
not  lymphocytes.   The  second  molecule  was  a  40,000  dalton  glycoprotein  assoc- 
iated with  60-m  and  was  found  on  both  guinea  pig  thymocytes  and  lymphocytes.  By 
structure,  chemical  composition,  association  with  6~-m,  and  tissue  distribution, 
the  first  molecule  is  an  attractive  candidate  for  the  guinea  pig  homologue  of 
the  murine  thymus -leukemia  (TL)  antigen,  whereas  the  second  fits  the  criteria 
for  the  guinea  pig  homologue  of  the  murine  Qa-2  antigen. 

We  also  examined  the  effects  of  a  goat  anti-guinea  pig  Bo-111  serum  on  a 
number  of  T  lymphocyte  functions  in  vitro.   Anti-g„-m  serum  produced  a  marked 
inhibition  of  the  response  of  peritoneal  exudate  T  cells  to  antigen  and 
mitogen  stimulation.   Surprisingly,  a  marked  activation  of  lymph  node  T 
lymphocyte  proliferation  was  observed  in  the  absence  of  antigen  or  mitogen 
stimulation.   The  stimulatory  effect  of  anti-B„-m  serum  was  specific  for  3„-m 
and  could  not  be  blocked  by  antisera  to  the  antigens  of  the  guinea  pig  MHC. 
These  studies  suggest  that  3?-m  may  play  some  critical  role  in  the  immune 
response  at  the  level  of  T  cell  activation. 

b)  Role  of  C4  in  the  Cellular  Immune  Response  -  Previous  studies  have 
demonstrated  that  the  structural  gene  for  guinea  pig  C4  was  linked  to  the 
GPLA  complex.   Because  of  the  demonstration  that  antisera  to  human  C4  inhibit 
the  human  mixed  leukocyte  reaction  and  the  response  to  the  mitogen,  phyto- 
hemagglutinin,  we  undertook  an  intensive  study  of  the  possible  role  of  C4  in 
the  afferent  phase  of  immune  recognition.   Antisera  to  C4  were  raised  in 
guinea  pigs,  rabbits  and  goats  and  tested  for  inhibition  of  the  proliferative 
response  to  antigens,  mitogens  and  alloantigens .   In  a  large  number  of  exper- 
iments no  inhibition  was  found  when  the  various  cultures  were  formed  in  the 
presence  of  high  titer  antisera  to  guinea  pig  C4.   Furthermore,  lymphocytes 
from  C4  deficient  guinea  pigs  responded  as  well  as  inbred  strain  13  lympho- 
cytes to  specific  antigen  or  mitogen  induced  proliferation,  indicating  a 
normal  capacity  of  T  cells  from  C4-deficient  animals  for  recognition  of 
antigenic  stimuli  and  for  proliferation.   Macrophages  from  C4D  animals  were 
able  to  effectively  stimulate  T  cell  proliferation,  suggesting  a  normal 
capability  for  antigen  or  mitogen  presentation.   These  data  indicate  that  the 
demonstrated  functional  association  of  C4  and  MHC-mediated  reactions  in  the 
human  does  not  generally  apply  to  another  species. 

c)  Antibodies  to  Guinea  Pig  Anti-Thrombin  III  Induce  T  Cell  Prolifera- 
tion -  In  the  course  of  studies  investigating  the  reaction  of  a  goat  anti- 
guinea  pig  immunoglobulin  serum  with  guinea  pig  lymphocytes ,  it  was  noted 
that  the  serum  reacted  with  both  T  and  B  cells  as  determined  by  indirect 
immunof luorescent  staining  and  complement  mediated  cytotoxicity.   In  addition, 
the  antiserum  (or  FCab')-  fragments)  stimulated  the  proliferation  of  T 
lymphocytes,  yet  had  no  mitogenic  effects  on  B  lymphocytes.   Absorption  of 


22-52 


Z01-AI-00040-05  LI 

the  antiserum  with  sepharose-bound  IgG  removed  all  detectable  reactivity  of 
the  antiserum  with  B  cell  or  serum  Ig,  but  did  not  reduce  its  capacity  to 
bind  to  or  stimulate  T  cells.   Immunoelectrophoretic  studies  revealed  that, 
in  addition  to  y-globulins,  the  serum  also  recognized  an  a  -globulin  which 
was  subsequently  characterized  as  a  68,000  dalton  glycoprotein  that  bore  no 
identifiable  Ig  determinants.   This  protein  was  then  purified  by  ion 
exchange  chromatography  and  immunoabsorbent  chromatography,  and  it  was  found 
that  the  cytotoxic  and  mitogenic  activity  of  the  antiserum  could  be  abrogated 
by  addition  of  the  purified  a  -globulin.   Functional  studies  and  purification 
on  heparin-sepharose  affinity  columns  demonstrated  that  the  a„-globulin  was 
anti-thrombin  III.   Studies  to  evaluate  the  importance  of  the  cross  reaction 
between  this  major  serum  protease  inhibitor  and  an  antigen  on  guinea  pig  T 
cells  are  in  progress.   The  relevant  antigenic  determinant  on  the  molecule 
was  resistant  to  reduction  and  alkylation  in  7m  guanidine  and  to  treatment 
with  2%  SDS  at  100  ,  yet  was  sensitive  to  periodate  oxidation,  suggesting  it 
was  carbohydrate  in  nature. 

d)  Production  of  Monoclonal  Antibodies  to  Guinea  Pig  Cell  Surface 
Antigens  -  In  order  to  obtain  more  useful  reagents  for  functional  studies  of 
the  role  of  la  antigens  in  immunocompetent  cell  interactions,  spleen  cells 
from  BALB/c  mice  immunized  to  the  la-positive  L„C  leukemia  of  strain  2  guinea 
pigs  were  fused  to  the  NS1  mouse  myeloma.   Culture  supernatants  reactive  with 
guinea  pig  la  antigens  were  obtained  by  screening  against  the  la-positive 
leukemia  cell  line  and  its  la-negative  variant.   Selected  cultures  were 
cloned  and  injected  into  pristane  primed  mice.   Ascitic  fluid  from  five 
independently  derived  hybrids  appeared  to  detect  la  antigens  in  that  they 
reacted  with  the  la-positive  leukemia  cells,  normal  spleen  cells,  and  to  a 
lesser  extent  thymus,  yet  did  not  react  with  the  la  negative  BZ-L„C  cell. 
Studies  of  the  molecular  characteristics  of  the  antigens  identified  by  these 
monoclonal  antibodies  are  in  progress.   In  addition,  studies  of  the  effect  of 
these  antisera  on  T  cell  and  macrophage  functions  are  also  in  progress. 
These  data  clearly  demonstrate  the  usefulness  of  the  hybridoma  technique  for 
the  production  of  monoclonal  antibodies  to  xenogeneic  antigens  if  a 
sufficiently  selective  screening  system  is  available. 

e)  Studies  of  the  Induction  of  Transplantation  Tolerance  Following 
Allogeneic  Spleen  Transplantation  -  Previous  studies  in  the  rat  by  Dr.  H. 
Bitter-Suermann  have  shown  that  immunocompetent  rat  spleen  allografts  could 
survive  indefinitely  in  the  absence  of  immunosuppression  and  that  accepted 
spleen  allografts  induced  a  donor  specific  unresponsiveness  for  skin  allo- 
grafts.  Because  of  our  experience  with  in  vitro  assays  of  immunologic 
function  in  the  guinea  pig,  we  have  collaborated  with  Dr.  Bitter-Suermann  to 
extend  his  experimental  observations  to  the  guinea  pig.   We  have  shown  that 
strain  13  spleens  can  be  transplanted  to  strain  2  guinea  pigs  and  that  in  the 
absence  of  immunosuppression  the  splenic  allograft  is  present  up  to  3  months 
following  grafting.   Preliminary  studies  suggest  that  recipients  of  spleen 
allografts  are  tolerant  of  skin  derived  from  the  donor  strain.   Further 
studies  of  the  serologic  makeup  of  the  grafted  spleen  and  the  immunologic 
mechanisms  involved  in  this  unusual  system  of  transplantation  tolerance  are 
in  progress. 


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Z01-AI-00040-05  LI 

PUBLICATIONS 

Clement,  L.T.,  Kask,  A.M.,  and  Shevach,  E.M.   Rapid  Purification  of  Detergent 
Solubilized  la  Antigens  by  Immunoabsorbent  Chromatography.   Immunochemistry 
15:  393-399,  1978. 

Shevach,  E.M.   The  Guinea  Pig  I-Region  -  A  Functional  Analysis  of  Ia-Ir 
Associations.   Seminars  in  Immunopathology  1_:  207-234,  1978. 

Clement,  L.T.,  Thomas,  D.W.,  Kask,  A.M.,  Shevach,  E.M.  Guinea  Pig  la  Are  Not 
Derivatized  on  Trinitrophenyl-Modif ied  Cells.   Nature  274 :  592-594,  1978. 

Yamashita,  U. ,  Shevach,  E.M.,  and  Thomas,  D.W.  The  Expression  of  la  Antigens 
on  Immunocompetent  Cells  in  the  Guinea  Pig.  III.  Functional  Selection  of  la 
Negative  T  Cells  by  In  Vitro  Culture.   J.  Immunol.  121:  390-396,  1978. 

Thomas,  D.W.  and  Shevach,  E.M.   Nature  of  the  Antigenic  Complex  Recognized 
by  T  Lymphocytes.   VI.   The  Effect  of  Anti-TNP  Antibody  on  T  Cell  Responses 
to  TNP-Conjugated  Macrophages.   J.  Immunol.  121:  1145-1151,  1978. 

Thomas,  D.W.  and  Shevach,  E.M.   Nature  of  the  Antigenic  Complex  Recognized 
by  T  Lymphocytes.   VII.   Evidence  for  an  Association  Between  TNP-Conjugated 
Macrophage  Membrane  Components  and  la  Antigens.   J.  Immunol.  121:  1152- 
1156,  1978. 

Clement,  L.T.  and  Shevach,  E.M.   Characterization  of  Major  Histocompatibility 
Antigens  on  Trinitrophenyl-Modif ied  Cells.   Molecular  Immunology  16:  67-76, 
1979. 

Schwartz,  B.D.,  Cigen,  R. ,  Berggard,  I.  and  Shevach,  E.M.   Guinea  Pig 
Homologues  of  TL  and  Qa-2  Antigens.   J.  Immunol.  121:  835-839,  1979. 

Yamashita,  U.,  Logdberg,  L.,  Berggard,  I.  and  Shevach,  E.M.   The  Activation 
of  Guinea  Pig  T  Lymphocytes  by  Anti-6?-microglobulin  Serum.   J.  Immunol.  122 : 
1427-1432,  1979. 

Yamashita,  U.  and  Shevach,  E.M.   The  Histocompatibility  Restrictions  on 
Macrophage  T-Helper  Cell  Interaction  Determine  the  Histocompatibility 
Restrictions  on  T-Helper  Cell  B-Cell  Interactions.   J.  Exp.  Med.  148: 
1171-1185,  1978. 

Stingl,  G.,  Katz,  S.I.,  Clement,  L.,  Green,  I.  and  Shevach,  E.M.   Immunolog- 
ical Functions  of  la-Bearing  Epidermal  Langerhans  Cell.   J.  Immunol.  121: 
2013-2055,  1978. 

Stingl,  G.,  Pichler,  W.J.  and  Shevach,  E.M.   Guinea  Pig  T  Cell  Subpopulations. 
Wiener  Klinische  Wochenschrif t  90:  741-744,  1978. 


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Z01-AI-00040-05  LI 

Shevach,  E.M.,  Chan,  C. ,  Thomas,  D.W.  and  Clement,  L.   What  is  the  Nature 
of  the  Antigenic  Complex  Recognized  by  T  Lymphocytes?   In,  Bach,  F. , 
Bonavida,  B.,  Vitetta,  E.  and  Fox,  C.F.  (Editors).   T  +  B  Lymphocytes 
Recognition  and  Function.   ICN-UCLA  Symposia  on  Molecular  and  Cellular 
Biology,  New  York,  N.Y.,  Academic  Press,  in  press,  1979. 

Burger,  R.  and  Shevach,  E.M.   Evaluation  of  the  Role  of  C4  in  the  Cellular 
Immune  Response  in  vitro.   J.  Immunol.  122:  2388-2394,  1979. 

Clement,  L.T.,  Kask,  A.M.,  Schwartz,  B.D.  and  Shevach,  E.M.   Analysis  of 
Guinea  Pig  Membrane  Proteins  Recognized  by  Heterologous  An ti- Lymphocyte 
Sera:   Specific  Recognition  of  la  Alloantigens.   Transplantation  27:  397-405, 
1979. 

Yamashita,  U.  and  Shevach,  E.M.   The  Role  of  the  I-Region  in  the  Control  of 
Macrophage-T  Cell  and  T-Cell-B  Cell  Interactions.   In  Quastel,  M.A.  (Editor) 
Cell  Biology  and  Biochemistry  of  Leukocyte  Function.   Academic  Press,  New 
York,  N.Y.   In  press,  1978. 

Braendstrup,  0.,  Werdelin,  0.,  Shevach,  E.M.  and  Rosenthal,  A.S.  Macrophage- 
Lymphocyte  Clusters  in  the  Immune  Response  to  Soluble  Protein  Antigen  In 
Vitro.   VII.   Genetically  Restricted  and  Nonrestricted  Physical  Interactions. 
J.  Immunol.  122:  1608-1613,  1979. 

Werdelin,  0.,  Braendstrup,  0.  and  Shevach,  E.M.   Specific  Absorption  of  T 
Lymphocytes  Committed  to  Soluble  Protein  Antigens  by  Incubation  on  Antigen- 
Pulsed  Macrophage  Monolayers.   J .  Immunol .   In  press. 

Shevach,  E.   The  Role  of  Antigenic  Determinants  in  Macrophage  Lymphocyte 
Interaction.   In  (Unanue,  E.  and  Rosenthal,  A.S.,  Editors)  Regulatory  Role 
of  Macrophages  in  Immunity,  Academic  Press,  New  York,  N.Y.   In  press. 


22-55 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  Of 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01-AI-00147-04  LI 


PERIOD  COVERED 

October  1,  1978  -  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

The  Mechanism  of  Activation  of  Thymus-Derived  Lymphocytes 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


Principal  Investigator:   Ronald  H.  Schwartz,  M.D.,  Ph.D. 


Others: 


Sr.  Investigator, 
LI/NIAID 

William  E.  Paul,  M.D. ,  Chief,  LI/NIAID 

Harley  Tse,  Ph.D.,  Guest  Worker,  LI/NIAID 

Dan  Longo,  M.D. ,  Clinical  Associate,  LI/NIAID 

Lou  Matis,  M.D.,  Clinical  Associate,  LCI  and  LI/NIAID 

David  Lebwohl,  COSTEP  Summer  Student 

Michiel  Ultee,  Graduate  Student,  Dept.  of  Biochemistry  and  Molecular 

Biology,  Northwestern  University 
Emanuel  Margoliash,  Chairman,  Dept.  of  Biochemistry  and  Molecular 

Biology,  Northwestern  University 


COOPERATING  UNITS  (if 

Department  of  Biochemistry  and  Molecular  Biology,  Northwestern  University, 
Evanston,  Illinois. 


LAB/BRANCH 

Laboratory  of  Immunology 


institute  and  LOCATION   National  institute  ot  Allergy  and  lnfectxous  Diseases. 
National  Institutes  of  Health,  Bethesda,  MD.  20205 


TOTAL  MANYEARS: 

2.5 


PROFESSIONAL: 

1.25 


1.25 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (a!)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


£  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

This  project  focuses  on  the  function  of  immune  response  (Ir)  genes  in  the 
activation  of  thymus-derived  (T) -lymphocytes ,  using  a  secondary  proliferation 
assay.   By  a  slope  analysis  of  cell  dose-response  curves,  we  have  shown  that 
three  different  cells  are  involved  in  the  proliferative  response,  a  nonimmune 
antigen-presenting  cell,  an  antigen  specific,  primed  T  lymphocyte,  and  a  non- 
immune, recruitable  T  lymphocyte.   Experiments  with  radiation  chimeras  demon- 
strated that,  of  these  three  cell  types,  only  the  antigen-presenting  cell  has  to 
possess  high  responder  Ir  genes.   However,  the  proliferating,  primed  T  lympho- 
cyte must  also  have  matured  in  a  high  responder  environment.   Studies  of  the 
proliferative  response  to  the  antigen,  pigeon  cytochrome  c_,  demonstrated  that 
two  complementing  Ir  genes  could  control  the  response  to  a  single  antigenic 
determinant  and  that  both  gene  products  must  be  present  in  the  same  antigen- 
presenting  cell  in  order  to  stimulate  a  response. 


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PHS-6040 
(Rev.    10-76] 


Z01  AI  00147-04  LI 

In  our  studies  of  the  role  of  Ir  gene  products  in  T-lymphocyte  immune 
responses,  we  have  utilized  the  proliferative  response  of  Peritoneal 
Exudate  T_-Lymphocyte-Enriched  Subpopulations  (PETLES),  an  assay  developed 
in  this  laboratory  several  years  ago.   In  addition  we  have  recently  set  up 
a  lymph  node  cell  proliferation  assay  which  also  measures  primed  T  lymphocyte 
function.   During  the  past  year  we  have  demonstrated  that  3  different  cells 
are  involved  in  the  proliferative  response,  that  immune  response  (Ir)  genes 
need  only  be  expressed  in  the  antigen-presenting  cell,  and  that  the  prolifera- 
tive response  to  pigeon  cytochrome  c_  involves  the  recognition  of  a  single 
antigenic  determinant  controlled  by  two  MHC-linked  Ir  genes. 

It  has  been  known  for  many  years  that  two  cell  types  are  involved 
in  the  proliferative  response  to  soluble  protein  antigens.   One  of  these 
cells  is  the  antigen-presenting  cell,  a  nonimmune,  radioresistant,  la- 
antigen  bearing  cell  which  lacks  Ig  and  Thy  1  determinants.   The  most 
likely  candidate  for  this  cell  type  is  one  in  the  monocyte-macrophage 
lineage.   The  other  known  cell  type  is  the  antigen-specific  primed  T 
lymphocyte  which  is  of  the  Ly  1  2  lineage.   However,  when  we  attempted  to 
do  log-log  plots  of  cell  dose-response  curves,  the  slope  for  several 
different  antigen-primed  populations  was  always  3,  suggesting  that  more  than 
two  cells  were  involved  in  the  response.   By  adding  different  cell 
populations  in  excess  over  the  primed  population,  we  could  convert  the  slope 
from  3  to  2,  thus  identifying  the  various  cells.   Both  irradiated  spleen 
cells  and  nylon  wool-passed  spleen  cells  from  nonimmune  mice  could  convert 
the  slope  from  3  to  2.   Addition  of  both  populations  in  excess  reduced 
the  slope  from  3  to  1,  suggesting  that  each  population  contained  a  different 
cell.   Because  the  irradiated  nonimmune  spleen  cells  are  known  to  possess 
antigen-presenting  activity  it  was  assumed  that  this  population  contained 
the  macrophage.   The  nylon  wool  passed  spleen  cells,  on  the  other  hand, 
are  depleted  of  macrophages  and  contain  mostly  nonimmune  T  lymphocytes. 
Because  its  filling  capacity  was  eliminated  by  irradiation,  we  concluded 
that  this  newly  identified  cell  type  was  probably  a  recruitable  (i.e.  non- 
antigen  specific)  T  lymphocyte  which  divided  in  response  to  mitogenic 
signals  from  the  stimulated  antigen-specific  T  lymphocyte. 

Our  previous  studies  on  the  localization  of  immune  response  genes 
in  the  responding  PETLES  population  had  demonstrated  a  requirement  for  the 
antigen-presenting  cell  to  come  from  a  high  responder  strain.   In  pur- 
suing these  studies  further,  we  have  now  demonstrated  that  the  T-lymphocyte 
does  not  have  to  be  from  a  genotypic  high  responder  provided  the  cells 
mature  in  the  environment  of  a  high  responder.   This  conclusion  was  reached 
from  studies  with  radiation  chimeras.   T-cell  depleted  bone  marrow  cells 
from  low  responder  mice  were  injected  into  lethally  irradiated  high 
responder  F  mice  and  allowed  to  develop  into  mature  T  cells.   Such 
chimeras  do  not  respond  to  the  antigen,  suggesting  the  requirement  for  at 
least  one  high  responder  cell  type.   Because  we  knew  that  the  antigen- 
presenting  cell  was  one  such  cell  type,  we  had  to  provide  this  cell  in  order 
to  assess  the  requirements  for  the  T-lymphocyte.   This  was  accomplished  by 
transferring  chimeric  spleen  cells  to  an  acutely  irradiated  F.  along  with 
T-cell-depleted  F..  bone  marrow.   These  animals  now  responded  to  the  antigen. 

22-57 


Z01  AI  00147-04  LI 

Thus,  only  high- responder  antigen-presenting  cells  were  required.   However, 
the  environment  in  which  the  T  cells  develop  was  also  critical.   When 
chimeras  of  the  type  [high  responder  F.  bone  marrow  into  lethally  irradiated 
low  responder  hosts]  were  created,  they  failed  to  respond  to  the  antigen 
even  though  they  possessed  high  responder  antigen-presenting  cells  derived 
from  the  bone  marrow.   Thus,  the  T  cell  must  learn  to  recognize  the 
appropriate  Ir  alleles  from  radioresistant  cells  of  the  host  when  they 
differentiate  from  marrow  stem  cells.   Once  a  high  responder  repertoire  is 
fixed,  the  T  cells  can  then  recognize  antigen  when  it  is  presented  on  a  high 
responder  macrophage. 

In  deciphering  the  mechanism  of  action  of  Ir  gene  products,  it  was 
deemed  necessary  to  investigate  the  types  of  antigenic  determinants 
recognized  by  T  lymphocytes  to  ascertain  whether  the  repertoire  of  re- 
ceptors on  these  cells  was  any  different  from  that  of  B  lymphocytes.   To 
facilitate  such  experiments  we  turned  to  the  small  globular  proteins  as 
antigens,  because  they  provided  us  with  probes  for  which  we  possessed  a 
thorough  understanding  of  the  amino  acid  sequence  and  a  precise  knowledge  of 
the  three-dimensional  structure.   Thus,  we  could  localize  the  antigenic 
determinants  by  cross-stimulation  with  species  variants  and  enzymatic  or 
chemical  cleavage  fragments  of  the  molecules.   For  the  last  few  years  we 
have  focused  on  the  T- lymphocyte  proliferative  response  of  BIO. A  mice  to 
pigeon  cytochrome  ji.      This  response  appears  to  be  directed  at  a  single 
determinant  comprised  of  three  amino  acid  residues  from  different  parts 
of  the  molecule:  the  isoleucine  at  position  3,  the  glutamine  at  position  100 
and  the  lysine  at  position  104.   These  three  residues  lie  next  to  each 
other  on  the  outer  surface  of  the  back  face  of  the  molecule.   Although  these 
results  suggest  that  T  cells  recognize  three  dimensional  determinants, 
cyanogen  bromide  cleavage  fragments  of  the  molecule  could  stimulate  the  T 
cells  primed  to  the  whole  molecule.   In  fact,  one  fragment,  residues  81-104, 
containing  only  two  of  the  three  critical  amino  acids  and  lacking  any  of  the 
a  helical  content  this  segment  possesses  in  the  native  structure,  stimulated 
better  than  the  whole  molecule  on  a  molar  basis.   Furthermore,  this  fragment 
was  immunogenic  and  the  response  was  under  the  control  of  two  MHC-linked  Ir 
genes,  just  as  was  the  response  to  the  whole  protein.   These  results  have 
raised  the  possibility  that  T-cell  receptors  recognize  antigen  fragments  on 
the  surface  of  macrophages  rather  than  intact  molecules.   Mixing  experiments 
demonstrated  that  many  of  the  responding  T  cell  clones  were  recognizing  the 
two  critical  amino  acids  found  in  the  fragment  as  part  of  the  same  deter- 
minant.  This  result  demonstrated  for  the  first  time  that  two  Ir  genes 
could  control  the  immune  response  to  a  single  antigenic  determinant.   Finally, 
antigen-presentation  experiments  showed  that  both  Ir  gene  products  must  be 
expressed  in  the  same  presenting  cell  in  order  to  generate  an  immune  response 
to  pigeon  cytochrome  <z. 

Significance  to  Biomedical  Research 

Our  attempts  to  understand  the  mechanism  of  T-lymphocyte  activation 
are  of  fundamental  importance  to  a  basic  understanding  of  how  the  immune 


22-58 


Z01  AI  00147-04  LI 

system  functions.   Since  this  system  plays  a  critical  role  in  the  bodies 
defense  mechanisms  against  infectious  diseases,  tumors  and  transplanted 
tissues,  achieving  our  goal  would  hopefully  provide  major  insights  into 
how  to  manipulate  the  immune  system  for  the  benefit  of  the  patient. 

PUBLICATIONS: 

Schwartz,  R.H.,  Yano,  A.,  Stimpfling,  J.H.  and  Paul,  W.E.:   Gene  complementa- 
tion in  the  T- lymphocyte  proliferative  response  to  poly  (Glu  Lys  Phe  ) .   A 
demonstration  that  both  immune  response  gene  products  must  be  expressed  in 
the  same  antigen-presenting  cell.   J.  Exp.  Med.  149:  40-57,  1979. 

Schwartz,  R.H.  ,  Merryman,  C.F.  and  Maurer,  P  .H. :  c.7GeneRcomplementation  in  the 
T- lymphocyte  proliferative  response  to  poly  (Glu  Lys  Tyr  ) :  evidence  for 
effects  of  polymer  handling  and  gene  dosage.   J.  Immunol.   123:  272-278,  1979. 

Rosenwasser,  L.J. ,  Barcinski,  M.A. ,  Schwartz,  R.H.  and  Rosenthal,  A.S.: 
Immune  response  gene  control  of  determinant  selection.   II.  Genetic  control 
of  the  murine  T-lymphocyte  proliferative  response  to  insulin.   J.  Immunol. 
123:  471-476,  1979. 

Schwartz,  R.H.,  Solinger,  A.M.,  Ultee,  M.E.,  Margoliash,  E.,  Yano,  A., 
Stimpfling,  J.H.,  Chen,  C,  Merryman,  C.F.,  Maurer,  P.H.  and  Paul,  W.E.: 
Ir  Gene  Complementation  in  the  Murine  T-lymphocyte  Proliferative  Response. 
In  T  and  B  Lymphocytes:  Recogntion  and  Function.   F.L.  Bach,  E.S.  Vitteta, 
B.  Bonavida  and  C.F.  Fox  (Eds.).   Academic  Press,  New  York.   In  press. 

Solinger,  A.M.,  Ultee,  M. ,  Margoliash,  E.  and  Schwartz,  R.H. :   The  T-lympho- 
cyte response  to  cytochrome  c_.   I.  Demonstration  of  a  T-cell  heteroclitic 
proliferative  response  and  identification  of  a  topographic  antigenic  deter- 
minant on  pigeon  cytochrome  _c  whose  immune  recognition  requires  two  com- 
plementing MHC-linked  Ir  genes.   J .  Exp .  Med .   In  press. 


22-59 


INDEX 
LABORATORY  OF  INFECTIOUS  DISEASES 


Z01  AI 
Project  Number 

Summary  Statement 

00021-10 

00022-20 

00024-19 
00026-12 

00027-12 
00028-21 

00175-02 

00179-01 


The  Etiology  of  Acute  Infectious 
Nonbacterial  Gastroenteritis  -  Kapikian 

Study  of  Nonbacterial  Respiratory  Diseases 
in  Children  -  Chanock 

Laboratory  Studies  of  Myxoviruses  -  Murphy 

Laboratory  and  Epidemiology  Studies  of  Viral 
Hepatitis  Agents  -  Purcell 

Basic  Studies  of  Mycoplasmas  -  Tully 

Study  of  Respiratory  Viruses  and  Mycoplasmas 
in  Volunteers  -  Murphy 

Laboratory  Studies  of  Paramyxoviruses  and 
Respiratory  Syncytial  Virus  -  Chanock 

Molecular  Biology  of  Respiratory  and 
Gastrointestinal  Viral  Pathogens 
Studied  by  DNA  Recombinant 
Technology  -  Lai 


Page 
23-1 

23-19 

23-39 
23-40 

23-52 
23-69 

23-76 

23-81 


23-95 


SUMMARY  STATEMENT  —  Annual  Report  of  the 

Laboratory  of  Infectious  Diseases 
National  Institute  of  Allergy  and  Infectious  Diseases 
October  1,  1978  to  September  30,  1979 

The  main  focus  of  the  research  programs  of  LID  continues  to  be  the  natural 
history  and  prevention  of  the  three  major  uncontrolled,  acute  infectious 
diseases  of  man — acute  respiratory  tract  disease,  viral  hepatitis  and  acute 
gastroenteritis.   Broad  advances  were  made  in  each  of  these  areas  during 
1978-79. 

Hepatitis 

Hepatitis  B  virus  (HBV)  -  Although  a  hepatitis  B  vaccine  prepared  from 
HBsAg  (surface  antigen)  purified  from  the  plasma  of  chronic  carriers  of 
the  antigen  is  feasible  and  probably  cost-effective  for  its  contemplated 
uses  in  the  United  States,  such  a  vaccine  probably  will  not  be  cost-effective 
for  the  people  most  in  need  of  a  vaccine  in  the  developing  world.   Therefore, 
additional  studies  on  alternative  methods  of  purification  and  inactivation, 
comparisons  of  monovalent  vs.  bivalent  or  polyvalent  vaccines,  studies  of 
different  combining  ratios  of  monovalent  vaccines  into  bivalent  preparations, 
different  vaccine  dosages,  different  vaccination  schedules,  different 
routes  of  administration,  studies  of  possible  adjuvant  and  their  interaction 
with  vaccine  preparations  and  further  characterization  of  the  immunizing 
antigens  themselves  are  underway.   Four  modified  vaccines  have  been  prepared. 
Two  of  these  have  been  extracted  with  ether-tween  80;  2  are  alum-precipitated. 
Preliminary  results  from  studies  in  volunteers  indicate  that  certain  of  the 
vaccines  are  much  more  immunogenic  than  any  previous  hepatitis  B  vaccine 
tested.   Thus,  they  produce  antibody  to  hepatitis  B  surface  antigen  in  two 
to  three  weeks  instead  of  two  to  three  months  (Purcell,  McAuliffe) . 

Hepatitis  A  virus  (HAV)-  Only  one  antigen,  hepatitis  A  antigen  (HAAg)  has 
been  associated  with  type  A  hepatitis  infections  to  date.   An  immunofluorescence 
assay  was  developed  for  hepatitis  A  antigen  that  has  permitted  us  to  study 
the  tissue  distribution  of  viral  infection  in  chimpanzees  and  marmosets. 
HAAg  was  detected  in  the  liver,  germinal  centers  of  lymph  nodes  and  spleen, 
and  basement  membrane  of  the  kidney.  The  latter  sites  probably  represent 
sequestration  of  antigen  released  from  the  liver  into  the  circulation. 
Interestingly,  viral  antigen  was  not  detected  in  the  intestines.  Thus,  we 
have  not  been  able  to  demonstrate  an  enteric  phase  of  multiplication  for 
the  hepatitis  A  virus. 

The  development  of  an  immunofluorescence  test  for  hepatitis  A  viral  antigen 
permitted  Provost  and  Hilleman  to  identify  hepatitis  A  virus  multiplication 
in  tissue  culture.  The  inoculum  for  their  tissue  culture  studies  was  a         . 
strain  of  HAV  that  had  been  passaged  in  marmosets  for  31  times.   Multiplication 
of  hepatitis  A  virus  in  tissue  culture  after  marmoset  passage  was  confirmed 


23-1 


in  LID.   Three  strains  of  HAV  have  been  serially  passaged  in  marmosets;  at 
least  two  of  these  (and  possibly  the  third)  produce  hepatitis  A  viral 
antigen  detectable  by  immunofluorescence  in  African  green  monkey  cells. 
Serial  passage  in  this  cell  line  is  currently  being  attempted,  and  the 
parameters  that  affect  growth  in  culture  (number  of  passages  in  marmosets, 
titer,  etc.)  are  being  examined. 

As  with  type  B  hepatitis,  pathologic  mechanisms  involved  in  type  A  hepatitis 
are  not  understood.   It  appears  that  type  A  hepatitis  rarely  if  ever  progresses 
to  chronic  disease.   However,  this  assessment  is  based  upon  a  limited 
serologic  analysis  of  chronic  hepatitis  patients.   It  is  now  possible  to 
directly  examine  liver  biopsies  from  patients  with  chronic  non-B  hepatitis 
to  determine  if  their  hepatocytes  contain  HAAg  using  the  immunofluorescence 
technique  developed  in  LID.  Such  studies  have  confirmed  that  HAV  is  not  an 
important  cause  of  chronic  hepatitis  (Feinstone,  Purcell) . 

Non-A,  non-B,  hepatitis  viruses  -  One  approach  to  the  identification  of 
non-A  non-B  hepatitis-associated  antigens  is  the  application  of  techniques 
useful  in  the  detection  and  identification  of  HBV  and  HAV:   immune  electron 
microscopy,  radioimmunoassays  and  immunofluorescence.   By  application  of 
radioimmunoassay  techniques,  a  serum  antigen  associated  with  two  cases  of 
well-characterized  non-A,  non-B  hepatitis  was  identified.   The  appearance 
and  disappearance  of  this  antigen  in  the  serum  was  temporally  related  to 
hepatitis  in  these  two  individuals,  and  the  antigen  was  shown  to  be  particulate 
and  biophysically  characterizable  by  ultracentrifugation  procedures. 
Preliminary  antibody  surveys  suggested  that  antibody  to  this  antigen  was 
widespread  among  humans  and  chimpanzees  and  that,  if  it  is  an  antigen 
associated  with  non-A,  non-B  hepatitis,  it  is  not  associated  with  the  major 
cause  of  this  disease  (Purcell,  Feinstone) . 

A  number  of  unsuccessful  attempts  to  transmit  non-A,  non-B  agents  to 
primates  were  carried  out  in  the  past.   However,  recently  two  successful 
transmission  studies  were  completed  simultaneously,  one  by  the  Bureau  of 
Biologies  and  the  other  by  the  Clinical  Center  Blood  Bank  in  collaboration 
with  our  laboratory.   In  both  studies  the  incubation  period  in  chimpanzees 
was  comparable  to  that  for  non-A  non-B  hepatitis  in  man,  and  both  biochemical 
and  histological  evidence  of  hepatitis  was  obtained.   In  our  collaborative 
study,  plasma  or  serum  from  patients  with  chronic  non-A  non-B  hepatitis  as 
well  as  from  patients  with  acute  infections  transmitted  disease  to  chimpanzees. 
Thus,  proof  of  chronic  carriage  of  non-A  non-B  viral  hepatitis  agents  has 
been  obtained  (Feinstone,  Purcell) . 

As  part  of  our  study  of  non-A,  non-B  hepatitis  in  chimpanzees,  serial  liver 
biopsies  have  been  obtained.   These  biopsies  were  studied  intensively  by 
thin  section  electron  microscopy  and  morphologic  evidence  for  two  types  of 
non-A,  non-B  hepatitis  was  observed.   One  produces  characteristic  cytoplasmic 
changes  that  consist  of  proliferation,  thickening  and  duplication  of  endoplasmic 
reticulum.   The  structures  so  formed  appear  to  be  cylinders  of  modified         , 
membrane  that  enclose  a  tube  of  rough  endoplasmic  reticulum.   Although  the 


23-2 


« 


structures  do  not  resemble  known  viruses,  they  do  "breed  true",  that  is, 
the  same  types  of  structures  are  seen  in  liver  biopsies  of  chimpanzees  that 
have  received  the  same  inoculum  or  that  have  participated  in  serial  transmission 
of  the  agent.   In  contrast,  the  other  non-A,  non-B  agent  produces  predominantly 
nuclear  changes  that  consist  of  shrinking,  heterogeneous  staining  of  the 
chromatin  and,  in  some  cells,  clusters  of  intranuclear  virus-like  particles. 
These  changes  also  "breed  true".   The  nature  of  the  structure  is  not  well 
understood.   Furthermore,  the  relationship  to  the  etiologic  agents  may  not 
be  a  direct  one.   Nevertheless,  they  provide  the  first  means  for  differentiating 
between  non-A,  non-B  agents  (Y.  Shimizu,  Feinstone,  Purcell) . 

Acute  Gastroenteritis 

Important  advances  were  made  last  year  in  our  understanding  of  the  etiological 
agents  of  acute  infectious  nonbacterial  gastroenteritis,  a  syndrome  that 
affects  a  broad  segment  of  the  population.   Equally  impressive  advances 
were  made  toward  our  goal  of  preventing  such  disease  by  immunoprophylaxis. 

Cultivation  of  type  2  human  rotavirus  in  vitro  -  Rotaviruses  are  now 
recognized  as  the  major  cause  of  serious  diarrheal  disease  of  early  life. 

They  are  responsible  for  significant  morbidity  in  the  USA  and  undoubtedly 
cause  a  major  share  of  the  many  millions  of  fatal  diarrheal  illnesses  that 
occur  in  developing  countries  each  year.   Although  laboratories  throughout 
the  world  have  sought  to  grow  these  viruses  serially  and  to  high  titer  in 
tissue  culture  all  attempts  until  now  have  failed.  Several  years  ago  we 
succeeded  in  passaging  a  type  2  human  rotavirus  strain  24  times  in  human 
embryonic  kidney  but  only  limited  viral  replication  occurred.   During  the 
past  year,  the  long  sought  after  goal  of  growing  human  rotavirus  to  high 
titer  in  culture  was  achieved. 

Initially,  42  stool  specimens  known  to  be  positive  for  rotavirus  by  electron 
microscopy  (EM)  were  tested  for  their  ability  to  infect  primary  African 
green  monkey  kidney  (AGMK)  cell  cultures.   Only  5  specimens  induced  fluorescent 
antibody  stainable  antigens  in  10%  or  more  of  cells.   In  order  to  amplify 
virus  that  was  efficient  in  initiating  infection  in  culture  and  hence 
increase  the  probability  of  the  emergence  of  a  cell  culture  adapted  mutant, 
a  human  type  2  rotavirus  (strain  "Wa")  was  administered  orally  to  gnotobiotic 
piglets.  These  animals  developed  mild,  transient  diarrhea.   Virus  was 
passaged  subsequently  10  times  in  gnotobiotic  piglets  in  collaboration  with 
Dr.  E.H.  Bohl,  Ohio  Agricultural  Research  and  Development  Center,  Wooster, 
Ohio.   The  development  of  diarrhea  was  variable  but  piglets  at  each  passage 
were  infected  as  indicated  by  the  detection  of  a  large  quantity  of  viral 
antigens  in  intestinal  contents. 

Attempts  were  made  to  initiate  serial  passage  of  the  "Wa"  strain  in  AGMK 
cells  using  a  stool  filtrate  derived  from  patient  "Wa"  and  intestinal 
aspirates  collected  at  the  first,  second,  third,  and  eleventh  passage  in 
piglets.  A  standardized  technique  was  used,  but  serial  passage  in  culture 
was  achieved  only  with  the  eleventh  passage  material  from  piglets.   Prior 
to  inoculation  of  AGMK  cells,  virus  suspension  was  incubated  with  trypsin 


23-3 


at  a  final  concentration  of  10  ug/ml  for  1  hour  at  38°C  since  this  procedure 
had  been  shown  to  enhance  the  infectivity  of  some  animal  rotaviruses  for 
tissue  culture.   Inoculated  AGMK  cell  cultures  were  centrifuged  at  low 
speed,  a  procedure  that  also  increases  the  infectivity  of  some  animal 
rotaviruses.   During  14  serial  passages  in  AGMK  cells  the  "Wa"  strain 
attained  a  titer  of  10  "   to  10    fluorescent  focus  (FF)  units  per  ml. 

Two  approaches  were  used  to  establish  the  identity  of  the  AGMK-adapted 
virus  as  a  human  rotavirus.   When  studied  by  polyacrylamide  gel  electrophoresis 
the  viral  RNA  pattern  of  the  tissue  culture  adapted  strain  was  identical  to 
a  previously  recognized  human  rotavirus  RNA  pattern.   It  differed,  however, 
from  the  patterns  of  six  animal  rotaviruses  that  grow  readily  in  cell 
culture.   Bovine,  simian  and  porcine  tissue  culture  adapted  rotaviruses 
were  also  distinct  from  the  culture-adapted  "Wa"  strain  when  tested  by 
neutralization  in  cell  culture.   Thus,  the  tissue-grown  "Wa"  strain  appears 
to  be  type  2  human  rotavirus  and  not  a  tissue  culture-adapted  animal  rotavirus 
contaminant  acquired  in  the  laboratory.   Cultivation  of  type  2  human  rotavirus 
in  tissue  culture  should  facilitate  a  more  detailed  examination  of  its 
properties  and  permit  us  to  manipulate  its  genome  with  the  intent  of 
developing  attenuated  mutants  for  use  in  prevention  of  serious  diarrheal 
disease  of  early  life  (Wyatt,  Kalica,  Kapikian,  Chanock) . 

Development  of  ts  mutants  of  calf  rotavirus  -  As  part  of  our  effort  to 
modify  the  virulence  of  rotavirus,  8  ts^  mutants  of  the  UK  strain  of  calf 
rotavirus  were  developed  using  5  azacytidine  or  nitrosoguanidine  as  mutagen. 
The  8  mutants  had  a  shutoff  temperature  for  plaque  formation  of  38°C  or 
39°C  and  appeared  to  be  relatively  stable  genetically.   It  is  our  intention 
to  use  these  and  subsequent  mutants  to  probe  the  function  of  individual 
genes  of  the  rotavirus  genome.   _ts  mutants  will  also  be  used  as  donors  of 
ts  genes  that  will  be  transferred  to  the  tissue  culture-adapted  human 
rotavirus  via  genetic  reassortment  in  an  attempt  to  attenuate  the  human 
virus.   Bovine  rotavirus  genes  will  probably  prove  to  be  host  restricted 
in  human  cells,  i.e.,  hr,  and  this  may  offer  another  mechanism  by  which 
genes  transferred  from  the  bovine  virus  could  bring  about  attenuation  of 
the  human  virus  (Greenberg) . 

Possible  use  of  animal  rotavirus  to  vaccinate  against  human  rotaviral 
disease  -  Last  year  we  reported  that  in  utero  infection  of  calves  with  a 
bovine  rotavirus  induced  resistance  to  challenge  at  birth  with  type  2 
human  rotavirus.  This  stratagem  was  necessary  since  calves  are  susceptible 
to  the  disease  producing  effects  of  human  rotavirus  only  during  the  first 
few  days  of  life.   Hence  the  need  to  immunize  in  utero  in  order  to  be  able 
to  challenge  with  the  human  virus  shortly  after  birth.   Completion  of  this 
study  during  the  past  year  indicated  that  the  bovine  rotavirus  was  sufficiently 
related  to  the  human  virus  to  be  considered  as  an  immunizing  agent  against 
human  rotaviral  disease.   Safety  testing  of  a  suspension  of  bovine  rotavirus 
is  currently  being  completed.  No  adventitious  agents  have  been  detected. 
Since  this  rotavirus  was  recovered  from  a  domestic  animal  in  the  UK,  additional 
safety  tests  are  being  performed  at  Plum  Island  Animal  Disease  Center, 
USDA,  in  collaboration  with  Dr.  C.A.  Mebus.   Disease  produced  in  calves  by 


23-4 


the  UK  strain  was  similar  to  that  seen  with  the  U.S.  bovine  rotavirus. 
However,  the  UK  isolate  induced  disease  in  gnotobiotic  piglets,  while  the 
U.S.  isolate  did  not.   When  testing  is  complete,  USDA  will  be  asked  for 
permission  to  release  this  strain  for  distribution  and  study  in  man  (Wyatt) . 

Norwalk  and  related  27  nm  viruses  -  A  radioimmunoassay  blocking  (RIABL) 
test  for  detection  of  antibody  to  the  Norwalk  agent  was  employed  to  continue 
our  survey  of  the  importance  of  this  virus  in  epidemic  gastroenteritis. 
Sixty-five  outbreaks  were  studied  serologically  and  20  (31%)  appeared  to  be 
associated  with  the  Norwalk  virus  as  indicated  by  seroresponse  of  affected 
individuals.   This  information  reaffirms  our  previous  conclusion  that  the 
Norwalk  virus  is  a  major  cause  of  epidemic  gastroenteritis.   The  new  Norwalk- 
associated  outbreaks  occurred  in  a  wide  variety  of  situations  including 
grade  schools,  summer  camps,  nursing  homes,  and  cruise  ships.   In  two 
Norwalk-associated  outbreaks,  originally  studied  by  the  CDC,  water  borne 
transmission  appeared  to  be  the  mode  of  spread.   Interestingly,  workers  in 
Australia  associated  Norwalk  virus  etiologically  with  several  very  large 
oyster  borne  outbreaks  of  acute  gastroenteritis;  this  was  confirmed  in  our 
laboratory  (Greenberg,  Kapikian) . 

Antibody  prevalence  to  the  Norwalk  virus  -  A  large  battery  of  age  stratified 
sera  from  adults  from  various  parts  of  the  world  was  studied  for  Norwalk 
antibody.  Prevalence  rates  were  stable  and  similar  in  adults  from  the  U.S., 
Switzerland,  Belgium,  Yugoslavia,  Ecuador,  Bangladesh,  and  Nepal  (approximately 
70%).   On  the  other  hand,  children  from  the  U.S.  and  Yugoslavia  acquired 
antibody  far  more  slowly  than  did  children  in  Ecuador  and  Bangladesh.   In 
Ecuador  and  Bangladesh  an  antibody  prevalence  of  greater  than  70%  was  found 
in  children  0-5  years  of  age.   This  high  prevalence  in  early  childhood 
suggests  that  Norwalk  virus  may  play  an  important  role  in  childhood 
gastroenteritis  in  underdeveloped  countries.   The  low  prevalence  of  antibody 
in  young  children  in  the  U.S.  is  in  keeping  with  our  failure  to  incriminate 
this  agent  as  a  significant  cause  of  infantile  gastroenteritis  (Greenberg) . 

Bacterial  gastroenteritis  -  The  solid-phase  microtiter  radioimmunoassay  for 
the  detection  of  E_^_  coli  heat  labile  enterotoxin  was  modified  to  a  blocking 
test  to  detect  antibodies  to  this  enterotoxin.   A  comparison  of  the  efficiency 
of  the  RIA  blocking  and  the  adrenal  cell  neutralization  tests  for  detecting 
serological  responses  in  individuals  experimentally  infected  with  toxigenic 
E.  coli  revealed  that  both  techniques  were  efficient  at  detecting  a  seroresponse 
in  ill  volunteers.   Nine  of  10  volunteers  who  became  ill  developed  an  RIA 
blocking  antibody  response,  and  7  had  a  serological  response  when  tested  by 
the  adrenal  cell  method.   Proportionately  fewer  non-ill  volunteers  responded 
when  tested  by  either  method.   RIA  blocking  antibody  titers  were  between  5- 
and  10-fold  higher  than  adrenal  cell  titers,  and  preexisting  antibody  was 
detected  somewhat  more  commonly  by  the  RIA  blocking  method.   When  paired 
sera  from  adults  with  naturally  occurring  diarrhea  in  Kenya  and  Bangladesh 
were  studied  it  was  found  that  both  methods  detected  a  significant  rise  in 
antibody  in  approximately  one-half  of  the  patients  shown  to  be  shedding 
toxigenic  E^_  coli.   The  age  prevalence  of  serum  anti-LT  was  investigated  by 


23-5 


screening  random  sera  obtained  at  Children's  Hospital,  Washington,  D.C.  and 
Junior  Village.   By  48  months  of  age,  over  50%  of  the  population  studied 
had  developed  antibody  to  E_;_  coli  LT.   In  addition,  it  was  found  that  over 
70%  of  a  group  of  University  of  Maryland  students  aged  17  through  26  had 
detectable  serum  anti-LT  (Greenberg,  Kapikian) . 

Influenza  Viruses 


ts  Mutants  for  Immunization  -  One  of  the  goals  of  the  LID  influenza  virus 
program  is  to  develop  a  safe,  effective  procedure  for  the  prevention  of 
influenzal  disease.   Although  inactivated  influenza  vaccines,  as  now 
formulated,  are  effective  they  do  not  provide  complete  protection  nor  do 
they  appear  to  retain  their  effectiveness  when  administered  annually.   For 
this  reason  we  have  attempted  to  develop  a  live  attenuated  vaccine  that 
would  mimic  natural  infection  in  its  greater,  broader  and  more  durable 
protective  effect.   We  sought  to  produce  mutant  influenza  viral  genes  which 
would  confer  a  satisfactory  level  of  attenuation  upon  any  recombinant  virus 
into  which  they  were  transferred  by  genetic  reassortment.   The  explosion  of 
new  information  concerning  the  structure  and  function  of  the  influenza 
virus  genome  provides  a  basis  for  deliberate  manipulation  of  its  genes  for 
this  purpose.  Influenza  viruses  bearing  specific,  identifiable,  attenuating 
mutations  represent  the  vaccine  strains  of  the  future  since  the  genetic 
basis  for  attenuation  can  be  monitored  directly  during  all  phases  of 
vaccine  development,  manufacture  and  utilization  in  man. 

Our  approach  has  been  to  develop  t_s  mutations  on  genes  that  code  for  the 
non-surface  proteins  of  the  virus  so  that  the  mutant  genes  could  be  transferred 
into  recombinant  viruses  bearing  the  surface  antigens  of  any  new  epidemic 
or  pandemic  influenza  virus.  It  is  implicit  in  this  approach  that  _ts  mutations 
are  primarily  responsible  for  the  attenuation  of  recombinant  viruses  bearing 
ts  genes.   During  the  past  year  convincing  evidence  was  obtained  that  this 
is  indeed  the  case.   When  a  series  of  ts-l[E]  and  ts-lA2  recombinants, 
previously  evaluated  in  volunteers,  was  genotyped  (i.e.,  the  parental 
origin  of  each  gene  in  the  recombinant  was  determined  by  polyacrylamide  gel 
electrophoresis)  it  was  shown  that  only  the  ts  genes  from  the  _ts  donor 
parent  segregated  with  the  property  of  attenuation.  This  finding  confirmed 
previous  observations  made  in  hamsters  which  indicated  that  restriction  of 
growth  of  _ts  recombinants  in  the  respiratory  tract  was  a  function  of  their 
ts  genes  (Markoff,  Massicot,  Murphy,  Chanock) . 

Initially  a  master  strain  containing  the  ts-l[E]  mutations  was  used  as  a 
donor  of  _ts_  genes.   These  ts  mutations  were  located  on  the  genes  coding  for 
the  P3  and  NP  proteins,  the  former  involved  in  cRNA  synthesis  and  the 
latter  in  vRNA  synthesis.  Recombinants  bearing  both  of  these  ts  genes  were 
satisfactorily  attenuated, immunogenic  and  genetically  stable  when  tested  in 
adults  who  had  detectable  neuraminidase  immunity  but  who  lacked  serum 
antibody  for  the  hemagglutinin  antigen.   However,  ts-l[E]  recombinants 
produced  mild  febrile  reactions  when  given  to  individuals  who  lacked  • 

immunity  to  both  viral  surface  antigens,  i.e.,  hemagglutinin  and  neuraminidase. 
Also  genetically  altered  (_ts  )  virus  was  recovered  from  some  doubly  seronegative 
volunteers  infected  with  a  ts-l[E]  recombinant. 

23-6 


When  immunity  to  both  surface  antigens  is  absent  only  the  degree  of  defectiveness 
of  the  vaccine  virus  determines  attenuation  and  in  this  situation  defectiveness 
must  be  greater  than  that  produced  by  the  ts-l[E]  mutations.   For  this 
reason  we  developed  the  ts-lA2  set  of  mutant  genes  that  specified  a  greater 
degree  of  defectivenss  than  the  ts-l[E]  mutations.   The  A/Udorn/72  ts-lA2 
donor  virus  and  its  recombinant  derivatives  were  more  temperature  sensitive, 
more  stable  genetically,  and  more  restricted  in  growth  in  the  respiratory 
tract  of  hamsters  than  the  ts-l[E]  parent  or  its  recombinants.   Further, 
unlike  the  ts-l[E]  recombinants,  the  ts-lA2  parent  and  its  recombinants 
were  completely  stable  genetically  during  growth  in  hamsters.   The  ts-lA2 
mutations  were  shown  during  the  past  year  to  be  located  on  the  genes  coding 
for  the  P3  and  PI  proteins,  both  of  which  are  involved  in  cRNA  synthesis, 
the  earliest  phase  of  viral  replication  (Massicot,  Murphy,  Markof f ) . 

To  date  the  ts-LA2  mutations  have  been  transferred  to  recombinants  bearing 
the  surface  antigens  of  A/Vic/75  (H3N2) ,  A/Alaska/77  (H3N2)  or  A/HK/77 
(H1N1)  virus.   Transfer  of  the  two  _ts  genes  from  the  A/Udorn/72  ts-LA2 
parent  to  a  total  of  11  such  recombinants  yielded  viruses  with  a  uniform 
set  of  properties:  (a)  37°C  shutoff  temperature  for  plaque  formation,  (b) 
10,000-fold  suppression  of  growth  of  virus  in  the  lungs  of  the  hamster,  (c) 
100-fold  restriction  of  viral  growth  in  the  hamster's  nasal  turbinates  and 
(d)  genetic  stability  after  replication  in  hamsters  (Murphy) . 

Based  on  these  observations  representative  A/Vic/75,  A/Alaska/77  and  A/HK/77 
recombinants  were  evaluated  in  adult  volunteers,  most  (A/Alaska/77)  or  all 
(A/Vic/75  and  A/HK/77)  of  whom  lacked  detectable  immunity  to  both  surface 
antigens.   These  recombinants  were  satisfactorily  attenuated  in  each 
instance.   The  A/Vic/75  and  A/Alaska/77  recombinants  were  effective  in 
stimulating  serum  hemagglutination- inhibiting  (HI)  antibodies,  but  the 
A/HK/77  recombinant  appeared  to  be  poorly  antigenic  when  measured  by  this 
criterion.  However,  when  tested  by  the  more  sensitive,  immuno globulin- 
specific  influenza  A  virus  ELISA  it  was  found  that  most  of  the  A/HK/77 
volunteers  had  developed  a  serologic  response.   In  every  instance  virus 
recovered  from  infected  adult  volunteers  retained  the  _ts  phenotype. 

The  A/Vic/75  ts-lA2  recombinant  was  satisfactorily  attenuated,  antigenic 
and  genetically  stable  when  tested  in  doubly  seronegative  children  by  Drs. 
Peter  Wright  and  David  Karzon  (Vanderbilt) .   In  contrast  the  A/Alaska/77 
ts-lA2  recombinant,  although  it  did  not  cause  illness,  was  unstable  genetically 
when  evaluated  in  a  doubly  seronegative  child  by  Drs.  H.W.  Kim  and  Robert 
Parrott  (Children's  Hospital,  D.C.).   Late  in  the  course  of  infection  this 
child  shed  virus  that  produced  plaques  in  culture  at  39°C  but  not  at  40°C. 
Hence  this  virus  was  less  _ts_  than  its  A/Alaska/77  ts-LA2  parent,  but  more 
ts  than  A/Alaska/77  wild  type  virus  (shutoff  temperature  of  greater  than 
40°C).   These  observations  highlight  one  of  the  central  dilemmas  inherent 
in  the  _ts  mutant  approach.   The  same  may  be  said  for  cold-adapted  (ca) 
recombinants  which  on  occasion  have  lost  (a)  the  ca_  phenotype  during  infection 
of  volunteers  and  (b)  the  _ts_  phenotype  during  experimental  infection  of        : 
hamsters  (Murphy,  Markof f) . 


23-7 


The  mechanism  for  genetic  alteration  leading  to  loss  of  the  t£  phenotype 
was  partially  elucidated  during  the  past  year.   Among  the  reoviruses,  which 
have  a  segmented  RNA  genome,  loss  of  the  ts_  phenotype  is  usually  the  result 
of  extragenic  suppressor  mutation  (Ramig  and  Fields,  1979).   This  can  be 
shown  by  backcrossing  the  _ts  "revertant"  virus  with  wild  type  virus  and 
recovering  _ts  progeny  bearing  the  original  ts_  mutation.   A 
similar  analysis  was  performed  with  the  Alaska/ 7 7-_ts-lA2  £s   isolate  by 
mating  it  with  Alaska/77  wild  type  virus.   Twenty-two  percent  of  progeny 
virus  from  this  mating  was  t_s  and  each  _ts_  clone  possessed  the  P3  _ts_  lesion 
present  in  the  ts-lA2  parent.   However,  the  ts_  segregants  possessing  the  P3 
ts  gene  were  less  temperature  sensitive  than  P3  _ts  segregants  derived  from 
mating  Udorn/72-ts-lA2  with  wild  type  virus.   This  indicates  that  two  types 
of  genetic  alteration  occurred  in  the  Alaska/ 77- ts-lA2  virus  during  replication 
in  a  seronegative  child.   One  type  involved  a  suppressor  mutation  in  another 
gene  (i.e.,  not  the  P3  gene)  that  partially  corrected  the  ts_  phenotype  of 
the  ts-lA2  virus.   The  second  type  was  an  alteration  affecting  the  P3  and 
PI  genes;  this  type  of  change  could  result  from  reversion  of  one  of  several 
distinct  mutations  or  it  could  represent  intragenic  suppression.   A  mutation 
probably  occurred  on  the  P3  gene  of  the  Alaska/ 7 7-_ts_-lA2  ts_  isolate  because 
this  segment  migrated  more  slowly  on  polyacrylamide  gel  than  the  corresponding 
RNA  segment  of  the  parent  Alaska/ 77- ts-LA2  recombinant.   This  observation 
favors  the  possibility  that  intragenic  suppression  was  responsible  for 
decreased  temperature  sensitivity  of  the  P3  ts_  segregants  derived  from  the 
ts   isolate  (Murphy) . 

The  cumulative  experience  indicates  that  acquisition  of  the  two  ts-lA2 
genes  by  H3N2  or  H1N1  subtype  virus  was  regularly  associated  with  a  level 
of  attenuation  satisfactory  for  individuals  lacking  antibody  to  both 
surface  antigens  of  the  virus.   However,  the  experience  with  the  A/Alaska/ 77 
ts-lA2  recombinant  indicated  that  genetic  stability  remains  a  formidable 
problem  that  must  be  resolved.      It  may  be  unrealistic  to  insist  that 
live  attenuated  vaccine  viruses  possess  _ts,  ca  or  host  restriction  (hr) 
mutations  that  are  completely  stable.   The  high  mutation  frequency  of 
single  stranded  RNA  viruses  may  make  this  a  difficult,  if  impossible  goal 
to  achieve.  For  example,  the  spontaneous  frequency  of  t_s  mutation  for 
influenza  A  and  VSV  viruses  has  been  observed  to  range  from  1-2%.   Perhaps 
we  should  focus  our  attention  upon  the  consequences  of  genetic  alteration 
that  occurs  in  attenuated  mutants  during  replication  in  vivo  and  determine 
whether  such  alteration  results  in  increased  virulence  and  whether  altered 
vaccine  virus  poses  a  threat  to  contacts  of  vaccinees.  Alteration  or  loss 
of  laboratory  markers  of  attenuation,  i.e.,  the  ts  or  ca  phenotype,  may  not 
necessarily  disqualify  vaccine  viruses  that  undergo  such  modification, 
especially  if  it  results  from  "suppression"  rather  than  true  reversion.   If 
complete  genetic  stability  were  an  absolute  requirement  for  vaccine  virus, 
live  poliovirus  vaccine  would  not  be  available  for  the  control  of  poliomyelitis. 
The  attenuated  poliovirus  vaccine  strains  frequently  undergo  a  modification 
in  ^s_  and  other  laboratory  markers  of  attenuation  during  viral  replication 
±n  man  without  causing  disease  in  vaccinees  except  on  very  rare  occasion. 
The  spectrum  of  change   in  markers  seen  in  polioviruses  recovered  from 
vaccinees  suggests  that  "suppression"  probably  plays  a  major  role  in  these 
modifications . 

23-8 


Cold-adapted  (ca)  Recombinants  for  Immunization  -  Two  ca  recombinants 
developed  by  Maassab  (Univ.  Michigan)  were  evaluated  in  doubly  seronegative 
adult  volunteers.   These  recombinants  both  possessed  the  maximum  number  of 
transferrable  za_  parental  genes  (i.e.,  PI,  P2,  P3,  NP,  M  and  NS)  yet  the 
A/Alaska/77  cold  recombinant  (CR-29)  had  a  39°C  shutoff  temperature,  while 
A/HK/77  CR35  was  more  temperature  sensitive  (37°C  shutoff).  Nonetheless 
both  CR-29  and  CR-35  were  satisfactorily  attenuated  and  antigenic  in  adults. 
Furthermore,  these  recombinants  retained  the  ts_  phenotype  during  replication 
in  volunteers  although  some  isolates  of  the  CR-35  exhibited  some  genetic 
drift  (37°C  shutoff  — >  39°C  shutoff  temperature)  (Murphy). 

Characterization  of  New  ts  Recombinants  -  Genetic  studies  of  136  new  _ts_ 
mutants  of  A/Udorn/72  virus  were  completed  this  year.   Several  unusual 
phenomena  were  observed  during  the  characterization  of  these  mutants. 
First,  56  of  the  _ts_  mutants  exhibited  host  dependent  temperature  sensitivity, 
i.e.,  they  were  t_s  when  tested  in  one  host  cell  but  not  _ts  when  evaluated 
in  another  host  cell  system.   This  type  of  host  dependent  mutation  was 
widely  distributed  throughout  the  viral  genome  and  did  not  exhibit  a 
predelection  for  a  particular  gene.   Second,  intracistronic  complementation, 
without  recombination,  occurred  with  _ts_  mutations  that  affected  the  genes 
coding  for  the  PI,  P2,  NP  and  NS  proteins. 

The  136  mutants  were  assigned  to  8  distinct,  non-overlapping  complementation 
recombination  groups.   This  number  of  groups  is  in  agreement  with  the 
number  of  influenza  A  virus  RNA  gene  segments.   This  large  suite  of  highly 
characterized  _ts_  mutants,  83  of  which  are  single  mutants,  should  prove 
useful  in  future  studies  of  gene  function  and  expression  particularly  in 
experiments  in  which  rescue  of  cloned  influenza  DNA  sequences  from  eukaryotic 
cells  will  be  attempted  (K.  Shimizu) . 

Use  of  Recombinant  DNA  Techniques  to  Study  Influenza  A  Virus  -  In  view  of 
the  medical  importance  of  influenza  viruses  there  is  a  need  to  broaden  our 
knowledge  of  the  genetics  of  these  viruses  in  order  to  design  ways  to  more 
effectively  prevent  pandemics  and  epidemics.   One  approach  is  to  use 
recombinant  DNA  technology  that  has  been  well-developed  and  has  proven 
valuable  in  elucidating  gene  organization  and  expression  in  other  appropriate 
host  systems.   Our  general  plan  for  this  approach  includes  two  phases.   The 
first  phase  involves  cloning  each  of  the  eight  influenza  gene  segments 
using  the  E_;_  coli  K12-plasmid  system   (approved  MUA  #91)  .   The 
second  phase  envisions  the  use  of  purified  clones  of  influenza  DNA  sequences 
to  examine  viral  gene  expression  in  animal  cell  culture  and  to  produce  viral 
RNA  (vRNA)  in  eukaryotic  cells.   To  carry  out  this  second  phase  we  propose 
to  utilize  defective  SV40  (lacking  the  late  region  of  the  genome  that  codes 
for  capsid  proteins)  for  construction  of  inf luenza-SV40  hybrid  DNA  molecules 
(submitted  MUA  #110).   Introduction  of  the  appropriate  influenza-SV40 
hybrid  DNA  molecules  into  eukaryotic  cells  may  lead  to  transcription  of 
cRNA  or  replication  of  vRNA  from  influenza  viral  DNA  depending  upon  its 
orientation  of  insertion.   Our  goal  is  to  devise  procedures  which  would 
permit  conversion  of  influenza  DNA  back  to  an  influenza  RNA  negative  strand 


23-9 


(i.e.,  vRNA)  and  eventually  transfer  such  RNA  into  an  influenza  virus.   In 
this  manner,  stable,  site-specific  mutations,  such  as  deletions,  induced  in 
the  cloned  DNA  might  be  transferred  back  to  the  influenza  virus.   Thus,  it 
may  be  possible  to  develop  stable  mutants  for  experimental  study  and  for 
use  in  immunoprophylaxis. 

A  procedure  was  devised  for  producing  double-stranded  DNA  sequences  corresponding 
to  each  of  the  influenza  virus  RNA  segments.   Negative  and  positive  strands 
of  influenza  RNA  segments  were  copied  separately  into  DNA  using  the  reverse 
transcriptase  of  avian  myeloblastosis  virus.   Influenza  virion  RNA  segments, 
which  are  negative  strands,  were  reverse- transcribed  into  their  complementary 
DNA  copies  in  the  presence  of  a  specific  priming  DNA  oligomer.  The  primer 
was  complementary  to  the  conserved  3 '-end  sequence  of  the  virion  RNA  segments 
and  the  DNA  products  of  the  reverse  transcriptase  enzyme  appeared  to  represent 
full-length  genomic  copies.  Similarly,  poly  A-containing  cytoplasmic  RNA's 
(positive  strands)  isolated  from  the  virus- infected  cells  were  transcribed 
into  DNA  sequences  in  a  reaction  mixture  in  which  oligo-(dT)  primer  was 
added.   The  single-stranded  DNA  molecules  from  both  preparations  were 
annealed  to  generate  double-stranded  DNA  segments  which  were  subsequently 
isolated  for  cloning  in  plasmid  PBR322  of  Ej_  coli  K-12. 

The  ±n   vitro  synthesized  double  stranded  influenza  DNA  segments  were  then 
inserted  into  plasmid  PBR322  at  the  specific  Pst  I  site  using  the  dG-dC 
tailing  technique.   The  hybrid  DNA  molecules  were  used  to  transform  recipient 
E.  coli  and  transf ormants  containing  influenza  gene  sequences  were  identified 
by  hybridization.   In  this  manner,  we  have  so  far  obtained  putative  full- 
length  influenza  virus  DNA  segments  corresponding  to  genes  coding  for  non- 
structural protein  (gene  VIII) ,  matrix  protein  (gene  VII) ,  neuraminidase 
(gene  VI) ,  and  hemagglutinin  (gene  IV) .   Cloning  of  other  genes  from  wild 
type  influenza  A/Udorn/72  (H3N2)  virus  is  now  underway.   Cloned  DNA  will  be 
analyzed  by  restriction  enzyme  cleavage  and  nucleotide  sequencing.  By 
comparing  such  information  for  corresponding  genes  derived  from  influenza 
viruses  belonging  to  different  subtypes  and  different  strains  within  a 
subtype  it  should  be  possible  to  define  the  genetic  basis  for  variability 
of  the  hemagglutinin  and  neuraminidase  antigens  as  well  as  some  of  the 
internal  proteins  (Lai,  Markof f ) . 

Respiratory  Syncytial  Virus 

RS  virus  continues  to  be  the  major  etiologic  agent  of  bronchiolitis  and 
pneumonia  of  early  life,  and  the  need  for  effective  immunoprophylaxis  was 
again  emphasized  last  year  by  the  occurrence  of  unusually  large  outbreaks 
of  serious  RS  virus  lower  respiratory  tract  disease  throughout  the  world. 

Studies  in  Inbred  Mice  -  Five  animal  models  of  RS  virus  infection  have  been 
studied  by  us,  including  the  chimpanzee,  cebus  monkey,  owl  monkey,  ferret 
and  cotton  rat.   None  of  these  species,  however,  is  inbred,  thus  precluding 
genetic  manipulation  and  certain  types  of  immunologic  study.  Furthermore,       •, 
few,  if  any  specific  immunologic  reagents  are  available  to  allow  study  of 
certain  aspects  of  RS  virus  pathogenesis  and  virus-host  immunologic  interaction. 


23-10 


In  an  effort  to  develop  a  model  for  experimental  RS  virus  infection  in  an 
inbred  species,  we  examined  twenty  strains  of  mice.  Intranasal  inoculation 
of  RS  virus  in  infant  mice  produced  infection  in  each  strain  examined. 
However,  there  was  wide  variation  among  the  inbred  strains  in  the  amount 
of  virus  recovered  from  the  nose  and  lungs.   The  most  resistant  strain, 
CBA/CaHN,  yielded  only  one-hundredth  the  quantity  of  virus  recovered  from 
the  nose  and  lungs  of  the  most  permissive  strain,  DBA/2N.   Ordering  of 
geometric  mean  nasal  and  pulmonary  titers  from  the  twenty  strains  demonstrated 
a  pattern  of  gradual,  incremental  increase  from  relatively  resistant  to 
relatively  permissive  strains.   If  level  of  viral  replication  were  controlled 
by  a  single  gene  with  few  alleles,  one  would  not  expect  to  observe  such  a 
shallow  linear  array  of  titers.   This  suggests  that  response  to  RS  virus 
infection  is  determined  by  a  combination  of  genes,  or  perhaps  a  single  gene 
with  multiple  alleles.  Since  there  was  no  overlap  between  the  virus  titers 
observed  for  the  strains  that  exhibited  the  lowest  and  highest  levels  of 
viral  growth  it  should  be  possible  to  analyze  the  genetic  control  of  viral 
replication  using  the  appropriate  crossbreeding  techniques.   In  addition  to 
the  opportunity  to  study  genetic  control  of  viral  replication,  the  availability 
of  an  inbred  animal  model  for  RS  virus  infection  offers  possibilities  for 
studies  which  were  not  feasible  previously.   For  instance,  in  vivo 
experiments  using  adoptive  transfer  of  immunologic  components,  including 
immune  cells,  can  now  be  performed.   Another  advantage  of  the  mouse  model, 
is  the  availability  of  a  large  number  of   specific  immunological  reagents 
that  will  permit  us  to  examine  individual  portions  of  the  immune  system  for 
their  role  in  recovery  from  infection,  and  resistance  to  infection  and 
possible  participation  in  immunopathology  (Prince,  Suf f in) . 

Further  in  vivo  Evaluation  of  ts-2  -  Two  seronegative  chimpanzees  were 
inoculated  with  a  safety-tested  ts-2  vaccine  suspension  prepared  by  Flow 
Laboratories.   This  vaccine  is  currently  being  evaluated  in  children.   One 
animal  was  successfully  infected  in  two  attempts  but  did  not  develop  any 
signs  of  illness,  despite  shedding  a  moderate  amount  of  virus  (10  *   pfu/ml 
of  swab  fluid)  from  the  upper  respiratory  tract.  The  unavailability  of 
further  seronegative  animals  precluded  additional  study  (Prince,  Suf fin) . 

In  vivo  Evaluation  of  ts-1  NG-1  and  ts-1  NG-16  Mutants  -  The  first  ts 
mutant  of  RS  virus  to  be  tested  in  man,  ts-1,  appeared  promising  when 
evaluated  in  adult  volunteers.   However,  tests  in  seronegative  infants 
showed  the  virus  to  possess  a  low  level  of  residual  virulence,  and  to 
exhibit  some  genetic  instability.   In  an  attempt  to  further  attenuate  ts-1, 
nitrosoguanidine  (NG)  was  used  to  remutagenize  the  ts-1  mutant.   Two  mutants, 
ts-1  NG-1  and  ts-1  NG-16,  were  recovered  from  the  progeny  of  NG  treated  ts- 
1  and  were  shown  to  exhibit  greater  temperature-sensitivity  and  genetic 
stability  than  ts-1.   These  two  mutants  were  evaluated  in  the  owl  monkey 
because  it  is  the  only  readily  available  experimental  animal  which  develops 
clinically  evident  disease  when  infected  with  wild  type  RS  virus.   Neither 
ts-1  NG-1  nor  ts-1  NG-16  differed  significantly  from  wild  type  virus  in 

either  duration  of  infection  or  peak  virus  titer.  However,  the  time  of 
onset  of  virus  shedding  and  the  time  of  peak  titer  of  both  mutants  were 
significantly  delayed  compared  to  wild  type  virus  suggesting  that  both 

23-11 


mutants  were,  nonetheless,  functionally  defective.   Finally,  both  mutants 
were  significantly  attenuated  compared  to  wild  type  virus.  These  observations, 
in  conjunction  with  previous  studies  showing  ts-1  NG-1  and  ts-1  NG-16  to  be 
more  defective  and  more  stable  genetically  than  the  parental  ts-1  mutant, 
suggest  that  they  are  potential  candidates  for  use  in  a  live  vaccine.   The 
fact  that  the  two  mutants  did  not  differ  significantly  from  each  other  in 
any  of  the  observed  parameters  suggests  that  both  should  be  subjected  to 
additional  in  vivo  testing  in  primates  and,  ultimately,  man  (Prince,  Suf f in) . 

Duration  of  Immunity  Following  Intranasal  Infection  of  Cotton  Rats  -  Man 
does  not  develop  lasting  immunity  to  RS  virus  infection.   During  the  past 
18  months  the  transient  nature  of  RS  virus  immunity  was  investigated  in 
cotton  rats.   Immunity  to  nasal  infection  was  temporary;  animals  were 
susceptible  to  nasal  reinfection  beginning  8  months  after  primary  infection. 
The  level  of  virus  replication  in  the  nose  increased  with  time  after  initial 
infection;  at  18  months  post-infection  animals  were  completely  permissive. 
In  contrast,  none  of  the  animals  examined  throughout  the  course  of  the  18 
month  experiment  were  susceptible  to  reinfection  of  the  lungs.   If  a  similar 
situation  obtains  in  man  vaccination  may  offer  greater  promise  of  protection 
than  had  previously  been  considered  possible  (Prince,  Suf fin) . 

Intramuscular  Immunization  with  Live  RS  Virus  -  Recently,  Buynak.  reported 
that  parenteral  administration  of  wild-type  RS  virus,  grown  in  human  diploid 
cells,  induced  the  development  of  serum  neutralizing  antibody  in  young 
children  without  causing  any  objective  signs  or  symptoms  of  disease.   These 
exciting  findings  clearly  required  amplification  and  extension,  because 
evidence  that  virus  replicated  following  intramuscular  (IM)  inoculation  was 
not  provided  in  the  original  report,  nor  was  the  possible  immunosuppressive 
effect  of  maternally  derived  passive  immunity  evaluated.   Following  IM 
inoculation  of  10  "   to  10  PFU  live  virus  was  not  recovered  from  the  local 
site  and  was  never  detected  in  the  nose  or  lungs  although  extensive  attempts 
were  made  over  a  prolonged  period.  Furthermore,  attempts  to  detect  viral 
antigens  at  the  site  of  IM  inoculation  were  unsuccessful.   However,  inactivation 
of  infectivity  of  three  strains  of  virus  by  the  minimal  UV  dose  markedly 
reduced  or  completely  ablated  their  antigencitity  and  protective  efficacy. 
Although  this  observation  does  not  constitute  unequivocal  evidence  for  the 
occurrence  of  viral  replication  after  IM  inoculation,  it  suggests  that 
limited  replication,  perhaps  restricted  to  an  abortive  cycle,  was  responsible 
for  stimulation  of  immunity  by  the  small  quantities  of  virus  employed. 

The  possibility  that  passive  immunity  might  interfere  with  the  effectiveness 
of  parenteral  immunization  with  live  RS  virus  was  examined  because  immuno- 
suppression could  pose  a  serious  obstacle  to  this  approach.   Thus,  the 
greatest  need  for  an  RS  virus  vaccine  is  in  the  first  few  months  of  life,  a 
time  when  infants  possess  a  moderately  high  level  of  maternally  derived  RS 
virus  serum  antibody.   We  attempted  to  simulate  these  conditions  by  administering 
live  RS  virus  IM  to  weanling  rats  possessing  passive  serum  antibody  derived 
from  their  immune  mother.   In  this  situation  an  immunosuppressive  effect  of 


23-12 


passive  immunity  was  observed.   Only  50%  of  inoculated  weanling  rats  were 
rendered  resistant  to  subsequent  IN  challenge  with  RS  virus.   This  suggests 
that  parenteral  immunization  with  live  virus  may  not  be  effective  in  protecting 
human  infants  against  RS  virus  during  their  period  of  greatest  vulnerability 
to  serious  RS  virus  disease,  i.e.,  the  first  3  months  of  life.   If  this  be 
the  case,  the  usefulness  of  live  IM  virus  vaccine  would  be  limited  to  individuals 
over  6  months  of  age  who  had  escaped  natural  infection  and  who  had  lost 
most  or  all  of  their  passive  maternally  derived  serum  antibody.   The  outlook 
for  IM  vaccination  of  individuals  who  had  been  infected  previously  is  also 
not  encouraging  since  Buynak's  study  indicated  that  seropositive  children 
respond  poorly  to  vaccine  (Prince,  Suffin,  Chanock) . 

Immunopathoarcheology  -  We  have  been  engaged  in  the  development  of  a  new 
method  of  enzyme-linked  immunohistologic  diagnostic  technology.   Because  of 
the  widely  recognized  difficulties  in  the  evaluation  of  peroxidase  staining 
due  to  endogenous  peroxidase  activity  in  tissues  and  inducibility  of 
endogeneous  peroxidase-like  activity  in  inflammatory  and  neoplastic  states 
a  method  of  enzyme-immunohistochemistry  was  developed  which  would  be 
unaffected  by  inflammatory  and  neoplastic  processes.  Our  search  for  an 
enzymatic  system  not  present  in  mammalian  tissues  led  us  to  the  consideration 
of  glucose  oxidase,  an  enzyme  derived  from  non-mammalian  sources,  as  a 
possible  candidate  for  this  purpose.   Earlier  work  had  shown  that  by  modification 
of  the  reaction  product  a  stable  preparation  could  be  obtaind  suitable  for 
immunohistochemistry.  Antisera  were  prepared  against  this  enzyme  in  rabbits, 
guinea  pigs  and  goats.   These  antisera  were  converted  into  a  soluble  enzyme 
antibody  complex  and  used  in  a  manner  similar  to  that  described  by  Sternberger. 
Currently  we  are  determining  the  critical  variables  for  respiratory  syncytial 
virus  antigen  preservation  during  the  fixation  and  embedment  processes. 
Subsequently  an  immunopathoarcheologic  survey  for  RS  virus  and  other  viral 
pathogens  will  be  performed  using  autopsy  material  collected  over  the  past 
20-30  years  (Suffin) . 

Mycoplasmas 

Efforts  to  assess  the  significance  of  pathogenic  spiroplasmas  are  centered 

around  the  biological  and  serological  characterization  of  organisms  within 

this  group,  on  development  of  appropriate  models  to  explore  pathogenicity 

and  host  response,  and  on  evaluation  of  various  techniques  to  determine  the 

possible  role  of  these  agents  in  human  disease.   Experimental  pathogenicity 

studies  on  chick  embryos  and  one-day-old  suckling  rats  with  a  number  of 

spiroplasmas  from  diverse  plant  and  insect  origin  have  suggested  a  number 

of  unique  virulence  markers  in  these  organisms.   Several  tick-derived 

spiroplasmas  (the  SMCA  group)  are  highly  pathogenic  for  the  chick  embryo 

when  as  few  as  1-10  spiroplasmas  are  administered,  and  most  strains  retain 

this  virulence  when  passaged  repeatedly  on  artificial  medium.   Spiroplasmas 

in  this  group  demonstrated  an  inverse  relationship  between  the  mortality 

rate  (LD,.)  and  the  occurrence  of  cataracts  in  suckling  rats.   Strains 

which  are  highly  virulent  by  intracerebral  challenge  to  rats  (when  given       . 

10  organisms  or  less)  do  not  induce  cataracts  in  survivors.   At  least  one 


23-13 


strain  (TP-2)  in  this  group,  exhibited  a  reversal  in  virulence  pattern 
after  repeated  passage  on  artificial  medium.   While  this  organism  showed  a 
decline  in  pathogenicity  for  the  chick  embryo,  there  was  enhanced  virulence 
for  the  suckling  rat.   Additional  studies  of  other  spiroplasmas  from  insects 
and  from  free-living  origins  snowed  these  organisms  to  have  increasing 
pathogenicity  for  the  chick  embryo  following  repeated  passage  in  the 
laboratory. 

The  new  serological  procedures  developed  in  the  Section  for  assessing  the 
interrelationships  of  cultivated  spiroplasmas  has  been  applied  with  notable 
success.   These  techniques  possess  the  necessary  specificity  and  sensitivity 
to  provide,  for  the  first  time,  some  understanding  of  those  spiroplasmas 
possessing  distinct  antigenic  determinants.  A  combined  def ormation/metabolism- 
inhibition  test  system,  utilizing  the  activity  of  specific  antiserum  to 
deform  helical  organisms  or  inhibit  metabolism  of  the  organism,  has  been 
adapted  to  the  microtiter  system.   The  analysis  of  spiroplasmas  has  shown 
that  the  tick-derived  SMCA  group  is  serologically  distinct  from  all  other 
known  spiroplasmas.   In  addition,  two  groups  of  spiroplasmas  recovered  from 
flowers,  and  the  sex-ratio  spiroplasmas  from  Drosophila  species  appear  to 
be  serologically  distinct.   Ttie  fifth  serological  group  consists  of  a  large 
number  of  plant  and  insect  strains  identical  to,  or  partially  related  to, 
Spiroplasma  citri.   Tne  results  of  this  analysis,  and  the  test  itself, 
should  provide  a  basis  for  continued  sero-epidemiological  study  of  spiroplasma 
antibody  in  man  and  other  vertebrates.   (Tully) 


23-14 


Honors  and  Awards 
Robert  M.  Chanock 
Director,  International  Reference  Laboratory  for  Respiratory  Viruses  other 

than  Influenza,  World  Health  Organization,  1973  -  present 
Director,  International  Reference  Laboratory  for  Mycoplasmas,  World  Health 

Organization,  1973  -  present. 
Editorial  Board,  Journal  of  Infectious  Diseases 
Associate  Editor,  American  Journal  of  Epidemiology 
Member,  Advisory  Board  Archives  of  Virology 
Invited  lecturer,  Johns  Hopkins  University,  "Epidemiology  and  Prevention 

of  Influenza",  November  1,  1978 
Invited  lecturer,  "Molecular  Genetics  of  Viruses"  course,  FAES  Graduate 

School,  NIH,  November  20,  1978 

Invited  participant,  Royal  Society  Symposium  on  Influenza  Virus  Genetic, 

London,  England,  February  21  &  22,  1979 
Invited  speaker,  Advances  in  Clinical  Medicine,  1979,  sponsored  by  the 

American  Physicians  Fellowship  and  the  Foundation  for  Advanced  Education 

in  the  Sciences,  Bethesda,  Maryland,  March  29-30,  1979 
Invited  consultant,  MRC  Committee  on  Viral  Vaccines,  London,  England. 

Invited  to  discuss  future  prospects  for  immunization  against 

RSV  disease,  February,  1979 
Chairman,  Section  on  Medical  Microbiology  and  Immunology,  National  Academy 

of  Sciences 
Appointed  member  Nominating  Committee,  National  Academy  of  Sciences,  1979 

Robert  H.  Purcell 

Invited  Participant  and  Co-chairman,  US-Japan,  Program  Symposium  on 

Viral  Hepatitis,  Tokyo,  Japan,  July  17-19,  1978. 
Invited  Participant,  Emposium  on  Viral  Hepatitis,  Taipai,  Taiwan, 

July  24-26,  1978. 
Invited  Speaker,  Annual  Meeting  of  The  Infectious  Diseases  Society 

of  America,  Atlanta,  Georgia,  October  5-6,  1978. 
Invited  Speaker,  16th  Annual  Briefing  on  "New  Horizons  of  Science", 

Council  for  the  Advancement  of  Science  Writing,  Gatlinburg,  Tenn., 

November  13-17,  1978. 
Visiting  professor,  Michigan  State  University  School  of  Medicine, 

East  Lansing,  Michigan,  January  30,  1979. 
Invited  Speaker,  "Update-Hepatitis",  Sponsored  by  The  New  York  Academy 

of  Gastroenterology,  New  York  City,  February  26,  1979. 
Invited  Consultant,  World  Health  Organization  Consultation  on  Hepatitis 

B  Vaccines,  Geneva,  March  12-14,  1979. 
Invited  Participant,  International  Symposium  on  Viral  Hepatitis, 

Munich,  April  5-6,  1979. 
Invited  Speaker,  Symposium  on  Frontiers  of  Science  and  the  Liver, 

Mt.  Sinai  Medical  Center,  New  York  City,  June  6,  1979. 


23-15 


Albert  Z .  Kapikian 

Invited  member,  International  Coronavirus  Study  Group,  1972 — 

Invited  member,  Study  Group  on  Reoviridae  of  International  Committee  for 

the  Toxonomy  of  Viruses,  1976 — 
Invited  member,  Rotavirus  subgroup  of  the  WHO/FAO  Comparative  Virology 

Reoviridae  Group,  197  7 — 
Invited  to  serve  as  World  Health  Organization  Temporary  Advisor  for 

Consultations  on  Rapid  Laboratory  Viral  Diagnosis  at  Institute  of 

Medical  Microbiology,  University  of  Gothenberg,  Gothenberg,  Sweden, 

Aug.  17,  18,  1978. 
Invited  to  serve  as  one  of  the  two  vice-chairmen  of  Workshop  "Aspects  of 

Gastroenteritis  Viruses"  at  the  Fourth  International  Congress  for  Virology, 

The  Hague,  The  Netherlands.  Aug.  30  -  Sept.  6,  1978.  Workshop  on  Aug.  30, 

1978. 
Invited  speaker  at  U.S.  and  Japanese  Panels  on  Virus  Diseases  of  the  U.S. -Japan 

Cooperative  Medical  Science  Program.  Meeting  at  Osaka,  Japan,  Oct.  3-5, 

1978. 
Invited  to  become  panel  member  of  the  United  States  Panel  on  Viral  Diseases  of 

U.S. -Japan  Cooperative  Medical  Science  Program,  1978- 
Invited  speaker  at  Children's  Hospital,  Washington,  D.C.,  Infectious  Diseases 

Conference,  Nov.  3,  1978. 
Invited  to  participate  in  a  Workshop  on  Infectious  Agents  in  Inflammatory  Bowel 

Diseases  at  Tarrytown  Conference  Center,  Tarrytown,  N.Y. ,  Nov.  17-19,  1978. 

sponsored  by  National  Foundation  for  Ileitis  and  Colitis,  Inc.  and  the 

American  Gastroenterological  Association.  Selected  as  one  of  the  members  of 

the  panel  on  cultivation  of  infectious  agents  for  session  III  on  Nov.  18, 

1978. 
Invited  speaker  at  Preventive  Medicine  Symposium  on  Recent  Developments  in 

Infectious  Diarrheas  at  33rd  Annual  Meeting  of  The  Society  of  Medical 

Consultants  to  the  Armed  Forces,  Washington,  D.C. 
Invited  to  present  a  lecture  of  Virology  '79  postgraduate  course  presented  by 

Institute  for  Medical  Research,  Copewood,  New  Jersey.   Presented  lecture  on 

Rotaviruses  and  the  Norwalk  Group  of  Agents  on  Jan.  11,  1979. 
Invited  to  make  brief  presentation  on  viral  diarrhea  to  Mr.  Joseph  Calif ano,  Jr. 

Secretary  of  Health,  Education  and  Welfare  on  March  8,  1979  in  Dr.  Donald 

Frederickson' s  office. 
Invited  to  attend  and  to  prepare  a  working  paper  on  "Epidemiology  of  diarrheas 

caused  by  parvovirus-like  agents"  for  the  World  Health  Organization 

Scientific  working  Sub-Group  Meeting  on  Rotavirus  and  Other  Viral  Diarrheas 

at  the  WHO  Regional  Office  for  the  Americas,  Washington,  D.C,  March  27-28, 

19/9. 
Elected  chairman  of  WHO  Scientific  working  Sub-Group  Meeting  on  Rotavirus  and 

Other  Viral  Diseases  at  WHO  Regional  Office  for  the  Americas,  Washington, 

D.C,  March  27-28,  1979. 
Invited  co-convener  of  symposium  Epidemiology  of  Enteric  Viruses  on  May  9,  1979 

at  American  Society  for  Microbiology  Annual  Meeting  -  U.S. -Japan  Inter- 
society  Microbiology  Congress,  Honolulu,  Hawaii.  ; 
Invited  to  present  lecture  on  Viral  Diarrhea  of  Tropical  Medicine  Course  on  July 

20,  1979  at  Walter  Reed  Army  Institute  of  Research,  Washington,  D.C 

23-16 


Joseph  G.  Tully 

Past-Chairman  and  Member,  Board  of  Director,  International  Organization  for 

Mycoplasmology  (IOM) 
Past-Chairman  and  Member  of  Board,  IOM  International  Research  Program  on 

Comparative  Mycoplasmology  (formerly  WHO/FAO  Board) 
Chairman,  Subcommittee  on  Taxonomy  of  Mycoplasmas,  American  Society  for 

Microbiology 
Course  Director,  International  Organization  for  Mycoplasmology  Course  on 

Mycoplasma  Techniques,  Bordeaux,  France,  September  3-21,  1979 
Co-Director,  Workshop  on  Detection  of  Mycoplasmas,  Cell  Science  Center, 

Lake  Placid,  N.Y. ,  November  16-20,  1978  and  August  20-23,  1979 
Member,  International  Subcommittee  on  the  Taxonomy  of  Mollicutes 
Invited  speaker,  Perspectives  in  Biology  and  Science,  Iowa  State  University, 

Ames,  Iowa,  May  2,  1979 
Invited  speaker,  Department  of  Microbiology,  University  of  Bordeaux 

Medical  School,  Bordeaux,  France  September  14,  1978 
Invited  speaker,  Bergey's  Manual  Roundtable,  American  Society  for  Microbiology, 

Los  Angeles,  Ca.,  May  5,  1979 
Invited  speaker,  National  Animal  Disease  Laboratory,  Ames,  Iowa,  May  3,  1979 
Invited  speaker,  Symposium  on  Mycoplasma  Infection  of  Cell  Cultures,  Tissue 

Culture  Association,  Seattle,  Washington,  June  12,  1979 
Invited  speaker,  Conference  on  Lethal  Yellowing  Disease  of  Palms,  Fort 

Lauderdale,  Florida,  August  13,  1979. 
Invited  speaker,  Conference  on  Current  Status  of  the  Agent  of  Contagious 

Caprine  Pleuropneumonia,  USDA,  Hyattsville,  Md.  July  12,  1979 

Brian  R.  Murphy 

Invited  speaker,  Royal  Society  Symposium  on  Influenza  Virus  Genetics, 
London,  England,  February  21  &  22,  1979 

Stephen  M.  Feinstone 

Invited  Participant,  American  Society  for  Microbiology  Symposium 

on  Epidemiology  of  Enteric  Viral  Infections.   May  1979. 
Invited  Participant,  Society  for  Epidemiologic  Research  Symposium 

on  Viral  Hepatitis,  June  1979. 
Invited  Participant  in  a  Symposium  of  the  American  Association  for 

Tissue  Banks,  May  1979. 
Invited  Contributor  to  the  Proceedings  of  a  Symposium  on  Frontiers 

of  Science  and  the  Liver  dedicated  to  Prof.  Hans  Popper 

Harry  B.  Greenberg 

Elected  member,  Infectious  Diseases  Society  of  America,  1979 
Invited  participant,  American  Society  for  Microbiology  meeting,  Los 
Angeles,  Ca.  and  Honolulu,  Hawaii,  May,  1979 


23-17 


Richard  G.  Wyatt 

Invited  presentation  on  Viral  Diarrhea  to  pediatric  staff,  housestaff, 

and  medical  students  at  Walter  Reed  Army  Medical  Center,  Washington. 
D.C. ,  February  12,  1979 

Invited  presentation  on  Viral  Diarrhea  to  students  participating  in  the 
Minority  Biomedical  Support  Program,  NIH,  February  28,  1979 

Consultant  to  Navajo  Community  College,  Tsaile,  Navajo  Nation  (Arizona) 

on  Minority  Biomedical  Support  Project:   "Gastroenteritis  in  Newborn 
Lambs  and  Young  Children  on  the  Navajo  Reservation",  June  19-22,  1979 


23-18 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANG 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  AI  00021-10  LID 


PERIOD  COVERED 


Octets   ig    1Q7S   t-o   September   30,    1979 


TITLE   OFPROJECT    C80   characters    or   less) 

THE  ETIOLOGY  OF  ACUTE  INFECTIOUS  NONBACTERIAL  GASTROENTERITIS 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:   Dr.  Albert  Z.  Kapikian 

Dr.  Harry  B.  Greenberg 

Dr.  Anthony  R.  Kalica 

Dr.  Richard  G.  Wyatt 

Dr.  Robert  H.  Yolken 

Dr.  Shigeo  Matsuno 

Other:   Dr.  Robert  M.  Chanock 
Mr.  W.  Lee  Cline 


LID,NIAID  Head,  Epidemiology  Section 

LID  NTAID  Medical  Officer 

LIDjNIAID  Research  Microbiologist 

LID,NIAID  Medical  Officer 

LID,NIAID  Research  Associate 

LID.NIAID  Guest  Worker 

LID,NIAID  Chief 

LID,NIAID  Bio.  Lab.  Tech.  (Micro) 


COOPERATING  UNITS  (if  any) 


see  next  page 


lab/branch 

Laboratory  of   Infectious   Diseases 


SECTION 

Epidemiology  Section 


INSTITUTE  AND  LOCATION 

National  Institute  of  Allergy  and  Infectious  Diseases,  Bethesda,  Maryland 


TOTAL  MANYEARS: 

155/12 


PROFESSIONAL: 

55/12 


100/12 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 

[J  (al)  MINORS   [J  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Objectives:  (1)  To  search  for  viruses  which  play  an  etiological  role  in 
the  syndrome  of  acute  infectious  non-bacterial  gastroenteritis  of  infants, 
children,  and  adults;  (2)  to  cultivate  (in  vitro)  the  viral  agents  of  acute 
infectious  nonbacterial  gastroenteritis;  (3)  to  study  the  biophysical, 
immunological  and  epidemiological  characteristics  of  such  agents;  (4)  to 
reproduce  the  syndrome  experimentally  for  the  purpose  of  a)  studying  the 
pathophysiological  and  immunological  responses  of  the  host,  and  b)  assaying 
the  infectivity  of  viruses  such  as  human  rotaviruses  and  the  Norwalk  and 
Norwalk-like  agents;  (5)  to  develop  effective  immunoprophylaxis  for  the 


viruses  of  acute,  infectious,  nonbacterial  gastroenteritis;  (6)  to 
develop  efficient  and  sensitive  assays  for  a)  detection  of  the  viral  and 
bacterial  agents  associated  with  gastroenteritis  and  b)  antibodies  to  these 
pathogens. 


23-19 


PHS-6040 
(Rev.    10-76) 


Z01  AI  00021-10  LID 

Cooperating  Units 

Children's  Hospital  National  Medical  Center,  Washington,  D.C.;  Johns 
Hopkins  University,  Baltimore,  Md.,  Johns  Hopkins  University  Center  for 
Medical  Research  and  Training,  Dacca,  Bangladesh,  Division  of  Geographic 
Medicine  of  the  Johns  Hopkins  University  School  of  Medicine  and  the 
Baltimore  City  Hospitals,  Baltimore  Md. ;  Center  for  Disease  Control, 
Atlanta,  Ga.;  Meloy  Laboratories,  Rockville,  Md.;  Plum  Island  Animal 
Disease  Center,  USDA,  New  York;  New  York  Blood  Center;  University  of 
Minnesota  School  of  Medicine;  University  of  Massachusetts;  University  of 
Virginia,  Charlottesville;  Bureau  of  Biologies,  FDA;  NINCDS;  Veterinary 
Resources  Branch,  DRS;  Cholera  Research  Laboratory,  Dacca,  Bangladesh; 
Royal  Children's  Hospital,  Melbourne, Australia;  Fairfield  Hospital,  Melbourne, 
Australia;  INISA  San  Jose  Costa  Rica;  INCAP,  Guatemala;  Tohoku  University, 
Sendai,  Japan;  Department  of  Veterinary  Science,  Ohio  Agricultural  Research 
and  Development  Center,  Wooster,  Ohio;  LBV,  NIAID;  LSD,  NIAID;  Duke  University, 
North  Carolina,  University  of  New  Mexico  VA  Hospital;  Center  for  Vaccine 
Development,  University  of  Maryland,  Baltimore,  Md. ;  UCLA;  University  of 
Rochester;  University  of  Vermont;  Walter  Reed  Army  Institute  of  Research; 
California  State  Health  Department,  Berkeley,  California;  Children's 
Hospital,  Philadelphia,  Pa.;  ICRR  Sydney,  Australia;  University  of  Melbourne, 
Melbourne,  Australia;  Regional  Virus  Laboratory  Ruchill  Hospital,  Glasgow, 
Scotland;  WHO  Collaborative  Gastroenteritis  Study  involving  numerous 
laboratories;  Agricultural  Research  Council  Institute  for  Research  in 
Animal  Diseases,  Compton  Newbury,  Berkshire,  England;  Gorgas  Memorial 
Laboratory,  Panama;  Medical  Research  Center,  Nairobi;  Department  of  the 
Royal  Tropical  Institute,  Amsterdam,  Holland. 


23-20 


Z01  AI  00021-10  LID 
Major  Findings 

Background  -  Important  advances  have  been  made  in  elucidating  the  etiological 
agents  of  acute  infectious  nonbacterial  gastroenteritis,  a  syndrome  that 
affects  a  broad  segment  of  the  population.   It  appears  that  viral 
gastroenteritis  consists  of  at  least  two  entities  with  distinct 
epidemiological  characteristics.   One,  designated  epidemic  viral 
gastroenteritis,  tends  to  occur  in  family  or  community -wide  outbreaks 
affecting  adults,  school-aged  children,  family  contacts  and  probably  young 
children  as  well.   The  illness  is  usually  self-limited  and  characteristically 
lasts  24-48  hours.   In  1972,  we  demonstrated  by  immune  electron  microscopy 
(IEM)  that  a  27  nm  particle  Norwalk  agent  —  was  associated  with  an  outbreak 
of  this  form  of  gastroenteritis  that  occurred  in  Norwalk,  Ohio.   In  addition, 
in  further  studies  by  our  laboratory,  particles  that  resemble  the  Norwalk 
agent  morphologically  have  also  been  associated  by  IEM  with  2  family 
outbreaks  of  gastroenteritis,  one  in  Hawaii  and  the  other  in  Montgomery 
County  (MC),  Md.   The  Norwalk  and  Hawaii  agents  appear  to  be  distinct, 
whereas  the  Norwalk  and  MC  agents  appear  to  be  related.   An  agent  designated 
Ditchling  or  "W"  has  been  associated  with  this  form  of  gastroenteritis  in 
studies  in  England. 

The  other  form  of  gastroenteritis  designated  sporadic  infantile 
gastroenteritis  has  been  associated  predominantly  with  a  severe  form  of 
diarrhea  affecting  infants  and  young  children,  that  often  necessitates 
hospitalization  and  parenteral  fluid  therapy.   It  is  evident  now  from  studies 
in  many  parts  of  the  world  that  the  70  nm  human  rotavirus  is  the  major  known 
etiological  agent  of  sporadic  infantile  gastroenteritis.   Before  the 
discovery  of  this  agent  by  Australian  investigators  in  1973,  the  etiology  of 
the  majority  of  cases  of  infantile  gastroenteritis  was  unknown. 

Our  gastroenteritis  studies  are  focused  on  the  rotaviruses  as  well  as  on  the 
Norwalk  group  of  viruses.   The  Norwalk  group  of  agents  have  not  been 
cultivated  conclusively  in  any  in  vitro  system  whereas  the  human  rotavirus 
had  been  grown  only  in  a  limited  fashion.   However,  in  spite  of  the  failure 
to  grow  the  Norwalk  agent,  our  studies  have  elucidated  the  importance  of  the 
Norwalk  virus  as  a  cause  of  epidemic  viral  gastroenteritis  in  various 
epidemiologic  settings.   In  addition,  a  major  breakthrough  has  been 
achieved  recently  with  the  successful  efficient  in  vitro  propagation  of  the 
human  rotavirus.   Highlights  of  research  activities  carried  out  with  LID 
staff  alone  or  in  collaboration  with  others  are  outlined  below. 

In  Vitro  Cultivation  of  Human  Rotavirus  -  A  major  effort  of  this 
laboratory  has  involved  attempts  to  propagate  efficiently  human  rotavirus. 
The  efficient  propagation  of  rotavirus  type  2  appears  now  to  have  been 
achieved.   New  approaches  were  used  in  attempts  to  cultivate  this 
agent.   Initially,  42  fecal  specimens  known  to  be  positive  for  rotavirus 
by  EM  were  tested  for  their  ability  to  infect  primary  African  green  monkey 
kidney  (AGMK)  cell  cultures  using  low-speed  centrifugation  to  enhance 
infectivity.   Rotaviral  antigens  were  detected  by  immunofluorescence  (FA). 
Thirty-one  of  the  42  specimens  were  positive  by  this  method  but  only  5 


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yielded  as  many  as  10%  FA  positive  cells.   One  strain  ("Wa")  detected  in  this 
manner  was  passaged  subsequently  11  times  in  gnotobiotic  piglets  in 
collaboration  with  Dr.  E.H.  Bohl,  Ohio  Agricultural  Research  and  Development 
Center,  Wooster,  OH.   This  approach  was  prompted  by  the  findings  of  Provost 
and  Hilleman  who  described  the  adaptation  of  hepatitis  A  virus  to  cell  culture 
after  31  passages  in  marmosets.   The  occurrence  of  diarrhea  was  variable  but 
piglets  at  each  passage  were  infected  based  on  finding  viral  antigens  in  the 
gut  contents.   After  11  passages  in  piglets,  the  "Wa"  strain  was  passaged  into 
primary  AGMK  cells  by  pretreating  virus  inoculum  with  trypsin  (10  ug/ml)  and 
centrifuging  the  inoculum  at  low-speed  onto  the  cells.   At  that  point  the 
virus  appeared  to  be  capable  of  in  vitro  growth.   Viral  titers  ranged  from 
1.1  x  10^  fluorescent  foci  (FF) /ml  to  3.9  x  106  FF/ml  during  the  subsequent 
14  passages.   Contamination  with  SV5  was  discovered  at  the  eleventh  passage 
(and  retrospectively  on  earlier  passages)  but  was  removed  by  ether  treatment. 
Beginning  with  "Wa"  strain  after  six  passages  in  AGMK,  the  virus  has  been 
plaqued  and  has  undergone  two  plaque  purifications. 

Two  approaches  were  used  to  establish  the  identity  of  this  isolate  as  a 
human  rotavirus.   Polyacrylamide  gel  electrophoresis  of  viral  RNA  was  performed; 
the  RNA  pattern  of  the  isolate  is  identical  to  a  previously  recognized  human 
rotavirus  RNA  pattern.   It  differs  however,  from  six  animal  rotaviruses  which 
grow  readily  in  cell  cultures,  with  which  it  was  compared.   Further,  serological 
testing  using  plaque  reduction  and  fluorescent  focus  neutralization  was 
performed.   The  human  "Wa"  isolate  appears  to  be  serologically  distinct  from 
the  bovine,  porcine,  and  simian  rotaviruses;  it  demonstrates  a  one-way  antigenic 
relationship  with  the  simian  rotavirus  SA-11 — a  finding  consistent  with  a 
previous  observation  made  by  IEM  by  Schoub,  et  al. 

Studies  on  the  Etiology  of  Non-Bacterial  Gastroenteritis  Among  Infants  and 
Young  Children  Admitted  to  Children's  Hospital,  Washington,  D.C.  -  These 
studies  are  carried  out  collaborative  with  Children's  Hospital  National 
Medical  Center.   Currently  the  stool  or  rectal  swab  specimens  are  examined 
by  EM  and  ELISA  at  Children's  Hospital  for  gastroenteric  agents  (Dr.  Brandt) 
whereas  the  routine  rotavirus  serologic  studies  (CF)  are  carried  out  at  the 
LID  (Mr.  Cline) .   The  clinical  data  and  specimens  are  furnished  by  Dr.  Kim 
and  her  staff  at  Children's  Hospital.   In  addition,  the  serotyping  of 
rotaviruses  has  been  performed  at  LID.   The  scope  of  the  study  was  outlined 
previously  and  essentially  involved  study  of  pediatric  patients  admitted  to 
Children's  Hospital  with  a  diarrheal  illness.   Limited  outpatient: studies 
were  also  performed. 

In  data  analyzed  by  Dr.  Brandt  for  the  period  Jan.  16,  1974  through  June, 
1978  the  importance  of  rotavirus  as  the  major  etiologic  agents  of  diarrheal 
illness  was  again  highlighted.   In  last  year's  annual  report  we  presented 
data  from  Jan.  1974  through  May,  1978.   The  description  in  this  annual  report 
represents  final  analysis  of  data  for  the  Jan.  1974  through  June  1978  period. 
From  January  16,  1974  through  June  1978,  specimens  from  604  inpatients  with 
gastroenteritis  were  tested  by  EM  and  ELISA  for  rotavirus  and  232  (38.4%) 
were  found  to  be  positive.   In  contrast  only  9  (1.7%)  of  522  control 
inpatients  studied  March,  1974  through  June  1978  were  rotavirus  positive  by 


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identical  techniques,  thus  demonstrating  the  statistically  significant 
association  of  rotavirus  infection  with  diarrheal  disease  (p<  .0001).   The 
pronounced  predilection  of  rotavirus  infection  for  the  cooler  months  of 
the  year  was  a  consistent  finding  during  this  period.   For  example,  the 
frequency  of  rotavirus  infections  in  patients  admitted  to  the  hospital  in 
January  was  striking  as  87(71%)  of  123  gastroenteritis  patients  shed  this 
agent.   Adenoviruses  (as  detected  by  EM  and/or  tissue  culture)  were 
associated  with  5.1%  of  the  diarrheal  episodes,  whereas  1.9%  of  the  controls 
was  adenovirus  positive  (p  <.05)  suggesting  that  these  viruses  play  a  role, 
albeit  small,  in  the  etiology  of  acute  enteric  disease.   It  was  noteworthy 
that  26  of  the  31  adenoviruses  from  inpatients  did  not  grow  when  tested  by 
routine  tissue  culture  methods.   Only  13  (2.2%)  gastroenteritis  inpatients  were 
found  to  shed  27nm  like  virus  particles  (six  also  excreted  human  rotavirus) , 
whereas  5(1.0%)  of  the  controls  shed  this  type  of  virus  (p  >.05)  thus, 
indicating  again  that  this  group  of  agents  is  not  an  important  etiologic 
agent  of  serious  gastroenteritis  in  infants  and  young  children  requiring 
hospitalization. 

From  November,  1975  -  June,  1978,  rotaviruses  were  detected  in  42  (21%)  of 
200  outpatients  with  diarrheal  disease  and  in  8  (2.8%)  of  290  outpatient 
controls.   Thus,  it  appeared  that  rotaviruses  caused  not  only  severe 
diarrheal  illnesses  requiring  hospitalization  but  also  milder  gastroenteritic 
illnesses  which  could  be  treated  on  an  outpatient  basis.   Adenoviruses  were 
found  in  2.5%  of  outpatients  with  gastroenteritis  and  in  0.3%  of  outpatient 
controls  (p  <.05)  again  supporting  the  contention  that  adenoviruses  may  play 
a  role  in  gastrointestinal  disease.   27  nm  particles  were  found  in  1 
outpatient  with  gastroenteritis  and  in  2  controls. 

Approximately  three  fourths  of  the  rotaviruses  detected  were  found  to  be 
type  2  which  was  prevalent  during  5  successive  epidemic  years  from  Jan.  1974 
through  June  1975.   Type  1  was  detected  in  the  last  4  successive  epidemic 
years  and  comprised  46%  of  the  human  rotavirus  infections  detected  among 
gastroenteritis  inpatients  during  the  year  1977-78.   Numerically,  human 
rotavirus  infection  was  detected  most  often  in  those  gastroenteritis 
inpatients  who  were  10  through  12  months  of  age.   The  group  of 
gastroenteritis  inpatients  with  the  highest  percentage  of  human  rotavirus 
infection  was  13  through  15  months  of  age.  The  excess  of  type  2  human 
rotavirus  infection  in  relation  to  type  1  infection  was  especially  large  in 
those  aged  7  through  24  months.   Lower  socioeconomic  status  or  greater 
crowding  appeared  to  be  associated  with  the  occurrence  of  rotavirus 
infection  earlier  in  life  and  earlier  in  the  epidemic  year. 

More  up  to  date  data  (up  to  April,  1979)  on  the  role  of  the  above  agents 
(minus  new  serotyping  data  which  is  not  available)  analyzed  by  Dr.  Brandt 
are  consistent  with  the  previous  years'  experiences. 

Incidence  of  Pediatric  Rotavirus  Gastroenteritis  Resulting  in  Admission  to 
Hospital  in  Washington  D.C.  Area  -~In  studies  carried  out  by  Dr.  Rodriguez 
at  the  Children's  Hospital  National  Medical  Center,  Washington,  D.C,  the 
incidence  of  rotavirus  gastroenteritis  requiring  admission  to  this  hospital 


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in  a  defined  population  15  yrs.  of  age  and  under  was  determined.   This  was 
a  major  achievement  as  the  importance  of  rotavirus  as  a  cause  of  pediatric 
gastroenteritis  has  been  ascertained  primarily  in  studies  of  infants  and 
children  hospitalized  with  this  disease.   However,  in  such  studies,  incidence 
rates  of  hospitalization  caused  by  rotavirus  infection  could  not  be 
derived  since  it  was  not  possible  to  define  precisely  the  population  from 
which  the  hospitalized  patients  came.   Thus,  the  incidence  of  rotavirus 
gastroenteritis  requiring  hospitalization  was  estimated  for  a  defined 
population  of  approximately  105,000  individuals  whose  health  care  was 
provided  by  Group  Health  Association  (GHA) ,  a  health  maintenance 
organization  in  the  Washington,  D.C.  area.   About  28%  were  15  yrs.  of  age  or 
under.   Almost  all  of  the  pediatric  hospitalizations  of  this  health 
maintenance  organization  occurred  at  the  Children's  Hospital.   Visits  to  the 
GHA  clinics  for  diarrhea  were  recorded  but  those  illnesses  were  not  studied 
for  viral  etiology.   However,  etiologic  studies  were  performed  for  GHA  infants 
and  children  who  were  admitted  to  Children's  Hospital  for  acute 
gastroenteritis.   Data  on  this  population  was  studied  for  the  period  Jan. , 
1977  through  March,  1979,  an  interval  encompassing  three  periods  of  rotavirus 
prevalence.   Approximately  29,000  individuals  15  yrs.  of  age  and  younger 
comprised  the  study  group  for  each  period.   On  the  average,  1  in  272  (3.7/1000) 
infants  under  1  yr.  of  age  and  1  in  451  (2.2/1000)  13-24  months  of  age  were 
hospitalized  for  rotavirus  disease  in  each  period  of  rotavirus  prevalence. 
The  incidence  of  rotavirus  gastroenteritis  requiring  hospitalization 
precipitously  after  2  years  of  age  and  such  illness  requiring  hospitalization  was 
not  detected  after  5  years  of  age.   The  role  of  other  agents  in  acute 
gastroenteritis  requiring  hospitalization  was  minimal  compared  to  that  of 
rotavirus.   It  was  striking  that  when  hospital  admission  patterns  for 
gastroenteritis  of  any  etiology  were  examined  by  month,  76%  (28/38)  of  such 
admissions  in  the  study  population  occurred  during  the  winter  months  of 
January,  February  and  March.   The  data  derived  from  studying  those  hospitalized 
from  the  GHA  population  suggest  that  at  least  50%  of  admissions  for  dehydrating 
gastrointestinal  illness  was  associated  with  rotavirus  infection. 

Study  of  Rotavirus  Infection  in  Adult  Volunteers  -  Data  from  the  initial 
phase  of  this  study,  reported  in  a  preliminary  fashion  last  year,  is  now 
complete.  A  safety  tested  inoculum  of  rotavirus  type  2  consisting  of  a 
dilution  of  a  2%  stool  filtrate  prepared  from  a  stool  rich  in  rotavirus 
particles  obtained  from  a  13  month  old  child  hospitalized  with 
gastroenteritis  was  administered  orally  to  18  volunteers  once  and  to  2  of 
these  volunteers  a  second  time,  about  19  months  after  initial  challenge. 
These  studies  were  carried  out  in  6  phases;  the  first  at  the  Clinical  Center, 
NIH  and  the  remainder  at  the  University  of  Maryland  Center  for  Vaccine 
Development  in  Baltimore,  Md.   The  first  study  began  in  November,  1974  and  the 
sixth  in  June,  1978.   In  the  initial  study,  2  volunteers  with  high  levels  of 
serum  rotavirus  antibody  were  selected  since  the  clinical  response  to 
rotavirus  infection  under  experimental  conditions  was  not  known.   Since 
illness  did  not  occur,  2  additional  volunteers  with  somewhat  lower  levels  of 
rotavirus  antibody  were  inoculated  in  a  second  study;  neither  developed 
illness.   Efforts  were  then  made  to  identify  volunteers  with  little,  if  any, 
rotavirus  serum  antibody.   It  soon  became  apparent  however,  that  almost  all 


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adults  possessed  such  antibody  detectable  by  at  least  one  of  the  assays 
employed.   Thus,  when  feasible,  individuals  with  the  lowest  serum  antibody 
titers  were  selected  for  inoculation.   Five  of  18  volunteers  shed  rotavirus 
in  their  stools  beginning  on  the  second,  third  or  fourth  day  after 
inoculation.   Although  only  5  shed  rotavirus,  15  of  the  18  developed  a 
four-fold  or  greater  increase  in  serum  antibody  to  rotavirus  as  detected 
by  at  least  one  of  the  assayed  employed.   ELISA  was  the  most  efficient 
of  the  serologic  assays.   Four  of  the  5  volunteers  who  shed  rotavirus 
developed  a  diarrheal  illness  which  began  2-4  days  after  inoculation.   The 
average  duration  of  diarrhea  was  2.5  days  with  a  range  of  1-4  days.   Two 
of  these  4  volunteers  also  vomited. 

Although  there  was  a  tendency  for  detectable  preinoculation  serum  antibody  as 
measured  by  CF,  IF  and  neutralization  (of  NCDV)  tests  to  be  associated  with 
resistance  to  diarrheal  illness  following  rotavirus  inoculation,  the  trend  was 
not  statistically  significant.   Analysis  of  the  relationship  between  serum 
antibody  detectable  by  these  methods  and  the  occurrence  of  rotavirus  shedding 
revealed  a  significant  association  only  for  detectable  IF  antibody  although 
a  similar  trend  was  again  observed  for  the  other  assays.   Thus,  by  selecting 
individuals  with  the  lowest  levels  of  serum  rotavirus  antibody,  an  attack 
rate  of  rotaviral  diarrhea  disease  of  38-60%  was  achieved.   Similar  analyses 
were  made  to  evaluate  the  protective  effect  of  pre-existing  rotavirus  type 
specific  serum  IgG  antibody  by  ELISA:   a  significant  association  was  not 
observed  for  the  presence  of  either  type  1  or  2  serum  IgG  antibody  and 
failure  to  shed  virus  or  develop  diarrheal  illness. 

However,  each  of  5  volunteers  without  detectable  rotavirus  type  2  intestinal 
fluid  IgA  antibody  shed  virus  indicating  that  the  presence  of  local  IgA 
rotavirus  antibody  correlated  with  resistance  to  virus  shedding  (p  <.05). 
Moreover,  4  of  5  volunteers  without  detectable  IgA  rotavirus  antibody  in 
intestinal  fluids,  but  none  of  10  with  such  antibody,  developed  a  diarrheal 
illness  after  inoculation  (p<  .05)  suggesting  that  the  presence  of  such 
antibody  was  associated  with  resistance  to  diarrheal  illness.   Rotavirus 
types  1  IgA  intestinal  fluid  antibody  did  not  correlate  with  resistance  to 
virus  shedding  or  illness.   Thus,  it  appears  that  local  intestinal  IgA 
rotavirus  antibody  correlates  well  with  resistance  to  rotavirus  shedding 
and  diarrhea  under  experimental  conditions.   In  addition,  4-fold  IgA  antibody 
increases  to  type  2  rotavirus  were  observed  in  intestinal  fluid  from  each  of 
the  5  volunteers  who  shed  rotavirus,  and  from  an  additional  volunteer  as 
well,  following  initial  challenge. 

Two  volunteers  who  developed  illness  and  laboratory  evidence  of  rotavirus 
infection  following  initial  administration  of  rotavirus  type  2  were  given  the 
same  inoculum  19  months  later.   Neither  developed  a  diarrheal  illness 
or  vomiting  after  second  inoculation  although  one  developed  mild  clinical 
manifestations.   Both  individuals  lacked  detectable  rotavirus  IgA  intestinal 
antibody  prior  to  initial  challenge  but  had  such  antibody  prior  to  second 
challenge.   One  (who  had  the  mild  clinical  manifestations)  developed  a 
4-fold  increase  in  intestinal  IgA  rotavirus  antibody  after  the  second 
inoculation  as  well. 

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Frequency  of  Detection  of  Rotavirus  and  Norwalk  Agent  in  Stools  of  Infants  and 
Children  with  Gastroenteritis  in  World  Health  Organization  Collaborative  Study 
In  a  WHO  collaborative  study,  specimens  were  obtained  from  pediatric  patients 
with  diarrheal  illnesses  from  various  parts  of  the  world  including:  Dakar, 
Senegal;  Entebbe,  Uganda;  Singapore;  Tunis,  Tunisia;  Kalubowila,  Sri  Lanka; 
Seoul,  Korea;  Kuala  Lumpur,  Malaysia;  Hong  Kong;  Cayenne,  French  Guiana; 
Bungui,  Central  African  Republic;  and  Kinshasha,  Zaire.   Specimens  were 
obtained  Feb.,  1976- July,  1978.   Dates  were  not  available  for  the  last  2 
locations.   A  total  of  438  specimens  were  examined  for  rotavirus  by  ELISA  and 
118  (27%)  were  positive.   Rotavirus  was  detected  in  each  of  the  countries 
with  a  range  of  7%  to  71%  being  positive.   In  contrast,  these 
stool  specimens  were  also  tested  for  Norwalk  antigen  by  RIA  and  none  was 
positive. 

Antibodies  Against  Rotavirus  in  Sera  from  Children  Living  in  the  Machakos 
District  of  Kenya  -  In  a  collaborative  study  with  Metselaar,  Sack,  and 
Muller  the  prevalence  of  rotaviral  CF  antibody  in  children  under  5  years 
of  age  living  in  the  Machakos  District  of  Kenya  was  determined.   Two  different 
antigens,  NCDV  and  "0"  were  used  and  the  superiority  of  "0"  antigen  over  NCDV 
was  confirmed.   The  percentage  of  children  with  rotavirus  antibody  rose 
rapidly  so  that  by  18-23  months  of  age  79%  had  such  antibody  indicating  that 
the  time  of  initial  rotavirus  infection  in  the  Kenya  population  was  similar  to 
that  observed  in  various  other  areas  of  the  world. 

Plaque  Reduction  Tests  with  Animal  Rotaviruses  -  Plaque  reduction  tests  to 
measured  serum  antibody  against  bovine  rotavirus  (UK  strain)  and  simian 
rotavirus  (SA11)  were  performed  regularly  to  characterize  immune  response, 
identify  specific  hyperimmune  sera  and  to  identify  rotavirus  isolates  such 
as  those  arising  from  genetic  reassortment  studies.  While  other  rotavirus 
strains  (porcine,  bovine-NCDV,  and  "0"  agent)  have  been  plaqued,  plaque 
reduction  tests  have  not  been  performed. 

Development  of  ts  Mutants  of  Calf  Rotavirus  -  As  part  of  our  effort  to 
modify  the  virulence  of  rotavirus,  8  _ts  mutants  of  the  UK  strain  of  calf 
rotavirus  were  developed  using  5  azacytidine  or  nitrosoguanidine  as  mutagen. 
The  8  mutants  had  a  shutoff  temperature  for  plaque  formation  of  38°C  or 
39°C  and  appeared  to  be  relatively  stable  genetically.   It  is  our  intention 
to  use  these  and  subsequent  mutants  to  probe  the  function  of  individual  genes 
of  the  rotavirus  genome.  T_s  mutants  will  also  be  used  as  donors  of  jts_  genes 
for  transfer  to  the  tissue  culture-adapted  human  virus.   Bovine 
genetic  reassortment  in  an  attempt  to  attenuate  the  human  virus.   Bovine 
rotavirus  genes  will  probably  prove  to  be  host  restricted  in  human  cells, 
i.e.,  hr,  and  this  may  offer  another  mechanism  by  which  genes  transferred  from 
the  bovine  virus  could  bring  about  attenuation  of  the  human  virus. 

Animal  Rotavirus  as  Potential  Vaccine  for  Humans  -  The  evaluation  of  the 
ability  of  bovine  rotavirus  to  protect  against  subsequent  challenge  with  human 
rotavirus  was  presented  in  a  preliminary  way  in  the  FY  1978  annual  report. 
In  the  past  year  this  study  has  been  finalized  and  published  in  Science.   In 
the  first  phase  of  the  study  2  calves  were  infected  in  utero  with  bovine 
rotavirus  (Cody  strain  from  Nebraska)  and  challenged  with  homologous  virus 


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shortly  after  birth.   Complete  resistance  to   disease  on  this  second  challenge 
was  observed,  while  two  control  animals  which  were  not  infected  in  vitro  but 
challenged  shortly  after  birth  became  ill.   For  the  second  phase~5  animals 
were  infected  with  bovine  rotavirus  in  utero  and  challenged  with  the  heterologous 
human  rotavirus  (type  2)  one  or  two  days  after  birth.   All  animals  were 
resistant  to  disease  in  contrast  to  eight  control  animals  which  received  human 
rotavirus  one  or  two  days  after  birth  without  having  been  exposed  previously  to 
bovine  rotavirus;  seven  of  the  control  animals  became  ill.   These  data  suggest 
that  the  bovine  virus  is  sufficiently  related  to  the  human  type  2  virus  to 
warrant  further  evaluation  of  the  former  as  a  live  vaccine. 

Further  studies  on  the  bovine  rotavirus  (UK  strain)  are  under  way  to 
ascertain  its  suitability  as  a  possible  vaccine  candidate  for  human  use.   This 
bovine  rotavirus  strain  was  chosen  because  it  has  been  cultivated  only 
in  primary  bovine  kidney  cell  cultures  and  would  likely  meet  the  requirements 
for  human  use.   It  is  serologically  closely  related  to  the  bovine  rotavirus 
(Cody)  from  Nebraska  which  was  evaluated  in  the  animal  cross-protection  studies, 
although  it  does  not  exhibit  an  identical  RNA  gel  electrophoresis  pattern.   A 
large  suspension  (approximately  2  liters)  of  this  plaque-purified  bovine 
rotavirus  underwent  successful  routine  safety-testing.   Additional  safety  testing 
is  being  carried  out  at  Plum  Island  Animal  Disease  Center  in  collaboration  with 
Dr.  C.A.  Mebus.   This  is  required  since  this  particular  rotavirus  strain 
originated  in  the  UK  from  a  domestic  animal.   No  adventitious  agents  have  been 
recognized  in  the  virus  suspension,  and  the  disease  it  produces  in  calves 
is  similar  to  that  seen  with  the  U.S.  bovine  rotavirus.   However,  the  UK  isolate 
does  induce  disease  in  gnotobiotic  piglets  (4  of  7)  while  the  U.S.  isolate  does 
not.   When  testing  is  complete,  USDA  will  be  asked  for  permission  to  release 
this  strain  for  distribution  and  further  study. 

Nonhuman  Primate.  Animal,  Models  for  Gastroenteritis  -  A  further  attempt  to 
induce  illness  with  human  rotavirus  (type  2)  in  2  young  seronegative  chimpanzees 
failed.   Both  animals  were  infected  based  on  virus  shedding  in  feces.  The 
evailability  of  a  satisfactory  nonhuman  primate  model  for  rotavirus  disease 
still  remains  a  goal. 

An  attempt  to  infect  2  chimpanzees  with  the  27  ran  Marin  County  agent  failed. 
An  IEM  antibody  response  was  not  observed  (see  later) . 

Analysis  of  Rotaviral  RNA  by  Gel  Electrophoresis  -  Continued  use  was  made  of  the 
technique  of  electrophoresis  of  rotaviral  RNA  for  detection  of  differences 
and/or  similarities  among  various  animal  and  human  strains.   It  was  evident  from 
previous  analysis  of  7  type  2  human  rotaviruses  that  three  different  RNA  patterns 
existed.   Since  only  one  ELISA  serotype  1  strain  was  included  in  the  original 
studies,  a  second  serotype  1  strain  0  268  from  Bangladesh)  was  compared  with 
the  original  type  1  strain  (DS-1  from  Children's  Hospital);  5  of  the  DS-1 
segments  had  a  different  migration  rate  from  the  corresponding  gene  segments  of 
strain  268.   Thus,  we  detected  at  least  two  RNA  patterns  for  ELISA  serotype  1 
human  rotaviruses. 

Several  calf  rotavirus  strains  from  Canada  (RS,C10,C27  and  C486)  were  growni 
in  cell  culture  and  RNA  was  prepared  for  comparisons  among  the  calf  rotaviruses. 
Two  of  these  (RS  and  CIO)  did  not  grow  well  enough  to  permit  further  study. 
Of  the  remainder,  C27  appeared  to  be  identical  to  NCDV  by  RNA  analysis 

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and  the  second  (C486)  appeared  to  differ  in  migration  of  at 
least  one  segment  (#5)  from  NCDV.   This  strain  (C846)  was  contaminated  by  a 
second  virus,  since  its  RNA  pattern  contained  a  second  less  abundant  #5 
segment.   Both  Canadian  calf  viruses  had  an  RNA  pattern  different  from  that  of 
the  UK  calf  rotavirus  pattern. 

Since  there  are  two  isolates  of  Nebraska  calf  diarrhea  virus  (NCDV) , 
namely  the  Cody  and  Lincoln  isolates,  a  comparison  of  their  RNA  patterns 
was  made  to  see  if  they  could  be  distinguished  on  this  basis.   Results 
from  the  comparison  showed  them  to  be  indistinguishable. 

RNA  analysis  was  also  used  to  characterize  a  putative  human  rotavirus  that 
<*rew  in  cell  culture  (described  earlier) .   Comparison  of  this  isolate 
(Wa  strain)  with  representatives  of  two  of  the  previously  described  human 
rotavirus  patterns  ("D"  strain,  Group  I  and  "L"  strain,  Group  II)  were  carried 
out.   The  "Wa"  RNA  pattern  was  identical  to  that  of  "L"  strain  and  differed 
from  that  of  the  "D"  strain  by  the  migration  of  segment  #5.   The  Wa  isolate  was 
further  shown  to  be  different  from  the  following  animal  rotavirus  RNAs  by 
difference  in  migration  of  3  to  7  of  the  11  segments:  SA-11,  "0"  agent,  OSU 
and  EE  porcine  rotaviruses, NCDV  and  UK  calf  rotaviruses.   Thus,  the  RNA 
analysis  showed  that  Wa  is  most  likely  a  human  rotavirus. 

In  addition,  the  methodology  for  preparing  individual  rotavirus  genes  or 
segments  was  developed.   50  to  150  ug  quantities  of  total  RNA  was  prepared  from 
cell  culture  grown  animal  rotavirus  strains  and  examined  by  gel 
electrophoresis.   After  gel  electrophoresis,  separated  segments  are  cut  out  of 
the  gel.   In  this  manner,  1  to  5  ug  quantities  of  segments  #1,  4,  5,  6,  10  and 
11  have  been  readily  prepared  from  SA-11,  NCDV  and  0  agent  RNAs. 
Since  they  do  not  resolve  well  by  electrophoresis,  mixtures  of  RNA 
segments  2  and  3  as  well  as  segments  7,8  and  9  have  also  been  prepared  from 
these  three  rotaviruses.   The  segments  have  been  examined  by  electron 
microscopy  and  are  intact,  although  some  RNA  breakage  has  occurred.   This 
technology  will  be  useful  for  translation  experiments  using  a  reticulocyte 
system,  and  S-35  methionine. 

Rotavirus  Hemagglutination  (HA)  and  Hemagglutination-Inhibition  (HI)  Studies  - 
Cell  culture  grown  simian  rotavirus  (SA-11)  and  calf  rotavirus  (C486 
from  Canada)  have  been  established  as  hemagglutinating  rotavirus  strains. 
Other  cell  culture  adapted  animal  rotavirus  isolates  were  tested  for  their 
ability  to  hemagglutinate.   The  virus  preparations  were  genetron  treated 
to  remove  possible  inhibitors  and  concentrated  50-fold  before  being 
assayed  in  a  phosphate  buffered  saline  HA  system.   SA-11  was  used  as  a 
positive  control  and  gave  optimal  HA  activity  with  Rhesus  monkey  red  blood 
cells.   NCDV  and  "0"  agent  were  found  to  hemagglutinate  Rhesus  monkey  and 
guinea  pig  cells,  whereas  the  UK  calf  virus  and  OSU  porcine  strain  were  HA 
negative.   Rhesus  monkey  erythrocytes  were  chosen  for  routine  HA  assays  since 
they  worked  as  well  as  human  "0"  cells  and  were  available  commercially. 

Shinozaki  and  coworkers  have  published  results  which  demonstrate  the 
existence  of  a  human  rotaviral  hemagglutinin  and  subsequently  have  indicated 


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that  Tris  buffered  saline  supplemented  with  Ca  and  Mg  ions,  and  chicken 
erythrocytes  provide  optional  hemagglutination  activity.   Other  workers  in 
Japan  (Sato  et  al.  and  Inaba  et  al. )  have  used  Tris-HCl  buffered  saline 
and  veronal  buffered  saline  with  chicken  cells  to  demonstrate  HA  activity 
for  NCDV.   The  Tris  buffered  saline  and  veronal  buffered  saline  were  used 
with  chicken  cells  and  monkey  cells  in  our  laboratory  to  assay  various 
preparations  containing  human  and  animal  rotaviruses. 

Fifty-fold  concentrates  of  the  cell  culture  adapted  animal  strains  and 
12  human  rotavirus  suspensions  prepared  by  genetron  treatment  and  5-fold 
concentration  of  gnotobiotic  calf  feces  collected  during  experimental 
infection  were  assayed  for  HA  activity  by  the  two  alternative  HA  systems 
mentioned  above.   The  human  rotavirus  preparations  represented  first  through 
third  passage  of  3  different  strains  ("D,"  "C"  and  "DS-1")  in  calves.  A  human 
hemagglutinin  could  not  be  demonstrated  by  these  methods  and  those  animal 
strains  which  were  negative  by  the  routine  method  used  in  our  laboratory 
were  also  negative  in  these  systems.   Compared  to  our  standard  method,  the 
Tris  buffered  saline  system  gave  lower  titers  and  the  veronal  buffered 
saline  system  gave  negative  assays  using  the  HA  positive  viruses,  SA-11,  NCDV 
and  "0"  agent. 

It  was  shown  by  others  (Cohen  et  al. ,  Hruska  et  al.  and  Estes  et  al.)  that  EDTA 
removes  the  outer  capsid  of  rotavirus.   A  preliminary  experiment  was  carried 
out  to  determine  the  effect  of  EDTA  on  the  HA  of  NCDV  and  SA-11.  SA-11  and 
NCDV  HA  appeared  to  have  different  sensitivities  to  EDTA.   EDTA  at 
concentrations  above  0.5  mM  destroyed  NCDV  HA  activity,  whereas  SA-11  HA  was 
reduced  only  2  to  4-fold  at  concentrations  of  1  to  5  mM  of  EDTA. 

The  fifty-fold  concentrates  of  SA-11,  NCDV  and  "0"  agent  were  used  as 
antigens  in  HI  tests  with  23  human  serum  pairs  from  children  ill  with 
diarrhea  (Children's  Hospital).  9  of  the  23  children  had  developed  an  4-fold 
increase  in  rotavirus  CF  antibody  and  each  of  the  23  had  demonstrated  a 
significant  rotavirus  type  specific  antibody  response  by  ELISA;  11  were  type  1 
and  12  were  type  2.   Six  of  the  23  demonstrated  a  significant  HI  antibody  rise 
to  SA-11  and  none  developed  an  antibody  response  to  either  NCDV  or  0  agent. 
Four  of  the  six  with  HI  responses  had  developed  antibody  responses  by  ELISA 
to  type  2,  and  two  to  type  1  rotavirus.   When  compared  to  CF,  2  of  the  HI  rises 
occurred  in  patients  who  did  not  develop  a  significant  CF  antibody  rise,  but  2 
who  were  negative  by  HI  were  positive  by  CF,  and  2  were  CF  and  HI  positive. 

Hyperimmune  guinea  pig  sera  to  SA-11  and  NCDV  were  prepared  by 
using  crude  infected  cell  culture  fluid  mixed  with  Freund's  imcomplete 
adjuvant  as  immunogen.   These  sera  did  not  cross  react  with  each  other  by 
HI  and  had  a  homologous  titer  of  1:512.   Hyperimmune  anti-  0  agent  serum  was 
made  in  this  manner,  but  in  one  way  tests  with  SA-11  and  NCDV  the  "0"  agent 
cross-reacted  with  the  anti-NCDV  serum  but  not  with  the  anti-SA-11  serum. 

Six  guinea  pig  sera  and  one  goat  hyperimmune  serum  prepared  against 
doubly  purified  (sucrose  and  CsCl  gradients)  human  rotavirus  failed  to 
show  HI  activity  against  any  of  the  three  HA  antigens.   However,  one  other 

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goat  (930)  which  was  hyperimmunized  with  sucrose  purified  human  rotavirus 
developed  HI  antibody  to  all  three  viruses;  also  human  immune  serum  globulin 
(Armour  Corp.)  had  HI  activity  against  all  three  antigens  at  a  titer  of  1:32. 

Studies  of  Rotavirus  Polypeptides  -  African  green  monkey  kidney  cells  were 
infected  with  an  MO I  of  10  of  SA-11  virus  and  at  intervals  from  2  to  4  hours 
post  infection  were  pulse  labeled  for  2  hours  with  S-35  methionine.   Cytoplasmic 
extracts  were  prepared  according  to  the  methods  employed  by  Ramig  et  al  and 
electrophoresed  on  discontinuous  tris-glycine  gels.  With  this  technique, 
seven  viral  polypeptides  were  detected  and  maximum  polypeptide  synthesis 
appeared  from  12  to  18  hours  post  infection.   A  high  background  of  labeled 
cellular  components  was  a  distinct  problem  and  probably  masked  viral 
polypeptides  which  were  present  in  small  quantity.  To  overcome  this, 
hyperimmune  anti-SA-11  and  anti-NCDV  guinea  pig  antisera  (as  described  in 
section  on  HA)  were  prepared  and  will  prove  useful  for  immunoprecipitation  of 
these  less  abundant  polypeptides. 

Experiments  were  carried  out  to  label  the  structural  polypeptides  of 
SA-11,  UK  calf,  and  NCDV  with  S-35  methionine  produced  during  infection  of 
African  green  monkey  kidney  or  CV-1  cells.   The  virus  was  harvested  and 
purified  on  shallow  CsCl  gradients.   Density  and  HA  activity  were  used  to 
assay  for  complete  virions  and  core  particles  for  the  purpose  of 
determining  the  inner  and  outer  capsid  polypeptides  for  each  of  the  three 
viruses.   However,  NCDV  and  UK  complete  virions  appear  to  be  sensitive 
to  the  combination  of  CsCl  and  centrifugation  since  no  detectable  complete 
virions  were  detected  in  CsCl  gradients  despite  a  relatively  high  HA 
titer  of  the  NCDV  starting  material.   SA-11  appeared  to  be  more 
resistant  to  centrifugation  in  CsCl  since  HA  positive  labeled  particles 
were  detected  at  densities  of  1.35  and  1.36  g/cc.   Gel  electrophoresis  of 
these  preparations  was  carried  out  on  a  discontinuous  tris-glycine 
system  for  comparison  with  the  continuous  phosphate  buffered  system 
used  in  earlier  studies.   The  results  with  S-35  labeled  SA-11  in  the 
continuous  gel  system  closely  resembled  those  achieved  in  our  earlier  work 
using  a  protein  staining  technique,  i.e.,  8  polypeptides  were  seen  for  the  HA 
positive  SA-11  fraction.   The  UK  and  NCDV  fractions  yielded  only  5  polypeptides 
in  the  continuous  gel  system  indicating  that  they  were  composed  of  labeled  core 
particles.   Gel  electrophoresis  of  these  samples  in  the  discontinuous  system 
gave  fewer  and  less  well  resolved  polypeptides  for  the  SA-11  virions  (i.e.,  7 
polypeptides)  and  about  the  same  results  for  the  core  SA-11,  NCDV  and  UK 
particles  (i.e.,  5  polypeptides). 

Role  of  Norwalk  Virus  in  Outbreaks  of  Non-Bacterial  Gastroenteritis  - 
The  development  of  a  radioimmunoassay  (RIA)  for  detection  of  Norwalk 
antigen  and  a  radioimmunoassay  blocking  (RIABL)  test  for  detection  of 
antibody  to  the  Norwalk  agent  were  described  in  the  last  two  years'  annual 
reports.  The  availability  of  the  RIA  and  RIABL  has  permitted  continued 
large  scale  epidemiologic  studies  of  Norwalk  infection. 

The  original  study  of  epidemic  gastroenteritis  has  now  been  considerably 
enlarged.  65  outbreaks  have  been  studied  serologically,  and  20  (31%)  appear  to 

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be  associated  with  the  Norwalk  virus  as  indicated  by  seroresponse  of  affected 
individuals.   This  information  reaffirms  our  previous  conclusion  that  the 
Norwalk  virus  is  a  major  cause  of  epidemic  gastroenteritis.   The  new 
Norwalk-associated  outbreaks  occurred  in  a  wide  variety  of  situations  including 
grade  school,  summer  camp,  nursing  home,  and  cruise  ship.   Interestingly, 
Norwalk  virus  was  etiologically  associated  with  a  very  large  oyster  borne 
outbreak  of  acute  gastroenteritis  in  Australia  by  Australian  investigators  and 
confirmed  in  our  laboratory.   In  two  Norwalk-associated  outbreaks  originally 
studied  by  the  CDC,  water  borne  transmission  appeared  to  be  the  mode  of  spread. 

Antibody  Prevalence  to  the  Norwalk  Virus  -  A  large  battery  of  age  stratified 
sera  from  adults  from  various  parts  of  the  world  was  studied  for  Norwalk 
antibody.   Prevalence  rates  were  stable  and  similar  in  adults  from  the  U.S., 
Switzerland,  Belgium,  Yugoslavia,  Ecuador,  Bangladesh,  and  Nepal  (approximately 
70%).   On  the  other  hand,  children  from  the  U.S.  and  Yugoslavia  acquired 
antibody  far  more  slowly  than  did  children  in  Ecuador  and  Bangladesh.   In 
Ecuador  and  Bangladesh  an  antibody  prevalence  of  greater  than  70%  was  found  in 
children  0-5  years  of  age.   This  high  childhood  prevalence  rate  may  indicate 
that  Norwalk  virus  plays  an  important  role  in  childhood  gastroenteritis  in 
underdeveloped  countries.   The  low  level  of  antibody  prevalence  in  young 
children  from  the  U.S.  is  in  keeping  with  the  failure  to  incriminate  this  agent 
as  a  major  cause  of  infantile  gastroenteritis  in  the  U.S. 

Norwalk  Virus  and  Traveler ' s  Diarrhea  -  In  collaboration  with  R.B.  Sack  and 
others  we  have  investigated  the  role  of  Norwalk  virus  in  traveler's  diarrhea. 
Serologic  studies  were  performed  on  sera  from  Peace  Corps  volunteers  in  Kenya, 
Morocco,  and  Honduras.   These  volunteers  were  involved  in  doxycycline  efficacy 
studies.   There  were  no  seroresponses  detected  in  Peace  Corps  workers  in  Kenya. 
In  Honduras  and  Morocco,  a  small  percentage  of  ill  individuals  (8-9%)  had 
evidence  of  Norwalk  infection.  (Three  individuals  in  the  Morocco  study  had 
serologic  evidence  of  Norwalk  infection  but  did  not  develop  diarrheal  illness; 
in  addition,  only  one  individual  in  the  Morocco  study  had  a  significant 
increase  in  rotavirus  antibody  but  did  not  develop  diarrheal  illness.)   These 
findings  imply  that  the  Norwalk  virus  is  one  of  the  minor  causes  of  tourista 
in  American  travelers.   In  an  interesting  study  of  tourista  in  Panamanians 
traveling  to  Mexico,  Norwalk  virus  infection  was  also  associated  with  traveler's 
diarrhea  (15%).   In  this  study,  rotavirus,  Norwalk  agent  and  Campylobacter 
were  more  important  than  toxigenic  E_  coli  as  possible  causes  of  traveler's 
diarrhea.   The  study  of  Panamanian  tourista  was  done  by  R.  Ryder  and  others. 

The  Role  of  Norwalk  Virus  in  Enteric  Gastroenteritis  -  Prospective  Family 
Studies  -  In  collaborative  studies  with  Dr.  W.  Rodriguez,  Children's  Hospital, 
Wash.,  D.C.,  Dr.  Mark  Gurwith,  UCLA  Medical  School  and  Dr.  R.  Guerrant,  Univ. 
of  Virginia,  we  studied  the  incidence  of  Norwalk  infection  in  families  followed 
prospectively  for  diarrheal  disease.   Preliminary  data  from  the  Rodriguez  and 
Guerrant  study  indicate  that  the  Norwalk  agent  might  be  associated  with 
between  6  and  30%  of  family  episodes. 

It  is  hoped  that  in  the  coming  months  further  analysis  of  this  serologic 
data  will  better  define  the  role  Norwalk  virus  plays  as  a  cause  of  endemic 

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sporadic  Infectious  diarrhea  in  both  children  and  adults  in  the  U.S.  and 
Canada.   Similar  prospective  studies  of  families  in  underdeveloped  countries 
are  needed. 

Detection  of  Norwalk  Virus  in  Vomitus  -  By  RIA,  we  examined  5  vomitus  specimens 
obtained  from  5  volunteers  experimentally  infected  with  the  Norwalk  virus. 
The  vomitus  specimens  were  obtained  during  acute  illness,  24-48  hours  after 
inoculation.   Four  of  the  5  specimens  were  positive  for  Norwalk  antigen  by  RIA. 
Following  100-fold  concentration  of  one  of  the  specimens,  the  Norwalk  particle 
was  visualized  by  IEM. 

Anti-viral  Therapy  for  Norwalk  Disease  -  In  a  collaborative  study  with  Dr. 
R.G.  Douglas  in  Rochester,  we  evaluated  the  therapeutic  efficacy  of  Bismuth 
Subsalicylate  (BSS  —  Pepto-Bismol)  for  Norwalk  gastroenteritis.   BSS  had 
previously  been  shown  to  be  an  effective  therapy  for  diarrhea  caused  by 
toxigenic  E  coli.   In  a  volunteer  study  involving  59  volunteers,  BSS  was  shown 
to  have  a  minor  but  significant  effect  on  the  course  of  Norwalk  virus 
gastroenteritis  reducing  both  the  severity  and  duration  of  cramps  but  not 
affecting  viral  shedding. 

Serologic  Tests  for  Relatedness  of  the  Norwalk  Virus  and  Other 
Gastrointestinal  Viruses  -  As  part  of  continuing  effort  to  find  other  viruses 
serologically  related  to  the  Norwalk  agent,  we  tested  antisera  raised  to  two 
new  candidate  animal  gastroenteritis  agents  that  morphologically  resemble 
Norwalk.   The  Newberry  agent  (a  calf  agent  from  Dr.  J.  Bridger,  England)  and 
several  dog  parvo-  and  calicivirus  isolates   (Dr.  L.  Binn,  Walter  Reed)  were 
examined  and  found  to  be  antigenically  unrelated  to  Norwalk  virus.   In  addition, 
antisera  to  an  astrovirus  (Dr.  McSwiggan,  England)  and  several  established 
caliciviruses  did  not  react  with  Norwalk  virus. 

Norwalk  and  Hawaii  Volunteer  Studies  -  23  volunteers  were  administered  the  8FIIA 
Norwalk  inoculum.   These  studies  were  carried  out  basically  for  two  reasons. 
Jejunal  secretions  were  gathered  so  that  the  local  immune  response  to  the 
Norwalk  virus  could  be  studied.   As  expected,  approximately  half  of  the 
inoculated  volunteers  became  ill.   Using  a  sensitive  RIABL ,we  could  find  no 
correlation  between  preexisting  serum  or  intestinal  fluid  antibody  titer  and 
protection.   We  also  found  that  a  subset  of  individuals  clinically 
resistant  to  Norwalk  illness  appeared  to  be  absolutely  refractory  to 
infection,  i.e.,  they  have  no  detectable  antibody  prior  to  or  after 
challenge  with  virus.   These  findings  may  indicate  that  a  proportion  of 
people  are  resistant  to  Norwalk  virus  on  a  genetic  or  physiologic  rather  than 
immunologic  basis.   A  similar  finding  had  been  made  by  Parrino  et  al  in 
Boston  in  a  collaborative  study  and  was  described  in  a  previous  annual  report. 

Using  stool  specimens  obtained  from  the  volunteer  studies,  we 

attempted  to  purify  whole  virion  and  a  soluble  viral  protein  found  in  stool. 

Norwalk  particles  are  present  in  small  amount  in  stool  so  that 

progress  has  been  slow.   Using  a  combination  of  isopycnic  and  rate  zonal 

ultracentrifugation,  iodination  and  polyacidamide  gel  electrophoresis,  we  have 

preliminary  evidence  showing  the  Norwalk  particle  contains  3  proteins, 


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2  approximately  70K  and  1  of  40-50K.   These  findings  most  closely  resemble  that 
described  for  parvoviral  proteins.  In  addition,  a  soluble  viral  protein 
was  found  in  stools  from  volunteers  infected  with  Norwalk  virus.   Using  gel 
filtration,  ion  exchange  chromatography  and  affinity  chromatography  we  have 
partially  characterized  this  protein.   It  has  a  molecular  weight  of 
approximately  45K  and  antibody  to  it  agglutinates  whole  virus. 

We  recently  carried  out  one  volunteer  study  with  the  Hawaii 

agent.   7  of  12  volunteers  became  ill.   Jejunal  secretions  were 

gathered  and  will  be  analyzed  in  the  future.   Stool  and  vomitus  specimens 

are  currently  being  looked  at  by  I EM  in  order  to  identify  particle  positive 

specimens.   These  will  be  used  in  the  development  of  a  Hawaii  RIA. 

Other  Agents  -  Another  27  nm  particle,  the  Marin  Co.  agent  (kindly  supplied  by 
Dr.  L.  Oshiro)  was  studied.   This  agent  was  clearly  associated  with  an 
outbreak  of  gastroenteritis  in  a  nursing  home  in  California.   It  is  not 
serologically  related  to  Hawaii  or  Norwalk  agents.   We  have  given  a  stool 
filtrate  preparation  of  the  agent  to  two  chimpanzees.   As  noted  before,  neither 
became  ill  or  seroconverted  by  IEM.   We  are  currently  safety  testing  this 
filtrate  for  future  studies  in  volunteers. 

Development  of  a  Solid-Phase  Microtiter  Radioimmunoassay  Blocking  Test  for 
Detection  of  Antibodies  to  Escherichia  coli  Heat-Labile  Enterotoxin  -  A 
solid-phase  microtiter  radioimmunoassay  for  the  detection  of  E_  coli  heat- 
labile  enterotoxin  was  described  in  a  previous  annual  report.   This  assay  was 
modified  into  a  blocking  test  to  detect  antibodies  to  E_  coli  heat-labile 
enterotoxin,  using  burro  antiserum  to  cholera  toxin  and  IgG  fraction  of 
this  serum  as  cross  reactive  antibody  to  _E  coli  heat-labile  toxin. 

A  comparison  of  the  efficiency  of  the  RIA  blocking  and  the  adrenal  cell 
neutralization  tests  for  detecting  serological  responses  in  volunteers 
experimentally  infected  with  toxigenic  E_  coli  revealed  that  both 
techniques  were  efficient  at  detecting  rises  in  ill  volunteers.   (The 
volunteer  studies  had  been  carried  out  by  Dr.  M.  Levine  et  al  of  the 
University  of  Maryland.)   Nine  of  10  volunteers  who  became  ill  developed  an 
RIA  blocking  antibody  response,  and  7  had  a  serological  response  when  tested 
by  the  adrenal  cell  method.   Proportionately  fewer  non-ill  volunteers  responded 
when  tested  by  either  method.   The  adrenal  cell  assay  detected  one  more 
seroresponse  in  the  non-ill  volunteers  than  did  the  RIA.   RIA  blocking  antibody 
titers  were  between  5-  and  10-fold  higher  than  adrenal  cell  titers,  and 
preexisting  antibody  was  detected  somewhat  more  commonly  by  the  RIA  blocking 
method. 

We  next  compared  the  efficiency  of  the  two  techniques  for  detecting 
seroresponses  using  paired  sera  from  adults  with  naturally  occurring 
diarrhea  in  Kenya  and  Bangladesh.   The  results  of  the  two  tests  were  similar. 
Both  methods  detected  significant  rises  in  antibody  in  just  under  one-half 
of  the  patients  shown  to  be  shedding  toxigenic  E_  coli  (5  of  12;  the  same  5 
individuals  developed  a  response  in  both  assays).   Of  14  patients  with  acute 
diarrhea  who  did  not  have  detectable  toxigenic  E_  coli  in  their  stools,  2 

23-33 


Z01  AI  00021-10  LID 

had  a  significant  antitoxin  rise  by  RIA  blocking  test  and  1  of  these  also 
developed  a  rise  by  adrenal  cell  neutralization  test.   Again  the  actual 
antibody  titer  of  these  sera  was  considerably  higher  in  the  RIA  blocking 
test  than  the  adrenal  cell  neutralization  assay;  however,  the  relative 
correlation  of  the  two  tests  was  high.   No  change  in  titer  was  detected  with 
the  RIA  blocking  test  when  four  paired  sera  from  volunteers  infected  with 
Norwalk  agent  or  four  paired  sera  from  children  with  naturally  acquired  human 
rotavirus  infection  were  tested. 

The  age  prevalence  of  serum  anti-LT  was  investigated  by  screening 
random  sera  obtained  at  Children's  Hospital,  Washington,  D.C.  and  Junior 
Village  over  the  past  15  years.   By  48  months  of  age,  over  50%  of  the 
population  studied  had  developed  antibody  to  E   coli  LT.  In  addition,  it  was 
found  that  over  70%  of  a  group  of  University  of  Maryland  students  aged  17 
through  26  had  detectable  serum  anti-LT. 

Because  the  RIA  blocking  technique  appeared  to  readily  detect  anti-LT 
in  pediatric  sera,  we  studied  the  acute  and  convalescent  bloods  obtained 
from  a  group  of  children  with  acute  diarrhea  seen  at  Children's  Hospital, 
Washington,  D.C.   This  group  of  patients  was  a  subset  of  a  previously 
studied  population.   They  were  patients  in  whom,  despite  extensive 
investigation,  human  rotavirus  could  not  be  implicated  as  a  cause  of  their 
diarrhea.   None  of  these  51  individuals  showed  evidence  of  a  serological 
response  to  E  coli  LT. 

Development  of  an  ELISA  for  Detection  and  Identification  of  Coxsackie 
A  Viruses  -  Many  of  the  Coxsackie  A  viruses  -  a  group  consisting  of  23 
serotypes  -  have  been  shown  to  cause  a  wide  variety  of  diseases.   However, 
many  strains  grow  poorly  if  at  all  in  conventional  cell  cultures  and  require 
suckling  mice  for  their  propagation.   The  difficulties  in  using  suckling  mice 
has  hampered  the  study  of  Coxsackie  virus  infections.   We  recently  developed 
an  ELISA  for  detecting  and  serotyping  Coxsackie  A  viruses  using  Coxsackie  virus 
type  specific  monkey  sera  as  precoat  (capture  antibody),  type  specific 
mouse  sera  as  second  antibody  (detector  antibody)  and  goat  anti-mouse 
globulin  conjugated  with  alkaline-phosphatase  as  indicator  antibody.   ELISA 
was  considerably  more  sensitive  than  CF  for  detecting  Coxsackie  virus. 
In  addition,  by  ELISA  we  were  able  to  correctly  type  strains  of  all  23 
recognized  serotypes  of  Coxsackie  A  virus.   With  22  of  the  23  viruses 
significant  positive  ELISA  reactions  were  noted  only  with  homotypic  sera. 
Some  cross  reactivity  was  found  with  Coxsackie  A-12  virus.   Coxsackie  A-12 
antigen  had  small  amounts  of  ELISA  reactivity  with  Coxsackie  A- 5  and  A-7 
antisera.   However,  the  Coxsackie  A-12  virus  cound  be  distinguished  from  the 
other  two  types  because  of  the  greater  P/N  value  of  the  homologous  reaction. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute 

Acute  infectious  nonbacterial  gastroenteritis  is  a  common  infectious  disease 
which  affects  a  broad  segment  of  the  population  and  was  the  second  most  common 
disease  observed  in  a  10  year  family  study  in  the  United  States.   In  addition, 
diarrheal  diseases  are  a  leading  cause  of  mortality  as  well  as  morbidity  in 

23-34 


Z01  AI  00021-10  LID 

developing  countries.   Ultimate  goals  of  prevension  and  therapy  have  been 
furthered  in  our  current  studies  by  (1)  the  detection  and  preliminary 
characterization  of  etiologic  agents  of  this  disease,  (2)  the  development  of 
sensitive,  efficient  and  rapid  assay  systems  by  which  the  epidemiologic 
importance  of  the  disease  can  be  determined,  (3)  the  in  vitro  cultivation  of 
type  2  human  rotavirus,  (4)  the  ability  to  induce  illness  in  human  volunteers 
and  experimental  animals  administered  the  human  rotavirus,  (5)  the 
elucidation  of  the  importance  of  intestinal  fluid  IgA  rotavirus  antibody  in 
preventing  rotavirus  illness  under  experimental  conditions,  (6)  development 
of  biophysical  and  serologic  methods  to  differentiate  the  human  rotavirus  from 
other  related  agents  and  (7)  the  elucidation  of  the  epidemiology  of  the 
rotavirus  and  Norwalk  viruses. 

Proposed  Course  of  Project 

In  future  studies  of  the  Epidemiology  Section  major  emphasis  will  be  given  to 
the  efficient  propagation  of  the  viral  gastroenteritis  agents  in  vitro,  to  the 
delineation  of  their  overall  importance  over  a  sustained  period  in  the  etiology 
of  gastroenteritis  in  various  populations,  to  the  immune  mechanisms  involved  in 
host-defense,  to  the  development  of  effective  methods  to  prevent  illness  due 
to  these  agents,  and  to  the  continued  search  for  other  etiologic  agents  of 
acute  infectious  gastroenteritis. 

Publications 

Wyatt,  R.G. ,  and  Kapikian,  A.Z.:   "Viral  Gastrointestinal  Infections  in 
Textbook  of  Pediatric  Infectious  Diseases.   R.D.  Feigin  &  J.D.Cherry 
(eds.),  W.B.,  Saunders  Co.,  Phila. ,  in  press. 

Greenberg,  H.B.,  Gebhard,  R.L.,  McClain,  C.J.,  Soltis,  R.D.,  and  Kapikian, 
A.Z.:   Antibodies  to  Viral  Gastroenteritis  Viruses  in  Crohn's  Disease. 
Gastroenterology.  76:  349-350,  1979. 

Rodriguez,  W.J.,  Kim,  H.W. ,  Brandt,  CD.,  Yolken,  R.H.,  Richard,  M. , 
Arrobio,  J.O.,  Schwartz,  R.H. ,  Kapikian,  A.Z.,  Chanock,  R.M. ,  Parrott, 
R.H.:  Common  Exposure  Outbreak  of  Type  2  Rotavirus  Gastroenteritis  With 
High  Secondary  Attack  Rate  Within  Families.  J.  Infect.  Pis.,  in  press. 

Yolken,  R.H. ,  Barbour,  B.A. ,  Wyatt,  R.G.,  Kapikian,  A.Z.:   Immune  Response 

to  Rotaviral  Infection-Measurement  by  Enzyme  Immunoassay.  JAVMA.  173: 

522-554,  1978. 

Hansen,  D.P.,  Kaminsky,  R.G.,  Bagg,  L.R.,  Kapikian  A. Z. ,  Slack,  R.C.B., 

Sack,  D.A. :   New  and  Old  Agents  in  Diarrhea:   A  Prospective  Study  of  an 

Indigenous  Adult  African  Population.   Am.  J.  Trop.  Med.  Hyg.  27(3):   609- 

615,  1978. 

Brandt,  CD.,  Kim,  H.W.,  Yolken,  R.H. ,  Kapikian,  A.Z.,  Arrobio,  J.O., 

Rodriguez,  W.J.,  Wyatt,  R.G.,  Chanock,  R.M.  and  Parrott,  R.H. :  Comparative 

Epidemiology  of  Two  Rotavirus  Serotypes  and  Other  Viral  Agents  Associated 

with  Pediatric  Gastroenteritis.  Amer.  J.  Epidemiol.,  in  press. 

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Z01  AI  00021-10  LID 

Yolken,  R.H.  ,  and  Kapikian,  A.Z.:  Rotavirus,  in  Principles  and  Practice 
of  Infectious  Diseases.  Mandell,  Douglas,  Bennett  (Eds.),  Wiley  &  Sons, 
in  press. 

Greenberg,  H.B.,  Wyatt,  R.G. ,  Kapikian,  A.Z. :  Norwalk  Virus  in  Vomitus. 
Lancet  1:  55,  1979  (letter). 

Metselaar,  D. ,  Sack,  D.A.,  Kapikian,  A.Z. ,  Muller,  A.S.:   Machakos  project 
studies  —  Agents  affecting  health  of  mother  and  child  in  rural  area  of 
Kenya.   XI.  Antibodies  against  rotavirus  of  children  living  in  the  Machakos 
district  of  Kenya.  Trop.  Geographical  Med.  30:  531-555,  1978. 

Sack,  R.B.,  Froehlich,  J.L.,  Zulich,  A.W. ,  Hidi,  D.S.,  Kapikian,  A.Z., 
Orskov,  F. ,  Orskov,  I.,  Greenberg,  H.B.:   Prophylactic  doxycycline  for 
traveler's  diarrhea.  Results  of  a  prospective  double-blind  study  of  Peace 
Corps  volunteers  in  Morocco.   Gastroenterology  76:  1368-1378,  1979. 

Kapikian,  A.Z.,  Barile,  M.F. ,  Wyatt,  R.G.,  Yolken,  R.H. ,  Tully,  j.G., 
Greenberg,  H.B.  ,  Kalica,  A.R.,  Chanock,  R.M. :   Mycoplasma  contamination  in 
cell  culture  of  Crohn's  disease  material.   Lancet,  in  press),  (letter). 

Kalica,  A.R. ,  Theodore,  T.S.:   Polypeptides  of  simian  rotavirus  (SA-11) 
determined  by  a  continuous  polyacrylamide  gel  electrophoresis  method. 
J.  Gen.  Virol.  43:  463-466,  1979. 

Kaplan,  J.E.,  Larrick,  J.W.,  Yost,  J.,  Calisher,  C.H.,  Farrell,  L. ,  Greenberg, 
H.B.,  Herrmann,  K.L.,  Sulzer,  A.J.,  Walls,  K.W. ,  Pederson,  L. :   Infectious 
disease  patterns  in  the  Waorani,  an  isolated  Amerindian  population.  Am.  J.  Trop, 
Med .  &  Hyg . ,  in  press. 

Greenberg,  H.B.,  Valdesuso,  J.,  Kapikian,  A.Z.,  Chanock,  R.M. ,  Szmuness, 
W. ,  Larrick,  J.,  Kaplan,  J.,  Gilman,  r.H. ,  Sack,  D.A. :   The  prevalence  of 
antibody  to  the  Norwalk  virus  in  various  parts  of  the  world.   Infect ■  Immun. , 
in  press. 

Yolken,  R.H. ,  Mata,L. ,  Urrutia,  J.J.,  Wyatt,  R.G.,  Chanock,  R.M.  and 
Kapikian,  A.Z.:   Secretory  antibody  directed  against  rotavirus  in  human 
mild — Measurement  by  means  of  enzyme-linked  immunosorbent  assay  (ELISA) . 
J.  Pediatr..  93:  916-921,  1978. 

Yolken,  R.H. ,  Wyatt,  R.G. ,  Barbour,  B.A. ,  Kim,  H.W. ,  Kapikian,  A.Z.,  and 
Chanock,  R.M. :   Measurement  of  anti-rotavirus  antibody  by  an  enzyme-linked 
immunosorbent  assay  (ELISA)  blocking  assay.   J.  Clin.  Microbiol.  8:   283- 
287,  1978. 

Chanock,  R.M.,  Wyatt,  R.G.,  and  Kapikian,  A.Z.:   Immunization  of  infants 
and  young  children  against  rotavirus  gastroenteritis — Prospects  and 
problems.   J.  Amer.  Vet.  Med.  Assoc.  173:  570-572,  1978. 


23-36 


Z01  AI  00021-10  LID 

VanKirk,  D.H.,  Kapikian,  A.Z.,  Wyatt,  R.  and  Kalica,  A.R. :   Viral  digestive 
tract  infections  in  CRC  Handbook  Series  in  Clinical  Laboratory  Science 
(Seligsan  editor-in-chief)  Section  H:   Virology  and  Rickettsiology  Volume 
1,  Part  2  (Section  Editoris  Hsiung  and  Green),  CRC  Press,  West  Palm  Beach, 
Fla,  pp  211-233,  1978. 

Kapikian,  A.Z.:   Identification  and  serology  of  rotavirus,  Norwalk  and 
Norwalk-like  agents.   In  Report  of  the  Seventy-Fourth  Ross 
Conference  on  Pediatric  Research.   Etiology,  Pathophysiology  and  Treatment 
of  Acute  Gastroenteritis,  Ross  Laboratories,  Columbus,  Ohio,  pp.  27-35, 
1978  (abridged  version) . 

Kapikian,  A.Z.,  Greenberg,  H.B.,  Cline,  W.L.,  Kalica,  A.R.,  Wyatt,  R.G., 
James,  H.D.  Jr.,  Lloyd,  N.L. ,  Chanock,  R.M. ,  Ryder,  R.W.  and  Kim,  H.W. : 
Prevalence  of  antibody  to  the  Norwalk  agent  by  a  newly  developed  immune 
adherence  hemagglutination  assay.   J.  Med.  Virology  2:  281-294,  1978. 

Kapikian,  A.Z. ,  Yolken,  R.H.  ,  Wyatt,  R.G.,  Chanock,  R.M. ,  Kim,  H.W. :  Viral 
diarrhea.   Etiology  and  control.   Amer.  J.  Clin.  Nutr.  31:  2219-2236, 
1978. 

Greenberg,  H.B. ,  and  Kapikian,  A.Z.:   Detection  of  Norwalk  agent  antibody 

and  antigen  by  solid-phase  radioimmunoassay  and  immune  adherence  hemagglutination. 

J.  Amer.  Vet.  Med.  Assoc.  173:  620-623,  1978. 

Kalica,  A.R. ,  Wyatt,  R.G. ,  Kapikian,  A.Z.:   Detection  of  differences  among 
human  and  animal  rotaviruses,  using  analysis  of  viral  RNA.   J.  Amer.  Vet.  Med. 
Assoc.  173:  531-537,  1978. 

Schieble,  J.H. ,  and  Kapikian,  A.Z. :  "Coronaviruses"  in  Diagnostic  Procedures 
for  Viral  and  Rickettsial  and  Chlamydial  Infections,  Fifth  Edition,  Lennette, 
E.H. ,  Schmidt,  N.J.  (Eds.),  in  press. 

Tyrrell,  D.A.J. ,  Alexander,  D.A.J. ,  Almeida,  J.D.,  Cunningham,  C.H. , 
Easterday,  B.C.,  Garwes,  D. J. ,  Hierholzer,  J.C.,  Kapikian,  A.Z.,  MacNaughton, 
M.R.,  and  Mcintosh,  K. :   Coronaviridae:  Second  Report.   Intervirology  10: 
321-328,  1978.  """  

Greenberg,  H.B.,  Levine,  M.M. ,  Merson,  M.H. ,  Sack,  R.B.,  Sack,  D.A., 
Valdesuso,  J.R. ,  Nalin,  D. ,  Hoover,  D.,  Chanock,  R.M. ,  and  Kapikian,  A.Z. : 
Solid-phase  microtiter  radioimmunoassay  blocking  test  for  detection  of 
antibodies  to  Escherichia  coli  heat-labile  enterotoxin.  J.  Clin.  Microbiol. 
9:  60-64,  1979. 

Kapikian,  A.Z.,  Yolken,  R.H. ,  Greenberg,  H.B.,  Wyatt,  R.G.,  Kalica,  A.R., 
Chanock,  R.M. ,  and  Kim,  H.W. :   "Viral  Gastroenteritis"  in  Lennette,  E.H., 
and  Schmidt,  N.J.  (Eds.),  Diagnostic  Procedures  for  Viral,  Rickettsial 
and  Chlamydial  Infections,  fifth  edition,  in  press,  1979.  , 


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Z01  AI  00021-10  LID 

Wyatt,  R.G.,  Yolken,  R.H. ,  Urrutia,  J.J.,  Mata,  L. ,  Greenberg,  H.B., 
Chanock,  R.M. ,  and  Kapikian,  A.Z. :   Diarrhea  associated  with  rotavirus  in 
rural  Guatemala:   A  longitudinal  study  of  24  infants  and  young  children. 
Amer.  J.  Trop.  Med.  Hyg.,  23:  325-328,  1979. 

Wyatt,  R.G.,Mebus,  C.A.,  Yolken,  R.H.,  Kalica,  A.R.,  James,  H.D.  Jr., 
Kapikian,  A.Z.,  and  Chanock,  R.M. :   Rotaviral  immunity  in  gnotobiotic 
calves:   Heterologous  resistance  to  human  virus  induced  by  bovine  virus. 
Science.  203:  548-550,  1979. 

Schoub,  B.D. ,  Kalica,  A.R.,  Greenberg,  H.B.,  Bertran,  D.M.,  Sereno,  M.M. , 
Wyatt,  R.G.,  Chanock,  R.M.  and  Kapikian,  A.Z.:   Enchancement  of  antigen 
incorporation  and  infectivity  of  cell  cultures  by  human  rotavirus.   J. 
Clinical  Microbiol.  9:  488-492,  1979. 

Greenberg,  H.B. ,  Valdesuso,  J.,  Yolken,  R.H. ,  Gangarosa,  E.,  Gary,  W. , 
Wyatt,  R.G.,  Konno,  1.,  Suzuki,  H. ,  Chanock,  R.M. ,  Kapikian,  A.Z.:  Role  of 
Norwalk  virus  in  outbreaks  o  non-bacterial  gastroenteritis.  J.  Infect. 
Pis.  139:  564-568,  1979. 


23-38 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Zol  AI  00022-?o 


PERIOD  COVERED 


October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

STUDY  OF  NONBACTERIAL  RESPIRATORY  DISEASES  IN  CHILDREN 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:   Robert  M.  Chanock 


LID/NIAID 


Chief 


COOPERATING  UNITS  (if  any) 

Children's  Hospital  National  Medical  Center,  Washington,  D.C. 


LAB/BRANCH 

Laboratory  of  Infectious  Diseases 


Respiratory  Viruses  Section 


INSTITUTE  AND  LOCATION 


National  Institute  of  Allergy  and  Infectious  Diseases,  Bethesda,  Maryland 


TOTAL  MANYEARS: 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  project  has  been  terminated. 


23-39 


PHS-6040 
(Rev.    10-76) 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00024-19  LID 


PERIOD  COVERED 


OCTOBER  1,  1978  TO  SEPTEMBER  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

LABORATORY  STUDIES  OF  MYXOVIRUSES 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

P.I.:   Brian  R.  Murphy,  M.D.   Medical  Officer  LID,  NIAID 

Robert  M.  Chanock.M.D.  Chief  LID,  NIAID 

Lewis  J.  Markoff,  M.D.  Medical  Officer  LID,  NIAID 

Susan  B.  Spring,  Ph.D.  Research  Microbiologist  LID,  NIAID 

Kazufumi  Shimizu,  M.D.  Visiting  Associate  LID,  NIAID 

Ching-Juh  Lai,  Ph.D.    Visiting  Scientist  LID,  NIAID 


COOPERATING  UNITS  (if  an/) 

Flow  Laboratories  (Control  t   NOl  AI  32521) 

NINCDS;  Bureau  of  Biologies,  FDA,  National  Cancer  Institute,  Immunology 
Branch 


LAB/BRANCH 

LABORATORY  OF  INFECTIOUS  DISEASES 


SECTION 

RESPIRATORY  VIRUSES  SECTION 


INSTITUTE  AND  LOCATION 

NATIONAL  INSTITUTE  OF  ALLERGY  AND  INFECTIOUS  DISEASES,  BETHESDA,  MARYLANE 


TOTAL  MANYEARS: 

129/12 


PROFESSIONAL: 

57/12 


72/12 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

D  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


(c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

ts  mutants  of  influenza  A  virus  were  produced  and  assigned  to  8  recombinantion 
groups.   Host  dependent  t_s  mutation  was  observed  and  found  to  be  widely 
distributed  through  the  viral  genome.   Intracistronic  complementation  was  also 
observed  for  _ts  mutations  affecting  the  PI,  P2,  NA  and  NP  genes.   The  mutations 
in  the  ts-lA2  master  donor  virus  were  shown  to  affect  the  genes  coding  for  the 
polymerase  1  (PI)  and  polymerase  3  (P3)  proteins.   Genotype  analysis  indicated 
that  transfer  of  the  PI  and  P3  ts_  genes  from  the  ts-lA2  donor  to  11  distinct 
virulent,  wild  type  viruses  led  to  a  defined  and  reproducible  level  of  attenuation 


of  these  recombinants  for  the  hamster's  respitatory  tract.   ts-lA2  recombinants 
were  stable  genetically  during  growth  in  experimental  animals.  However,  one 
doubly  seronegative  child  shed  _ts  virus  late  in  the  course  of  infection  with 
a  ts-lA2  recombinant.   In  this  instance  shift  from  the  t_s  to  _ts_  phenotype  was 
shown  to  be  associated  with  an  extragenic  suppressor  mutation. 


>3- 


PHS-6040 
(Rev.  10-76) 


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Z01  AI  00024-19  LID 

Project  Description 

Production  and  characterization  of  ts-lA2  recombinant  viruses  -  When  immunity 
to  both  surface  antigens  of  influenza  A  virus  is  lacking,  only  the  degree 
of  defectiveness  of  the  vaccine  virus  determines  attenuation  and  in  this 
situation  defectiveness  must  be  greater  than  that  specified  by  the  ts-l[E] 
lesions.   For  this  reason  we  constructed  a  recombinant  (1A2)  with  a  set  of 
ts  lesions  that  specified  a  greater  degree  of  defectiveness  than  that  seen 
with  the  ts-l[E]  recombinants.   Initially  ts  mutants  of  wild  type  influenza 
A  virus  were  produced  by  chemical  mutagenesis.   Next,  viruses  with  a  single 
ts  lesion  were  identified  among  the  _ts  progeny  of  mutagenized  virus  or  were 
produced  by  segregating  recombinants  with  single  lesions  from  mutants 
bearing  two  or  more  _ts_  lesions.   The  single  _ts_  mutants  were  characterized 
as  to  complementation-recombination  group  and  were  evaluated  for  degree  of 
growth  restriction  and  genetic  stability  in  vivo  (in  the  hamster).   In  this 
manner  we  identified  the  2  most  defective  and  stable  ts_  lesions  that  were 
genetically  distinct,  i.e.,  located  on  different  RNA  segments  of  the  genome. 
Finally,  we  combined  the  2  lesions  into  a  single  virus  by  genetic  recombination. 
The  resulting  recombinant  virus  (designated  1A2)  that  had  the  A/Udorn/72 
hemagglutinin  and  the  desired  two  _ts  lesions  (complementation  groups  1  and 
5)  was  more  restricted  in  replication  at  37°C  than  either  ts_  parent  or  the 
ts-l[E]  recombinants.   Genetic  evidence  suggested  that  the  1A2  _ts_  lesions 
were  located  on  the  gene  coding  for  P3  protein  and  the  gene  coding  for  PI 
protein;  both  of  the  affected  genes  are  believed  to  code  for  large  proteins 
throught  to  be  involved  in  the  synthesis  of  complementary  RNA. 

Genotyping  of  ts-lA2  recombinants  -  Background  studies  were  thus  undertaken 

to  ascertain  which  RNA  segments  of  the  Udorn/72-ts-lA2  virus  bear  the  _ts_ 

lesions.   The  _ts_  lesion  in  the  group  1  had  been  shown  to  be  on  RNA  segment 

1  which  codes  for  the  P3  polymerase.   To  identify  the  RNA  segment  that 

bears  the  group  5  ts  lesion  the  HK/68-ts-315  virus  (the  donor  of  the  group 

5  _ts  lesion  to  the  _ts_-LA2  virus)  was  crossed  with  the  a/WSN/1933  (H0N1) 

wild  type  virus  and  WSN  _ts_  progeny  were  genotyped  by  polyacrylamide  gel 

electrophoresis  to  determine  which  315  gene  co-segregated  with  the  group  5 

ts  phenotype.   By  analyzing  these  WSN  X  ts-315  recombinants  and  another  set 

of  recombinants,  the  group  5  _ts  lesion  was  shown  to  co-segregate  with  the 

RNA  2  segment  which  codes  for  the  PI  polymerase  protein.   The  gel  electrophoretic 

data  thus  confirm  the  complementation  analysis  which  together  demonstrate 

that  the  Udorn/72-ts-LA2  virus  has  jts_  lesions  on  its  RNA  1  and  RNA  2  segments 

which  code  for  P3  and  PI  polymerase  respectively. 

The  acquisition  of  a  _ts  gene(s)  by  a  wild-type  has  been  consistently 
associated  with  a  reduced  level  of  replication  of  the  virus  in  hamsters' 
lung  and  with  reduced  virulence  in  man.   This  consistant  association  has 
led  to  the  conclusion  that  _ts  genes  are  responsible  for  the  attenuation. 
However,  non-ts  genes  present  in  the  _ts_  donor  virus  can  also  be  transferred 
to  recombinants  bearing  wild  type  surface  antigens.  Since  non-ts  genes  can 
also  affect  attenuation,  it  is  necessary  to  examine  the  importance  of  these 


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genes  as  well  as  the  _ts_  genes  in  reduction  of  virulence.   A  study  was 
therefore  undertaken  to  determine  which  of  the  Udom/72-ts-lA2  genes,  i.e., 
ts  or  non-ts,  were  responsible  for  attenuaton.   The  Udorn/72-ts-lA2  virus 
had  been  mated  (last  year's  annual  report)  with  A/Victoria/75  wild  type 
virus  and  six  Vic/75- ts-lA2  recombinant  viruses  were  isolated.   One  had 
only  the  P3  jts  lesion  and  the  remaining  five  possessed  both  _ts_  lesions 
(i.e.,  _ts  PI  and  _ts  P3)  .   The  single  lesion  virus  was  100-fold  reduced  in 
its  replication  in  the  hamster's  lung,  whereas  the  Vic/75-ts  recombinants 
that  possessed  both  _ts  lesions  exhibited  a  10,000  fold  reduction  of  pulmonary 
viral  replication.   The  six  Vic/75- ts-LA2  recombinants  were  genotyped  to 
determine  which  gene(s)  present  in  the  Udorn/72-ts-lA2  virus  conferred  the 
property  of  marked  restriction  of  pulmonary  virus  replication  on  the  Vic/ 75- 
ts-lA2  recombinants.   Each  of  the  five  Vic/75-ts-lA2  recombinants  that 
received  both  of  the  ts-lA2  ts  genes  exhibited  the  same  properties  in  vitro 
and  in  vivo  and  were  indistinbuishable  in  this  regard  from  their  Udorn/72- 
ts-lA2  parent.   The  Udorn/ 72-ts-lA2  genes  at  all  other  loci  segregated 
independently  of  these  in  vitro  and  ui  vivo  properties.   These  observations 
indicated  that  the  two  ts-lA2  ts  genes  were  responsible  for  attenuation  and 
suggest  that  the  acquisition  of  the  two  ts-lA2  ts  lesions  by  a  virus  can 
effect  a  predictable  level  of  restriction  of  replication  in  hamsters,  and, 
by  inference,  attenuation  in  man. 

A/Alaska/77-ts-lA2  (H3N2)  recombinants  -  The  ability  of  the  ts_  genes  of  the 
Udorn/ 72-ts-lA2  virus  to  reproducibly  attenuate  additional  influenza  A  wild 
type  viruses  within  the  H3N2  subtype  was  evaluated  further  by  mating  this 
donor  virus  with  the  A/Alaska/77  wild  type  virus.   The  four  Alaska/77  ts- 
1A2  recombinant  viruses  that  received  the  P3  and  PI  _ts_  genes  of  the  Udorn/72- 
ts-lA2  virus  had  a  37°C  shutoff  temperature  and  exhibited  restriction  of 
growth  in  pulmonary  and  nasal  turbinate  tissues  similar  to  their  attenuated 
Udorn/ 72-ts-lA2  parent.   Eleven  additional  Alaska/ 77-ts-lA2  recombinants 
were  obtained  and  fell  into  two  groups:  (1)  segregants  with  the  group  1 
(P3)  lesion  and  (2)  segregants  with  the  group  5  (PI)  _ts_  lesion. 

The  relative  contribution  of  the  _ts_  P3  and  PI  genes  to  attenuation  and 
genetic  stability  of  the  Udorn/ 72-ts-IA2  virus  and  that  of  its  recombinants 
was  estimated  by  evaluating  shutoff  temperature,  level  of  replication  in 
hamsters,  and  genetic  stability  of  ts-lA2  single  lesion  segregant  viruses. 

The  Alaska/ 77  ts  P3  segregants  were  not  a  homogenous  group  of  viruses  with 
respect  to  shutoff  temperature  or  level  of  replication  in  vivo.   P3  segregants 
had  a  37°,  38°,  or  39°C  shutoff  temperature.   The  existence  of  _ts  P3  segregants 
with  a  different  shutoff  temperature  allowed  us  to  examine  the  varying 
effects  of  _ts  mutation  in  one  gene  on  replication  in  vivo.   The  two  _ts  P3 
segregants  with  a  37°C  shutoff  temperature  were  more  restricted  in  replication 
in  the  lungs  and  nasal  turbinates  than  the  38°C  ts_  P3  segregant.   This 
suggests  that  the  level  of  temperature  sensitivity  of  a  virus  with  a  ts_ 
lesion  in  the  P3  gene  is  a  major  determinant  of  growth  restriction  in  vivo. 
These  data  also  suggest  that  the  defectiveness  of  a  ts  mutation,  i.e., 


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Z01  AI  00024-19  LID 

temperature  sensitivity,  at  a  given  genetic  locus  specifies  the  extent  to 
which  replication  is  restricted  in  vivo.   Lung  isolates  from  19  hamsters 
infected  with  _ts  P3  segregants  retained  the  _ts_  phenotype,  as  did  44  previously 
studied  isolates  from  A/Vic/75  P3  segregants.   Thus  a  total  of  63  isolates 
from  the  lungs  of  hamsters  infected  with  tjs  P3  segregants  derived  from  the 
Udorn/72-ts-lA2  parent  retained  their  temperature  sensitivity.   This  demonstrates 
the  high  level  of  genetic  stability  of  the  _ts  P3  gene  present  in  the  Udorn/72- 
ts-lA2  parent  and  its  recombinants. 

Similar  observations  were  made  for  the  Alaska/77-ts-lA2  ts  PI  segregants. 

The  38° C  shutoff  J^s_  PI  segregant  (clone  6)  was  more  restricted  in  replication 

in  the  hamster's  lungs  than  the  39°C  (clone  33)  shutoff  segregant.   Interestingly, 

and  for  reasons  that  are  not  clear,  the  38°C  _ts  P3  segregant  (clone  249) 

was  less  restricted  in  pulmonary  replication  than  the  38°C  _ts  PI  segregant. 

A  loss  of  the  _ts  phenotype  after  in  vivo  replication  was  seen  with  the  ts_ 

PI  clone  6  segregant  and  the  Hong  Kong/68-ts-315  parent.   In  general,  the 

level  of  replication,  genetic  stability  after  in  vivo  replication,  and 

shutoff  temperature  of  the  _ts_  PI  segregants  were  similar  to  that  of  the 

Hong  Kong/ 68- ts- 315  (ts  PI  donor)  virus  used  to  produce  the  Udorn/72  ts-lA2 

virus. 

The  observations  just  described  confirms  that  the  Udorn/72-ts-lA2  donor 
virus  has  two  genes  with  _ts_  lesions.   The  _ts_  P3  gene  appears  to  confer  on 
recombinants  a  greater  degree  of  restriction  of  replication  in  the  nasal 
turbinates  and  lungs  and  a  greater  degree  of  genetic  stability  than  does 
the  _ts  PI  gene.   The  acquisition  of  both  ts-LA2  ts  gene  genes  are  needed  to 
confer  a  predictable  set  of  genetic  and  biological  properties  on  viruses 
within  the  H3N2  subtype. 

Four  of  the  15  Alaska/77-ts-lA2  recombinants  were  genotyped  and  it  is  clear 
that  the  assignment  of  _ts_  genes  by  complementation-recombination  analysis 
accurately  identified  the  parental  origin  of  the  P3  and  PI  ts_  genes  in  the 
ts-lA2  recombinants.  Each  of  15  Alaska/77  ts-lA2  recombinants  is  currently 
being  genotyped  and  these  genetic  patterns  will  be  compared  to  shutoff 
temperature  and  replication  in  vivo.   In  this  way  we  hope  to  better  understand 
the  genetic  mechanism(s)  underlying  the  heterogeneity  of  the  P3  and  PI 
segregant  viruses. 

A/Hong  Kong/ 7 7  ts-lA2  (H1N1)  recombinants  -  Transfer  of  the  P3  and  PI  ts 
genes  present  in  the  Udorn/72-ts-lA2  donor  virus  to  two  H3N2  wild  type 
viruses  imposed  a  similar  level  of  restriction  of  pulmonary  viral  replication 
in  the  hamster  on  each  of  nine  H3N2-ts-lA2  recombinants.   A  study  was 
undertaken  to  determine  if  the  transfer  of  the  two  ts-lA2  ts  genes  into  a 
virus  belonging  to  a  different  influenza  A  subtype,  the  influenza  A/Hong 
Kong/123/77  (H1N1)  wild  type  virus,  would  result  in  restriction  of  replication 
in  vitro  and  in  vivo  comparable  to  that  observed  with  the  two  H3N2  viruses. 
Eleven  HK/77-ts-lA2  recombinants  were  obtained  and  these  fell  into  three        . 
subsets.   One  subset  had  both  ts  lesions  of  the  Udorn/72-ts-lA2  virus  and  a 


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Z01  AI  00024-19  LID 


37° C  shutoff  temperature  like  their  _ts_  parent.   The  second  subset  consisted 
of  two  clones  with  the  P3  _ts_  lesion  and  a  37°C  shutoff  temperature.   The 
third  subset  contained  7  clones  that  possessed  the  PI  _ts_  lesion  and  had  a 
37°,  38°,  or  39°C  shutoff  temperature.   When  mated  with  each  other,  the 
HK/77-ts-lA2  recombinants,  in  general,  interacted  as  expected,  i.e.,  genetic 
interaction  between  the  P3  and  PI  _ts  segregants  and  a  lack  of  interaction 
between  viruses  that  shared  a  _ts  lesion.   However,  unexpectedly,  genetic 
interaction  occurred  between  one  recombinant  and  4  others  that  each  possessed 
the  PI  _ts_  lesion.   This  suggested  that  intracistronic  complementation 
occurred  between  certain  PI  j^s_  segregants.   These  recombinants  are  being 
genotyped  to  determine  if  they  indeed  derived  their  PI  gene  from  the  Udorn/72 
ts-lA2  parent. 

The  HK/77  (H1N1)  wild  type  virus  was  attenuated  to  the  anticipated  level  by 
acquisition  of  the  two  _ts_  genes  from  the  Udorn/72-ts-lA2  parent.   The  two 
HK/77-ts-lA2  recombinant  viruses  (clones  92  and  144)  that  received  the  P3 
and  PI  _ts  genes  of  the  Udorn/72-ts-lA2  virus  had  a  37°C  shutoff  temperature. 
The  clone  144  was  examined  in  some  detail  and  was  found  to  exhibit  restriction 
of  growth  in  pulmonary  and  nasal  turbinate  tissues  similar  to  that  of  its 
attenuated  Udorn/72-ts-lA2  parent.   All  isolates  obtained  from  the  nasal 
turbinates  of  hamsters  infected  with  parent  and  recombinant  ts-lA2  viruses 
were  ts_.   Thus,  segregation  of  the  two  ts-lA2  genes  into  eleven  separate 
Udorn/72  (H3N2) ,  Vic/75  (H3N2) ,  Alaska/77  (H3N2)  and  HK/77  (H1N1)  ts-lA2 
recombinants  was  regularly  associated  with  (1)  37°C  restriction  of  plaque 
formation  (2),  marked  restriction  of  replication  in  the  lungs  of  hamsters, 
and  (3)   a  100-fold  restriction  of  replication  in  the  nasal  turbinates,  and 
(4)  genetic  stability  after  replication  in  hamsters.   Since  it  was  demonstrated 
that  the  P3  and  PI  _ts_  genes  of  the  ts-lA2  virus  were  responsible  for  the 
attenuation  of  the  Vic/75- ts-lA2  recombinant,  it  is  reasonable  to  assume 
that  these  two  _ts_  genes  were  responsible  for  the  attenuation  of  the  Alaska/77 
and  HK/77  ts-lA2  viruses  in  the  present  studies.   Thus,  the  two  ts-lA2  ts 
genes  attenuated  influenza  A  viruses  belonging  to  two  distinct  subtypes  to 
a  specific  and  predictable  level. 

Characterization  of  ts  Virus  Isolated  from  a  Child  Who  Received  Alaska/72- 
ts-LA2  Virus  -  Evidence  for  Suppressor  Mutation  -  A  seronegative  child 
given  a  Alaska/ 77  ts-lA2  recombinant  that  had  a  37°C  shutoff  temperature 
for  plaque  formation  (clone  190)  shed  virus  on  days  7,  8,  and  9  that  produced 
plaques  efficiently  at  39°C.   However,  this  altered  virus  failed  to  produce 
plaques  at  40°C,  a  temperature  at  which  wild  type  Alaska/ 77  virus  produced 
plaques  efficiently. 

Among  the  reoviruses,  which  also  have  a  segmented  RNA  genome,  loss  of  the 
ts  phenotype  is  usually  the  result  of  extragenic  suppressor  mutation  (Ramig 
and  Fields,  1979).   This  can  be  shown  by  backcrossing  the  _ts_  "revertant" 
virus  with  wild  type  virus  and  recovering  ts  progeny  virus  belonging 
to  the  original  complementation  -  recombination  group.  A  similar  analysis 
was  performed  for  the  Alaska/ 77- ts-lA2  ts   isolate  by  mating  it  with 


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Z01  AI  00024-19  LID 

Alaska/77  wild  type  virus.   Twenty-two  percent  of  progeny  virus  from  this 
mating  was  _ts  and  each  _ts  clone  possessed  the  P3  ts_  lesion  present  in  the 
ts-lA2  parent.   However,  the  t_s_  segregants  possessing  the  P3  _ts_  gene  were 
less  temperature  sensitive  than  P3  ts_  segregants  derived  from  mating  Udorn/72- 
ts-LA2  with  wild  type  virus;   this  indicates  that  two  types  of  genetic 
alteration  occurred  in  the  Alaska/ 7  7-_ts-LA2  virus  during  replication  in  a 
seronegative  child.   One  type  involved  a  suppressor  mutation  in  another 
gene  (i.e.,  not  the  P3  gene)  that  partially  corrected  the  _ts  phenotype  of 
the  ts-lA2  virus.   The  second  type  was  an  alteration  affecting  the  P3  and 
PI  genes;  this  type  of  change  could  result  from  reversion  of  one  of  several 
distinct  mutations  or  it  could  represent  intragenic  suppression.   A  mutation 
probably  occurred  on  the  P3  gene  of  the  Alaska/ 77-ts-lA2  ts   isolate  because 
this  segment  migrated  more  slowly  on  polyacrylamide  gel  than  the  corresponding 
RNA  segment  of  the  parent  Alaska/77-ts-lA2  recombinant.   This  observation 
favors  the  possibility  that  intragenic  suppression  was  responsible  for 
decreased  temperature  sensitivity  of  the  P3  _ts  segregants  derived  from  the 
ts  isolate. 

The  observation  that  the  tjs  virus  was  isolated  from  the  first  seronegative 
child  who  received  the  Alaska/ 77-ts-lA2  virus,  whereas  77  isolates  from  11 
children  who  shed  Vic/75-ts-lA2  remained  _ts_  raised  the  possibility  that  the 
Alaska/ 7 7-ts_-LA2  virus  was  already  genetically  altered  before  being  administered 
to  volunteers.   This  was  possible  even  though  the  Alaska/ 77- ts-lA2  recombinant 
had  the  same  level  of  temperature  sensitivity  as  the  Vic/75-ts-lA2  recombinant. 
Vic/75-ts-LA2,  Alaska/77-ts-lA2,  and  HK/77  (HlNl)-ts-LA2  viruses  were 
therefore  inoculated  at  a  multiplicity  of  infection  of  .001  onto  MDCK 
monolayer  cultures  and  incubated  at  34°  and  37°C.   Each  virus  caused 
cytopathic  effects  (CPE)  at  34°.   At  37°C  none  of  20  HK/77-ts-lA2,  2  of  20 
Vic/75-ts-LA2  and  10  of  20  of  Alaska/77  ts-lA2  virus-infected  cultures 
developed  CPE  after  3  to  4  days.   Only  cultures  infected  with  Alaska/ 7 7-ts- 
1A2  virus  yielded  virus  at  37°C  that  produced  plaques  at  38°C.   In  a  separate 
experiment  each  of  11  ts-lA2  recombinants  (H3N2  and  H1N1)  containing  the 
two  ts-lA2  lesions  were  evaluated  in  the  same  way  and  only  the  Alaska/77- 
ts-lA2  virus  clone  190  produced  CPE  at  37°C.   These  preliminary  results 
indicate  that  the  Alaska/ 77-ts-LA2  clone  190  virus  readily  changed  from  a 
37°C  to  a  39°C  shutoff  mutant  in  tissue  culture  after  1  passage  at  the 
restrictive  temperature  of  37°C.   This  suggests  that  this  virus  underwent 
genetic  change  during  recombination  and/or  passage  and  that  this  change 
increased  the  probability  that  other  changes  could  occur  (?  intragenic 
and/or  extragenic  suppression)  resulting  in  a  decrease  in  temperature 
sensitivity.  The  simple  in  vitro  technique  used  to  demonstrate  this  phenomenon 
may  prove  useful  in  identifying  "labile"  ts-lA2  recombinants  not  suitable 
for  study  in  man. 

Production  of  a  New  ts-l[E]-like  Donor  Virus  -  It  has  been  suggested  that 
perhaps  two  donor  viruses  might  be  needed  for  effective  immunoprophylaxis 
against  influenza  A  virus.   One  donor,  such  as  the  ts-lA2,  would  be  used  in 


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(a)  young  children  who  lack  experience  with  influenza  A  virus  and  (b)  in 
all  individuals  at  the  time  of  a  pandemic  involving  a  shift  in  both  HA  and 
NA  antigens.   The  other  donor,  similar  to  the  ts-l[E]  virus,  would  be  used 
during  interpandemic  periods  in  children  and  adults  who  have  some  NI  and  HI 
immunity.   The  HK/68-ts-l[E]  virus  was  suitably  attenuated  in  this  situation, 
but  showed  significant  tendency  to  lose  its  temperature  sensitivity  in  the 
hamster's  lungs,  in  doubly  seronegative  children,  and  after  passage  in 
tissue  culture,  organ  culture  or  eggs.   The  ts-l[E]  virus  has  _ts  lesions  on 
the  P3  and  NP  genes  and  has  a  38° C  shutoff  temperature.   Segragants  from 
the  ts-l[E]  virus  that  contained  the  P3  or  NP  ts_  gene  had  a  39°C  shutoff 
temperature  and  were  less  stable  genetically  than  the  parental  ts-l[E] 
virus  (shutoff  temperature  38°C)  after  replication  in  the  hamster's  lungs 
indicating  that  the  ts-l[E]  donor  virus  possessed  two  unstable  ts_  genes. 
For  this  reason  we  produced  a  series  of  recombinant  "ts-l[E]-like"  viruses 
that  contained  _ts  P3  and  NP  genes  that  were  more  stable  than  those  found  in 
the  ts-l[E]  donor  virus.   The  single  lesion  donors  of  the  _ts_  P3  and  NP 
genes  were  more  stable  genetically  than  the  double  lesion  ts-l[E]  master 
strain.  Three  recombinant  viruses,  clone  20,  53  and  55,  were  isolated; 
these  viruses  possessed  _ts  P3  and  _ts_  NP  genes  and  had  a  37°,  38°,  or  39°C 
shutoff  temperature.  Current  studies  are  directed  at  evaluating  the  genetic 
stability  of  these  recombinant  virus  in  the  hamsters  to  determine  if  they 
are  indeed  more  stable  than  the  ts-l[E]  virus. 

Identification  of  a  Simian  Species  Permissive  for  Influenza  A  Virus  - 
Attenuation  of  new  strains  of  influenza  A  virus  can  be  accomplished  rapidly 
by  the  transfer  of  attenuating  genes  from  an  attenuated  donor  virus  to  new 
epidemic  or  pandemic  strains  by  genetic  reassortment.   However,  evaluation 
of  such  recombinants  for  their  usefulness  in  immunoprophylaxis  of  influenza 
has  proceeded  slowly  since  volunteers  remain  the  most  reliable  indicator  of 
satisfactory  attenuation.   For  this  reason  it  would  be  advantageous  to 
identify  a  simian  host  in  which  recombinants  could  be  evaluated  in  a 
preliminary  manner  prior  to  tests  in  man.   Such  a  simian  species  would  be 
especially  useful  for  evaluation  of  host  range  mutants  since  infection  of  a 
susceptible  subhuman  primate  species  should  most  closely  approximate  that 
of  man.   The  virulence  of  three  cloned  influenza  A  viruses  for  man  and  for 
three  readily  available  species  of  monkeys  (owl,  squirrel  and  cebus)  was 
compared  in  an  attempt  to  identify  a  species  of  monkey  that  could  be  used 
to  investigate  the  genetic  basis  of  attenuation  of  influenza  A  viruses  for 
man.   Of  the  three  species  tested  squirrel  monkeys  developed  mild  illness 
confined  to  the  upper  respiratory  tract  in  response  to  three  different 
viruses  belonging  to  the  H3N2  or  H1N1  subtypes.   This  susceptibility  was 
accompanied  by  a  high  level  of  virus  shedding.   Each  of  the  nine  squirrel 
monkeys  that  shed  greater  than  10  "   TCID,-n/ml  of  virus  developed  illness, 
whereas  those  that  shed  less  remained  well.   None  of  the  cebus  monkeys 
attained  this  level  of  shedding,  while  2  owl  monkeys  did  so  without  evidence 
of  illness. 


Previous  studies  indicated  that  squirrel  monkeys  developed  clinically 
apparent  influenzal  illness  when  given  A/New  Jersey/76  (Hswine  Nl)  or  an 
A/Aichi/68  (H3N2)  virus.   These  findings  in  combination  with  the  present 
observations  indicate  that  the  squirrel  monkey  regularly  develops  objective 

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upper  respiratory  illness  after  infection  with  influenza  A  viruses  belonging 
to  the  HO-Hl-Hswine  or  H3N2  subtypes.  In  contrast,  the  cebus  monkey  appears 
to  respond  less  reproducibly  to  influenza  A  virus  infection  in  that  only  1 
of  3  H3N2  viruses  and  1  of  2  HO-Hl-Hswine  viruses  induced  illness.   Thus, 
the  squirrel  monkey  appears  to  be  a  moderately  permissive  primate  host  in 
which  to  investigate  the  genetic  basis  of  virulence  of  human  influenza  A 
viruses. 

Use  of  ELISA  to  Measure  the  Antibody  Response  to  Influenza  A  and  B  Viruses  - 
An  important  aspect  in  the  evaluation  of  a  live  influenza  A  vaccine  strain 
is  determination  of  the  human  infectious  dose  50  (HID,.  ) .   Infection  with  a 
vaccine  strain  is  ascertained  by  isolation  of  virus  from  the  vaccinee 
and/or  by  detection  of  a  rise  of  antibody  titer  in  the  serum  or  nasal  wash. 
As  candidate  mutant  vaccine  strains  of  greater  defectiveness  have  been 
developed  our  ability  to  detect  infection  in  vaccinees  by  virus  isolation 
has  decreased.   Thus,  increased  reliance  has  been  placed  on  immunological 
methods  for  detection  of  infection.   The  hemagglutination-inhibition  (HI) 
and  neuraminidase-inhibition  (NI)  techniques  are  the  most  widely  used 
methods  to  measure  serum  antibody.   In  some  of  our  recent  vaccine  trials, 
we  detected  a  serum  HI  antibody  rise  in  less  than  50%  of  vaccinees.   This 
indicated  the  need  to  develop  more  sensitive  tests  for  detection  of  a 
seroresponse  to  highly  attenuated  vaccine  virus.  Recently,  radioimmunoassays 
and  enzyme-linked  immunosorbant  assays  (ELISA)  were  shown  to  be  more  sensitive 
than  conventional  serologic  tests  for  detection  of  serum  antibody  to  influenza 
virus.   For  this  reason  the  simpler  ELISA  was  adapted  to  detect  rises  in 
serum  antibody  in  vaccinees  who  received  an  influenza  A  wild  type  or  vaccine 
virus. 

Pre  and  post-vaccination  sera  were  diluted  and  assayed  on  plates  containing 
whole,  zonally  purified,  inactivated  virus.   The  area  between  the  pre  and 
post  immunization  curves  (optical  density  versus  serum  dilution)  was 
determined.   Areas  greater  than  50  were  indicative  of  an  antibody  rise. 
ELISA  was  more  sensitive  for  detecting  an  antibody  response  in  recipients 
of  live  virus  vaccine  than  were  HI  and  NI  tests.   With  H1N1  and  H  swine  Nl 
viruses,  ELISA  was  superior  to  NI  and  HI  for  detection  of  an  antibody 
response.  This  was  also  true  for  the  H3N2  virus  tested.   Of  interest,  an 
anti-human  IgG  conjugate  was  not  able  to  detect  all  rises  by  ELISA.   Only  3 
of  8  H1N1  vaccinees  developed  a  response  when  tested  with  an  anti-IgG 
conjugate,  whereas,  each  of  the  8  vaccinees  developed  a  response  detectable 
with  an  anti-IgM  conjugate.   Since  these  vaccinees  lacked  prior  experience 
with  H1N1  surface  antigens,  the  predominance  of  IgM  antibody  in  their 
primary  response  was  not  unexpected. 

Use  of  whole  virus  as  antigen,  in  the  ELISA  measures  antibody  responses  to 
the  surface  glycoproteins  (the  hemagglutinin  and  neuraminidase)  as  well  as 
to  the  matrix  protein  and  nucleoprotein.   Antibodies  to  the  hemagglutinin 
and  neuraminidase  are  associated  with  resistance  to  influenza  A  virus, 
whereas  the  other  antibodies  do  not  appear  to  contribute  to  resistance.   To 
measure  only  those  antibodies  associated  with  resistance,  hemagglutinin  and 


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neuraminidase  specific  ELISA  were  developed.   Wells  of  a  microtiter  plate 
were  coated  with  200  ug  of  purified  hemagglutinin  from  the  A/USSR/77  (H1N1) , 
A/Texas/77  (H3N2) ,  or  B/Hong  Kong/72  virus  produced  in  collaboration  by  Dr. 
Michael  Phalem  of  BOB,  FDA.   Sera  from  vaccinees  were  assayed  using  an 
ELISA  and  HI  test  against  homologous  and  heterologous  hemagglutinins. 
Rises  were  detected  by  ELISA  and  HI  only  against  homologous  antigens. 
However,  the  ELISA  was  more  sensitive  in  detecting  rises  than  the  HI  test 
with  each  of  the  three  hemagglutinins;  an  ELISA  rise  of  1.5  log  occurred 
for  each  1.0  log  HI  rise.   Sera  from  children  who  had  not  been  exposed  to 
influenza  A  or  B  virus  possessed  ELISA  titers  of  _<1:20  and  post-infection 
sera  registered  titers  of  1:640  to  1:20,460.   The  ELISA  therefore  greatly 
extends  the  range  of  detection  of  antibody  compared  to  the  conventional  HI 
test.   There  is  a  good  correlation  between  HI  and  ELISA  titers  with  HI 
titers  of  1:8  corresponding  to  ELISA  titers  of  1:160  to  1:640.   Modifications 
of  the  ELISA  to  detect  neuraminidase  antibody  and  to  detect  nasal  wash 
antibody  are  in  progress. 

Genotyping  of  influenza  A  virus  recombinants  -  New  _ts  mutants  derived  by  5- 
FU  mutagenesis  of  influenza  A  Udorn/72  virus  were  isolated  and  characterized 
as  previously  described.   These  were  assigned  to  complementation  groups 
represented  by  seven  HK/68  prototype  _ts  mutants  and  two  new  complementation 
groups.   One  of  these  mutants,  Udorn/72-ts-368,  was  evaluated  for  the  locus 
of  its  _ts_  lesion  by  a  mating  with  WSN/33  wild  type  virus.   We  selected  for 
recombinant  viruses  that  were  ts_  (39°C  titer/34°C  titer  10   )  and  retained 
the  WSN/33  hemagglutinin  (HO).   Ten  such  recombinants  were  cloned  and 
evaluated  by  polyacrylamide  gel  electrophoresis  (PAGE)  in  the  presence  of 
6M  Urea,  as  previously  described.   All  of  these  viruses  retained  RNA  6  of 
the  Ud/72-ts-368  parent  virus,  and  one  recombinant  clone  218,  was  shown  to 
have  received  seven  RNA  segments  from  the  WSN/33  wild  type  parent  and  only 
one,  RNA  6,  from  its  Ud/72  _ts  parent.   RNA  6  is  known  to  represent  the  NP 
gene  of  Ud/72  virus  under  these  conditions  of  electrophoresis.   Clone  218 
was  further  evaluated  for  efficiency  of  plaque  formation  and  in  complementation- 
recombination  assays.   In  each  instance,  it  behaved  exactly  as  its  Ud/72- 
ts-368  parent,  indicating  that  mutation  in  the  Ud/72  RNP  gene  of  clone  218 
accounts  for  all  of  its  properties  as  a  _ts  virus.   These  results  were 
consistent  with  earlier  findings  by  others  that  HK/68  _ts  mutant  R8,  our 
prototype  complementation  group  2  mutant,  also  bears  its  ts_  lesion  in  the 
NP  gene. 

Attempts  were  made  to  determine  the  locus  or  loci  of  the  _ts_  lesion  or 
lesions  of  several  other  newly  derived  Ud/72  _ts  mutants,  in  particular 
those  that  bear  _ts_  lesions  not  represented  among  our  previously  existing 
prototypes.   We  were  not  successful  in  obtaining  true  ts_  recombinants  (39°C 
titer/34°C  titer  10  ) ,  when  we  mated  these  viruses  with  WSN/33  wild  type 
virus.   This  may  be  due  to  the  phenomenon  of  gene  incompatibility.   Other 
approaches  to  this  problem  are  under  consideration,  such  as  mating  the 
Ud/72-ts  viruses  with  a  different  wild  type  influenza  A  strain  as  well  as 
mating  them  with  other  ts_  viruses  that  have  a  lesion  at  a  known  locus  and 
selecting  for  ts  recombinants. 


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Characterization  of  ts  mutants  of  influenza  A/Udorn/72  (H3N2)-  Genetic 
studies  of  influenza  A/Udorn/72  (H3N2)  virus  _ts  mutants,  isolated  from 
virus  mutagenized  with  ICR191  (an  acridine-based  compound) ,  nitrous  acid  or 
ultraviolet  light,  were  continued.   Since  we  noticed  that  temperature 
sensitivity  of  mutants  varied  significantly  from  test  to  test,  we  had  to 
reevaluate  the  temperature  sensitivity  of  each  mutant  in  several  tests.  If 
a  mutant, had  a  plaque  titer  40°/34°  _^10   in  more  than  a  half  of  the  tests 
and  <L0   in  at  least  one-fourth  of  the  tests,  the  mutant  was  retained  for 
further  study.  Using  this  criterion  136  _ts_  mutants  were  retained  for  further 
investigation.   Eighty  mutants  were  _ts  on  both  RMK  and  MDCK  cells,  3  were 
ts  on  only  RMK  cells,  and  53  were  not  _ts_  on  RMK  cells  but  were  _ts  on  MDCK 
cells. 

The  83  RMK  ts  mutants  were  arranged  into  13  complementation  groups,  groups 
1  to  13,  by  extensive  complementation  assay  on  RMK  cells  at  40° C.   The 
progeny  of  the  crosses  between  prototypes  of  the  13  groups  were  examined 
for  emergence  of  wild  type  (ts  )  recombinants  to  determine  if  each 
complementation  group  also  represented  a  recombination  group.   The  progeny 
from  the  pairwise  crosses  involving  the  _ts_  mutants  of  groups  1  and  11,  2 
and  12,  4  and  6,  and  5,  7,9  and  12  were  predominantly  _ts  virus,  whereas 
the  progeny  from  the  other  crosses  were  predominantly  ts_  virus.   On  the 
basis  of  these  findings,  complementation  groups  1  and  11  were  assigned  to 
complementation-recombination  group  C,  groups  2  and  12  to  group  F,  groups  4 
and  6  to  group  B,  and  groups  5,  7,  9  and  12  to  group  H.   Complementation 
groups  3,  8,  10  and  13  were  assigned  to  complementation-recombination 
groups  A,  D,  E  and  G,  respectively.  The  prototype  of  group  12  seemed  to 
possess  _ts  lesions  on  two  genes  corresponding  to  complementation-recombination 
groups  F  and  H.   Thus,  we  obtained  8  complementation-recombination  groups, 
A  to  H  in  the  order  which  RNA  migrates  during  polyacrylamide  gel 
electrophoresis;  A  was  least  mobile  etc.   The  number  of  the  complementation- 
recombination  groups,  8,  is  in  good  agreement  with  the  number  of  influenza 
A  gene  RNA  segments. 

Complementation  between  _ts  mutants  which  belong  to  the  same  complementation- 
recombination  group  can  be  explained  by  intracistronic  complementation. 
Mapping  the  _ts_  locus  of  each  complementation  group  to  its  viral  RNA  segment 
provided  direct  evidence  for  occurrence  of  intracistronic  complementation. 
The  mapping  was  done  by  gel  electrophoresis  of  RNA  from  ts_  recombinants 
produced  by  crossing  an  A/Udorn/72  ts_  mutant  and  an  A/WSN/33  ts  mutant 
whose  corresponding  RNA  segments  could  be  distinguished  by  this  method. 
Each  of  the  independent  _ts_  recombinants  should  have  a  WSN  RNA  segment 
corresponding  to  the  Udorn  RNA  segment  on  which  the  ts  lesion  resides. 
Using  this  method  complementation  group  3  of  complementation-recombination 
group  A  was  mapped  to  RNA  1  which  codes  for  P3  protein.   Groups  4  and  6 
belonging  to  group  B,  were  mapped  to  RNA  2  which  codes  for  PI  protein, 
although  we  could  not  exclude  the  possibility  that  group  6  has  other  _ts 
lesions  on  RNAs  7  and  8.   Groups  1  and  11  of  group  C,  were  mapped  to  RNA  3 
which  codes  for  P2  protein.   Group  8  of  group  D,  was  mapped  to  RNA  4  which 
codes  for  HA  protein.   Group  10  of  group  E,  was  mapped  to  RNA  5  which  codes 


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for  NA  protein.   Group  2  of  group  F,  was  mapped  to  RNA  6  which  codes  for  NP 
protein.   Group  15  of  group  G,  was  mapped  to  RNA  7  for  M  protein  or  RNA  2 
for  PI  protein.   Groups  5,  7  and  9  all  belonging  to  group  H,  were  mapped  to 
RNA  for  NS  protein  although  there  is  a  possibility  that  group  5  might  have 
additional  _ts  lesions  on  RNA  3  and  6.   The  prototype  of  group  12,  which  is 
thought  to  have  two  _ts_  lesions  belonging  to  groups  F  and  H,  was  mapped  to 
RNAs  6  and  8.   It  was  shown  that  intrasistronic  complementation  could  occur 
with  _ts  mutations  that  affect  the  gene  coding  for  PI,  P2,  NP  or  NS  protein. 
It  was  also  demonstrated  that  each  of  the  8  complementation-recombination 
groups  corresponded  to  one  of  the  8  genomic  RNA  segments  without  overlap, 
although  the  mapping  of  group  G  on  RNA  7  is  not  yet  conclusive.   New  prototypes 
of  the  8  complementation-recombination  groups  A  to  H,  were  selected.   The 
new  prototypes  were  more  stable  than  the  corresponding  prototypes  of  the 
original  13  complementation  groups.   The  new  prototypes  also  failed  to 
complement  with  the  prototypes  of  complementation  groups  which  belonged  to 
their  respective  complementation-recombination  group.   The  progeny  of 
pairwise  crosses  of  these  8  mutants  were  examined  for  temperature  sensitivity. 
Each  of  the  crosses  yielded  predominantly  _ts_  virus.   This  confirmed  that 
the  8  new  prototypes  represent  8  non-overlapping  complementation-recombination 
groups.   The  remainder  of  the  mutants  were  crossed  with  the  8  prototypes  to 
locate  the  _ts  lesions  that  corresponded  to  complementation-recombination 
groups  A  to  H.   Each  of  the  remaining  mutants  had  one  or  more  _ts  lesions 
represented  within  the  8  complementation-recombination  groups.   This  indicated 
that  the  8  groups  covered  all  complementation  groups  present  in  the  83  ts 
mutants.   Sixty-one  of  the  83  mutants  appeared  to  be  single  lesion  mutants: 
13  mutants  were  group  A  mutants;  16,  group  B;  group  C;  1  group  D;  1  group 
E;  16,  group  F;  1  group  G;  and  4,  group  H.   Twenty-one  mutants  had  _ts_ 
lesions  of  two  groups  and  one  mutant  had  _ts  lesions  of  three  groups. 

Some  pairs  of  mutants  that  complemented  on  RMK  cells  failed  to  complement 
each  other  in  MDCK  cells.   This  host  dependency  of  complementation  was  also 
seen  in  crosses  between  the  8  prototypes.   Therefore,  we  had  to  choose 
another  set  of  prototypes  for  assay  on  MDCK  cells.   The  t_s  lesions  of  133 
MDCK  jts_  mutants  were  assigned  to  the  8  complementation-recombination  groups, 
A  to  H,  by  performing  crosses  with  the  8  prototypes  selected  for  assay  on 
MDCK  cells.   There  were  85  single  lesion  mutants:  22  mutants  of  them  were 
group  A  mutants;  22,  B;  17,  C,  1,  D;  2,  E;  16,  F;  2,  G;  and  5,  H.   No 
mutant  complemented  all  of  the  8  prototypes  suggesting  that  there  are  not 
more  than  8  complementation-recombination  groups  in  influenza  A  virus. 

Sixteen  mutants  showed  distinct  host  dependency  of  temperature  sensitivity. 
These  mutants  plaqued  equally  on  MDCK  and  RMK  cells  at  34°C.  However  at 
40°C  MDCK/RMK  plaque  titer  was  < 10~  ,  and„plaque  titer  40°/34°C  on  MDCK 
cells  was  <_10~  but  on  RMK  cells  was  >_10   .  Thus,  the  16  mutants  appeared 
to  be  temperature  dependent,  host  range  (td-hr)  mutants.  The  16  td-hr 
mutants  did  not  share  a  common  td-hr  lesion  but  instead  fell  into  8  groups 
by  complementation  assay  on  MDCK  cells  at  40° C.   The  involvement  of  host- 
dependent  suppressor  mutation  in  this  td-hr  phenomenon  was  examined  by 
segregation  analysis  in  which  a  host  independent  _ts  marker  from  a  td-hr 


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mutant  (SP392)  was  sought  during  a  mating  with  host  independent  _ts  mutants. 
The  results  indicated  that  a  suppressor  mutation  was  not  involved  in  SP392 
and  that  a  host  dependent  _ts_  mutation  was  responsible  for  the  temperature 
dependent  host  range  restriction.   Although  we  did  not  do  this  test  with 
all  of  the  td-hr  mutants,  our  results  strongly  suggested  that  each  influenza 
gene  can  undergo  td-hr  mutation. 

Seven  mutants,  3  of  group  A,  4  of  group  B,  1  of  group  C  and  2  of  group  F, 
had  a  shutoff  temperature  of  37°C  or  38°C  on  both  of  RMK  and  MDCK  cells. 
Therefore,  these  7  mutants  have  potential  for  use  as  vaccine  strains  or  as 
a  donor  of  a  _ts  lesion  to  construct  stable  multi-lesion  ts_  recombinants. 

Publications 

Murphy,  B.R.,  Wood,  F.T.,  Jr.,  Massicot,  J.G.,  Chanock,  R.M. :   Temperature- 
sensitive  mutants  of  influenza  A  virus.   XV.  The  genetic  and  biological 
characterization  of  a  recombinant  influenza  virus  containing  two  ts_ 
lesions  produced  by  mating  two  complementing,  single  lesion  ;ts  mutants. 
Virology  88:  231-243,  1978. 

Murphy,  B.R. ,  Wood,  F.T.,  Jr.,  Massicot,  J.G.,  Chanock,  R.M. :   Temperature 
sensitive  mutants  of  influenza  virus.   XVI.  Transfer  of  the  two  ts_ 
lesions  present  in  the  Udorn/72-ts-lA2  donor  virus  to  the  Victoria/3/75 
wild  type  virus.   Virology  88:   244-251,  1978. 

Grizzard,  M.B.,  London,  W.T.,  Sly,  D.L.,  Murphy,  B.R.,  James,  W.D., 
Parnell,  W.P. ,  and  Chanock,  R.M. :   Experimental  production  of  respiratory 
tract  disease  in  cebus  monkeys  following  infection  with  influenza  A/Victoria/ 
5/75  or  influenza  A/New  Jersey/76  virus.   Infect.  Immun.  21:   201-205, 
1978.  s 

Chanock,  R.M. .  Murphy,  B.R.,  Spring,  S.B.,  Markoff,  L.J.,  Richardson, 
L.S.,  and  Belshe,  R.B. :   The  use  of  live  mutants  for  the  prevention  of 
respiratory  tract  disease.   Acta  Pathologica  et  Microbiologica  Scandinavica, 
in  press,  1979. 

Markoff,  L. J. ,  Murphy,  B.R. ,  Kendal,  A.J.,  and  Chanock,  R.M. :  Probable 
association  of  plaque  size  with  neuraminidase  subtype  among  H3N2  influenza 
A  viruses.   Arch.  Virol. ,  in  press. 


23-51 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00026-12  LID 


PERIOD    COVERED 

October  1,  1978  through  September  30,  19  79 


TITLE  OF  PROJECT  (80  characters  or  less) 

Laboratory  and  Epidemiologic  Studies  of  Viral  Hepatitis  Agents 


NAMES,  LABORATORY  ANO  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:   R.H.  Purcell  Head,  Hepatitis  Viruses  Section 

R.  Daemer  Research  Microbiologist 

S.  Feinstone  Staff  Scientist 

M.  Canese  Visiting  Scientist 

Y.  Moritsugu  Visiting  Scientist 

M.  Rizzetto  Visiting  Scientist 

I.  Gust  Visiting  Scientist 

McAuliffe  Research  Associate 

Johnson  Research  Associate 

Ticehurst  Research  Associate 

Shimizu  Visiting  Associate 

„  ,   B.  Hansson  Visiting  Fellow 

Other: p. Hoiiand,  H. Alter  (CC, Blood  Bank,  NIH)  K. Soike(Delta  Pr 
J.L.Gerin(MAN  Laboratory)  ,,W.London(NINCDS)  D. Lorenz,E. Tabor ,R. 

COOPERATING  UNITS  (if  any 

Above 


V. 
R. 
J. 
Y. 


D.Sly    (Meloy  Labs) 


AND    ALL   OTHER 

LID,NIAID 
LID,NIAID 
LID,NIAID 
LID,NIAID 
LID,NIAID 
LID,NIAID 
LID,NIAID 
LID,NIAID 
LID,NIAID 
LID,NIAID 
LID, N I AID 
LID,NIAID 
imate  Center) 
Gerety(FDA) 


lab/branch 
Laboratory  of   Infectious  Diseases 


SECTION 

Hepatitis  Viruses  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland 


TOTAL  MANYEARS 


13 


PROFESSIONAL: 

12 


CHECK  APPROPRIATE  BOX(ES) 
K(a)   HUMAN  SUBJECTS 

□  (al)  MINORS   Q  (a2)  INTERVIEWS 


3  (b)  HUMAN  TISSUES 


D  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords)      ,   . 

This  project  consists  of  continuing  studies  of  the  chemistry,  structure, 
epidemiology,  immunology  and  pathology  of  the  human  hepatitis  viruses. 
The  goal  of  such  studies  is  the  control  of  human  viral  hepatitis  by  application 
of  the  most  appropriate  methods,  including  active  and  passive  immunization, 
chemotherapy  and  interdiction  of  spread  of  the  viruses.   Progress:   Partial 
biophysical  and  biochemical  characterization  of  hepatitis  A  virus  has  been 
achieved  and  studies  of  hepatitis  type  A  in  non-human  primates,  using  defined 
pools  of  virus,  are  almost  complete.   There  is  preliminary  evidence  for  successful 
cultivation  of  hepatitis  A  virus  in  vitro.   An  inactivated  subunit  vaccine  for 
hepatitis  type  B  has  been  developed  and  is  undergoing  extensive  tests  of 
safety  and  efficacy  in  man.   A  third  hepatitis  B  antigen,  e_  antigen,  is  being 
characterized  and  its  relationship  to  infectivity  is  being  explored.  Evidence 
that  populations  of  hepatitis  B  viruses  may  contain  defective  interfering 
particles  has  been  obtained,  and  this  finding  is  being  utilized  in  renewed 
attempts  to  isolate  the  virus.   A  newly  recognized  clinical  syndrome,  type 
non-A,  non-B  hepatitis  has  been  further  defined  and  attempts  to  identify  an 
etiologic  agent  intensified  through  transmission  stud-jp^  in_  chimpanzees'. 


PHS-6040 
(Rev.  10-76) 


23-52 


Z01  AI  00026-12  LID 


Project  Description 


Objectives:   (1)   To  collect  clinical  material  of  high  potential  infectivity 
for  use  in  laboratory  studies,   (2)  To  develop  and  test  in  vitro  procedures 
and  animal  systems  for  use  in  laboratory  studies  of  viral  hepatitis,  (3)  To 
propagate  the  viruses  of  hepatitis  in  culture  or  in  experimental  animals, 
(4)   To  study  the  biochemistry,  biophysics,  immunology  and  epidemiology  of 
hepatitis-associated  antigens;  (5)  To  develop  vaccines  or  other  control 
measures  for  viral  hepatitis;  (6)  To  develop  chemo therapeutic  approaches  to 
the  control  of  chronic  viral  hepatitis. 

Methods  Employed 

(1)  Longitudinal  collection  of  clinical  specimens  from  hepatitis  patients. 

(2)  Ultracentrifugal,  electrophoretic  and  column  chromatographic  purification 
of  hepatitis  viruses  and  related  antigens. 

(3)  Inoculation  of  non-human  primates  for  the  determination  of  infectivity 
and  clinical  spectrum  of  hepatitis  viruses. 

(4)  Microscopic  examination  of  hepatitis  viruses  and  virus-infected  tissue 
by  light  microscopy,  fluorescence  microscopy,  and  electron  microscopy. 

(5)  A  wide  variety  of  tissue  culture  techniques. 

(6)  A  wide  variety  of  immunologic  techniques  for  measuring  humoral  and 
cellular  immune  responses. 

(7)  Application  of  DNA  recombinant  techniques  to  the  study  of  the  hepatitis 
viruses. 

Major  Findings 

(1)   Type  B  Hepatitis 

HBsAg 

Because  of  its  potential  usefulness  as  the  raw  material  for  hepatitis  B 
vaccine,  HB  Ag  is  being  intensively  studied.   Preparation  of  two  pilot  lots 
of  vaccine,  one  subtype  adw  and  the  other  subtype  ayw,  has  been  completed 
by  Dr.  J.  Gerin,  in  a  collaborative  program. 

Although  a  hepatitis  B  vaccine  prepared  from  HB  Ag  purified  from  the  plasma 
of  chronic  carriers  of  the  antigen  is  feasible  and  probably  cost-effective 
for  its  contemplated  uses  in  the  United  States,  such  a  vaccine  probably 
will  not  be  cost-effective  for  the  people  most  in  need  of  a  vaccine  in  the 
developing  world.  Therefore,  additional  studies  on  alternative  methods  of 
purification  and  inactivation,  comparisons  of  monovalent  vs.  bivalent  or 


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Z01  AI  00026-12  LID 

polyvalent  vaccines,  studies  of  different  combining  ratios  of  mononvalent 
vaccines  into  bivalent  preparations,  different  vaccine  dosages,  different 
vaccination  schedules,  different  routes  of  administration,  studies  of 
possible  adjuvants  and  their  interaction  with  vaccine  preparations  and 
further  characterization  of  the  immunizing  antigens  themselves  are  being 
carried  out. 


Four  modified  vaccines  have  been  prepared.   Two  of  these  have  been  extracted 
with  ether-tween  80;  2  are  alum-precipitated.   These  vaccines  are  currently- 
being  evaluated  for  immunogenicity  in  volunteers.   Preliminary  results 
indicate  that  at  least  certain  of  the  vaccines  are  much  more  immunogenic 
than  any  previous  hepatitis  B  vaccine  tested.   Thus,  they  produce  antibody 
to  hepatitis  B  surface  antigen  in  two  to  three  weeks  instead  of  two  to 
three  months.   Additional  testing  of  the  best  of  these  vaccines  will  be 
carried  out  in  voluteers  to  determine  optimum  immunization  schedules. 

There  have  recently  been  several  reports  of  antibodies  to  polymerized  human 
albumin  occurring  during  or  after  HBV  infection.   Other  studies  have  demonstrated 
that  this  reactivity,  rather  than  being  antibody,  is  associated  with  receptor 
sites  for  polymerized  albumin  on  the  surface  of  HBsAg  particles.   Furthermore, 
there  is  a  correlation  between  the  detection  (or  titer)  of  such  receptor 
sites  and  the  presence  of  HBeAg.   Thus,  the  presence  of  the  receptor  sites, 
like  the  presence  of  HBeAg,  may  correlate  with  relative  infectivity.   Dr. 
Hansson  has  studied  these  receptor  sites  and  has  developed  a  solid  phase 
radioimmunoassay  that  is  highly  sensitive  and  specific.   The  assay  is  so 
sensitive  that  he  is  able  to  detect  the  presence  of  HBsAg  associated  with 
the  receptor  sites  with  a  greater  sensitivity  than  the  best  direct  tests 
for  HBsAg.   The  significance  of  these  receptor  sites  remains  obscure,  but 
they  may  in  some  way  be  related  to  the  attachment  of  hepatitis  B  virions  to 
hepatocytes.   Alternatively,  they  may  bind  albumin  in  vivo  and,  thereby 
make  it  more  closely  resemble  "self".   Indeed,  removal  of  albumin  from 
HBsAg  of  hepatitis  B  vaccines  appears  to  markedly  improve  its  ability  to 
stimulate  the  production  of  antibodies. 

Other  HBV  Antigens 

HBeAg  -  Current  interest  in  HB  Ag  centers  on  the  association  between  its 
detection  in  liver  cell  nuclei  and  the  prognosis  and  relative  infectivity 
of  chronic  hepatitis  type  B  patients.   A  commercial  test  for  anti-HBc, 
based  upon  a  radioimmunoassay  first  described  by  us  is  now  available. 

Recently,  interest  has  centered  on  the  detection  of  anti-HBc  of  the  IgM 
class.  The  detection  of  such  antibody  would  provide  a  means  for  diagnosis 
of  a  recent  hepatitis  B  virus  infection,  and  there  is  some  evidence  that 
prolongation  of  the  IgM  anti-HBc  response  may  correlate  with  the  development 
of  chronic  HBV  infection.   Dr.  Hansson  is  developing  a  test  for  anti-HBc 
that  is  specific  for  IgM  antibody;  this  test  is  being  evaluated  with  serial 
serum  samples  from  our  extensive  collection  of  experimental  HBV  infections 
in  chimpanzees. 

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Z01  AI  00026-12  LID 

HBeAg  -  There  has  been  much  interest  in  this  non-particulate  hepatitis 
antigen  because  of  its  association  with  chronic  hepatitis  and  relative 
infectivity  of  blood  from  HB  Ag  positive  type  B  hepatitis  patients. 
Firm  proof  that  HB  Ag  is  a  hepatitis  virus  gene  product  is  lacking,  but 
circumstantial  evidence  strongly  points  in  that  direction.  There  have 
been  reports  that  HB  Ag  is  a)   on  the  surface  of  hepatitis  B  virions,  b) 
an  idiotypic  IgG  molecule  and  c)   a  modified  lactic  acid  dehydrogenase. 
Probably  none  of  these  is  correct:   it  is  probably  either  a  nonstructural 
viral  gene  product  or  excess  viral  DNA  polymerase  that  is  never  assembled 
into  complete  virus.   We  have  recently  developed  procedures  for  the 
purification  of  HB  Ag,  and  preparations  in  which  host  serum  proteins 
cannot  be  detected  have  been  prepared.   Plasmapheresis  units  obtained 
from  chimpanzees  chronically  infected  with  HBV  have  served  as  raw  material 
for  the  purification. 

Extensive  attempts  at  purification  and  analysis  of  partially  purified 
HBeAg  indicate  that  it  is  a  molecule  of  17,000-19,000  daltons  molecular 
weight  that  may  occur  in  larger  forms  or  as  complexes  with  other  proteins. 
Absolute  purification  has  so  far  not  been  possible,  and  Dr.  McAuliffe  is 
attempting  to  produce  specific  antibody  through  the  use  of  the  mouse 
hybridoma  system. 

"Delta  antigen"  -  This  antigen  was  discovered  several  years  ago  by 
Rizzetto  in  Italy.   It  appears  to  be  another  hepatitis  B  antigen  found, 
like  HB  Ag,  in  the  nucleus  of  infected  liver  cells.   It  has  been  detected 
only  by  immunofluorescence  to  date,  but  its  existence  has  been  confirmed 
by  others.   Because  of  the  importance  of  thoroughly  evaluating  vaccine 
recipients  for  all  possible  markers  of  hepatitis  B  virus  infection,  we 
plan  to  characterize  this  new  antigen  and  determine  its  relationship  to 
type  B  hepatitis.   Dr.  Rizzetto  is  spending  a  year  in  Dr.  Gerin' s  laboratory 
and  LID  for  this  purpose.   Preliminary  results  indicate  that  delta 
antigen  has  a  world-wide  distribution,  although  the  highest  prevalence 
of  antibody  to  this  antigen  occurs  in  parts  of  Italy.   In  addition,  the 
prevalence  of  antibody  appears  to  be  much  higher  among  HBsAg  positive 
carriers  who  have  had  opportunities  for  multiple  exposures,  e.g.,  hemophiliacs 
and  parenteral  drug  users.   Seroepidemiologic  data  and  data  obtained 
from  transmission  studies  in  chimpanzees  suggest  that  delta  antigen  may 
be  a  defective  non-A,  non-B  agent  that  requires  the  presence  of  hepatitis 
B  virus  for  multiplication.   Attempts  to  discover  a  virion  distinct  from 
the  hepatitis  B  virion  are  in  progress.   Activities  include  studying  the 
development  of  delta  antigen  and  anti-delta  antigen  in  experimentally 
infected  chimpanzees,  determining  its  relationship  to  other  hepatitis  B 
antigens,  attempting  to  purify  the  antigen  from  infected  liver  tissue 
(human  and  chimpanzee)  and  the  preparation  of  reagents.   This  work  is 
progressing  rapidly:   a  radioimmunoassay  for  delta  antigen  and  antibody 
has  been  developed  and  is  being  applied  to  seroepidemiological  studies. 


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Z01  AI  00026-12  LID 
Hepatitis  B  Virus 

Q 

Because  HBV  occurs  in  titers  of  over  10  infectious  particles/ml  in  the 
plasma  of  certain  chronically  infected  individuals,  it  is  possible  to 
purify  these  particles  by  large  volume  ultracentrifugation  and  characterize 
them.   Recent  collaborative  studies  carried  out  with  Dr.  Gerin  have 
revealed  that  HB  virions  are  heterogeneous:  three  populations  of  particles 
can  be  separated  by  isopynic  banding  in  cesium  chloride.  The  lowest 
density  population  lacks  DNA  polymerase  activity  and  probably  most  if 
not  all  nucleic  acid.   The  highest  density  population  is  strongly  positive 
for  polymerase  activity  and  contains  double-stranded  circular  DNA  that 
can  be  extracted  and  characterized.   Most  interesting  is  the  intermediate 
density  population  which,  itself,  is  heterogeneous  and  which  is  weakly 
polymerase  positive  and  contains  nucleic  acid  with  a  molecular  weight 
only  80-90%  of  that  of  the  highest  density  particles.   Thus,  these 
particles  have  all  of  the  biophysical  characteristics  of  defective 
interfering  particles,  entities  that  in  other  classical  virus  systems 
have  been  shown  to  inhibit  fully  infectious  virus  synthesis  in  vitro  and 
to  modulate  virus  infection  in  vivo  in  certain  laboratory  animal  model 
systems,  converting  a  rapidly  progressive  infection  to  a  persistent  or 
chronic  one.   Such  a  defective  interfering  particle  system  has  never 
been  described  for  a  human  disease.   This  finding  has  important  implications 
for  the  two  following  topics. 

Isolation  of  HBV  in  vitro 


Despite  many  attempts  over  many  years  by  many  investigators,  HBV  has 
never  been  successfully  isolated  and  serially  transmitted  in  vitro.   The 
reason  for  this  is  not  known  and  may  be  related  to  a  number  of  factors 
including  limited  host  range  (HBV  is  known  to  grow  only  in  hepatocytes 
of  humans  and  certain  species  of  apes  closely  related  to  man) .  However, 
one  reason  for  failure  to  isolate  the  virus  may  be  that  the  virus  is 
always  found  in  the  presence  of  a  vast  excess  (up  to  a  million-fold  more 
particles)  of  viral  coat  material  (HB  Ag)  as  well  as  a  variable  concentration 
of  defective  interfering-like  particles.  The  former  can  compete  for 
receptor  sites  on  sensitive  cells,  and  the  latter  can  inhibit  the  synthesis 
of  infectious  virus  when  they  coinfect  cells  with  intact  virions.   To 
examine  this,  preparations  of  high  density  and  intermediate  density  HB 
virions  have  been  purified  by  isopynic  and  rate  zonal  centrifugal  procedures 
under  conditions  of  high  containment  in  Dr.  Gerin' s  laboratory.   These 
preparations  are  being  titered  for  infectivity  in  chimpanzees;  they  are 
being  used  for  attempts  to  isolate  HBV  in  a  number  of  tissue  culture 
cell  lines. 

Coincident  with  these  studies  is  the  development  of  reagents  for  identifying 
all  of  the  markers  of  HBV  infection.   These  assays  are  being  used  in 
evaluating  various  clones  of  hepatocytes  derived  from  human  or  chimpanzee 
liver.   In  collaboration  with  researchers  at  Columbia  University,  we  are 
attempting  to  produce  hybridoma  cell  lines  derived  from  fusions  of 


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Z01  AI  00026-12  LID 

hepatoma  cells  and  normal  hepatocytes.   Hopefully  the  resulting  cell 
hybrid  will  have  the  immortality  of  the  hepatoma  cells  but  retain  the 
metabolic  patterns  and  virus  sensitivities  of  the  hepatocytes.   Cell 
lines  that  can  be  certified  as  being  of  liver  origin  will  be  inoculated 
with  partially  purified  HBV  that  is  being  titered  in  chimpanzees.   These 
inoculated  cultures  will  be  monitored  with  the  various  assays  available 
in  the  laboratory  including  radioimmunoassays  for  HB  Ag  and  HB  Ag,  HBV- 
specific  DNA  polymerase  enzyme  activity,  visualization  of  HB  Ag  and 
HB  Ag  particles  and  HB  virions,  immuno fluorescent  detection  of  HB  Ag  or 
HB  Ag  and  detection  of  intracellular  HBV  genome  by  nucleic  acid  homology 
techniques. 

Although  HBV  has  not  been  successfully  cultured  in  vitro,  recently  a 
hepatoma  cell  line  was  isolated  that  synthesizes  HB  Ag.   This  cell  line, 
derived  in  South  Africa,  has  been  distributed  to  others,  and  we  are 
conducting  a  detailed  characterization.   It  is  recognized  that  this  cell 
line  probably  cannot  serve  as  a  source  for  hepatitis  B  vaccine  because 
of  its  malignant  origin,  but  it  may  yield  important  information  on  HBV- 
cell  interaction.   Attempts  to  recover  the  virus  by  cocultivation  and 
induction  with  anti-metabolites  have  been  unsuccessful  to  date,  but  data 
from  several  laboratories  indicate  that  the  cell  line  may  contain  up  to 
three  virus  genomes  per  cell. 

Although  only  HBsAg  can  be  detected  in  this  cell  line,  it  is  not  known 
whether  small  quantities  of  infectious  virus  are  produced.   Such  production 
would  make  the  cell  line  considerably  more  hazardous  than  if  infectious 
virus  were  not  produced.   To  determine  the  infectivity  of  this  cell  line 
we  have  inoculated  sero  negative  chimpanzees  with  harvested  tissue 
culture  fluid  and  with  viable  hepatoma  cells.   This  experiment  is  still 
in  progress. 


Pathology 

Although  much  has  been  learned  about  hepatitis  B  virus  and  its  antigens, 
the  method  by  which  it  produces  disease  is  still  poorly  understood. 
There  is  some  evidence  that  HBV  is  not  cytopathogenic  but,  rather, 
produces  tissue  damage  through  the  host's  immune  response  to  infection. 
Thus,  it  has  been  postulated  that  the  host1  s  humoral  or  (more  probably) 
cellular  immune  response  to  viral  antigens  or  modified  liver  cell  antigens 
may  lead  to  cell  damage  or  death.   Without  going  into  detail  here,  there 
is  evidence  both  for  and  against  this  hypothesis.   However,  attempts  to 
prove  the  immunological  nature  of  viral  hepatitis  have  been  unconvincing 
for  a  number  of  reasons,  including  difficulty  in  measuring  and  interpreting 
cellular  immune  responses  in  man.   It  is  unlikely  that  this  question  can 
be  adequately  answered  until  autologous  target  cells  infected  with  HBV 
can  be  maintained  in  vitro  and  lymphocytoxicity  between  these  cells  and 
lymphocytes  obtained  prior  to  infection,  during  the  acute  phase  of 
illness  and  during  convalescence  can  be  studied  in  vitro.  This  is  not 


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Z01  AI  00026-12  LID 

technically  feasible  at  present,  but  studies  of  the  possible  role  of 
anti-idiotype  antibodies  in  modulating  HBV  infection  are  feasible  and 
will  be  attempted.   At  present  attempts  to  demonstrate  a  cellular  immune 
response  in  chimpanzees  with  traditional  in  vitro  methods  are  being 
repeated.  In  these  experiments  lymphocytes  are  being  harvested  at  weekly 
intervals,  and  skin  tests  with  purified  HBsAg  are  being  applied. 

However,  there  is  an  alternative  explanation  for  the  variable  course 

of  type  B  hepatitis:   the  infection  may  be  modulated  by  the  ratio  of 
defective  interfering-like  particles  and  fully  infectious  virus.   This 
hypothesis  is  amenable  to  experimental  analysis.   We  plan  to  experimentally 
infect  chimpanzees  and  obtain  serial  plasmapheresis  units  from  the 
animals  throughout  the  course  of  infection.   These  plasmapheresis  units 
will  be  analyzed  for  the  ratio  of  the  various  density  populations  of  HB 
virions  as  a  function  of  phase  of  the  disease  and  the  results  correlated 
with  analyses  of  serum  HB  Ag  and  HB  Ag  titers,  serum  liver  enzyme  levels, 
intrahepatic  HB  Ag  and  HB  Ag  and  histologic  evidence  of  liver  damage. 
If  the  results  of  these  experiments  warrant  it,  consideration  will  be 
given  to  combining  purified  heavy  density  and  intermediate  density  HB 
virions  in  different  ratios  and  inoculating  chimpanzees  to  determine  if 
the  clinical  course  of  type  B  hepatitis  in  the  animals  can  be  predicted. 

Meyer  zum  Buschenfelde  produced  a  chronic  hepatitis  in  rabbits  by  exhaustive 
immunization  with  liver  tissue.   He  demonstrated  that  an  antigen  on  the 
surface  of  hepatocytes  (liver  specific  protein,  LSP)  was  responsible  for 
initiating  this  immunological  disease.  A  similar  LSP-anti-LSP  system  was 
shown  to  be  present  in  cases  of  human  chronic  hepatitis.   In  independant 
studies  and  in  studies  in  collaboration  with  our  laboratory,  Meyer  zum 
Buschenfelde  has  shown  that  such  anti-LSP-positive  chronic  hepatitis 
patients  appear  to  have  a  disease  distinct  from  hepatitis  B  virus- 
induced  chronic  hepatitis.   However,  this  has  not  been  rigorously  proven, 
and  the  published  concern  that  hepatitis  B  vaccine  hypothetically  contaminated 
with  LSP  might  lead  to  chronic  hepatitis  in  vaccine  recipients  has  led 
us  to  begin  studying  LSP  in  more  detail.   LSP  appears  to  be  an  extremely 
labile  membrane-associated  antigen  that  has  not  been  purified,  although 
one  group  in  England  believes  that  they  have  effected  a  purification. 
Earlier  reports  of  various  assays  for  LSP  are  now  suspect,  and  the 
demonstration  of  LSP  on  liver  cell  surfaces  by  indirect  immunofluorescence 
appears  to  be  the  only  reliable  assay  method  at  present.   We  have  recently 
established  this  assay  in  the  laboratory  and  are  beginning  to  study  this 
antigen-antibody  system.   There  is  a  great  need  for  the  development  of 
more  sensitive  and  less  cumbersome  tests  for  measuring  LSP  and  anti-LSP 
and  for  the  preparation  of  hyperimmune  serum.  If  sensitive  assays  such 
as  radioimmunoassays  can  be  developed  for  LSP  and  anti-LSP,  they  will  be 
applied  to  serial  serum  samples  from  chimpanzees  and  humans  with  type  B 
hepatitis  (and  other  types  of  hepatitis)  to  determine  if  there  is  evidence 
for  this  type  of  immunological  response  in  viral  hepatitis.   The  results 
will  go  a  long  way  toward  resolving  the  question  of  whether  hepatitis  is 
an  immunological  disease. 

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Antivirals  and  Chemotherapy  of  Type  B  Hepatitis 

With  the  almost  simultaneous  reports  of  modification  of  chronic  hepatitis 
B  virus  infection  in  humans  or  chimpanzees  by  three  laboratories  (ours, 
Merigan1 s  at  Stanford,  and  Desmyter's  in  Belgium)  in  1976  a  new  era  in 
type  B  hepatitis  research  began.   Both  interferon  and  an  interferon 
inducer  were  shown  to  markedly  diminish  all  of  the  markers  of  hepatitis 
B  virus  infection  when  they  were  administered  in  sufficient  doses  and 
for  a  sufficiently  long  period  of  time.  Subsequent  studies  have  shown 
that  interferon  is  not  the  "magic  bullet"  for  treatment  of  chronic 
hepatitis,  and  there  is  still  insufficient  information  about  the  toxicity 
of  certain  interferon  inducers  to  use  them  in  large  doses  in  humans,  but 
the  studies  did  show  that  the  treatment  of  chronic  hepatitis  can  be 
approached  from  the  standpoint  of  treating  the  virus  rather  than  the 
host.  All  previous  treatment  regimens  have  been  concerned  with  suppressing 
the  host's  immune  response  with  steroids  and  antimetabolites  in  an 
attempt  to  diminish  the  clinical  manifestations  of  disease.   These 
studies  have  recently  been  critically  reviewed  by  the  Washington,  D.C., 
VA  liver  group  and  their  conclusion  was  that  steroids  and  antimetabolites 
have  not  been  shown  to  be  effective  in  the  treatment  of  chronic  type  B 
hepatitis,  a  finding  in  agreement  with  the  observations  of  others. 

Further  evidence  that  chemotherapy  holds  promise  for  the  treatment  of 
chronic  type  B  hepatitis  comes  from  recent  studies  by  Merigan  in  which 
the  antiviral  drug  adenine-arabinoside  (Ara-A)  was  also  shown  to  markedly 
diminish  the  titer  of  the  various  markers  of  hepatitis  B  virus  infection 
in  two  patients  with  chronic  type  B  hepatitis.   However,  there  was 
significant  toxicity  associated  with  treatment  with  this  drug. 

An  important  aspect  of  the  interferon/ interferon- inducer  studies  is  that 
a  chronic  virus  infection  was  modified.   In  in  vitro  and  in  vivo  studies 
of  the  action  of  interferon  this  substance  has  been  found  to  be  relatively 
ineffective  after  infection  of  cells  has  taken  place:   its  primary  mode 
of  protection  is  by  preventing  spread  of  virus  from  infected  cells  to 
uninfected  cells.   This  has  very  important  implications  for  the  understanding 
of  chronic  type  B  hepatitis  and  suggests  that  such  chronic  hepatitis  B 
virus  infection  is  an  ongoing  disease  in  which  infected  cells  produce 
new  virus,  die  and  are  replaced  by  new  cells  that  are  subsequently 
infected  with  virus  and  pass  through  the  same  cycle.   Interruption  of 
that  cycle  of  synthesis  of  new  virus  and  transmission  to  uninfected 
cells  may  be  the  key  to  terminating  chronic  type  B  hepatitis.   This 
becomes  even  more  significant  in  light  of  one  of  the  key  observations  in 
our  interferon- inducer  study  in  chimpanzees:   we  were  able  to  show  a 
marked  increase  in  the  ratio  of  defective  (polymerase-negative)  hepatitis 
B  virions  to  polymerase-positive  virions  as  a  result  of  treatment  of  the 
chimpanzees  with  interferon  inducer  and  at  a  time  that  coincided  with 
the  fall  in  titer  of  the  other  markers  of  hepatitis  B  virus  infection. 
This  would  be  consistent  with  our  hypothesis  that  chronic  type  B  hepatitis 
is  a  defective- interfering  virus  system  in  which  modulation  of  the 


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infection  can  be  achieved  by  altering  the  ratios  of  infectious  and 
defective  particles.   Verification  of  this  is  technically  possible  by 
the  methods  described  above  and  by  treating  chronically  infected  chimpanzees 
with  interferon,  interferon  inducers  or  other  antivirals  and  monitoring 
the  ratios  of  different  hepatitis  B  virions  by  sophisticated  isopycnic 
banding  procedures. 

Recently,  Nordenfelt  has  shown  that  another  antiviral  agent,  phosphonoformic 

acid,  inhibits  the  DNA  polymerase  of  HBV  in  vitro.  We  are  examining  the 

in  vivo  effects  of  phosphonoformic  acid  in  chimpanzees.   Preliminary 

studies  indicate  that  PFA  may  exert  an  antiviral  effect  on  HBV  in  chronically 

infected  chimpanzees  in  the  absence  of  significant  toxicity.   More 

detailed  toxicity  data  are  being  obtained  in  treated  chimpanzees,  with 

the  hope  of  testing  this  drug  in  patients  chronically  infected  with  HBV. 

Other  Studies 

Although  much  has  been  learned  about  the  epidemiology  of  type  B  hepatitis 
in  recent  years,  there  are  several  intriguing  questions  still  unanswered. 
One  stems  from  the  discovery  that  approximately  0.1%  -  0.5%  of  the 
United  States  population  are  carriers  of  HB  Ag;  a  proportion  of  these 
have  associated  chronic  persistent  hepatitis  or  chronic  active  hepatitis, 
the  latter  a  life-threatening  disease.  What  is  totally  unknown,  however, 
is  what  proportion  of  the  population  has  evidence  of  chronic  hepatitis 
of  any  etiology.   There  is  evidence  that,  as  with  hypertension,  there  is 
a  large  reservoir  of  undiagnosed  disease  not  recognized  because  it  has 
not  been  systematically  sought.   In  collaboration  with  others,  we  are 
initiating  a  study  of  the  Framingham  population  for  evidence  of  occult 
chronic  hepatitis.  When  identified,  such  patients  will  be  evaluated  and 
the  etiology  of  their  hepatitis  determined.   If  the  results  of  initial 
surveys  warrant  it,   we  would  attempt  long-term  followup  of  the  Framingham 
population  in  an  attempt  to  determine  the  incidence  and  outcome  of  such 
disease  by  etiology. 

The  rapid  progress  made  in  the  development  and  application  of  DNA  recombinant 
technology  to  virology  has  very  significant  implications  for  the  study 
of  hepatitis  viruses.   Already,  the  genome  of  HBV  has  been  cloned  and 
sequenced  in  its  entirety.   In  addition,  the  location  of  the  genes  that 
code  for  HBsAg  and  probably  HBcAg  have  been  determined.   A  cloned  HBV 
genome  is  being  made  available  to  our  hepatitis  program.   The  availability 
of  large  quantities  of  pure  HBV  DNA  will  make  possible  difinitive  studies 
of  the  virus  and  its  relationship  to  the  infected  hepatocyte,  as  well  as 
its  relationship  to  hepatic  cell  carcinoma.   Ultimately,  the  translation 
of  gene  products  from  the  HBsAg  gene  of  the  HBV  genome  may  provide  an 
inexpensive  and  safe  hepatitis  B  vaccine. 

Several  variants  of  HBV  are  being  examined  in  the  chimpanzee  model 
system.   Among  these  are  strains  of  HBV  that  have  been  obtained  from 
patients  with  Gianotti-Crosti  syndrome.   This  disease,  most  common  in 


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Italy,  is  a  papular  dermatitis  that  occurs  in  young  children  acutely 
infected  with  certain  strains  of  HBV.   The  disease  was  introduced  into 
Japan  several  years  ago  when  an  Italian  freighter  docked  at  a  Japanese 
port;  the  disease  has  spread  in  a  concentric  pattern  from  the  harbor  of 
the  city  since  that  time.   Young  chimpanzees  have  been  inoculated  with 
two  strains  of  HBV  associated  with  the  disease;  they  are  being  followed 
for  evidence  of  the  characteristic  skin  rash.   Should  it  occur,  it  would 
be  characterized  and  further  characterization  of  the  virus  performed  to 
determine  the  nature  of  these  variant  viruses. 

Recently  we  have  carried  out  seroepidemiologic  studies  of  hepatitis  B 
(and  A)  virus  infection  in  isolated  island  communities  of  the  South 
Pacific.   Both  viruses  were  found  to  be  endemic  in  this  setting.   Significant 
differences  in  prevalence  were  observed  on  different  islands,  but  these 
differences  could  not  be  related  to  island  size,  culture  or  geography. 
The  only  exception  to  this  was  the  observation  that  hepatitis  A  virus 
infection  apparently  could  not  be  sustained  on  a  relatively  small  island 
(Ponape)  where  the  susceptible  (seronegative)  proportion  of  the  population 
was  depleted. 


(2)   Hepatitis  A  Virus 

Characterization  of  the  virus 

Although  hepatitis  A  virus  (HAV)  has  recently  been  isolated  in  vitro, 

sufficient  particulate  viral  antigen (presumably  HAV)  has  been  available 

to  a  very  few  laboratories  to  begin  biophysical  and  biochemical  characterization. 

Two  preliminary  studies  indicate  that  the  viral  particles  have  peptides 

similar  to  those  of  picornaviruses.   Although  definitive  nucleic  acid 

studies  have  not  been  completed,  two  studies  suggest  that  the  viral 

nucleic  acid  is  RNA  but  possibly  of  lower  molecular  weight  than  that  of 

picornaviruses. 

Antigen(s) 

Only  one  antigen,  hepatitis  A  antigen  (HAAg)  has  been  associated  with 
type  A  hepatitis  infections  to  date.   Four  serologic  techniques  for 
detecting  this  antigen  and  antibody  to  it  have  proven  most  useful  for 
serologic  studies  of  type  A  hepatitis  infection:   these  are  immune 
electron  microscopy,  immune  adherence  hemagglutination,  radioimmunoassay 
and  enzyme-linked  immunoassay.  Recently  we  have  developed  an  immunofluorescence 
assay  for  hepatitis  A  antigen  that  has  permitted  us  to  study  the  tissue 
distribution  of  viral  infection  in  chimpanzees  and  marmosets.   HAAg  was 
detected  in  the  liver,  germinal  centers  of  lymph  nodes  and  spleen,  and 
basement  membrane  of  the  kidney.   The  latter  sites  probably  represent 
sequestration  of  antigen  released  from  the  liver  into  the  circulation. 
Interestingly,  viral  antigen  has  not  yet  been  detected  in  the  gut. 


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However,  only  animals  intravenously  infected  had  been  examined.   However, 
we  recently  studied  gut  tissue  obtained  sequentially  from  marmosets 
experimentally  infected  with  HAV  via  the  oral  route. 

The  small  and  large  intestines  of  marmosets  killed  sequentially  during 
the  incubation  period  and  acute  phase  of  experimental  hepatitis  A  virus 
infection  were  examined  for  hepatitis  A  viral  antigen  by  immunofluorescence 
and  radioimmunoassay.   Although  antigen  could  be  detected  by  both  techniques 
in  the  liver  and  bile  after  an  appropriate  incubation  period,  evidence 
of  virus  multiplication  could  not  be  found  in  any  part  of  the  intestine 
or  mesenteric  lymph-nodes  in  any  of  the  animals.   Thus,  we  have  not  been 
able  to  demonstrate  an  enteric  phase  of  multiplication  for  the  hepatitis 
A  virus. 

The  development  of  an  immunofluorescence  test  for  hepatitis  A  viral 
antigen  permitted  Provost  and  Hilleman  to  identify  hepatitis  A  virus 
multiplication  in  tissue  culture.   The  inoculum  for  their  tissue  culture 
studies  was  a  strain  of  HAV  that  had  been  passaged  in  marmosets  for  31 
times.   We  have  confirmed  the  multiplication  of  hepatitis  A  virus  in 
tissue  culture  after  marmoset  passage.   Three  strains  of  HAV  have  been 
serially  passaged  in  marmosets;  at  least  two  of  these  (and  possibly  the 
third)  produce  hepatitis  A  viral  antigen  detectable  by  immunofluorescence 
in  African  green  monkey  cells.   Serial  passage  in  this  cell  line  is 
currently  being  attempted,  and  the  parameters  that  affect  growth  in 
culture  (number  of  passages  in  marmosets,  titer,  etc.)  are  being  examined. 

As  with  type  B  hepatitis,  hepatitis  A  virus  infection  leads  to  the 
development  of  IgM  antibodies  early  in  infection,  followed  by  the  development 
of  IgG  antibodies.   Because  anti-HAV  appears  early  in  the  acute  phase  of 
illness,  serologic  confirmation  of  infection  is  difficult  to  obtain 
unless  both  acute  phase  and  late  convalescent  phase  sera  are  available 
for  comparison  of  titer.  Even  then,  acute  phase  titers  are  apt  to  be  as 
high  or  higher  than  convalescent  titers  when  tested  by  radioimmunoassay. 
Recently  several  laboratories  have  developed  excellent  radioimmunoassays 
for  detection  of  IgM  anti-HAV.   Dr.  Hansson  has  developed  such  a  test  in 
our  laboratory  and  has  evaluated  its  sensitivity  and  specificity  with 
serial  serum  samples  from  experimentally  infected  chimpanzees.   He  has 
used  the  test  to  confirm  that  several  epidemics  of  viral  hepatitis  were, 
indeed,  caused  by  HAV.   He  is  presently  attempting  to  modify  the  test  to 
permit  specific  detection  of  IgA  anti-HAV.   This  latter  test  would  have 
additional  value  as  a  diagnostic  test  and  would  provide  another  means  of 
looking  for  evidence  of  intestinal  multiplication  of  this  virus:   intestinal 
multiplication  should  lead  to  detectable  secretory  anti-HAV  in  intestinal 
contents. 

Pathology 

As  with  type  B  hepatitis,  pathologic  mechanisms  involved  in  type  A 
hepatitis  are  not  understood.   It  appears  that  type  A  hepatitis  rarely 


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if  ever  progresses  to  chronic  disease.   However,  this  assessment  is 
based  upon  a  limited  serologic  analysis  of  chronic  hepatitis  patients. 
It  is  now  possible  to  directly  examine  liver  biopsies  from  patients  with 
chronic  non-B  hepatitis  to  determine  if  their  hepatocytes  contain  HA  Ag. 
Such  studies  have  confirmed  that  HAV  is  not  an  important  cause  of  chronic 
hepatitis. 

(3)   Non-A,  Non-B,  Hepatitis  Viruses 

Antigens 

One  approach  to  the  identification  of  non-A,  non-B  hepatitis-associated 
antigens  is  the  application  of  techniques  useful  in  the  detection  and 
identification  of  HBV  and  HAV:   immune  electron  microscopy,  radioimmunoassays 
and  immunofluorescence.  By  application  of  radioimmunoassay  techniques, 
we  have  identified  a  serum  antigen  associated  with  two  cases  of  well- 
characterized  non-A,  non-B  hepatitis.   The  appearance  and  disappearance 
of  this  antigen  in  the  serum  was  temporally  related  to  the  hepatitis  in 
these  two  individuals,  and  the  antigen  was  shown  to  be  particulate  and 
biophysically  characterizable  by  ultracentrifugation  procedures. 
However,  insufficient  quantities  of  serum  were  available  for  more  detailed 
analysis.   Very  preliminary  antibody  surveys  suggested  that  antibody  to 
this  antigen  was  widespread  among  humans  and  chimpanzees  and  that,  if  it 
is  an  antigen  associated  with  non-A  non-B  hepatitis,  it  is  not  associated 
with  the  major  cause  of  this  disease.   Carriers  of  this  antigen  are 
being  sought  among  populations  known  to  have  a  high  prevalence  of  HB  Ag 
carriage  and  HAV  infection  in  order  to  collect  sufficient  material  for 
large-scale  purification  and  characterization. 

Transmission  of  non-A  non-B  agents  to  chimpanzees 

A  number  of  unsuccessful  attempts  to  transmit  non-A  non-B  agents  to 
primates  were  carried  out  in  past  years.   However,  recently  two  successful 
transmission  studies  were  completed  simultaneously,  one  by  the  Bureau  of 
Biologies  and  the  other  by  the  Clinical  Center  Blood  Bank  in  collaboration 
with  our  laboratory.   In  both  studies  the  incubation  period  in  chimpanzees 
was  comparable  to  that  for  non-A  non-B  hepatitis  in  man,  and  both  biochemical 
and  histologic  evidence  of  hepatitis  was  obtained.   In  our  collaborative 
study,  plasma  or  serum  from  patients  with  chronic  non-A  non-B  hepatitis 
as  well  as  from  patients  with  acute  infections  transmitted  disease  to 
chimpanzees.   Thus,  proof  of  chronic  carriage  of  non-A  non-B  viral 
hepatitis  agents  has  been  obtained. 

In  a  collaborative  confirmatory  study,  serial  plasmapheresis  units  and 
serial  liver  biopsies  were  obtained  from  experimentally  infected  chimpanzees. 
Fluorescein-labeled  convalescent  serum  from  the  original  non-A  non-B 
patients  and  from  experimentally  infected  chimpanzees  have  been  prepared 
and  are  being  tested  against  acute  phase  frozen  liver  biopsies  in  an 
attempt  to  identify  a  viral  antigen.   In  addition,  the  plasmapheresis 
units  will  be  subjected  to  ultracentrifugal  separations  and  studied  by 

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radioimmunoassay  and  immune  electron  microscopy  techniques  in  a  search 
for  viral  antigens.   Also,  previously  infected  chimpanzees  are  being 
cross-challenged  and  pools  of  infectious  plasma  are  being  identified  and 
aliquoted  for  subsequent  infectivity  titrations.   There  is  now  a  very 
high  probability  that  the  application  of  the  many  techniques  developed 
for  the  study  of  HBV  and  HAV  will  rapidly  lead  to  the  identification  and 
characterization  of  non-A  non-B  agents.   The  obvious  first  approach  to 
control  of  non-A  non-B  hepatitis  will  be  the  development  of  tests  for 
identifying  blood  donors  capable  of  transmitting  non-A  non-B  agents. 
Several  approaches  should  be  considered:   It  may  be  possible  to  identify 
a  serum  antigen  that  can  be  detected  in  a  manner  similar  to  HBs  Ag. 
Alternatively,  it  may  be  possible  to  detect  an  antibody  associated  with 
recent  or  chronic  non-A,  non-B  infection,  such  as  anti-HBc  in  HBV  infections. 
Finally,  it  might  be  possible  to  develop  or  identify  some  non-serologic 
test  that  has  a  strong  positive  correlation  with  presence  of  non-A  non- 
B  agents.  No  matter  what  direction  this  research  takes  there  will  be  a 
need  for  large  quantities  of  viral  antigen  for  the  development  of  serologic 
tests,  for  the  characterization  of  the  agents  and  for  seroepidemiologic 
studies.   These  will  most  likely  come  from  large-volume  purification  of 
antigens  from  plasma  of  chronic  carriers  (one  such  known  infectious 
carrier  is  already  being  plasmapheresed  on  a  regular  basis)  or  from 
extraction  of  antigens  from  liver  tissue  of  experimentally  infected 
chimpanzees  in  a  manner  similar  to  that  used  for  production  of  hepatitis 
A  viral  antigen  in  marmosets. 

Other 

As  part  of  our  study  of  non-A,  non-B  hepatitis  in  chimpanzees,  serial 
liver  biopsies  have  been  obtained.   Dr.  Shimizu  has  intensively  studied 
biopsies  by  thin  section  electronmicroscopy  and  has  found  morphologic 
evidence  for  two  types  of  non-A,  non-B  hepatitis.   One  produces  characteristic 
cytoplasmic  changes  that  consist  of  proliferation,  thickening  and  duplication 
of  endoplasmic  reticulum.   The  structures  so  formed  appear  to  be  cylinders 
of  modified  membrane  that  enclose  a  tube  of  rough  endoplasmic  reticulum. 
Although  the  structures  do  not  resemble  known  viruses,  they  do  "breed 
true",  that  is,  the  same  types  of  structures  are  seen  in  liver  biopsies 
of  chimpanzees  that  have  received  the  same  inoculum  or  that  are  part  of 
serial  transmission  studies  of  the  agents.   In  contrast,  the  other  non- 
A,  non-B  agent  produces  predominantly  nuclear  changes  that  consist  of 
shrinking,  heterogeneous  staining  of  the  chromatin  and,  in  some  cells, 
clusters  of  intranuclear  virus-like  particles.   These  changes  also 
"breed  true".  The  nature  of  the  structure  is  not  well  understood. 
Furthermore,  the  relationship  to  the  etiologic  agents  may  not  be  a 
direct  one.   Nevertheless,  they  provide  the  first  means  for  differentiating 
between  non-A,  non-B  agents.   Attempts  to  partially  purify  and  prepare 
antibodies  to  these  structures  are  currently  in  progress. 

The  development  of  tests  for  antigens  and  antibodies  associated  with 
non-A  non-B  hepatitis  will  provide  the  opportunity  for  seroepidemiologic 


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studies  similar  to  those  carried  out  for  types  A  and  B  hepatitis.   These 
tests,  plus  the  pools  of  virus  that  are  being  titered  and  certified  for 
infectivity  in  chimpanzees  will  provide  the  means  for  attempts  to  isolate 
the  virus (es)  in  vitro. 

Woodchuck  Virus 

Summers  recently  reported  the  discovery  of  a  virus  of  woodchucks  that 

resembles  HBV  in  many  respects.   Among  these  are  presence  of  three 

morphological  forms  very  similar  to  those  of  HBV,  presence  of  a  DNA 

dependent  DNA  polymerase  enzyme  activity,  a  genome  consisting  of  circular 

double- stranded  DNA  with  a  single- stranded  region,  serologic  cross  - 

reactivity  with  the  HBsAg  and  HBcAg  of  HBV  and  an  association  with  acute 

and  chronic  hepatitis  and  hepatic  cell  carcinoma.  The  last  association 

makes  this  virus  of  particular  interest,  because  the  woodchuck  may  serve 

as  a  useful  animal  model  for  hepatic  carcinogenesis.  Studies  of  the 

woodchuck  virus  are  being  carried  out  in  collaboration  with  Dr.  Gerin, 

who  is  characterizing  the  agent  biophysically,  biochemically  and  immunologically. 

A  colony  of  woodchucks  is  being  established  and  monitored  for  infection. 

Approximately  20-30%  of  wild-caught  woodchucks  have  had  evidence  of 

infection  with  woodchuck  virus,  either  at  time  of  capture  or  during  the 

first  three  months  of  captivity.   Thus,  the  woodchuck  virus  appears  to 

be  widespread  in  nature.   Plasma  from  these  naturally- infected  woodchucks 

has  been  used  as  a  source  of  raw  materials  for  development  of  reagents. 

Most  exciting  has  been  the  development  of  hepatic  cell  carcinoma  in  two 
animals  that  were  chronically  infected  with  the  woodchuck  virus.   Preliminary 
studies  of  alpha-fetoprotein  levels  indicate  that  woodchucks,  like  man, 
develop  elevated  levels  of  alpha-fetoprotein  when  hepatic  cell  carcinoma 
is  present. 

Histopathologic  evaluation  of  liver  biopsies  and  autopsy  material  from 
normal  woodchucks,  chronically  infected  with  the  woodchuck  virus  and 
woodchucks  with  hepatic  cell  carcinoma  is  being  performed  by  Professor 
Hans  Popper. 

Publications 

Dienstag,  J.L.,  Mathiesen,  L.R.,  and  Purcell,  R.H. :   Test  methods  and 
animal  models  for  hepatitis  A  virus  infection.   In  Vyas,  G.N. ,  Cohen, 
S.N.,  Shmid,  R.  (Eds):   Viral  Hepatitis,  Philadelphia,  Pa.,  Franklin 
Institute  Press  1978,  pp.  13-30. 

Feinstone,  S.M.,  Moritsugu,  Y.,  Shih,  J.W.K.,  Gerin,  J.L.,  and  Purcell, 
R.H.   Characterization  of  hepatitis  A  virus.    In  Vyas,  G.N.,  Cohen,  S.N., 
Schmid,  R.,(Eds):   Viral  Hepatitis,  Philadelhpia,  Pa.,  Franklin  Institute 
Press  1978,  pp.  41-48. 

Hess,  G.,  Shih,  J.W.K. ,  Gerin,  J.L.,  Purcell,  R.H.,  and  Meyer  zum  Buschenf elde, 
K.H.:   Hepatitis  B  antigens:   Lack  of  relationship  to  liver  specific 
protein  (LSP) :   J.  Med.  Virol. ,  in  press,  1978. 

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Purcell,  R.H.,  Gerin,  J.L.:   Hepatitis  B  virus  vaccines:   A  status  report. 
In  Vyas,  G.N.,  Cohen,  S.N.,  Schmid,  R. (Eds) :   Viral  Hepatitis,  Philadelhpia, 
Pa.,  Franklin  Institute  Press,  pp.  491-506. 

Purcell,  R.H.,  Gerin,  J.L.:  Hepatitis  B  vaccines:  On  the  threshold.  Am. 
J.  Clin.  Path.,  70:   159-169,  1978. 

Purcell,  R.H.,  Dienstag,  J.L. :  Experimental  hepatitis  A  virus  infection. 
In  Oda,  Toshitsugu,  (Ed):  Hepatitis  Viruses,  Tokyo,  Japan,  University  of 
Tokyo  Press  1978,  pp.  3-12. 

Purcell,  R.H.,  and  Alter,  H. J. :   Transfusion  associated  hepatitis  in  the 
United  States.   In  Oda,  Toshitsugu,  (Ed):   Hepatitis  Viruses,  Tokyo, 
Japan,  University  of  Tyokyo  Press  1978,  pp.  315-322. 

McAuliffe,  V.J.,  and  Purcell,  R.H.:   Current  status  of  tests  for  HBeAg  and 
anti-HBeAg.   In  Vyas,  G.N.,  Cohen,  S.N.,  Schmid,  R.  (Eds):   Viral  Hepatitis, 
Philadelphia,  Pa.,  Franklin  Institute  Press,  1978,  pp.  161-172. 

Smith,  D.,  Gribble,  T.J.,  Yeager,  A.,  Greenberg,  H.B.,  Purcell,  R.H., 
Robinson,  W. ,  Schwartz,  H.C.:   Spontaneous  resolution  of  severe  aplastic 
anemia  associated  with  viral  hepatitis  A  in  a  six-year-old  child:  Am.  J. 
Hematology,  5:   247-252,  1978. 

Shimizu,  Y.K.,  Mathiesen,  L.R.,  Lorenz,  D.,  Drucker,  J.,  Feinstone,  S.M., 
Wagner,  J. A.,  Purcell,  R.H. :   Localization  of  hepatitis  A  antigen  in  liver 
tissue  by  peroxidase-conjugated  antibody  method:   Light  and  electron 
microscopy  studies:   J .  Immun .  5:  1215-1671,  1978. 

Purcell,  R.H.,  Feinstone,  S.M.,  Wong,  D.C.,  Alter,  H.J.:   Detection  of  a 
novel  antigen  in  two  cases  of  non-A,  non-B  hepatitis,  a  recently  recognized 
persistent  infection  of  probable  viral  origin.   Proceedings  of  the  1978 
ICN-UCLA  Symposium  on  Persistent  Viruses,  pp.  535-549. 

Feinstone,  S.M.,  Purcell,  R.H.,  Barker,  L.F.:   Viral  Hepatitis,  in  Lennette, 
E.H.,  and  Schmidt,  N.J.  (eds.),  Diagnostic  Procedures  for  Viral  and 
Rickettsial  Infections,  New  York,  American  Public  Health  Assoc,  Inc.,  in 
press,  1978. 

Alter,  H.J.,  Purcell,  R.H.,  Feinstone,  S.M. ,  Holland,  P.V.,  and  Morrow, 
A.G.:   Non-A,  Non-B  Hepatitis:   A  review  and  interim  report  of  ongoing 
prospective  study.  In  Vyas,  G.N.,  Cohen,  S.N.,  Schmid,  R. (Eds) :   Viral 
Hepatitis,  Philadelphia,  Pa.,  Franklin  institute  Press  1978,  pp.  359-370. 

Mathiesen,  L.R.,  Feinstone,  S.M. ,  and  Purcell,  R.H.:   Present  methods  for 
detection  of  hepatitis  A  antigen  and  antibody.   Acta  Pathologica  et 
Microbiologica  Scandinavica,  in  press,  1978. 


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Purcell,  R.H.,  and  Gerin,  J.L. :   Hepatitis  B  vaccines:   a  review  of  progress. 
Acta  Pathologica  et  Microbiologica  Scandinavica,  in  press,  1978. 

Murphy,  B.L.,  Maynard,  J.E.,  Bradley,  D.W.,  Ebert,  J.W.,  Mathiesen,  L.R., 
and  Purcell,  R.H. :   Immunofluorescence  of  Hepatitis  A  Virus  Antigen  in 
Chimpanzees.   Infect.  Immun. ,  21:   663-665,  1978. 

Cherubin,  C.E.,  Nair,  S.R.,  Dienstag,  J.L.,  Purcell,  R.H.,  and  Szmuness, 
W. :   Antibody  to  Hepatitis  A  and  B  in  Children  in  New  York  City.  Pediatrics 
61:   781,  1978. 

Ziegler,  J.L.  Adamson,  R.H. ,  Barker,  L.F.,  Fraumeni,  J.F.,  Jr.,  Gerin,  J., 
and  Purcell,  RH. :  International  workshop  on  hepatitis  B  and  liver  cancer: 
Special  Report.   J.  Natl.  Cancer  Inst.  60:   717,  1978. 

Taylor-Robinson,  D.,  Purcell,  R.H.,  London,  W.T.,  and  Sly,  D.L. :   Urethral 
Infection  of  Chimpanzees  by  Ureaplasma  Urealyticum:   J.  Med.  Microbiol. 
11:   197-201,  1978. 

Murphy,  B.,  Tabor,  E.,  McAuliffe,  V.,  Williams,  A.,  Maynard,  J.,  Gerety, 
R. ,  and  Purcell,  R. :   Third  Component,  HBeAg/3,  of  Hepatitis  B  e  Antigen 
System,  Identified  by  Three  Different  Double-Diffusion  Techniques.  Journal 
of  Clinical  Microbiology,  8:  349-350,  1978. 

Mathiesen,  L.R. ,  Drucker,  J.,  Lorenz,  D.,  Wagner,  J.,  Gerety,  R.J.,  and 
Purcell,  R.H. :   Localization  of  Hepatitis  A  Antigen  in  Marmoset  Organs 
during  Acute  Infection  with  Hepatitis  A  Virus.   Journal  of  Infectious 
Diseases,  138:  369-377,  1978  ~~~ 

Hess,  G.,  Arnold,  W. ,  Meyer  zum  Buschenfelde,  K.H.,  Purcell,  R.H.:  [Anti- 
HBc  titer  in  HBeAg  (e-antigen)  and  anti-HBe  (anti-e)  positive  asymptomatic 
hepatitis  B  surface  antigen  (HBsAg)  carriers].   Verh  Dtsch  Ges  Inn  Med  84; 
943-5,  1978. 

Wong,  D.C.,  Purcell,  R.H.  and  Rosen,  L:   Prevalence  of  Antibody  to  Hepatitis 
A  and  Hepatitis  B  Viruses  in  Selected  Populations  of  the  South  Pacific. 
American  Journal  of  Epidemiology,  in  press,  1978. 

Hansson,  B.G.,  Purcell,  R.H. :  Sites  that  Bind  Polymerized  Albumin  on 
Hepatitis  B  Surface  Antigen  Particles:  Detection  by  Radioimmunoassay. 
Infection  and  Immunity,  in  press,  1979. 

Galbraith,  R.M.,  Dienstag,  J.L.,  Purcell,  R.H.,  Gower,  P.H.,  Zuckerman, 
A.J.,  Williams,  R. :   Non-A,  Non-B  Hepatitis  Associated  with  Chronic  Liver 
Disease  in  a  Haemodialysis  Unit.   Lancet,  in  press,  1979. 

Mathiesen,  L.R.,  Hardt,  F.,  Dietrichson,  0.,  Purcell,  R.H.,  Wong,  D., 
Skinhoj,  P.,  Nielsen,  J.O.,  Zoffmann,  H.,  Iversen,  K. :   The  Role  of  Acute 
Hepatitis  Type  A,  B  and  Non-A,  Non-B  in  the  Development  of  Chronic  Active 
Liver  Disease.   Scand  J.  Gastroenterology,  in  press,  1979. 


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Berman,  M. ,  Alter,  H.J.,  Ishak,  K. ,  Purcell,  R.H.,  Jones,  A.:  The  Chronic 
Sequelae  of  Non-A/Non-B  Hepatitis.  Annals  of  Internal  Medicine,  in  press. 
1979.  -  — 

Dienstag,  J.L.,  Bhan,  A.K.,  Alter,  H.J.,  Feinstone,  S.M. ,  Purcell,  R.H. : 
Non-A,  non-B  Hepatitis:   Possible  Masking  of  Virus  Antigen  within  Immune 
Complexes.   Lancet,  in  press,  1979. 


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SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  AI  00027-12  LID 


PERIOD    COVERED 


October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

BASIC  STUDIES  OF  MYCOPLASMAS 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:   Joseph  G.  Tully,  Ph.D. 
Other:   David  L.  Rose 


LID,  NIAID      Head,  Mycoplasma  Section 
LID,  NIAID      Research  Microbiologist 


cooperating  units  (if  any)  State  Univ .  N.Y.,  Stony  Brook,  N.Y.  (.Dr.  David  Williamson) 
USDA,  Beltsville,  Md.  (Dr.  R.  Whitcomb) ;  British  Ministry  Overseas  Develop., 
Jamaica  (Dr.  Eden-Green);  Univ.  Fla.,  Fort  Lauderdale,  Fla.  (Dr.  R.  McCoy); 
Children's  Hospital,  Washington,  D.C.(Dr.  H.Kim);  BOB,  FDA  (Dr.  Barile) ;  Univ. 


lab/branch       Bordeaux  (.France)  (Dr. 
LABORATORY  OF  INFECTIOUS  DISEASES 


tiove) 


SECTION 

MCYOPLASMA  SECTION 


INSTITUTE  ANO  LOCATION 

NATIONAL  INSTITUTE  OF  ALLERGY  AND  INFECTIOUS  DISEASES 


TOTAL  MANYEARS: 

3/12 


PROFESSIONAL: 

2/12 


1/12 


CHECK  APPROPRIATE  BOX(ES) 
G  (a)   HUMAN  SUBJECTS 


□  (b)  HUMAN  TISSUES 


S'(c 


□  (a1 )  MINORS 


(a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

These  efforts  cover  both  basic  and  applied  aspects  of  mycoplasmas  and  related 
wall-free  prokaryotes,  including  their  neurotoxins,  antigens  and  other  biological 
factors  involved  in  virulence,  their  immunological  interrelationships ,  and  their 
possible  role  in  human  disease  or  diseases  of  uncertain  etiology.   Current 
projects  of  interest  concern  the  characterization  and  serological  interrelationsh 
of  an  expanding  group  of  helical  mycoplasmas  (spiroplasmas)  being  isolated  from 
plants  and  a  variety  of  insects,  especially  ticks.   Recent  studies  have  confirmed 
that  many  of  these  new  organisms  possess  overt  pathogenicity  for  vertebrates 
(embryonated  chicken  eggs  and  suckling  rats) ,  suggesting  that  they  may  represent 
important  pathogens  for  man.   Further  sero-epidemiological  studies  in  man  seem 
warranted.   Recent  collaborative  studies  on  the  acholeplasmas  (non-sterol- 
requiring  mycoplasmas)  isolated  from  plant  and  animal  sources  suggest  some 
alteration  in  the  concept  that  these  organisms  are  parasitic.   The  occurrence  of 
free-living  acholeplasmas  in  plants,  some  species  of  which  had  previously  only 
been  found  in  vertebrates,  raises  important  questions  about  vector  transmission 
and  other  host-parasite  relationships  of  these  organisms. 


lp: 


PHS-6040 
(Rev.    10-76) 


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Z01  AI  00027  -12  LID 
Project  Description 

Studies  directed  to  various  aspects  of  the  biology  and  pathogenicity  of 
helical  mycoplasmas  (spiroplasmas)  have  continued.   Our  characterization 
of  these  agents  as  the  first  known  spiroplasmas  with  pathogenicity  for 
vertebrates  has  opened  up  a  number  of  areas  for  developing  the  possible 
role  of  these  agents  in  human  disease.   Current  work  is  directed  to  three 
broad  areas,  covering  (1)  the  biological  characterization  of  spiroplasmas, 
including  their  serological  and  biochemical  interrelationships;  (2) 
development  of  appropriate  models  to  study  their  pathogenicity  and  host 
response,  which  includes  not  only  small  laboratory  animals  but  primates, 
and  (3)  evaluation  of  various  techniques  (primary  isolation  in  culture, 
serological,  histological,  etc.)  to  assess  the  possible  role  of  spiroplasmas 
in  acute  and  chronic  human  disease.   We  are  also  concerned  with  other 
mycoplasmas,  particularly  with  the  acholeplasmas  (nonsterol-requiring 
mycoplasmas) ,  since  these  strains  have  been  recovered  frequently  from 
contaminated  cell  cultures  and  new  and  previously  documented  species  of 
the  genus  Acholeplasma  are  being  isolated  from  plants,  insects,  and  a  wide 
variety  of  free-living  sources  at  present. 

(A)   Pathogenicity  of  spiroplasmas  for  vertebrates 

Studies  on  the  virulence  of  spiroplasmas  for  embryonated  chicken  eggs  and 
suckling  rats  have  been  expanded  with  the  availability  of  a  number  of  new 
spiroplasmas  from  different  hosts.   The  overall  results  of  this  testing 
program  are  suggestive  of  the  presence  of  some  unique  virulence  markers  in 
spiroplasmas.   The  most  pathogenic  group  of  spiroplasmas  is  the  suckling 
mouse  cataract  cluster,  including  strains  SMCA,  GT-48 ,  and  TP-2.   These 
strains,  for  the  most  part,  are  highly  pathogenic  for  the  chick  embryo, 
showing  LD,-n  values  of  1-10  organisms/egg.   In  a  few  instances  (the  TP-2 
strain) ,  an  organism  may  show  a  decline  in  virulence  with  continued  passage 
on  artificial  medium  or  after  purification  by  filter-cloning  techniques. 
The  pathogenicity  of  these  strains  for  suckling  rats  is  more  interesting. 
The  classical  SMCA  strain  always  has  shown  rather  low  mortality  for  the 
suckling  rat,  with  intracerebral  challenge  levels  of  10   or  10   organisms/ 
rat  required  for  an  LD   dose.   However,  the  survivors  of  this  challenge 
level,  and  those  animals  receiving  dosages  of  at  least  two  logs  less, 
showed  a  very  high  incidence  of  bilateral  cataracts.   In  contrast,  suckling 
rats  challenged  with  the  GT-48  strain  have  never  been  observed  to  have 
cataracts.  Since  the  mortality  rate  in  suckling  rats  receiving  the  GJ-48 
strain  is  much  higher  (usually  1  LD5Q  dose  of  this  strain  is  near  10 
organisms  or  less) ,  it  is  thought  that  the  absence  of  cataracts  may  have 
some  relationship  to  surviving  a  massive  intracerebral  challenge  of  spiro- 
plasmas.  It  is  also  possible  that  some  toxic  component  of  the  SMCA  strain 
is  present  in  sufficient  amounts  in  high  challenge  levels  of  the  organism 
and  information  on  soluble  components  of  these  spiroplasmas  seems  warranted. 
Finally,  the  most  unique  observations  were  made  on  the  freshly-isolated 
TP-2  strain.   Early  passages  of  this  strain  had  a  pattern  of  pathogenicity 
very  similar  to  that  of  the  SMCA  strain  (high  virulence  for  the  chick 
embryo,  low  mortality  for  suckling  rats,  and  occurrence  of  cataracts  in 


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Z01  AI  00027-12  LID 

surviving  rats) .   After  purification  and  cultivation  in  artificial  medium 
for  25  passages,  this  strain  showed  a  completeQreversal_in  this  pattern  of 
virulence.   The  egg  LD    titer  dropped  from  10  '  ,-to  10  '   while  the  LD,-n 
titer  for  rats  increased  from  less  than  10   to  10  organisms.   The  occurrence 
of  cataracts  disappeared  with  the  increase  of  mortality  in  suckling  rats. 
Thus,  the  TP-2  strain,  with  the  observed  changes  in  virulence  pattern, 
offers  an  important  model  to  study  the  mechanism  for  this  transition  in 
pathogenicity.   Pathogenicity  tests  with  other  spiroplasmas  for  the  two 
animal  models  outlined  above  have  also  led  to  some  new  insights  regarding 
the  importance  of  these  organisms  in  induction  of  disease.   Strains  recovered 
from  the  honey  bee  (BC  and  KC  strains)  have  been  found  to  show  increasing 
virulence  for  the  chick  embryo  following  passage  on  the  SP-4  culture 
medium.   When  both  strains  were  carried  for  30^40  passages  on  thisRmedium, 
the  egg  LD,.   titer  increased  from  less  than  10   to  greater  than  10   (that 
is,  the  dose  required  to  kill  embryos  changed  from  10  organisms/egg  to 
less  than  10  organisms/egg.   While  the  honey  bee  spiroplasmas  had  been 
previously  shown  to  be  highly  pathogenic  for  bees,  the  results  reported 
here  indicate  that  vertebrates  are  also  very  susceptible  to  infections 
with  this  organism.   Although  only  limited  pathogenicity  studies  have  been 
done  with  the  free-living  flower  spiroplasmas  (OBMG  and  BNR1  strains) , 
the  early  results  indicate  that  these  organisms  are  also  pathogenic  for 
the  chick  embryo.   Studies  with  higher  passage  lines  of  these  strains 
remains  to  be  done.   Pathogenicity  tests  with  other  groups  of  spiroplasmas, 
representing  members  of  the  Spiroplasma  citri  serological  complex,  have 
not  demonstrated  virulence  for  chick  embryos  or  suckling  rats. 

(B)   Serological  tests  with  spiroplasmas 

In  early  studies  with  spiroplasmas,  it  appeared  that  conventional  mycoplasma 
serological  tests  were  less  suitable  for  these  organisms,  not  only  in 
assessing  serological  relationships  among  individual  strains  but  in  evaluating 
serological  response  to  these  organisms.   As  noted  in  the  last  report,  we 
have  developed  a  combined  deformation/metabolism-inhibition  (DF/MI) 
serological  test  for  spiroplasmas  and  have  now  applied  this  test  to  an 
analysis  of  about  twenty  strains  isolated  from  a  variety  of  sources.   The 
deformation  test  is  based  upon  the  observation  that  helical  spiroplasmas 
in  the  presence  of  specific  antibody  become  deformed.   The  metabolism- 
inhibition  test,  previously  developed  in  LID  by  other  investigators  for 
conventional  mycoplasmas,  is  based  upon  the  ability  of  a  specific  antiserum 
to  inhibit  metabolism  (in  this  case,  glucose  fermentation)  of  spiroplasmas 
grown  in  broth  cultures.   A  number  of  test  modifications  were  made  for  the 
spiroplasmas  and  the  addition  of  fresh  complement  was  required  in  the 
system.   A  combined  DF/MI  procedure  has  been  adapted  to  microtiter  plates 
and  a  frozen,  standardized  spiroplasma  antigen  prepared  for  all  strains 
examined.   The  DF  procedure  has  the  advantage  of  rapid  results  (final 
readings  in  1  hour),  although  it  requires  darkfield  microscopy.   The  MI 
procedure  requires  no  special  equipment  but  final  readings  are  only 
available  after  3-7  day  incubation  period.   The  results  of  the  serological 
analysis  of  spiroplasmas  by  DF/MI  has  clarifed  a  number  of  major  questions 
about  the  interrelationships  of  these  twenty  strains  recovered  from  a 


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Z01  AI  00027-12  LID 

number  of  sources  by  other  investigators.   Strains  isolated  from  cactus 
and  lettuce  plants  appear  to  be  identical  to  isolates  of  Spiroplasma  citri 
involved  in  citrus  stubborn  disease.   Spiroplasmas  from  ticks  (277F  strain), 
honey  bees  (BC3  and  KC3  strains) ,  and  from  corn  stunt  disease  (E-275  and 
Miss,  strains),  all  appear  to  be  members  of  a  large  S_^  citri  complex  and 
strains  in  this  grouping  all  share  a  number  of  major  serological 
determinants.   We  have  assigned  serogroup  numbers  to  major  members  of  the 
complex.  The  SMCA  group  (SMCA,  GT-48,  and  TP-2  strains),  the  flower  spiro- 
plasm  group  I  (OBMG  and  BNR1  strains) ,  the  flower  spiroplasma  group  II 
(Powder  puff  strain) ,  and  the  sex-ratio  spiroplasmas  from  Drosophila 
species  all  appear  to  be  serologically  distinct  groups  of  spiroplasmas. 
Further  comparison  of  these  spiroplasmas  by  the  conventional  mycoplasma 
growth  inhibition  technique  have  also  shown  similar  results,  indicating 
that  this  procedure  may  also  be  applied  to  serological  analysis  of  spiro- 
plasmas.  The  results  of  this  analysis  have  provided  data  important  to  our 
continued  sero-epidemiological  study  of  spiroplasma  antibody  in  the  serum 
of  man  and  other  vertebrates.   (Williamson,  State  Univ.,  of  N.Y.;  Whitcomb, 
USDA) . 

(C)   Studies  on  acholeplasmas 

We  have  continued  our  interest  in  the  acholeplasmas  (nonsterol-requiring 
mycoplasmas)  since  these  organisms  occur  frequently  in  a  wide  variety  of 
animal  hosts  and  are,  therefore,  important  contaminants  of  tissue  culture 
systems.   Two  unclassified  acholeplasmas,  previously  isolated  from  cell 
cultures  and  contaminated  bovine  serum,  are  being  characterized.   Serological 
tests,  with  a  variety  of  techniques,  and  biochemical  studies  indicated 
that  these  two  strains  (72-043  and  S2)  are  not  related  to  other  classifed 
species  in  the  genus  Acholeplasma.   DNA-DNA  hybridization  techniques  were 
performed  on  the  two  strains  and  comparisons  were  made  between  the  radio- 
labeled DNA  of  these  organisms  and  DNA  obtained  from  a  collection  of 
organisms  in  the  genus  Acholeplasma  and  genus  Mycoplasma.   Control  systems 
also  included  reciprocal  DNA-DNA  hybridization  between  A^  laidlawii  and  A. 
granularum,  where  previous  studies  with  less  sensitive  hybridization 
techniques  (cellulose  membrane  procedure)  had  shown  partial  relationships 
(at  about  34%  level) .  The  results  of  our  study  showed  that  the  new  achole- 
plasmas were  not  genetically  related  to  any  of  the  previously  classified 
acholeplasmas,  although  they  were  closely  related  to  each  other  (at  about 
80-85%  level) .   A^  laidlawii  and  A^  granularum  shared  relatedness  values 
of  about  23%  in  reciprocal  tests,  confirming  earlier  judgement  that  these 
organisms  were  distinct  species  but  distantly  related.  Efforts  are  in 
progress  to  complete  the  biological  and  serological  characterization  of 
the  72-043  and  S2  strains  as  new  species  of  Acholeplasma.   (Aulakh  and 
Barile,  FDA). 

Other  areas  of  acholeplasma  research  involve  two  collaborative  studies  in 
which  we  are  supplying  assistance  in  the  serological  analysis  of  strains 
recovered  from  plant  sources.   In  one  study  (Eden-Green,  Jamaica),  a 
number  of  acholeplasmas  have  been  recovered  from  crowns  of  palms  infected 
with  lethal  yellowing  disease.   We  have  typed  these  strains  as  A^  axanthum 
and  A^  oculi.   In  extended  studies,  35  isolations  of  acholeplasmas  from 
252  samples  obtained  from  infected  crowns  (positive  isolations  in  13.8%) 

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have  been  made.   Acholeplasmas  were  not  isolated  from  69  samples  from 
healthy  trees.   Of  the  35  isolations  made,  about  70%  of  the  strains  were 
A.  axanthum  and  30%  are  A^  oculi.   These  observations  are  the  first  clear 
illustration  that  acholeplasmas  might  represent  true  saphrophytes .   The 
results  are  also  of  interest  since  all  previous  isolations  of  both  A. 
axanthum  and  k^  oculi  have  been  from  animal  tissues  or  serum.   It  was  not 
fully  established  at  present  whether  the  recovered  acholeplasmas  were 
involved  in  the  etiology  of  lethal  yellowing  disease,  but  the  observations 
have  relevance  to  the  ecology  of  the  acholeplasmas.   The  second  collaborative 
study  involves  serological  comparisons  of  a  number  of  acholeplasmas  (and  a 
few  sterol-requiring  mycoplasmas)  recovered  from  tropical  flowering  trees 
(McCoy,  Univ.  Fla.).   Isolates  recovered  from  two  separate  tree  flowers 
have  been  found  to  share  a  number  of  properties  of  animal  acholeplasmas, 
including  ability  to  grow  in  a  serum-free  culture  medium,  fermentation  of 
glucose,  etc.   The  organisms  have  not  been  shown  to  be  serologically 
related  to  any  other  acholeplasma  or  mycoplasma  from  animal  hosts.  As 
noted  above,  the  occurrence  of  free-living  acholeplasmas  and  mycoplasmas 
is  unique  and  the  addition  of  typing  antisera  for  these  organisms  to  a 
collection  of  reference  sera  might  serve  a  useful  epidemiological  purpose. 

(D)  Mycoplasmas  from  clinical  specimens 

The  SP-4  medium  developed  for  isolation  and  cultivation  of  the  pathogenic 
suckling  mouse  cataract  spiroplasmas  was  found  to  increase  the  efficiency 
of  isolation  of  Mycoplasma  pneumoniae  from  human  throat  specimens  (see 
last  years  report)  This  medium  was  also  useful  for  cultivation  of  a  number 
of  other  fastidious  mycoplasmas.  The  current  study  was  designed  to  recover 
spiroplasmas  and  mycoplasmas  from  throat  specimens  collected  at  the  Children's 
Hospital  National  Medical  Center,  Washington,  D.C.  Bacterial  overgrowth  in 
the  throat  specimens  was  a  major  problem,  contrary  to  observations  made  on 
throat  specimens  from  Marine  recruits,  where  the  usual  bacterial  inhibitors 
in  mycoplasma  medium  were  sufficient  to  prevent  contamination.  The  problem 
was  resolved  by  the  addition  of  500  units/ml  of  polymyxin  to  the  transport 
and  culture  medium,  amounts  which  have  been  found  to  have  no  obvious 
effect  on  mycoplasmas  or  spiroplasmas.   Studies  have  been  made  on  98 
throat  specimens  and  the  M^_  pneumoniae  isolation  rate  was  about  10%  on 
these  samples.  Another  196  specimens  from  throat  swabs  are  under  test  now. 
A  small  number  of  spinal  fluids  have  been  tested  in  this  study,  without 
recovery  of  mycoplasma  or  spiroplasmas.  (Kim,  Children's  Hospital). 

(E)  Other  mycoplasma  research 

A  number  of  other  research  and  collaborative  projects  have  been  completed 
or  are  in  progress  within  the  section.   These  include: 

(a)   A  collaborative  study  with  investigators  at  the  University  of  Bordeaux, 
France  on  the  use  of  two  dimensional  slab  gel  electrophoresis  to  determine 
the  interrelationships  of  spiroplasmas  recovered  from  various  sources. 
The  results  show  this  technique,  which  is  essentially  a  finger-print  of 
radiolabeled  proteins  of  the  individual  spiroplasma,  to  be  an  effective 
procedure  to  distinguish  specific  spiroplasma  groups. 

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Z01  AI  00027-12  LID 

(b)  A  service  type  study,  performed  on  a  part-time  basis,  to  assess  the 
occurrence  of  mycoplasma  contamination  of  cell  cultures  (mostly  continuous 
cell  lines)  in  NIAID  laboratories.   This  testing  program  covers  culture 
procedures  and  two  indirect  staining  methods  (DNA  and  specific  immuno- 
fluorescence tests  with  anti-M.  hyorhinis  conjugate)  on  indicator  cell 
lines  (Vero  cells) .   Eighty-seven  tissue  culture  specimens  (from  ten 
principal  investigators)  have  been  examined  from  October  1978  to  June 
1979,  with  32  of  the  specimens  being  positive  for  one  or  more  mycoplasmas 
(positive  rate  of  36%) .   This  rate  is  not  necessarily  the  true  incidence 
of  mycoplasma  contamination,  since  a  number  of  known  contaminated  cell 
lines  were  examined  at  various  passage  levels  so  that  the  investigator 
could  obtain  a  clean  cell  line.   However,  two  points  should  be  emphasized: 
1)  mycoplasma  detection  procedure  must  involve  the  use  of  indicator  cells 
since  some  mycoplasmas  (M.  hyorhinis)  can  lose  the  ability  to  grow  on 
artificial  culture  medium  and  these  strains  can  only  be  detected  by  the  FA 
procedure,  and  2)  mycoplasma  contamination  will  compromise  the  results  of 
any  study  performed  on  infected  cell  cultures. 

(c)  The  taxonomic  characterization  of  the  SMCA  group.   The  results  of  the 
serological  analysis  of  spiroplasmas  (noted  above)  has  confirmed  the 
unique  and  distinct  status  of  the  SMCA  group  of  spiroplasmas.   Data  on  the 
biological  and  morphological  features  of  this  group  of  spiroplasmas  can 
now  be  applied  to  a  full  characterization  of  the  organisms  (Whitcomb, 
USDA) . 


Publications 

Rose,  D.L.,  Tully,  J.G.,  and  Langford,  E.V.:  Mycoplasma  citelli,  a  new 
species  from  ground  squirrels.  Int.  J.  Systematic  Bacteriol.  28:  567- 
572,  1978.  '" 

Williamson,  D.L.,  Whitcomb,  R.F.,  and  Tully,  J.G.:   The  spiroplasma 
deformation  test,  a  new  serological  method.   Current  Microbiol.  1:  203- 
207,  1978. 

Tully,  J.G.:  Special  features  of  the  acholeplasmas .  In  Barile,  M.F.  and 
Razin,  S.  (Eds.)  The  Mycoplasmas,  New  York,  Academic  Press,  1979,  Vol.  1, 
pp.  431-449. 

Tully,  J.G.,  Rose,  D.L. ,  Whitcomb,  R.F.,  and  Wenzel,  R.P.:  Enhanced 
isolation  of  Mycoplasma  pneumoniae  from  throat  washings  with  a  newly 
modified  culture  medium.  J.  Infect.  Pis.  139:  478-482,  1979. 

Aulakh,  G.S.,  Tully,  J.G. ,  and  Barile,  M.F.:   Differentiation  among  some 
acholeplasmas  by  nucleic  acid  homology.   Current  Microbiol.  2:   91-94, 
1979. 

Tully,  J.G.,  and  Whitcomb,  R.F.  (Eds.):   The  Mycoplasmas,  Volume  II,  Human 
and  Animal  Mycoplasmas,  New  York,  Academic  Press,  1979,  pp.  509. 


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Z01  AI  00027-12  LID 

Whitcomb,  R.F.,  and  Tully,  J.G.,  (Eds.):  The  Mycoplasmas,  Volume  III, 
Plant  and  Insect  Mycoplasmas,  New  York,  Academic  Press,  1979, pp.  334. 

Mouches,  C,  Vignault,  J-C,  Tully,  J.G.,  Whitcomb,  R.F.,  and  Bove,  J.M.: 
Characterization  of  spiroplasmas  by  one  and  two  dimensional  protein  analysis 
on  polyacrylamide  slab  gels.   Current  Microbiol.  2:  69-74,  1979. 

Rose,  D.L.,  Tully,  J.G.  and  Wittier,  R.G. :   Taxonomy  of  some  swine 
mycoplasmas:  Mycoplasma  suipneumoniae  Goodwin  et  al.  1965,  a  later  objective 
synonym  of  Mycoplasma  hypopneumoniae  Mare  &  Switzer  1965,  and  the  status 
of  Mycoplasma  flocculare  Meyling  and  Friis  1972.   Int.  J.  Systematic 
Bacteriol.  29:  83-91,  1979. 

Freundt,  E.A.,  Whitcomb,  R.F.  et  al. :   Proposal  of  minimum  standards  for 
description  of  new  species  of  the  class  Mollicutes.   Int.  J.  Systematic 
Bacteriol.  29:  172-180,  1979. 

Eden-Green,  S. ,  and  Tully,  J.G.:   Isolation  of  Actio leplasma  species  from 
coconut  palms  affected  by  lethal  yellowing  disease  in  Jamaica.   Current 
Microbiol.  2:  311-316,  1979. 

Williamson,  D.L.,  Tully,  J.G.,  and  Whitcomb,  R.F.:   Serological  relationships 
of  spiroplasmas  as  shown  by  combined  deformation  and  metabolism  inhibition 
tests.   Int.  J.  Systematic  Bacteriol.  (in  press). 

Adegboye,  D.S.,  Addo,  P.B.,  Ogunkoya,  A.B.,  and  Rose,  D.L.:   The  occurrence 
of  Mycoplasma  canis  in  two  colonial  morphological  forms.   Vet.  Record,  (in 
press) . 

Tully,  J.G.,  and  Whitcomb,  R.F.:   The  genus  Spiroplasma.   In  Starr,  M.P., 
Stolp,  H.,  Truper,  H.G.,  Balows,  A.,  and  Schlegel,  H.G.  (Eds.):   The 
Prokaryotes,  New  York,  Springer-Verlag,  1979  (in  press). 

Chanock,  R.M.  and  Tully,  J.G. :   The  Mycoplasmas.   In  Davis,  B.D.,  Dulbecco, 
R.A. ,  Eisen,  H.  and  Ginsburg,  H.  (Eds.)  Microbiology,  3rd  edition,  Hagerstown, 
Md.,  Harper  &  Row,  1980  (in  press). 


23-75 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZU1  AI  00028-  21  LID 


PERIOD  COVERED 


OCTOBER  1,  1978  TO  SEPTEMBER  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

STUDY  OF  RESPIRATORY  VIRUSES  AND  MYCOPLASMAS  IN  VOLUNTEERS 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


P.I.:   Brian  R.  Murphy,  M.D. 

Robert  M.  Chanock,  M.D. 
Lewis  J.  Markoff,  M.D. 


Medical  Officer 

Chief 

Medical  Officer 


LID,  NIAID 
LID,  NIAID 
LID,  NIAID 


COOPERATING  UNITS  (if  any) 

Flow  Laboratories,  Rockville,  Md 


University  of  Maryland  School  of 
Baltimore,  Md . ;  Children's  Hospital  National  Medical  Center,  Washing 
University  of  Rochester,  New  York;  Vanderbilt  University,  Nashville. 

Houston ,  Texas 


m 


Lty 

lor  Colls 


Medicine, 

;ton,  D.C. 

Tenn. ; 


if  Medi 


LABORATORY  OF  INFECTIOUS  DISEASES 


RESPIRATORY  VIRUSES  SECTION 


NSTITUTE  AND  LOCATION 

NATIONAL  INSTITUTE 


m 


nv  AT.T.FRnv  ANT)  TNFF.CTTOIIS  DISEASES.  BETHESDA,  MARYLAND 


ear;,: 


36/12 


PROFESSIONAL: 

12/12 


OTHER: 

12/12 


CHECK  APPROPRIATE  BOX(ES) 
Xx(a)   HUMAN  SUBJECTS 

□  (a!)  MINORS   □  (a2)  INTERVIEWS 


~3   (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  response  of  adult  volunteers  to  a  series  of  ts  or  cold-adapted  recombinants, 
was  evaluated.   The  A/Alaska/7  7-ts-lA2  (H3N2)  and  A/HK/123/77-_ts-lA2  (HINI) 
viruses,  each  of  which  had  a  37°C  shutoff  temperature,  were  satisfactorily 
attenuated  and  induced  an  immunological  response  in  greater  than  84%  of  the 
volunteers.   Virus  shed  by  each  of  the  volunteers  retained  the  t_s  phenotype. 
A/Alaska/77  (H3N2)  cold-adapted  recombinant  virus  which  possessed  a  39°C 
shutoff  temperature  for  plaque  formation  was  also  suitably  attenuated  and 
immunogenic  in  volunteers.   The  A/HK/77  cold-adapted  recombinant  possessed  a 
37°C  shutoff  temperature  and  was  satisfactorily  attenuated  and  immunogenic 
in  volunteers.   The  virus  shed  by  volunteers  retained  the  _ts  and  cold- 
adapted  properties.   These  studies  indicate  that  the  two  master  strains  of 
influenza  A  virus  can  confer,  via  the  mechanism  of  gene  transfer  a  satisfactory 
level  of  attenuation,  immunogenicity,  and  genetic  stability  on  virulent 
viruses  belonging  to  two  influenza  A  subtypes. 


PHS-6040 
(Rev.    10-76) 


23-76 


Z01  AI  00028-21  LID 

Project  Description 

Evaluation  of  Cold-Adapted  Influenza  A  Virus  Recombinants  in  Adults  - 
Recombinant  viruses  produced  by  mating  the  A/AA/6/60  cold-adapted  donor 
virus  and  cloned  wild  type  A/Alaska/77  (H3N2)  or  A/Hong  Kong/123/77 
(H1N1)  viruses  were  evaluated  in  adults  for  reactogenicity  and  immunogenicity. 
The  Alaska/77  CR-29  clone  2  virus  had  a  39° C  shutoff  temperature  for 
plaque  formation  and  the  HK/77-CR-35  recombinant  had  a  37°C  shutoff 
temperature.   This  was  so  despite  the  fact  that  both  viruses  received  all 
six  non-glycoprotein  genes  (i.e.,  PI,  P2,  P3,  NP,  M,  and  MS)  from  the 
AA/6/60  donor  virus.   The  basis  for  and  significance  of  this  difference  in 
shutoff  temperatures  remains  unknown. 

The  CR-29  virus  (H3N2)  was  administered  at  a  dose  of  10   TCID   to  24 
seronegative  adults  (all  with  pre- inoculation  serum  HI  antibody  titer  of 
<1:8  and  19  of  24-with  NI  titer  of  <1:4).   Eleven  shed  virus  in  a  titer 
ranging  from  10  "   to  10    TCID,n  per  ml  of  nasopharyngeal  wash  fluid  for 
an  average  duration  of  0. 71  days  which  is  significantly  less  than  the 
interval  of  4.5  days  observed  for  volunteers  who  received  wild  type  virus. 
Each  of  the  17  isolates  retained  the  _ts  phenotype.   Twelve  volunteers 
developed  a  rise  in  serum  HI  antibody  and  11  had  a  rise  in  serum  NI  antibody. 
Sixteen  volunteers  had  a  rise  of  HI  and/or  NI  antibody.   Two  volunteers 
became  ill;  one  was  febrile  and  the  other  had  a  cold  with  pharyngitis  and 
systemic  symptoms.   The  virus  was  clearly  more  attenuated  than  wild  type 
virus  which  (a)  produced  illness  in  50%  of  the  volunteers  at  a  dose  of 
10  "  TCID,.^  and  (b)  was  shed  longer  and  in  larger  quantity. 

The  CR-35  (H1N1)  recombinant  was  administered  at  a  dose  of  10    ,  10   , 
10  "  ,  or  10    to  24,  5,  11,  and  10  doubly  seronegative  volunteers, 
respectively.   These  studies  were  done  in  collaboration  with  the  University 
of  Maryland  (Dr.  Levine) ,  University  of  Rochester  (Dr.  Douglas)  and  Baylor 
University  (Dr.  Couch).   The  human  infectious  dose  50  for  the  virus  was 
approximately  10  '  TCID__.   At  doses  of  10  *-  or  higher  96  to  100%  of  the 
volunteers  were  infected.   At  a  dose  of  10  *  TCID^,  13%  of  24  volunteers 
had  mild,  afebrile  upper  respiratory  tract  illness  in  contrast  to  an  83% 
incidence  of  influenzal  illness  in  volunteers  who  received  wild  type 
virus.   Illness7was  not  observed  at  lower  doeses.   Sixty  percent  of  volunteers 
who  received  10  '   or  10    TCID^  shed  virus.   Each  virus  isolate  retained 
the  _ts  and  cold-adapted  (ca)  phenotypes.   The  shutoff  temperature  for 
plaque  formation  of  isolates  from  6  volunteers  who  shed  virus  for  6  to  9 
days  was  determined.   Of  33  isolates,  25  had  a  shutoff  temperature  similar 
to  the  parental  virus,  7  were  38° C  shutoff  temperature  mutants,  and  2  were 
39°C  mutants.   These  results  indicate  the  high  degree  of  genetic  stability 
of  this  recombinant.   However,  it  should  be  noted  that  some  genetic  drift 

(37°C  shutoff  >39°C  shutoff)  occurred.   At  doses  of  100  HID   or  higher 

greater  than  96%  of  volunteers  had  an  immunological  response.   Thus  the 
CR-35  virus  was  satifactorily  attenuated  and  immunogenic  at  doses  in  the 
range  of  100  HID   's. 


23-77 


Z01  AI  00028-21  LID 

Evaluation  of  ts-lA2  Influenza  A  Recombinants  in  Adults  -  An  influenza  A 
virus  recombinant  bearing  the  surface  antigens  of  the  A/Alaska/6/77 
(H3N2)  wild  type  virus  and  the  two  ts  genes  of  the  A/Udorn/72-ts-lA2 
(H3N2)  virus  was  evaluated  for  attenuation,  antigenicity,  and  transmissibility 
in  28  adults  volunteers  all  of  whom  had  a  preinoculation  serum  hemagglutination- 
inhibiting  (HI)  antibody  titer  of  <1:8  and  18  of  whom  also  had  a  serum 
neuraminidase-inhibiting  (NI)  antibody  titer  of  <1:4.   The  Alaska / 77- ts_- 
1A2  recombinant,  which  had  a  37°C  shutoff  temperature  for  plaque  formation 
and  _ts  mutations  on  the  genes  coding  for  the  PI  and  P3  polymerase  proteins, 
infected  86%  of  the  vaccinees  when  administered  at  a  dose  of  10  *  TCID,... 
Only  3%  of  the  vaccinees  developed  symptoms  in  contrast  to  50%  of  volunteers 
who  received  10  *  TCID,-n  of  wild  type  virus.   Vaccinees  shed  virus  for  a 
shorter  interval  and  at  a  lower  titer  than  the  volunteers  who  received 
wild  type  virus.  Each  ts-lA2  isolate  retained  the  jts  phenotype  indicating 
that  the  recombinant  was  stable  genetically  in  seronegative  adults.   An 
immunological  response,  as  measured  by  a  rise  in  serum  HI  and/or  NI  antibody, 
was  detected  in  71%  of  the  vaccinees  and  87%  of  the  recipients  of  wild 
type  virus.   Transmission  of  vaccine  virus  to  susceptible  contacts  was  not 
observed.   The  two  ts-lA2  ts  genes  have  now  been  transferred  to  two  variants 
within  the  H3N2  subtype,  the  Vic/75  and  Alaska/77  viruses,  and  have  rendered 
the  resulting  recombinants  satisfactorily  attenuated  for  seronegative 
adults. 

The  Alaska/ 7 7-ts-lA2  virus  was  given  to  children  by  Drs.  Kim  and  Parrott 
at  the  Children's  National  Medical  Center  in  Washington,  D.C.   The  virus 
was  administered  to  seropositive  children  who  behaved  like  the  adults 
mentioned  above.   One  seronegative  child  was  also  studied.   This  individual 
shed  virus  for  10  days.   Virus  shed  on  the  7th,  8th  and  9th  day  produced 
plaques  at  39°C,  a  temperature  restrictive  for  the  ts-lA2  recombinant 
administered.   On  day  2,  virus  present  in  the  nasopharyngeal  wash  fluid 
had  a  37°C  shutoff  temperature;  on  day  3,  38°C  shutoff  temperature;  and  on 
days  7,  8,  and  9  the  virus  had  a  40°C  shutoff  temperature.  This  indicated 
that  the  A/Alaska/77  ts-lA2  recombinant  gradually  lost  a  considerable 
amount  of  its  temperature  sensitivity  during  the  later  period  of  replication 
in  vivo.   A  characterization  of  these  isolates  and  the  role  of  suppressor 
mutation  in  loss  of  the  _ts  phenotype  are  described  in  Project  No.  28-18. 

The  Udorn/72-ts-lA2  virus  was  mated  with  the  A/Hong  Kong/123/77  (H1N1) 
wild  type  virus  and  a  HK/77-ts-lA2  recombinant  (clone  144)  was  isolated 
which  had  a  37°C  shutoff  and  the  two  ts-lA2  ts  lesions.   Doubly  serongative 
adults  (i,en,  no  detectable  HI  or  NI  serum  antibody)  were  administered 
10   ,  10   or  10   TCID.  of  HK/77-ts-lA2  vaccine  virus  or  10  "   of  wild 
type  virus.   Thej.50%  human  infectious  dose„n  of  the  vaccine  virus  was 
approximately  10  '  TCID^.   It  was  necessary  to  use  ELISA  to  detect  an 
immunological  response  in  the  vaccinees  (see  Project  24-18)  and  to  accurately 
estimate  the  HID--.   Of  6  volunteers  infected  with  wild  type  virus  5 
developed  febrile  or  systemic  illness  and  shed  virus  for  5.8  days  with  a 
peak  mean  titer  of  10  '  TCID   /ml  of  nasal  wash.   Eighty-six  percent  of  23 


23-7E 


Z01  AI  00028-21  LID 

fi  s 

volunteers  who  received  30  HID   '  s  (10  "  TCID   )  of  the  ts-lA2  recombinant 

became  infected.   Illness  developed  in  3  volunteers,  1  febrile  illness 
(100. 2°F)  of  12  hours  duration  and  2  mild  upper  respiratory  tract  illnesses. 
Only  25%  of  the  vaccinees  shed  virus  with  an  average  duration  of  1.7  days 
and  peak  mean  titer  of  10  "  TCID,.-.   Each  of  the  virus  isolates  recovered 
retained  the  _ts  phenotype.  These  results  indicate  that  the  HK/77-ts-lA2 
virus  was  satisfactorily  attenuated,  immunogenic,  and  genetically  stable 
in  doubly  seronegative  volunteers.  Preliminary  results  from  the  challenge 
of  HK/77-ts-lA2  and  CR-35  vaccinees  with  wild  type  virus  ((performed  by 
Dr.  Douglas  at  the  University  of  Rochester)  indicates  that  both  vaccines 
induce  significant  resistance  to  infection  and  illness. 

Publications 

Chanock,  R.M. ,  and  Murphy,  B.R.:   Genetic  approaches  to  control  of 
influenza.   Persp.  Biol.  Med.  22:  S37-S48,  1979. 

Murphy,  B.R.,,  Chanock,  R.M. ,  Levine,  M.M. ,  Van  Blerk,  G.A. ,  Berquist, 
E.J.,  Douglas,  R.G.,  Betts,  R.F.,  Couch,  R.B.,  Cate,  T.R.  Jr.:  Temperature- 
sensitive  mutants  of  influenza  A  virus.   XVII.   Evaluation  of  the  Vic/75- 
ts-lA2  temperature-sensitive  recombinant  virus  in  seronegative  adult 
volunteers.   Infect.  Immun. ,  23:  249-252,  1979. 

Murphy,  B.R. ,  Holley,  H.P.,  Jr.,  Berquist,  E.J.,  Levine,  M.M. ,  Spring, 
S.B.,  Maassab,  H.F. ,  Kendal,  A. P.,  Chanock,  R.M. :   Cold-adapted  variants 
of  influenza  A  virus.  III.  Evaluation  in  adult  seronegative  volunteers 
of  A/Scotland/840/74  and  A/Victoria/3/75  cold-adapted  recombinants 
derived  from  the  cold-adapted  A/Ann  Arbor /6/60  strain.   Infect.  Immun. 
23:  253-259,  1979. 

Kim,  H.W. ,  Brandt,  CD.,  Arrobio,  J.O.,  Murphy,  R.M. ,  Chanock,  R.H. ,  and 
Parrott,  R.H. :   Influenza  A  and  B  virus  infection  in  infants  and  young 
children  during  the  years  1975-1976.   Amer.  J.  Epidemiol.,  109:  464-479, 
1979. 

Murphy,  B.R. ,  Markoff,  L.J. ,  Chanock,  R.M. ,  Spring,  S.B.,  Maassab,  H.F., 
Kendal,  A. P. ,  Cox,  N. J. ,  Levine,  M.M. ,  Douglas,  R.G.  Jr.,  Betts,  R.F., 
Couch,  R.B.  and  Cate  T.R.  Jr.:   Genetic  approaches  to  attenuation  of 
influenza  A  viruses  for  man.   Phil.  Trans.  R.  Soc.  Lond. ,  in  press. 

Douglas,  R.G.  Jr.,  Markoff,  L.J.,  Murphy,  B.R.,  Chanock,  R.M. ,  Betts, 
R.F.,  Hayden,  F.G. ,  Levine,  M.M. ,  VanBlerk,  G.A.,  Sotman,  S.B.,  and 
Nalin,  D.R. :   Evaluation  of  live  Vic/75-ts-l[E]  influenza  A  virus  vaccines 
in  adult  volunteers:   Role  of  hemagglutinin  immunity  in  protection 
against  illness  and  infection  caused  by  influenza  A  virus.   Infect. 
Immun.,  in  press. 


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Z01  AI  00028-21  LID 

Markoff,  L.J.,  Thierry,  F.,  Murphy,  B.R.,  and  Chanock,  R.M. :  Relationship 
of  genotype  of  recombinants  of  influenza  A/Hong  Kong/68-ts_-l [E]  virus 
used  as  live  virus  vaccines  to  virulence  in  man.   Infect.  Immun. ,  in 
press. 

Murphy,  B.R. ,  and  Chanock,  R.M. :   The  present  status  of  live  influenza  A 
virus  vaccine.   Monographs  in  Pediatrics,  in  press. 


23-G0 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRADURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  AI  00175-02  LID 


PERIOD  COVERED 


OCTOBER  1,  1978  TO  SEPTEMBER  30.  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

LABORATORY  STUDIES  OF  PARAMYXOVIRUSES  AND  RESPIRATORY  SYNCYTIAL  VIRUS 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


P.I.   Robert  M.  Chanock,  M.D. 
Gregory  A.  Prince,  Ph.D. 
Stephen  C.  Suffin,  M  D. 


Chief 

Sr.  Staff  Fellow 

I. P. A. 


LID,  NIAID 
LID,  NIAID 
LID,  NIAID 


COOPERATING  UNITE 


if  any) 


LAB/BRANCH 

LABORATORY  OF  INFECTIOUS  DISEASES 


RESPIRATORY  VIRUSES  SECTION 


'NSTITUTNATIDONALAT INSTITUTE  OF  ALLERGY  AND  INFECTIOUS  DISEASES,  BETHESDA,  MARYLAND 


TOTAL  MANYEARS: 

66/12 


PROFESSIONAL: 

30/12 


36/12 


CHECK  APPROPRIATE  BOX(ES) 
C  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


(c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Variation  in  level  of  growth  of  RS  virus  was  detected  for  20  strains  of  inbred 
mice  tested.   The  difference  in  level  of  replication  between  the  most  permissive 
and  restrictive  strain  of  mice  was  sufficiently  large  that  it  should  now  be 
possible  to  analyze  genetic  control  of  viral  replication  by  the  appropriate  cross- 
breeding techniques.   ts-2,  ts-1  NG-1  and  ts-1  NG-16  mutants  were  shown  to  be 
highly  defective  and  attenuated  when  studied  in  primates  suggesting  that  these 
mutants  may  prove  useful  in  provention  of  human  RS  virus  disease.   IM  inoculationl 
of  virus  appears  to  initiate  an  aborative  cycle  of  replication  locally  leading 
to  an  immunologic  response.   Circulating  antibody  acts  to  suppress  this  response. 
Development  of  a  glucose  oxidase  linked  antibody  staining  technique  offers  the 
possibility  of  examining  paraffin  embedded,  formalin  fixed  tissues  for  presence  of 
£S  antigens.   In  this  manner  an  immunopathoarcheologic  survey  of  RS  virus  and  other 
viral  pathogens  can  be  performed  using  autopsy  material  collected  previously. 


PHS-6040 
(Rev.  10-76) 


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Z01  AI  00175-02  LID 

Project  Description 

This  year  the  respiratory  syncytial  virus  program  gave  special  emphasis  to 
7  areas  of  research:   1)  genetic  studies  of  the  virus,  utilizing  temperature 
sensitive  mutants;  2)  continued  development  of  animal  models  of  RS  virus 
infection  in  which  to  study  patterns  of  viral  growth  and  pathogenesis  of 
disease;  3)  ^Ln  vivo  evaluation  in  experimental  animals  of  _ts_  mutants  and 
cold-adapted  mutants  that  appeared  to  be  promising  vaccine  candidates;  4) 
in  vivo  evaluation  in  experimental  animals  of  bovine  respiratory  syncytial 
virus  as  a  potential  vaccine  candidate;  5)  immunological  studies  of  RS 
virus  infection  in  experimental  animals;  6)  studies  of  RS  virus  infection 
in  the  immunosuppressed  host;  7)  development  of  new  or  improved  diagnostic 
techniques  for  the  study  of  RS  virus  infection  in  humans  and  experimental 
animals. 

Major  Findings 

Genetics  of  Respiratory  Syncytial  Virus 

Background  -  RS  virus  is  the  major  etiologic  agent  of  bronchiolitis  and 
pneumonia  of  early  life,  and  the  need  for  effective  immunoprophylaxis  has 
been  clearly  established  in  studies  performed  throughout   the  world.  This 
virus  has   a  non-segmented  genome  and  does  not  undergo  genetic  reassortment. 
Initially  a  series  of   7  ts_  mutants  of  RS  virus  were  produced  by  chemical 
mutagenesis  with  the  expectation  that  their  temperature  sensitivity  would 
restrict  growth  in  the  target  organ,  the  lungs. 

Complementation  Analysis  of  Additional  ts  Mutants  of  RS  Virus  -  The 
complementation  studies  reported  last  year  were  continued  and  the  Glasgow 
mutants  have  been  further  characterized.   As  stated  previously,  at  least 
6-8  complementation  groups  are  likely  to  exist,  based  upon  the  probable 
molecular  weight  of  the  RS  virus  genome  and  the  existence  of  from  six  to 
eight  viral  specified  polypeptides.   Using  six  prototype  ts_  mutants  isolated 
by  Dr.  Craig  Pringle  and  his  collaborators  (Glasgow,  Scotland),  and  three 
prototypes  isolated  in  this  laboratory,  we  have  defined  six  complementation 
groups.   The  three  NIH  mutants  represent  groups  we  have  designated  Groups 
A,  B,  and  C;  three  Glasgow  mutants  represent  Groups  D,  E,  and  F.   The 
remaining  three  Glasgow  mutants  have  been  characterized  by  our  laboratory 
as  belonging  to  Group  A.   Experiments  varying  adsorption  period  and  incubation 
times  showed  complementation  between  the  two  mutants  we  had  placed  in 
Group  E  last  year,  thus  establishing  Group  F  by  our  method  this  year. 

Isolation  of  New  ts  Mutants  Additional  _ts_  mutants  are  being  isolated  and 
characterized  in  this  laboratory  in  a  continuing  search  for  new  complementation 
groups  and  possible  vaccine  candidiates.   Mutagenization  of  wild  type  RS 
virus  strain  A2  (F-059,  Flow  Laboratories)  was  carried  out  using  5-f luorouracil 
and  5-azocytidine  in  the  growth  medium.   Of  412  clones  screened  for  temperature 
sensitivity  during  this  year,  18  were  ts.   These  mutants  are  now  being 
characterized  in  vitro  by  complementation  analysis,  temperature  shift 


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Z01  AI  00175-02  LID 

experiments  to  determine  the  time  at  which  their  ts_  lesions  are  expressed, 
and  growth  at  subrestrictive  temperature  to  determine  their  genetic 
stability.   Potential  vaccine  candidates  will  then  be  tested  in  vivo, 
initially  in  the  cotton  rat,  then  in  primates. 

After  extensive  characterization  of  new  mutants  from  Glasgow  and  NIH,  none 
was  shown  to  share  NIH  Group  B  (ts2  prototype)  t£  lesion  which  is  characterized 
by  non-syncytial  plaque  morphology  and  a  defect  in  adsorption-penetration 
of  the  host  cell.   However,  one  new  NIH  mutant,  tslll,  despite  its  syncytial 
plaque  morphology  and  early  genetic  lesion  similar  to  Group  C,  appears  to 
belong  to  a  new  complementation  group.  Additional  in  vitro  tests  currently 
in  progress  will  allow  us  to  identify  it  definitively. 

Animal  Models  of  Respiratory  Syncytial  Virus  Infection 

Owl  Monkey  -  Pilot  studies  during  the  preceding  year  indicated  that  RS 
virus  infection  of  owl  monkeys  (Aotis  sp.)  produced  a  disease  characterized 
by  rhinorrhea.   Further  evaluation  of  RS  virus  infection  in  owl  monkeys 
was  undertaken  this  year.   Intranasal  inoculation  of  wild  type  RS  virus 
produced  upper  respiratory  tract  disease  in  each  of  seven  animals.   Virus 
was  recovered  from  nasopharyngeal  swab  specimens  fpr  a  mean  period  of  11 
days,  reaching  a  peak  titer  (geometric  mean)  of  10  '   plaque-forming  units 
per  milliliter.   Serum  neutralizing  antibody  to  RS  virus  began  to  appear 
14  days  after  inoculation,  reaching  peak  titer  by  28  days.   A  combined 
disease  score  was  calculated  for  each  animal,  which  formed  the  basis  for 
subsequent  analysis  of  the  virulence  of  ts  mutants  in  owl  monkeys.   Because 
of  the  extreme  scarcity  of  chimpanzees,  the  only  other  experimental  animal 
known  to  develop  clinically  evident  disease  from  RS  virus,  the  owl  monkey 
has  become  the  model  of  choice  for  experimental  study  of  virulence. 

Inbred  Mice  -  Five  animal  models  of  RS  virus  infection  have  been  described 
by  this  laboratory,  including  the  chimpanzee,  cebus  monkey,  owl  monkey, 
ferret  and  cotton  rat.   None  of  these  species,  however,  is  inbred,  thus 
precluding  genetic  manipulation  and  certain  types  of  immunologic  study. 
Furthermore,  few,  if  any  specific  immunologic  reagents  are  available  to 
allow  study  of  certain  aspects  of  RS  virus  pathogenesis  and  virus-host 
immunologic  interaction.   In  an  effort  to  develop  a  model  for  experimental 
RS  virus  infection  in  an  inbred  species,  we  examined  twenty  strains  of 
mice.  Intranasal  inoculation  of  RS  virus  in  infant  mice  produced  infection 
in  each  strain  examined.   However,  there  was  wide  variation  among  the 
inbred  strains  in  the  amount  of  virus  recovered  from  the  nose  and  lungs. 
The  most  resistant  strain,  CBA/CaHN,  yielded  only  one-hundredth  the  quantity 
of  virus  recovered  from  the  most  permissive  strain,  DBA/2N.   This  difference 
was  seen  in  comparisons  of  nasal  and  pulmonary  tissues  from  the  two  strains. 
Ordering  of  geometric  mean  nasal  and  pulmonary  titers  from  the  twenty 
strains  demonstrated  a  pattern  of  gradual,  incremental  increase  from 
relatively  resistant  to  relatively  permissive  strains. 

If  level  of  viral  replication  were  controlled  by  a  single  gene  with  few 
alleles,  one  would  not  expect  to  observe  such  a  shallow  linear  array  of 

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Z01  AI  00175-02  LID 

titers.   This  suggests  that  response  to  RS  virus  infection  is  determined 
by  a  combination  of  genes,  or  perhaps  a  single  gene  with  multiple  alleles. 
The  possibility  that  the  H-2  haplotype  might  have  an  association  with 
resistance  or  permissiveness  was  examined  by  evaluating  the  haplotype  of 
each  strain.   No  connection  between  H-2  haplotype  and  extent  of  RS  virus 
infection  was  evident,  however. 

The  nature  of  the  genetic  factors  governing  susceptibility  to  RS  virus 
infection  in  mice  is  not  known.   Our  preliminary  survey  suggests  that 
multiple  factors  are  involved,  but  does  not  distinguish  between  control  by 
several  genes  or  multiple  allelic  control  within  a  single  gene.    Since 
there  was  no  overlap  between  the  virus  titers  observed  for  the  strains 
that  exhibited  the  lowest  and  highest  levels  of  viral  growth  it  should  be 
possible  to  analyze  the  genetic  control  of  viral  replication  using  the 
appropriate  crossbreeding  techniques. 

In  addition  to  the  opportunity  to  study  genetic  control  of  viral  replication, 
the  availability  of  an  inbred  animal  model  for  RS  virus  infection 
offers  possibilities  for  studies  which  were  not  feasible  previously. 
For  instance,  in  vivo  experiments  using  adoptive  transfer  of  immunologic 
components,  including  immune  cells,  can  now  be  performed.   Recent  experiments 
using  immune  cell  transfer  in  mice  have  demonstrated  the  importance  of 
cellular  immunity  in  recovery  from  influenza  virus  infection.   The  similarities 
between  influenza  and  RS  viruses  suggest  that  cellular  immunity  may  also 
serve  such  a  function  for  the  latter  virus. 

Another  advantage  of  the  mouse  model,  in  comparison  with  other  models  of 

RS  virus  infection,  is  the  existence  of  a  large  number  of   specific  immunological 

reagents.   These  include  antisera  directed  against  mouse  immunoglobulins 

(with  heavy-  or  light-chain  as  well  as  whole  molecule  specificity) ,  interferon, 

theta  antigen,  Ly  1,  2  and  3  T  cell  antigens,  specific  histocompatibility 

antigens  and  macrophages.   Such  reagents  should  allow  the  eventual  definition 

of  those  components  of  the  immune  system  responsible  for  recovery  from  RS 

virus  infection,  as  well  as  immunity  to  subsequent  infection. 

Finally,  the  existence  in  certain  inbred  mice  of  well-defined  immunologic 
defects  gives  this  model  unique  advantages.   Animals  lacking  T-cell  dependent, 
B-cell  dependent,  and  both  T-  and  B-cell  dependent  immune  function  have 
been  described.   Efforts  to  introduce  such  specific  defects  into  the 
mouse  strains  most  susceptible  to  RS  virus  infection  are  currently  in 
progress,  in  cooperation  with  Dr.  Carl  Hanson,  Veterinary  Resources  Branch, 
Division  of  Research  Services,  NIH.   Elucidation  of  the  role  of  these 
immunologic  components  in  recovery  from  and  immunity  to  infection  would 
greatly  increase  our  understanding  of  disease  caused  by  RS  virus. 

Cotton  Rat  -  Last  year's  report  described  the  pathogenesis  of  RS  virus 
infection  in  cotton  rats.   Since  that  time,  the  cotton  rat  has  become  the 
major  animal  model  used  in  this  laboratory.   Despite  the  potential  advantages 
of  the  inbred  mouse  model,  the  cotton  rat  will  remain  a  major  tool  for  RS 


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Z01  AI  00175-02  LID 

virus  research,  since  it  is  approximately  one  hundred  fold  more  permissive 

for  RS  virus  than  the  most  permissive  mouse  strain.  Furthermore,  as  noted 

in  last  year's  report,  the  cotton  rat  remains  uniformly  susceptible  to 

both  nasal  and  pulmonary  infection  throughout  its  life.   Preliminary  studies  in  in'. 

suggest  that  adult  mice  may  be  as  much  as  one  hundred  fold  less  permissive 

than  infants  of  the  same  strain.   Therefore,  we  have  initiated  efforts  to 

develop  an  inbred  cotton  rat,  in  cooperation  with  Dr.  Carl  Hanson  of  NIH.  The 

inbreeding  program  currently  is  in  the  third  generation  of  brother-sister 

mating,  and  will  continue  until  an  inbred  line  has  been  achieved  (probably  4-5 

years) . 

In  Vivo  Evaluation  of  Mutant  Viruses 

Cotton  Rat  Nasal  Histopathology  as  a  Marker  of  Virulence  -  As  described 
earlier,  the  owl  monkey  is  now  used  by  this  laboratory  for  evaluating 
virulence  of  vaccine  candidates.   However,  the  expense  and  relative  scarcity 
of  this  primate  preclude  its  use  for  routine  screening  of  the  many  potential 
vaccine  mutants  being  isolated.   Therefore,  we  have  examined  the  cotton  rat  as 
a  potential  model  for  such  virulence  testing.   Histopathologic  examination  of 
nasal  tissues  from  cotton  rats  inoculated  with  wild  type  RS  virus,  as 
described  in  last  year's  report,  demonstrated  a  moderately  severe,  focal 
rhinitis.   This  finding  was  confirmed  in  more  extensive  studies  this  year, 
both  with  the  Long  and  A-2  strains  of  RS  virus.  Furthermore,  the 
histopathologic  response  of  cotton  rats  to  the  ^s_  mutants  tested  correlated 
with  the  response  of  chimpanzees  to  the  same  mutants.   Specifically,  the  ts-1 
and  _ts_  7  mutants,  which  had  caused  rhinorrhea  in  chimpanzees,  produced  nasal 
lesions  in  cotton  rats.  Furthermore,  the  ts-2  mutant  and  wild  type  bovine  RS 
virus,  neither  of  which  produced  clinical  signs  of  illness  in  chimpanzees, 
failed  to  produce  lesions  in  cotton  rat  nasal  tissues.   We  thus  envision  a 
protocol  for  virulence  testing  which  would  progress  from  cotton  rats  to  owl 
monkeys  and,  ultimately,  man. 

Further  in  vivo  Evaluation  of  ts-2  -  Two  seronegative  chimpanzees  were 
inoculated  with  a  safety-tested  ts-2  vaccine  prepared  by  Flow  Laboratories. 
This  vaccine  is  currently  being  used  in  initial  human  trials.   One  animal  was 
successfully  infected  in  two  attempts  but  did  not  develop  any  signs  of 
illness,  despite  shedding  a  moderate  amount  of  virus  (10  "   pfu/ml  of  swab 
fluid)  from  the  upper  respiratory  tract.  The  unavailability  of  further 
seronegative  animals  precluded  additional  study. 

The  same  lot  of  ts-2  vaccine  (Ft512)  was  evaluated  in  cotton  rats.   Animals 
inoculated  intranasally  with  10  pfu  were  examined  daily  for  evidence  of  viral 
replication  in  nose  and  lungs.   Of  six  animals  initially  screened  (twelve 
tissue  specimens) ,  a  single  tissue  yielded  virus  at  a  titer  of  less  than  10 
pfu  per  gram,  indicating  that  ts-2  is  minimally  infective  in  this  animal,  as 
was  observed  in  the  chimpanzee.   However,  when  cotton  rats  inoculated 
intranasally  with  ts-2  were  subsequently  challenged  intranasally  with  wild 
type  RS  virus,  a  protective  effect  of  the  vaccine  was  seen,  despite  its 
apparently  low  infectivity.   Of  21  animals  inoculated  with  ts-2,  six  were 


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completely  protected  against  pulmonary  infection  by  wild  type  virus. 
Furthermore,  comparision  of  growth  of  virus  in  the  lungs  of  vaccinated  and 
control  animals  showed  a  highly  significant  level  of  protection  (p  <.001). 
However,  the  level  of  viral  replication  in  the  nose  of  the  two  groups  of 
animals  showed  no  significant  difference.   A  third  group  of  animals, 
inoculated  intranasally  with  ts-2,  then  reinoculated  by  the  same  route  two 
weeks  later,  exhibited  no  further  resistance  than  the  group  that  received  a 
single  inoculation  of  ts-2. 

These  observations  provide  the  first  experimental  evidence  that  ts-2  is 
effective  in  protecting  against  pulmonary  infection  by  wild  type  RS  virus. 
Furthermore,  they  suggest  that  the  nose  and  the  lungs  may  be  regarded  as 
immunologically  separate,  independent  organs  with  regard  to  RS  virus 
infection.   Hence,  failure  to  detect  resistance  in  the  upper  respiratory  tract 
can  not  be  extrapolated  to  indicate  lack  of  resistance  in  the  lungs. 


A  final  area  of  in  vivo  evaluation  of  ts-2  involves  evaluation  of  temperature- 
competent  (ts  )  progeny  of  ts-2.   We  previously  reported  that  some  isolates  of 
virus  from  chimpanzees  infected  with  high  doses  of  ts-2  differed  from  the 
input  virus  in  their  ability  to  form  syncytial  plaques  at  a  temperature 
restrictive  for  plaque  formation  by  ts-2.   The  properties  of  these  ts_ 
viruses,  especially  virulence,  thus  assume  considerable  importance  as  regards 
the  safety  and  potential  usefulness  of  the  ts-2  mutant  in  immunoprophylaxis. 
Using  the  cotton  rat,  temperature-competent  viruses  isolated  from  two 
chimpanzees  inoculated  with  ts-2  were  examined  to  determine  their  infectivity, 
pathogenicity  and  immunogenicity .   Both  "revertant"  viruses  were  as  infectious 
for  this  host  as  wild  type  RS  virus.   Furthermore,  both  were  immunogenic,  and 
protected  cotton  rats  from  subsequent  challenge  by  wild  type  RS  virus. 
However,  one  of  the  two,  despite  its  similarity  to  wild  type  virus,  failed  to 
induce  histopathology  in  the  cotton  rat's  nose.   This  virus,  previously 
inoculated  into  two  chimpanzees,  also  had  failed  to  produce  disease.   This 
suggests  that  temperature-competent  progeny  of  ts-2  virus,  are  not  necessaruly 
virulent. 

The  second  "revertant"  virus,  however,  did  cause  histopathologic  changes  in 
cotton  rat  nasal  tissues.   This  virus,  nevertheless,  was  recovered  from  a 
chimpanzee  inoculated  with  100-fold  more  virus  than  given  the  other 
chimpanzee.  When  inoculated  into  a  seronegative  chimpanzee,  this  second 
"revertant"  caused  rhinitis,  again  demonstrating  the  correlation  between 
disease  in  primates  and  histopathology  in  cotton  rats.  Since  current  human 
trials  employ  a  viral  dose  approximately  one  hundredfold  lower  than  that  given 
the  chimpanzee  that  yielded  the  virulent  "revertant",  shift  to  a  _ts   phenotype 
may  not  be  a  problem  when  the  mutant  is  studied  in  children.  It  is  likely  the 
cotton  rat  will  prove  helpful  in  evaluating  this  phenomenon  during  vaccine 
trials  in  man. 


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In  vivo  Evaluation  of  ts-1  NG-1  and  ts-1  NG-16  Mutants  -  The  first  ts  mutant 
of  RS  virus  to  be  tested  in  man,  ts-1,  appeared  promising  when  evaluated  in 
adult  volunteers.   However,  tests  in  seronegative  infants  showed  the  virus  to 
possess  a  low  level  of  residual  virulence,  and  to  exhibit  some  genetic 
instability.   In  an  attempt  to  further  attenuate  ts-1,  which,  unlike  ts-2, 
appears  to  be  nearly  as  infective  as  wild  type  virus,  nitrosoguanidine  (NG) 
was  used  to  remutagenize  the  ts-1  mutant.   The  two  mutants  evaluated  in  this 
study,  ts-1  NG-1  and  ts-1  NG-16,  were  recovered  from  the  progeny  of  NG  treated 
ts-1  and  were  shown  to  exhibit  greater  temperature-sensitivity  and  genetic 
stability  than  ts-1.   We  used  the  owl  monkey  to  test  these  two  mutants  in  vivo 
because  it  is  the  only  experimental  animal  which  develops  clinically  evident 
disease  when  infected  with  wild  type  RS  virus  and  which  is  also  available  in 
sufficient  numbers  to  permit  statistically  valid  comparison  with  wild  type 
virus. 

In  evaluating  ts-1  NG-1  and  ts-1  NG-16  in  vivo,  three  phenomena  were  examined. 
Pattern  of  infection  was  assessed  by  examining  the  time  of  onset  of  shedding 
of  infectious  virus,  duration  of  shedding,  peak  viral  titer  and  time  of  peak 
titer.   Virulence  was  evaluated  by  a  composite  disease  score,  consisting  of 
the  sum  of  daily  disease  scores  for  each  animal.   Finally,  antigenicity  was 
evaluated  in  terms  of  the  time  of  onset  and  the  peak  titer  of  serum 
neutralizing  antibody.   Neither  ts-1  NG-1  nor  ts-1  NG-16  differed 
significantly  from  wild  type  virus  in  either  duration  of  infection  or  peak 
virus  titer.   This  would  suggest  that  both  mutants  produced  an  infection  that 
was  comparable  in  extent  to  that  of  wild  type  virus,  a  desirable  property  for 
a  vaccine  strain.   However,  the  time  of  onset  of  virus  shedding  and  the  time 
of  peak  titer  of  both  mutants  was  significantly  delayed  compared  to  wild  type 
virus  suggesting  that  both  mutants  were,  nonetheless,  functionally  defective. 

Though  capable  of  producing  extensive  infection  both  mutants  were 
significantly  attenuated  compared  to  wild  type  virus.   That  is,  the  composite 
disease  scores  were  significantly  lower  for  both  ts-1  NG-1  and  ts-1  NG-16 
infected  animals.  Reduced  virulence,  like  high  infectivity,  is  another 
property  desirable  for  a  potential  vaccine  strain.  Finally,  both  mutants  were 
nearly  as  antigenic  as  wild  type  virus,  when  various  aspects  of  the  humoral 
antibody  response  were  measured.   Again,  antigenicity  equivalent  to  that 
produced  by  wild  type  virus  is  desirable  for  a  vaccine  candidate. 

These  observations,  in  conjunction  with  previous  studies  showing  ts-1  NG-1  and 
ts-1  NG-16  to  be  more  defective  than  the  ts-1,  parental  mutant,  and  more 
genetically  stable  both  in  vitro  and  in  vivo,  suggest  that  they  are  potential 
candidates  for  use  in  a  live  vaccine.   The  fact  that  the  two  mutants  did  not 
differ  significantly  from  each  other  in  any  of  the  observed  parameters 
suggests  that  both  should  be  subjected  to  additional  in  vivo  testing  in 
primates  and,  ultimately,  man. 

Cold-adapted  ts-1  in  a  Chimpanzee  -  In  a  different  approach  to  further 
attenuation  of  ts-1  mutant,  the  virus  was  passaged  28  times  at  24°C,  then 
inoculated  intranasally  into  a  seronegative  chimpanzee.   This  animal  developed 

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extensive  rhinorrhea  and  shed  virus  for  an  extended  period  of  time,  indicating 
that  low  temperature  passaged  virus  was  as  virulent  as  its  ts-1  parent. 

Bovine  Respiratory  Syncytial  Virus 

Background  -  In  1971  a  strain  of  RS  virus,  antigenically  related  but 
genetically  distinct  from  human  RS  virus,  was  isolated  from  cattle  in  Europe. 
This  agent,  known  as  bovine  respiratory  syncytial  (BRS)  virus,  was 
subsequently  identified  in  bovine  herds  throughout  the  world.   The  successful 
use  of  vaccinia  virus,  a  naturally  occurring  bovine  virus,  in  the 
immunoprophylaxis  of  smallpox  raised  the  possibility  of  using  BRS  virus  as  a 
naturally  occurring  vaccine  against  its  human  counterpart. 

Chimpanzees  -  Two  chimpanzees  were  each  inoculated  intranasally  with  BRS 
virus.   Only  one  shed  BRS  virus.   The  quantity  of  virus  was  small  (less  than 
10  pfu/ml  of  nasopharyngeal  swab  specimen)  and  was  detected  only  in  roller 
tube  cultures.   Six  weeks  later  the  two  animals  were  challenged  intranasally 
with  human  RS  virus.   Neither  differed  from  human  RS  virus-inoculated  control 
chimpanzees  either  in  peak  viral  titer  or  extent  of  disease. 

Owl  Monkeys  -  Six  of  9  monkeys  were  infected  with  BRS;  three  shed  a  small 
quantity  of  virus  and  three  developed  a  low  level  rise  in  serum  neutralizing 
antibody.   These  monkeys  did  not  exhibit  resistance  when  challenged 
subsequently  with  human  RS  virus. 

Cotton  Rats  -  One  group  of  animals  was  inoculated  intranasally  (i.n.);  the 
second  received  virus  intramuscularly  (i.m.);  and  the  third  was  inoculated 
i.m.  twice  (2  week  intervals) .  BRS  virus  was  recovered  from  the  nose  and  lungs 
of  each  of  three  animals  from  the  first  group,  but  at  a  low  titer  (maximum 
10  '   pfu/gm) .   Animals  receiving  BRS  virus  i.n.  showed  the  most  consistent 
antibody  response,  with  84%  developing  serum  neutralizing  antibody  against  human 
RS  virus.   Animals  that  received  BRS  virus  i.m.  showed  a  less  consistent 
response. 

When  challenged  intranasally  with  human  RS  virus,  the  three  groups  of  cotton 
rats  previously  inoculated  with  BRS  virus  showed  significant  protection  when 
compared  to  control  animals.   The  greatest  protection  was  induced  by  IN 
inoculation  of  BRS.  The  mechanism  by  which  BRS  virus  stimulates  immunity 
against  human  RS  virus  in  cotton  rats  was  not  clear.   It  did  appear,  however, 
that  serum  neutralizing  antibody  was  not  responsible.   Although  many  animals 
inoculated  with  BRS  virus  failed  to  develop  detectable  antibody,  every  animal, 
regardless  of  the  route  of  administration  of  BRS  virus,  exhibited  some 
resistance  to  subsequent  human  RS  virus  challenge.   In  several  instances, 
complete  pulmonary  immunity  was  seen  in  animals  lacking  detectable  antibody, 
suggesting  that  another  portion  of  the  immune  system  was  the  effector  of 
resistance. 


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In  spite  of  these  promising  findings  in  the  cotton  rats,  the  results  of 
experiments  in  primates  were  disappointing.   BRS  virus  may  be  more  infectious 
for  cotton  rats,  and  not  capable  of  attaining  a  level  of  replication  in 
primates  sufficient  to  induce  an  effective  immune  response. 

Studies  involving  tissue  culture  suggest  that  primates  are  relatively  non- 
permissive  hosts  for  BRS  virus  infection.   Infected  bovine  turbinate  and 
bovine  embryonic  kidney  cells  each  yielded  from  10  "   to  10    pfu  of  BRS 
virus  per  ml.   In  contrast,  human  embryonic  kidney,  human  diploid  lung 
fibroblast  and  African  green  monkey  kidney  cells  were  relatively  or  completely 
resistant  to  BRS  virus. 

Immunological  Studies  in  Animal  Models 

Maternal-to-Infant  Transfer  of  Immunity  -  Previous  studies  demonstrated  the 
transfer  of  immunity  from  a  previously  infected  mother  to  offspring  (ferrets) . 
In  the  ferret,  immunity  was  conferred  by  factors  present  in  colostrum  and 
milk.   A  preliminary  experiment  was  performed  in  cebus  monkeys  to  study 
maternal-to-infant  transfer  of  immunity.   Cebus  monkeys  were  used  because 
they  are  the  only  primate  species  available  which  could  be  sacrificed,  and 
initial  findings  in  cotton  rats  showed  differential  protection  in  the  nose 
and  lungs.   Infants  of  seropositive  and  seronegative  cebus  monkeys  were 
challenged  with  wild  type  RS  virus,  then  sacrificed  and  examined  for 
virus.   The  results,  though  preliminary,  suggested  that  the  lower  respiratory 
tract  of  infants  nursed  by  an  immune  mother  was  partially  or  totally 
protected,  whereas  the  upper  respiratory  tract  was  not  protected. 

Additional  data  obtained  in  cotton  rats  confirmed  last  year's  observations. 
That  is,  infants  of  immune  cotton  rats  receive  complete  immunity  to  pulmonary 
infection,  but  incomplete  immunity  to  nasal  infection.   This  immunity  is 
not  long  lived,  and  initial  observations  suggest  that  it  may  last  only  4-5 
weeks.   Preliminary  experiments  with  foster  nursing  indicate  that  the 
immunity  is  transferred  through  the  milk,  rather  than  the  placenta.   A 
related  experiment  involved  the  passive  administration  of  RS  virus  antiserum, 
obtained  from  postinfection  cotton  rats,  to  infant  cotton  rats.   Compared 
with  control  animals,  which  received  an  equivalent  volume  of  normal  cotton 
rat  serum  by  the  same  route  (intraperitoneally) ,  animals  which  received 
antiserum  showed  significant  resistance  against  subsequent  pulmonary 
infection,  but  not  against  nasal  infection. 

Duration  of  Immunity  Following  Intranasal  Infection  of  Cotton  Rats  -  It 
is  known  that  man  does  not  develop  lasting  immunity  to  RS  virus  infection. 
During  the  past  18  months  the  transient  nature  of  RS  virus  immunity  was 
investigated  in  cotton  rats.   Immunity  to  nasal  infection  was  temporary; 
animals  were  susceptible  to  nasal  reinfection  beginning  8  months  after 
primary  infection.  The  level  of  virus  replication  in  the  nose  increased 
with  time  after  initial  infection;  at  18  months  post-infection  animals 


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were  completely  permissive.  In  contrast,  none  of  the  animals  examined 
throughout  the  course  of  the  18  month  experiment  were  susceptible  to 
reinfection  of  the  lungs.   If  a  similar  situation  obtains  in  man  vaccination 
may  offer  greater  promise  of  protection  than  had  previously  been  considered 
possible. 

Immunity  in  Cotton  Rats  Studied  by  Parabiosis  -  A  surgical  technique  was 
devised  by  which  animals  were  parabiosed  and  maintained  alive  for  at  least 
eleven  days,  the  duration  of  the  experiments.   Since  there  are  no  inbred 
cotton  rats,  experiments  were  restricted  to  parabiotic  pairs  of  same-sex 
littermates,  in  order  to  minimize  immunological  differences.   Three  types 
of  pairs  were  formed:   immune- to- immune  (immune  being  animals  parabiosed 
21  days  following  intranasal  infection  by  wild  type  RS  virus) ,  control-to- 
control,  and  immune-to-control.   Immune- to- immune  pairs  were  immune  to 
reinfection  both  in  the  nose  and  the  lungs.   Control-to-control  pairs, 
were  susceptible  to  infection  in  both  organs.   Immune-to-control  pairs, 
however,  showed  a  surprising  pattern.   The  immune  partner  remained  immune 
to  reinfection  in  both  organs.   The  control  partner,  however,  exhibited 
complete  resistance  in  the  lungs  but  not  in  the  nose. 

Intramuscular  Immunization  with  Live  RS  Virus  -  Recently,  Buynak  and  his 
colleagues  at  the  Merck  Institute  for  Therapeutic  Research  reported  that 
parenteral  administration  of  wild-type  RS  virus,  grown  in  human  diploid 
cells,  induced  the  development  of  serum  neutralizing  antibody  in  young 
children  without  causing  any  objective  signs  or  symptoms  of  disease. 
These  exciting  findings  clearly  required  amplification  and  extension, 
because  evidence  that  virus  replicated  following  intramuscular  (IM)  inoculation 
was  not  provided  in  the  original  report,  nor  was  the  possible  immunosuppressive 
effect  of  maternally  derived  passive  immunity  in  parenteral  immunization 
addressed.   The  former  issue  requires  resolution  because  a  virus  preparation 
that  failed  to  replicate  and  served  only  as  an  inactivated  antigen  might 
induce  potentiation  of  disease.   Since  an  RS  virus  vaccine  is  most  urgently 
needed  during  the  first  few  months  of  life,  the  issue  of  immunosuppression 
by  maternally  derived  serum  antibody  represents  a  potential  obstacle  to 
success  of  the  parenteral  vaccine  approach  in  this  age  group. 

These  two  issues  were  examined  using  cotton  rats  as  the  experimental  model 
of  infection.  This  rodent  is  permissive  for  RS  virus  and  supports  a  moderately 
high  level  of  viral  replication  in  the  upper  and  lower  portions  of  the 
respiratory  tract. 

First,  the  mechanism  of  induction  of  immunity  was  examined  in  an  attempt 
to  determine  whether  viral  replication  at  the  site  of  inoculation  or 
within  the  respiratory  tract  was  responsible  for  the  resistance  observed 
after  IM  inoculation  of  10  '   to  10  PFU  of  live  virus.   Virus  was  not 
recovered  from  the  local  site  of  inoculation  after  5  min.  and  was  never 
detected  in  the  nose  or  lungs  at  any  time  after  IM  inoculation.   Furthermore, 
attempts  to  detect  viral  antigens  at  the  site  of  IM  inoculation  were 
unsuccessful.   However,  inactivation  of  infectivity  of  three  strains  of 


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virus  by  the  minimal  UV  dose  required  for  inactivation  markedly  reduced  or 
completely  ablated  their  antigencitity  and  protective  efficacy.   Although 
this  observation  does  not  constitute  unequivocal  evidence  for  the  occurrence 
of  viral  replication  after  IM  inoculation,  it  suggests  that  limited  replication, 
perhaps  restricted  to  an  abortive  cycle,  was  responsible  for  stimulation 
of  immunity  by  the  small  quantities  of  virus  employed. 

The  issue  of  viral  replication  is  of  more  than  academic  interest,  because 
an  inactivated,  antigenic  RS  virus  vaccine  used  previously  in  a  series  of 
trials  did  not  stimulate  immunity  but  did  induce  a  state  of  altered  reactivity 
such  that  disease  was  enhanced  when  vaccinees  underwent  natural  infection. 
This  disease  potentiation  effect  could  not  be  ascribed  to  tissue  culture 
or  medium  constituents  in  the  vaccine,  since  a  comparison  group  of  vaccinees 
who  received  a  parainfluenza  virus  vaccine  prepared  in  the  same  manner  as 
the  RS  virus  vaccine  did  not  exhibit  potentiation  of  disease  when  infected 
naturally  with  RS  virus.   Hence  the  mechanism  by  which  a  small  quantity  of 
live  RS  virus  stimulated  an  immunological  response  must  be  resolved  to  be 
certain  that  it  differs  from  that  of  inactivated  vaccine.   As  cited  above, 
this  seems  to  be  the  case,  since  it  appears  that  live  virus  undergoes 
limited  replication  following  parenteral  inoculation. 

Second,  the  possibility  that  passive  immunity  might  interfere  with  the 
effectiveness  of  parenteral  immunization  with  live  RS  virus  was  examined 
because  immunosuppression  could  pose  a  serious  obstacle  to  this  approach. 
Thus,  the  greatest  need  for  an  RS  virus  vaccine  is  in  the  first  few  months 
of  life,  a  time  when  infants  possess  a  moderately  high  level  of  maternally 
derived  RS  virus  serum  antibody.   We  attempted  to  simulate  these  conditions 
by  administering  live  RS  virus  IM  to  weanling  rats  possessing  passive 
serum  antibody  derived  from  their  immune  mothers.   In  this  situation  an 
immunosuppressive  effect  of  passive  immunity  was  observed.   Only  50%  of 
inoculated  weanling  rats  were  rendered  resistant  to  subsequent  IN  challenge 
with  RS  virus.   This  suggests  that  parenteral  immunization  with  live  virus 
may  not  be  effective  in  protecting  human  infants  against  RS  virus  during 
their  period  of  greatest  vulnerability  to  serious  RS  virus  disease,  i.e., 
the  first  3  months  of  life.   If  this  be  the  case,  the  usefulness  of  live 
IM  virus  vaccine  may  be  limited  to  individuals  over  6  months  of  age  who 
have  escaped  natural  infection  and  who  have  lost  most  or  all  of  their 
passive  maternally  derived  serum  antibody.   The  outlook  for  IM  vaccination 
of  individuals  who  have  undergone  prior  infection  is  not  encouraging; 
Buynak's  study  indicated  that  seropositive  children  respond  poorly  to 
vaccine. 

RS  Virus  Infection  in  the  Immunosuppressed  Host 

Humans  -  Using  immuno enzyme  technology  developed  in  this  laboratory,  which 
enables  us  to  detect  RS  virus  antigen  in  paraffin-embedded  tissues,  we 
have  found  that  RS  virus  infection  in  the  immunocompromised  is  far  more 


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extensive  than  in  normal  individuals.   In  one  immunodef icient  infant  and 
two  immunosuppressed  adults  RS  viral  antigen  was  found  in  large  amounts  in 
the  respiratory  tract  as  well  as  other  tissues.  Dissemination  of  virus  to 
non-respiratory  tissues  had  not  been  recognized  previously. 

Cotton  Rats  -  A  regimen  was  devised  whereby  cotton  rats  received  cyclophosphamide 
in  small,  repeated  doses,  namely  50  mg/kg  three  times  a  week.   After  three 
weeks,  when  the  white  cell  count  had  dropped  and  stabilized  at  a  reduced 
value,  the  animals  were  infected  intranasally  with  RS  virus.  Drug  treatment 
was  continued  on  the  same  basis  following  infection.  Viral  titer  in  nose 
and  lungs  remained  at  maximal  level  as  long  as  drug  treatment  continued 
(at  least  58  days).   In  some  animals  virus  was  detected  in  the  kidneys. 
In  normal  cotton  rats,  RS  virus  was  never  found  beyond  the  respiratory 
tract. 

Efficiency  of  coupling  reactions  and  use  of  fluorescence  substrates 
in  enzyme-linked  immunosorbant  assays'   Numerous  enzyme-linked  immunosorbant 
assays  nave  been  described  using  many  different  enzyme  systems  as  well  as 
many  different  methods  of  coupling  or  alternately,  uncoupled  enzyme  conjugates. 
We  have  explored  the  effects  of  various  coupling  reactions  using  bi- 
functional  reagents,  periodate  cross-linking  procedures,  and  antibody 
enzyme  soluble  complex  methods.   In  the  evaluation  of  these  coupling 
reactions  three  enzyme  systems  were  employed:   alkaline  phosphatase, 
horseradish  peroxidase,  and  glucose  oxidase.   The  one-step  glutaraldehyde 
coupling  procedures  appeared  to  be  optimal  for  enzyme  linked  immunosorbant 
assay  conjugates.   Enzymatic  sensitivities*  when  measured  against  concentrations 
of  cholera  toxin  ranging  from  10   to  10    grams/ml  were  approximately 
equal  for  the  three  enzyme  systems  when  coupled,  with  one-step  glutaraldehyde 
procedure.   This  enabled  the  detection  of  10   ~  grams/ml  of  cholera  toxin. 
The  development  of  additional  sensitivity  in  this  assay  system  is  under 
investigation  with  the  use  of  fluorescence  substrates. 

Extension  of  ongoing  enzyme-linked  immunosorbant  assay  procedures:  As 
reported  last  year  we  began  to  measured  serum  IgG  antibody  specific  for 
respiratory  syncytial  virus.   This  year  we  have  extended  our  capabilities 
to  include  the  measurement  of  serum  IgA  as  well  as  the  IgA  and  IgG  present 
in  the  nasal  washings  of  patients.  Current  sensitivity  levels  in  nasal 
washings  are  4  X  10   grams/ml  of  IgA. 

Immunohist oar cheo logy 

This  laboratory  has  been  engaged  in  the  development  of  a  new  method  of 
enzyme-linked  immunohistologic  diagnostic  technology.   Because  of  the 
widely  recognized  difficulties  in  the  evaluation  of  peroxidase  staining 
due  to  endogenous  peroxidase  activity  in  tissues  and  inducibility  of 
endogeneous  peroxidase-like  activity  in  inflammatory  and  neoplastic  states 
we  developed  a  method  of  enzyme- immunohistochemistry  which  would  be  unaffected 
by  inflammatory  and  neoplastic  processes.  Our  search  for  an  enzymatic 
system  not  present  in  mammalian  tissues  led  us  to  the  consideration  of 


23-92 


Z01  AI  00175-02  LID 

glucose  oxidase,  an  enzyme  derived  from  non-mammalian  sources,  as  a  possible 
candidate  for  this  purpose.   Earlier  work  had  shown  that  by  modification 
of  the  reaction  product  a  stable  preparation  could  be  obtaind  suitable  for 
immunohistochemistry.  Antisera  were  prepared  against  this  enzyme  in  rabbits, 
guinea  pigs  and  goats.   These  antisera  were  converted  into  a  soluble 
enzyme  antibody  complex  and  used  in  a  manner  similar  to  that  described  by 
Sternberger.   Currently  we  are  determining  the  critical  variables  for 
respiratory  syncytial  virus  antigen  preservation  during  the  fixation  and 
embedment  processes. 

The  initial  application  of  this  new  methodology  was  in  a  cooperative  study 
with  the  Center  for  Disease  Control.   After  obtaining  human  material  from 
which  Legionella  pneumophila  was  identified  optimization  of  the  testing 
procedure  was  conducted  using  visible  bacteria  as  antigenic  moities. 
After  conclusion  of  this  optimization  procedure,  110  coded  specimens  of 
tissue,  some  from  patients  with  legionnaires  disease,  were  sent  to  us.   We 
identified  19  specimens  positive  for  Legionella  pneumophilia.   Upon  breaking 
the  blind  code,  there  was  complete  correlation  with  the  bacteriologic 
findings.   Additional  applications  include  antigenic  analysis  of  the 
materials  obtained  from  the  1978-1979  Naples,  Italy  epidemic  outbreak  as 
well  as  materials,  obtained  from  many  sources  across  the  United  States,  of 
documented  fatal  RS  virus  illnesses.   Additional  testing,  as  well  as 
production  of  infected  tissue  suitable  for  detection  of  respiratory  syncytial 
virus,  parainfluenza  types  1,  2,  3  and  4,  viruses  and  human  rotavirus  are 
currently  underway.   The  methodology  has  already  been  extended  to  the 
detection  of  immunoglobulins  G,  A,  and  M,  fibrinogen,  albumin,  and  C3. 

Experimental  Aerosol  Infection 

Using  the  cotton  rat  as  the  susceptible  host,  infection  via  the  aerosol 
route  with  respiratory  syncytial  virus  has  been  undertaken.   The  virus  is 
largely  destroyed  by  the  process,  however,  infections  were  established 
reproducibly  in  cotton  rats  and  demonstrate  that  the  cotton  rat  is  sensitive 
to  infection  with  as  few  as  25  plaque  forming  units  per  animal.   Moreover, 
examination  of  the  time  course  of  infection  by  this  means  of  inoculation 
with  a  small  quantity  of  virus  demonstrate  that  the  peak  titers  occur  not 
at  day  4,  as  with  intranasal  infection,  but  at  day  7  or  later.   Currently 
the  effect  of  age  and  sex  on  viral  titer  are  being  examined. 

Publications 

Belshe,  R.B.,  Richardson,  L.S.,  Prevar,  D.A.,  Camargo,  E.,  Chanock, 
R.M. :   Growth  and  genetic  stability  of  4  temperature-sensitive  (ts) 
mutants  of  respiratory  syncytial  (RS)  virus  in  newborn  ferrets.  Arch. 
Virology  58:  313-321,  1978. 


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Z01  AI  00175-02  LID 

Richardson,  L.S.,  Belshe,  R.B.,  London,  W.T.,  Sly,  D.L.,  Prevar,  D.A., 
Camargo,  E. ,  and  Chanock,  R.M. :   Evaluation  of  five  temperature  sensitive 
mutants  of  respiratory  syncytial  virus  in  primates.   Viral  shedding, 
immunologic  response  and  associated  illness.   J.  Med.  Virology,  3:   91- 
100,  1978. 

Belshe,  R.B.,  Richardson,  L.S.,  London,  W.T.,  Sly,  D.L.,  Camargo,  E., 
Prevar,  D.A.,  and  Chanock,  R.M. :   Evaluation  of  five  temperature-sensitive 
mutants  of  respiratory  syncytial  virus  in  primates.   II.   Genetic  analysis 
of  virus  recovered  during  infection.   J.  Med.  Virology  3:   101-110, 
1978. 

Prince,  G.A. ,  Jenson,  A.B. ,  Horswood,  R.L. ,  Camargo,  E.  and  Chanock, 
R.M. :  The  pathogenesis  of  respiratory  syncytial  virus  infection  in 
cotton  rats.  Amer.  J.  Pathol.  93:   185-205,  1978. 

Prince,  G.A. ,  Potash,  L. ,  Horswood,  R.L.,  Camargo,  E. ,  Suffin,  S.C., 
Johnson,  R.A. ,  and  Chanock,  R.M. :  Intramuscular  inoculation  of  live 
respiratory  syncytial  virus  induces  immunity  in  cotton  rats.  Infect. 
Immun.  23:  723-728,  1979. 

Suffin,  S.C.,  Prince,  G.A. ,  Muck,  K.B.,  and  Porter,  D.D.:   Immunoprophylaxis 
of  respiratory  syncytial  virus  infection  in  the  infant  ferret.   J. 
Immunol.  123:  10-14,  1979. 

Suffin,  S.C.,  Prince,  G.A. ,  Muck,  K.B.,  and  Porter,  D.D.:   Ontogeny  of 
the  humoral  immune  response  in  the  ferret.   J.  Immunol.:  123:  6-9,  1979. 

Chanock,  R.M. :   Parainfluenza  viruses.   In,  Lennette,  E.H.  (Ed.), 
Diagnostic  Procedures  for  Viral,  Rickettsial  and  Chlamydial  Infections, 
Fifth  Edition,  American  Public  Health  Association,  Inc.,  New  York,  1979 

Chanock,  R.M. ,  Kim,  H.W. ,  Brandt,  CD.,  and  Parrott,  R.H. :  Respiratory 
Syncytial  Virus.   In,  Evans,  A.S.  (Ed.),  Viral  Infections  of  Humans, 
Plenum  Press,  Inc.,  Boston,  1979,  in  press. 


23-94 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00179-01  LID 


PERIOD  COVERED 


OCTOBER  1,  1978  THROUGH  SEPTEMBER  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

MOLECULAR  BIOLOGY  OF  RESPIRATORY  AND  GASTROINTESTINAL  VIRAL  PATHOGENS 
STUDIED  BY  DNA  RECOMBINANT  TECHNOLOGY 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


P.I.  Ching-Juh  Lai,  Ph.D.     Visiting  Scientist 
Lewis  J.  Markoff,  M.D.    Medical  Officer 


LID,  NIAID 
LID,  NIAID 


Associate  Investigator:   Brian  R.  Murphy,  M.D.,  Medical  Officer,  LID, NIAID 


COOPERATING  UNITS  (if  any) 

None 


LAB/BRANCH 

LABORATORY  OF  INFECTIOUS  DISEASES 


RESPIRATORY  VIRUSES  SECTION 


INSTITUTE  AND  LOCATION 

NATIONAL  INSTITUTE  OF  ALLERGY  AND  INFECTIOUS  DISEASES,  Bethesda,  Maryland 


TOTAL  MANYEARS: 

48/12 


PROFESSIONAL: 

24/12 


24/12 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   Q  (a2)  INTERVIEWS 


Q  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

k   procedure  was  devised  for  producing  double-stranded  DNA  sequences 
corresponding  to  each  of  the  influenza  virus  RNA  segments.   Negative  and 
positive  strands  of  influenza  RNA  segments  were  copied  separately  into  DNA 
using  the  reverse  transcriptase  of  avian  myeloblastosis  virus.   The  DNA  products 
□f  the  reverse  transcriptase  enzyme  appeared  to  represent  full-length  genomic 
copies.   The  single-stranded  DNA  molecues  from  both  preparations  were  annealed 
to  generate  double-stranded  DNA  segments  which  were  subsequently  isolated  for 
cloning  in  plasmid  PBR322  of  E.  coli  K-12.  The  in  vitro  synthesized  double 
stranded  influenza  DNA  segments  were  than  inserted  into  plasmic  PBR322  at  the 
specific  Pst  I  site  using  the  dG-dC  tailing  technique.  The  hybrid  DNA  molecues 
were   used  to  transform  recipient  E_;_  coli  and  transf ormants  containing  influenza 
gene  sequences  were  identified  by  hybridization.   In  this  manner,  we  have  so  far 
btained  putative  full-length  influenza  virus  DNA  segments  corresponding  to  genes 
oding  for  non-structural  protein  (Gene  VIII),  matric  protein  (gene  VII), 
euraminidase  (gene  VI)  ,  and  hemagglutinin  (gene  IV)  .   Cloning  of  other  genes  froijn 
ild  type  influenza  A/Udorn/72  (H3N2)  virus  is  now  underway. 


PHS-6040 
(Rev.  10-76) 


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Z01  AI  00179-01  LID 

Objectives 

(a)   To  employ  recombinant  DNA  technology  to  study  the  genes  of  influenza 
virus  and  their  functions;  (b)  to  determine  the  antigenic  determinants  of 
the  viral  surface  proteins  and  elucidate  the  pattern  of  antigenic  variation 
among  influenza  virus  strains  by  analysis  of  cloned  DNA  sequences;  (c)  to 
study  the  viral  proteins  synthesized  in  E^  coli  (harboring  influenza  virus 
genes)  in  relation  to  their  antigenic  properties;  (d)  to  examine  cloned 
viral  DNA  for  gene  expression  in  eukaryotic  cells;  (e)  to  develop  procedures 
for  conversion  of  cloned  viral  DNA  back  to  the  viral  RNA  with  the  intent  of 
isolating  or  constructing  mutants  for  use  in  experimental  studies  and 
possibly,  immunoprophylaxis. 

Methods 

(a)  Influenza  viral  DNA  synthesis  -  Reverse-transcription  of  the  viral  RNA 
segments  using  (1)  a  specific  DNA  primer  which  is  complementary  to  the  3'- 
end  of  virion  RNA  segments  or  (2)  oligodeoxythymidylate  primer. 

(b)  Cloning  viral  DNA  copies  in  the  E^  coli-plasmid  PBR322  system. 

(c)  Analysis  of  influenza  DNA  molecules  by  restriction  enzyme  cleavage  and 
nucleotide  sequencing. 

(d)  Radioimmunoassay  for  viral  peptides  synthesized  in  E^  coli  containing 
cloned  influenza  viral  genes. 

(e)  Transfection  or  cloning  of  influenza  viral  DNA  (from  plasmid  PBR  322) 
in  eukaryotic  cells  using  early  region  of  SV40  as  vector. 

(f )  Biochemical  (nucleic  acid  hybridization) ,  immunologic  (radioimmunoassay 
or  ELISA)  and  genetic  (gene  rescue  of  ts_  genes  of  defined  _ts  mutants) 
analysis  of  transcription  and  expression  of  influenza  genes  in  eukaryotic 
cells  transformed  by  cloned  influenza  viral  DNA. 

Background 

The  genome  of  influenza  virus  consists  of  several  separate  RNA  segments. 
The  virion  RNA  segments  are  negative  strands  according  to  current  nomenclature, 
that  is,  their  complementary  RNA  strands  become  associated  with  the  polyribosomes 
and  are  translated  to  produce  viral  specific  proteins.   So  far,  eight 
discrete  virion  RNA  species  have  been  identified  by  gel  electrophoresis. 
Results  from  genetic  complementation-recombination  and  biochemical  analysis 
have  demonstrated  that  each  influenza  RNA  segments  codes  for  a  specific 
peptide  product  that  is  responsible  for  the  respective  viral  function. 
Temperature-sensitive  mutants  in  all  eight  complementation-recombination 
groups  have  been  isolated  and  characterized. 


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Z01  AI  00179-01  LID 

Segmentation  of  the  influenza  viral  genome  is  responsible  for  the  high  rate 
of  recombination  observed  during  coinfection  by  different  influenza  A  virus 
strains  within  the  same  type.  Recombination  of  influenza  virus  genes 
presumably  occurs  by  free-exchange  of  RNA  segments  as  a  result  of  gene 
reassortment  during  infection.   The  mechanism  of  assortment  that  gives  rise 
to  a  proper  gene  constellation  in  each  infectious  virus  particle  is  not 
clear. 

Another  important  feature  of  influenza  virus  is  antigenic  variation,  or  the 
emergence  of  viral  subtypes  (antigenic  shift) .   Other  phenomena  involving 
antigenic  variation  of  a  lesser  extent  within  a  subtype  (antigenic  drift) 
are  also  observed.   Such  antigenic  instability  results  in  epidemiologically 
significant  differences  in  surface  antigens  (hemagglutinin  and  neuraminidase) 
seen  in  the  influenza  A  virus  subtypes.   When  this  type  of  change  is  abrupt 
and  extensive  (antigenic  shift)  a  pandemic  ensues.   As  a  consequence,  the 
continuing  change  of  influenza  strains  has  rendered  ineffective  efforts  to 
vaccinate  man  against  major  antigenic  variants. 

In  view  of  the  medical  importance  of  influenza  viruses  there  is  a  need  to 
broaden  our  knowledge  of  the  genetics  of  these  viruses  in  order  to  design 
ways  to  prevent  pandemics.   One  approach  is  to  use  recombinant  DNA  technology 
that  has  been  well-developed  and  has  proven  valuable  in  elucidating  gene 
organization  and  expression  in  other  appropriate  host  systems.   Our  general 
plan  for  this  approach  includes  two  phases.   The  first  phase  involves 
cloning  each  of  the  eight  influenza  gene  segments  using  the  E_;_  coli  K12- 
plasmid  system   (approved  MUA  #91) .   The  second  phase  of  development  involves 
the  use  of  purified  clones  of  influenza  DNA  sequences  to  examine  viral  gene 
expression  in  animal  cell  culture  and  to  produce  vRNA  in  eukaryotic  cells. 
To  carry  out  this  second  phase  we  propose  to  utilize  defective  SVAO  (lacking 
the  late  region  of  the  genome  that  codes  for  capsid  proteins)  for  construction 
of  influenza-SV40  hybrid  DNA  molecules  (submitted  MUA  #110) .   Introduction 
of  the  appropriate  influenza-SV40  hybrid  DNA  molecules  into  eukaryotic 
cells  may  lead  to  transcription  or  translation  of  influenza  viral  DNA 
depending  upon  its  orientation  of  insertion.   Our  goal  is  to  devise  procedures 
which  would  permit  conversion  of  influenza  DNA  back  to  an  influenza  RNA 
negative  strand  and  eventually  transfer  such  RNA  into  an  influenza  virus. 
In  this  manner,  stable,  site-specific  mutations,  such  as  deletions,  induced 
in  the  cloned  DNA  might  be  transferred  back  to  the  influenza  virus.   Thus, 
it  may  be  possible  to  develop  stable  mutants  for  experimental  study  and  for 
use  in  immunoprophylaxis. 

Major  Findings 

(a)   Influenza  virus  double-stranded  DNA  synthesis  in  vitro 

In  this  study  we  devised  a  procedure  for  obtaining  double-stranded  DNA 
sequences  corresponding  to  each  of  the  influenza  virus  RNA  segments.   This 
approach  should  be  generally  applicable  to  negative-stranded  RNA  viruses 
containing  segmented  genomes.   Specifically,  the  procedure  involves  separate 


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Z01  AI  00179-01  LID 

synthesis  of  DNA  copies  from  both  the  negative  and  the  positive  strands  of 
RNA  segments  using  the  reverse  transcriptase  of  avian  myeloblastosis  virus. 
Influenza  virion  RNA  segments,  which  are  negative  strands,  were  reverse- 
transcribed  into  their  complementary  DNA  copies  in  the  presence  of  a  specific 
priming  DNA  oligomer.  The  primer  is  complementary  to  the  conserved  3 '-end 
sequence  of  the  virion  RNA  segments  and  the  DNA  products  of  the  reverse 
transcriptase  enzyme  appear  to  represent  full-length  genomic  copies.  Similarly, 
poly  A-containing  cytoplasmic  RNA's  (positive  strands)  isolated  from  the 
virus-infected  cells  were  also  transcribed  to  form  DNA  sequences  in  a 
reaction  mixture  in  which  oligo-(dT)  primer  was  added.   The  single-stranded 
DNA  molecules  from  both  preparations  were  annealed  to  generate  double- 
stranded  DNA  segments  which  were  subsequently  isolated  for  cloning  in 
plasmid  PBR322  of  E.  coli  K-12. 

(b)   Influenza  gene  segments  cloned  in  E_;_  coli  plasmid 

As  our  first  step  in  this  project  to  obtain  influenza  DNA  clones  in  a 
plasmid,  the  in  vitro  synthesized  influenza  DNA  segments  were  inserted  into 
plasmid  PBR322  at  the  specific  Pst  I  site  using  the  dG-dC  tailing  technique. 
The  hybrid  DNA  molecules  were  used  to  transform  recipient  E^_  coli  and 
transformants  containing  influenza  gene  sequences  were  identified  by 
hybridization.   In  this  manner,  we  have  so  far  obtained  putative  full- 
length  influenza  virus  DNA  segments  corresponding  to  genes  coding  for  the 
non-structural  protein  (gene  VIII) ,  the  matrix  protein  (gene  VII) ,  the 
neuraminidase  protein  (gene  VI) ,  and  hemagglutinin  (gene  IV) .   Cloning  of 
other  genes  from  wild  type  influenza  A/Udorn/72  (H3N2)  virus  will  be  initiated 
shortly.   Other  experiments  described  in  this  report  are  in  progress. 


23-c 


LABORATORY  OF  MICROBIAL  IMMUNITY 

1979  Annual  Report 
Table  of  Contents 


Z01-A1 


Project  Number  Page 

Summary  Statement   24-1 

0013^-17         Control  of  Immunoglobulin  Synthesis  in  Mice.  — 

Asof sky  24-7 

00135-05         Properties  of  Immunoglobulin  Secreting  Cells.  -- 

Morse 24-13 

00136-07         Functional  Activities  of  Subpopulat ions  of  Thymus- 

Derived  Cells.  —  Mathieson  24-15 

00137-13         Biology  of  Graf t-Versus-Host  Reactions.   -- 

Asofsky 24-19 

00138-05         Viruses  and  the  Immune  Response.  —  Morse  24-20 

00139-14         The  Immunologic  Response  of  Animals  to  Trypanosoma! 

Antigens.  --  Finerty  24-23 

00140-14  Immunology  of  Malaria.  --  Finerty  24-25 

00141-05  Immune  Responses  to  Malaria  and  Related  Intracellular 

Protozoa.  —  Taylor 24-27 

00142-06         Development  of  Thymus-Der i ved  (T)  Suppressor  and 

Amplifier  Cell  Function.  —  Baker  24-32 

00143-10         Genetic  Control  of  the  Antibody  Response  to  Type  III 

Pneumococcal  Polysaccharide  (SSS-lll).  —  Baker  ....24-33 

00144-15         Regulation  of  the  Antibody  Response  to  Type  III 

Pneumococal  Polysaccharide  (SSS-lll).  --  Baker  24-36 

00145-12         Mode  of  Action  of  Thymus-Der i ved  (T)  Suppressor  and 

Amplifier  Cells.  --  Baker  24-39 

00146-06  Immunological  Studies  on  Components  Isolated  from 

Bacteria,  Parasites  and  Plants.  --  Baker  24-41 

00153-03  In  Vi  tro  Response  of  Human  Peripheral  Lymphocytes  to 

Infectious  Organisms.   --  Asofsky 24-44 

00158-03         The  Immune  Response  to  Entamoeba  Antigens.  -- 

Finerty 24-45 


Z01-A1 
Project  Number  Page 

00186-06  Pathogenesis  of  Autoimmunity  in  Inbred  Strains  of 

Mice.   --  Chused 2*4-46 

00187-06  Studies  in  Sjogren's  Syndrome.  --  Chused  24-49 


PHS-NIH 
SUMMARY  STATEMENT 

ANNUAL  REPORT  OF  THE  LABORATORY  OF  MICROBIAL  IMMUNITY 

NATIONAL  INSTITUTE  OF  ALLERGY  AND  INFECTIOUS  DISEASES 

October  1,  1978  to  September  30,  1979 

Richard  Asofsky,  M.D. 
Chief,  Laboratory  of  Microbial  Immunity 


Research  in  this  Laboratory  is  concerned  with  the  differentiation  of  lymphoid 
cells  and  with  basic  mechanisms  controlling  cellular  and  humoral  immunity  to 
microbial  and  tissue  antigens.   Selected  microorganisms  or  their  products  are 
used  in  this  work,  and  include  viruses,  blood  parasites,  bacterial  poly- 
saccharides and  1 i popolysaccharides .   Tissue  antigens  include  constituents  of 
integrated  leukemia  viruses  which  are  present  on  cell  walls  and  histocompati- 
bility antigens.  Assembly  and  control  of  synthesis  of  immunoglobulins  is 
studied  in  cloned  lines  of  neoplastic  plasma  cells,  B  lymphocytes,  or  in 
somatic  hybrids  between  neoplastic  and  normal  cells.   Somatic  hybridization 
is  also  used  to  examine  the  repertoire  of  some  antibody  responses.   Hetero- 
geneity of  thymus  derived  (T)  cells  is  examined,  using  al loant igenic  markers 
of  T  cell  differentiation  and  certain  lectins.   Several  genetic  studies  are 
in  progress,  including  the  identification  and  distribution  in  inbred  mice  of 
genes  controlling  leukemia  viruses,  those  controlling  differentiation  of  T 
lymphocytes,  and  those  controlling  antibody  formation  to  pneumococcal  poly- 
saccharide. 

I .   IMMUNE  RESPONSE  GENES  MAY  BE  ON  DIFFERENT  CHROMOSOMES 

Previous  studies  conducted  in  this  laboratory  have  shown  that  at  least  five 
autosomal  genes  influence  the  capacity  of  bone  marrow-derived  precursors  of 
antibody-forming  cells  (B  cells)  to  make  an  antibody  response  to  Type  III 
pneumococcal  polysaccharide  (SSS-lll);  these  genes  act  in  a  complementary  or 
additive  manner  and  none  appear  to  be  linked  to  the  major  histocompatibility 
(H-2)  or  the  immunoglobulin  allotype  complex.   Congen ic-res istant  strains 
of  mice  having  chromosomal  segments  from  a  high-responding  strain  on  a  low- 
responding  background  strain  were  used  to  isolate  the  genes  involved  and 
to  assign  such  genes  to  well-defined  genetic  linkage  groups  or  chromosomes. 
The  results  obtained  showed  that  the  introduction  of  segments  from  chromo- 
somes 17,  9  and  k,    as  well  as  chromosome  segments  bearing  the  H-23  and  H-27 
loci  appeared  to  increase  immune  responsiveness  to  SSS-lll.   Thus,  genes 
governing  the  capacity  of  B  cells  to  respond  to  SSS-lll  may  be  located  on 
different  chromosomes.   It  remains  to  be  established  whether  the  effects 
of  these  genes  are  specific  for  the  antibody  response  to  SSS-lll  or  influence 
responsiveness  to  other  antigens. 

(Dr.  P.  J.  Baker;  B.  Prescott;  Mr.  G.  Caldes;  Ms.  D.  F.  Amsbaugh;  and 
P.  W.  Stashak) 


2^-1 


I  I .  THYMUS-DERIVED  HELPER  CELLS  REQUIRED  FOR  INCREASED  I MMUNOGEN I C I TY 
OF  THE  HIPOTEICHOIC  ACID  OF  STAPHYLOCOCCUS  AUREUS 

The  antibody  response  to  the  lipoteichoic  acid  (LTA)  of  S .  aureus  was 
examined  by  a  newly  developed  procedure  in  which  erythrocytes,  sensitized 
with  per iodate-act i vated  LTA,  were  used  for  the  detection  of  serum  antibody 
and  antibody-producing  plaque-forming  cells  (PFC) .   Serum  antibody  and  PFC 
were  found  in  mice  immunized  with  heat-killed  S.  aureus.   The  specificity  of 
such  antibody  was  affirmed  by  inhibition  tests  using  purified  LTA;  here, 
larger  amounts  of  unrelated  polysaccharide  and  protein,  extracted  from 
S.  aureus  membranes,  were  without  effect.   LTA-specific  PFC  were  first 
detected  two  days  after  immunization  with  heat-killed  bacterial  cells; 
maximal  numbers  were  attained  by  day  four.   But,  no  PFC  were  found  in 
mice  given  purified  LTA  over  a  10,000-fold  range  of  immunizing  doses.   Prior 
immunization  with  LTA  did  not  reduce  the  PFC  response  to  heat-killed  bacterial 
cells;  thus  LTA  was  not  tolerance- inducing.   Mice  pretreated  with  a  carrier 
known  to  activate  thymus-der i ved  (T)  helper  lymphocytes  produced  a  PFC 
response  to  LTA  only  when  immunized  with  LTA  bound  to  the  same  carrier. 
This  suggests  that  carrier-specific  T  cells  are  needed  to  initiate  an 
antibody  response  to  LTA.   Since  an  antibody  response  can  be  elicited  in 
mice  given  killed  bacterial  cells,  other  cell  wall  and/or  cell  membrane 
constituents  may  play  an  important  role  as  immunologically  active 
carriers  in  this  regard. 

(Dr.  P.  J.  Baker,  Dr.  B.  Prescott;  Mr.  G.  Caldes,  Ms.  D.  F.  Amsbaugh; 
and  Mr.  P.  W.  Stashak,  with  Dr.  P.  R.  Beining  and  Ms.  G.  M.  Flannery 
(Un  i v.  Scranton) . 

III.    INTRATHYMIC  DIFFERENTIATION  OF  T  CELLS  STUDIED 

Examination  of  thymocyte  subpopulat ions  fractionated  by  peanut  lectin 
agglutination  has  identified  functionally  and  phenotypical 1 y  distinct  sub- 
sets of  cells.   These  subsets  are  presumably  precursors  for  peripheral  T 
cells  with  similar  function  and  phenoty_pe.   Initially,  a  minor  subset  of 
phenotyp ical 1 y  differentiated  Lyt  1  23  cells  were  demonstrated  in  the  thymus 
with  immunofluorescence  by  flow  microfluorometry  (FMF) .   Using  peanut  lectin 
fractionation,  it  was  shown  subsequently  that  this  population  could  be  en- 
riched from  10%  in  a  population  of  normal  thymocytes  to  50-70%  of  peanut  non- 
agglutinated  (PNA  )  thymocytes.   PNA  thymocytes  consist  of  a  population 
roughly  comparable  to  cortisone  resistant  thymocytes  in  size  and  mitogen  re- 
activity.  In  addition  to  the  enrichment  for  Lyt  1  23  ,  in  the  PNA  cells,  a 
population  of  Lyt  123   cells  is  consistently  observed  in  the  PNA  fraction. 
Subsequent  analysis  of  this  Lyt  123   fraction  has  demonstrated  a  further  sub- 
set of  these  cells  which  are  eliminated  with  an  antisera  to  a  marker,  CTL , 
on  cytotoxic  lymphocytes  and  cytotoxic  lymphocyte  precursors.   The  PNA  cells 
which  are  eliminated  with  this  antisera  account  for  part  but  not  all  of  the 
cytotoxic  precursor  activity,  while  all  peripheral  differentiated  T  lympho- 
cytes with  cytotoxic  precursor  activity  can  be  eliminated  with  this  antisera. 
These  fundings  suggest  the  presence  of  a  differentiation  pathway  in  which 
both  phenotype  and  function  for  cytotoxic  lymphocytes  are  developed  in  a  sub- 
set of  PNA  Lyt  123   thymocytes.   These  analyses  have  demonstrated  further 


2*t-2 


that  extremely  small  populations,  in  this  case  less  than  \%   of  the  normal 
thymocytes,  can  be  identified  with  suitable  pre-enr ichment  techniques  and  FMF 
analysis.   (Drs.  B.  J.  Mathieson,  R.  Asofsky,  I.  Betel,  and  M.  Mage  (NCl), 
Ms.  P.  Campbell  and  S.  0.  Sharrow  (NCI)). 

IV.  GENETIC  STUDIES  OF  MOUSE  LEUKEMIA  VIRUSES 

Murine  leukemia  viruses  (MuLV)  are  highly  polymorphic.   The  mouse  genome 
includes  about  50  sequences  related  to  the  genetic  information  coding  for  the 
different  classes  of  MuLV  (termed  ecotropic,  xenotropic  and  amphotropic) . 
Some  of  these  sequences  code  for  complete  infectious  MuLV  of  the  various 
classes  whereas  some  code  for  only  isolated  portions  of  the  intact  virus. 
Studies  of  xenotropic  MuLV  (X-MuLV)  present  in  all  strains  of  mice,  have 
shown  that  expression  of  X-MuLV  as  infectious  virus  or  as  virus  coded  gp70 
on  the  surface  of  lymphocytes  varies  considerably  from  strain  to  strain 
(Morse,  Chused,  Boehm-Tru i tt ,  Mathieson,  Sharrow  and  Hartley)  and  in  different 
lymphoid  tissues  (Morse,  Chused,  Sharrow  and  Hartley)  and  that  expression  in 
these  two  modes  is  not  coordinate. 

Tests  for  infectious  virus  demonstrated  that  NZB  mice  have  two  loci  coding 
for  X-MuLV  and  that,  as  for  ecotropic  MuLV,  these  loci  are  dominant  in  crosses 
with  NFS  and  other  strains  of  mice  (Chused  and  Morse).   By  comparison,  F/St 
mice  appear  to  have  a  single  locus  for  infectious  X-MuLV  with  the  locus  be- 
having as  a  recessive  in  genetic  crosses  with  several  strains  of  mice.  Further- 
more, NZB's  produce  10-fold  more  X-MuLV  in  spleen  than  thymus  whereas  the 
ratio  is  reversed  in  F/St  spleen  and  thymus  (Morse,  Chused,  Sharrow,  Hartley; 
Morse,  Wolford,  Hartley-unpublished).   Finally,  in  NZB's,  the  levels  of  in- 
fectious X-MuLV  produced  by  T  cells  is  affected  by  genes  governing  T  cell 
differentiation  -  thymocytes  produce  less  virus  than  peripheral  T  cells. 
(Morse,  Chused,  Sharrow  and  Hartley). 

Analyses  for  X-MuLV  cell  surface  antigen  expression  (gp70's  termed  XenCSA) 
showed  that  disparate  levels  of  XenCSA  in  strains  DBA/2  (high  XenCSA)  and 
C57BL/6  (B6)  are  governed  by  a  semidominant  gene  on  chromosome  k   at  or  near 
Fv-1  (Morse,  Chused,  Taylor,  Hartley  and  Sharrow).   These  results  have  been 
confirmed  in  crosses  between  B6  and  the  high  XenCSA  strain  C3H  and  have  been 
extended  to  show  that  XenCSA  is  under  independent  genetic  control  from 
another  cell  surface  gp70  marker,  Gfx~.  Increasing  awareness  of  the  biology 
of   X-MuLV  should  be  of  great  help  in  defining  their  role  in  leukemo- 
genesis,  particularly  in  regard  to  the  formation  of  recombinants  with 
ecotropic  MuLV.   (Morse,  Stockert,  Chused,  Obata,  Kozak  and  Taylor). 

V.  IgD  ON  B-CELL  LYMPHOMAS 

Five  lymphomas  were  identified  previously,  which  had  cell  surface  immuno- 
globulin, as  indicated  by  staining  with  fluoresce  in-labeled  anti-immuno- 
globulin.   Cell  surface  I gM  was  found  on  four;  L10A,  X16C,  K46 ,  and  BAL  17. 
These  cell  lines  also  synthesize  but  do  not  secrete  monomeric  IgM.   Fluorescein- 
labeled  anti-6  stains  all  five  lines.   The  presence  of  IgD  has  also  been  shown 
by  labeling  surface  molecules  with  '25|5  then  precipitating  cell  lysates  with 
anti-6,  and  analyzing  the  labeled  products  by  pol yacry lamide  gel  electro- 
phoresis in  SDS.   Finally  these  cell  lines  synthesize,  but  do  not  secrete  IgD. 
(Drs.  Kim,  McKeever,  Laskov  (not  NIH)  and  Asofsky;  Mr.  Nero). 

2^-3 


VI .   NONSPECIFIC  SYNTHESIS  OF  IMMUNOGLOBULIN  STUDIED 

When  animals  are  immunized  with  many  antigens,  increased  synthesis  of  immuno- 
globulin (Ig)  far  exceeds  the  synthesis  of  specific  antibody.   The  reverse- 
plaque  assay  has  been  used  to  examine  this  increase.   I sotype-restr icted 
antisera  were  used  to  develop  the  reverse  plaques.   Certain  mitogens  such  as 
E  Col i  LPS  cause  increases  largely  in  I gM  producing  cells.   Primary  in- 
fection with  nonlethal  Plasmodium  yoel i  i  produces  a  parallel  increase  in  all 
classes,  whereas  secondary  infection  produces  increases  mainly  in  the  IgG 
classes.   Stimulation  with  aggregated  human  IgG  or  sheep  red  blood  cells 
cause  increase  mainly  in  IgGl.   Animals  deprived  of  T  cells  did  not  show  these 
increases  (except  for  stimulation  with  LPS).   The  use  of  more  than  one  anti- 
gen has  shown  that  the  precursors  of  the  reverse  plaques  must  belong  to  sets 
of  B  lymphocytes  which  overlap  but  which  are  not  identical.   It  therefore 
appears  that  nonspecific  stimulation  of  B  cells  occurs  via  stimulation  of 
T  cells  (Drs.  Rosenberg  and  Chiller  (not  NIH);  Drs .  Rosenberg,  Taylor, 
Weinbaum  and  Mr.  Evans). 


24-4 


ADMINISTRATIVE 

Dr.  Thomas  M.  Chused  joined  the  Laboratory  this  year.   He  has  continued  his 
research  on  autoimmune  diseases.   The  NIAID  fluorescence-activated  cell  sorter 
has  been  installed  together  with  a  PDP-11/34  computer.   The  sorter  is  a  two- 
laser,  2  color  instrument.   Dr.  Chused  is  now  programming  the  computer  to 
analyze,  display  and  print  data  from  experiments.   Also  joining  the  Laboratory 
this  year  were  Dr.  Diane  W.  Taylor,  a  Staff  Fellow,  Dr.  Wendy  F.  Davidson,  a 
Visiting  Fellow  and  Drs.  Janice  Longstreth,  Florence  Rol lwagen  and  Joyce 
Schroer,  all  NIH  post-doctoral  fellows.   Dr.  Sanford  Stone  was  moved  in  July 
from  OSD  to  LMI  where  he  will  be  head  of  the  Section  on  Experimental  Auto- 
immun  i  ty . 

Dr.  Paul  E.  McKeever  completed  three  very  productive  years  as  a  Research 
Associate.   He  is  now  a  senior  investigator  in  the  Neurosurgery  Branch, 
NINCDS.   Dr.  Carol  Ludwig  completed  her  assoc iatesh ip,  and  is  now  with  OD, 
NIA. 


24-5 


24-6 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGEI 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  Al  001 3^-1 7  LMI 


FERIOD   COVERED 

October  I,  1978  to  September  30,  1979 


TITLE   OF    PROJECT   (30   characters   or   less,) 


Control  of  Immunoglobulin  Synthesis  in  Mice 


NAMES,    LABORATORY    AND    INSTITUTE   AFFILIATIONS,    AND 
PROFESSIONAL   PERSONNEL   ENGAGED   ON   THE   PROJECT 


TITLES   OF    PRINCIPAL    INVESTIGATORS   AND   ALL   OTHER 


PI  : 

R. 

Asofsky 

OTHER: 

P. 

E.  McKeever 

P. 

Munoz 

K. 

J.  Kim 

C. 

Kanel lopoulos-Langevin 

Y. 

J.  Rosenberg 

C. 

B.  Evans 

B. 

J.  Fowlkes 

W. 

J.  Logan 

G. 

B.  Nero 

G. 

M.  Shearer 

R. 

B.  Levy 

D. 

H.  Sachs 

R. 

Laskov* 

J. 

M.  CH flier** 

Laboratory  Chief 
Research  Associate 
Research  Associate 
Visiting  Associate 
Visiting  Associate 
Visiting  Fellow 
Biological  Lab  Tech 
Microbiolog  ist 
Biological  Lab  Tech 
Biological  Lab  Tech 
Senior  Investigator 
Post-doctoral  Fellow 
Section  Head 


LMI /Nl Al D 

LMI/NIAID 

LMI/NIAID 

LMI/NIAID 

LMI/NIAID 

LMI/NIAID 

LMI/NIAID 

LMI/NIAID 

LMI/NIAID 

LMI/NIAID 

IB/NCI 

IB/NCI 

IB/NCI 


COOPERATING  UNITS  (if  any) 

*  R.  Laskov,  Dept.  of  Experimental  Medicine  S  Cancer  Research,  Hadassah  Univ, 

Medical  Center,  Jerusalem,  Israel 
**  J.  M.  Chiller,  National  Jewish  Hospital,  Denver,  Colorado 


LAB/BRANCH 

Laboratory  of  Microbial  Immunity 


SECTION 

Experimental    Pathology   Section 


INSTITUTE   AND   LOCATION 
Nl Al P.    NIH.    Bethesda.    Maryland    20205 


TOTAL  MANYEARS: 

72/12 


PROFESSIONAL: 

5V12 


18/12 


CHECK  APPROPRIATE  B0X(E3) 
C  (a)   HUMAN  SUBJECTS 

G  (al)  MINORS   []  (a2)  INTERVIEWS 


C  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

SAMM  368,  a  myeloma  wh  ich  secretes  lgG2b  and  IgA  paraproteins  s  imul taneously , 
shows  cytoplasmic  segregation  of  the  y_   and  a  heavy  chains,  and  rapid  assembly 
of  lgG2b  monomers  and  IgA  mul t  imers ,  the  latter  bonded  to  J  chains  in  a  late 
stage  of  assembly. 

Several  B-cel 1  tumors  have  surface- IgD  as  wel 1  as  surface  I gM  by  immunof 1 uores- 
cence.   Synthesis  of  IgD  has  been  demonstrated  in  these  tumors. 


24-7 


PHS-6040 
(Rev.  10-76) 


Project  No.  Z01  Al-0013^-17  LM I 


Project  Description: 


As  in  the  past  several  years,  considerable  attention  was  given  to  studies 
of  neoplastic  lymphoid  cells.  Work  was  also  done  on  the  control  of  synthesis 
of  immunoglobulins  of  different  classes  (isotypes)  following  stimulation  of 
mice  with  different  antigens. 

A.  Intracellular  Assembly  of  Two  Classes  of  Immunoglobulin  in 
Individual  Plasma  Cells 

Individual  plasma  cells  of  the  myeloma  SAMM  368  spontaneously  secrete  I gA 
and  lgG2b  in  the  absence  of  experimental  hybridization.  Immunoglobulin  bio- 
synthesis and  secretion  by  cell  lines  established  in  vi  tro  from  SAMM  368 
was  studied.   The  cells  secrete  I gA  predominantly  as  multimers  with  disulfide 
bonded  J-chains  and  noncovalent ly  bonded  light  chains.   They  simultaneously 
secrete  lgG2b  only  as  a  monomer  having  two  disulfide  bonded  light  chains. 
The  similarities  in  subunit  composition  made  by  this  plasmacytoma  and  single 
immunoglobulins  of  the  same  isotypes  show  that  a  cell  can  fully  assemble  and 
secrete  two  multichain  immunoglobulins  in  a  manner  similar  to  cells  which 
secrete  single  immunoglobulins.   SAMM  368  synthesizes  and  secretes  fully 
assembled  I gG2b  more  rapidly  than  I gA,  and  secretes  larger  amounts  of  I gG2b 
than  IgA,  suggesting  regulatory  factors  of  Ig  synthesis  in  addition  to  those 
used  by  cells  secreting  single  immunoglobulins. 

The  y2b  and  a-chains  of  SAMM  368  are  segregated  to  the  appropriate  immuno- 
globulin isotype,  thereby  avoiding  secretion  of  y2b-a  mixed  molecules. 
Similar  segregation  of  heavy  chains  occurs  among  cytoplasmic  precursors  of 
I gG2b  and  IgA.   This  indicates  that  segregation  of  heavy  chains  occurs  at  an 
early  stage  in  biosynthesis  prior  to  the  point  of  regulation  by  proteolytic 
enzymes.   J-chains  is  added  late  in  biosynthesis  only  to  the  IgA.   The 
reliability  and  speed  with  which  SAMM  368  synthesizes  two  complete  multichain 
proteins  without  making  inappropriately  mixed  molecules  favors  chemical  con- 
formation and  cellular  compartmental i zat i on  as  factors  critical  to  biosyn- 
thesis of  two  immunoglobulins  by  a  single  cell. 

Two  different  K-chains  are  present  on  the  IgA  and  I gG2b  of  SAMM  368  growing 
in  vivo.   IgA  and  lgG2b  secreted  in  vi  tro  has  similar  K-chains  with  major 
Tsoelectric  point  of  6.8,  possibly  reflecting  different  regulatory  factors 
in  vi  tro  than  in  vivo.  (Drs.  McKeever  and  Asofsky,  Mr.  Nero  (student)) 

B.  B  Cel 1  Tumors 

Previously,  we  reported  the  establishment  of  six  BALB/c  B  cell  lymphoma  lines 
expressing  different  surface  markers:   (1)  KkG,    L10A,  Xl6c  and  BALENTL  17  are 
lgM+  la+  Fc+,  C3  receptor",  (2)  A20  is  I g+  la+,  Fc+,  C3  receptor".   The  pre- 
sence of  S-lgD  from  these  tumors  was  indicated  by  analysis  of  surface  immuno- 
globulins following  labeling  of  surface  molecules  with  '25|  using  the  lacto- 
peroxidase  method.   Further,  flow  microfluorometry  following  staining  with 


24-8 


Project  No.   Z01  Al  001 3^-1 7   LM I 

several  kinds  of  fluorescei n- labeled  anti-6  antibodies,  including  a  hybridoma 
product,  showed  I gD  on  the  surface  of  cells  of  at  least  four  cell  lines. 
Tumors  K46,  XI 6C  and  L10A  were  induced  to  secrete  I gM  when  fused  with  a  drug- 
marked  igG2b  producing  MPC-11  myeloma  cell  line.   There  was  no  suppression  of 
the  parent  I gG2b  synthesis  in  the  hybrids.   (Drs.  Laskov,  Kim  and  Asofsky). 

Biosynthesis  and  surface  expression  of  I gM  and  I gD  has  been  studied  in  spon- 
taneous lymphomas  of  BALB/c  mice.   Lymphomas  K46  and  L10A  synthesize  i gM  mono- 
mers and  halfmers.   A  molecule  with  the  molecular  weight  expected  of  I gD  is 
usually  detected  by  biosynthetic  labelling.   This  molecule  was  best  detected 
with  ant i -6  antisera  and  sensitive  radioiodine  surface  labelling  techniques. 
Light  chains  on  the  I gM  and  I gD  of  K46  have  similar  isoelectric  points.   The 
light  chains  attached  to  I gM  halfmers  have  different  isoelectric  points  than 
light  chains  on  I gM  monomer  of  K46,  suggesting  post-trans lational  modification 
of  this  light  chain.   (Drs.  McKeever,  Kim  and  Asofsky,  Mr.  Nero  (student)) 

We  have  also  pursued  the  work  on  B  cell  lymphoma  lines  as  models  for  the 
study  of  normal  B  cell  subpopulat ions  since  these  in  vi  tro  cell  lines  express 
different  surface  immunoglobulin  classes  and  several  other  B  cell  markers  in- 
cluding la  and  LyM  antigens,  and  Fc  (igG)  receptors.   Three  different  cell 
lines  were  selected  for  further  investigation  of  the  possible  relationship 
between  Fc  (IgG)  receptors  and  alloantigens  on  their  surface.   Three  different 
patterns  of  inhibition  by  the  alloantisera  tested  (anti-la,  anti-H-2D  and 
anti-LyM)  were  observed.   Further  studies  of  the  mechanism(s)  of  the  blocking 
of  Fc  (IgG)  receptors  by  all  antibodies  have  been  delayed  by  the  difficulty 
in  obtaining  reasonable  amounts  of  F(ab')-  or  Fab  fragments  of  mouse  allo- 
antibodies  after  digestion.   Inhibition  experiments  performed  in  the  presence 
of  sodium  azide  or  after  mild  reduction  and  alkylation  of  al loant ibodies 
suggest  that  these  inhibitions  are  not  due  only  to  Fc  portions  of  al loant ibodies . 
Mixing  experiments  using  A20  ( I a+)  and  M12  (la")  mixed  1/1  and  incubated  with 
anti-la  serum  resulted  in  a  50%    inhibition,  suggesting  that  al loant ibodies 
block  Fc  receptors  only  on  the  cell  to  which  they  are  bound.   The  differences 
we  have  observed  between  the  relationships  of  alloantigens  and  Fc  receptors 
in  these  cell  lines  may  reflect  their  origin  from  different  distinct  B  cell 
subpopulat ions . 

C.   T  Cel 1  Tumors 


Ten  BALB/c  T  cell  lines  which  expressed  restricted  patterns  of  Lyt  differen- 
tiation antigens  were  investigated  for  the  possible  immunological  functions: 
Two  Lyt  1   Lyt  2+  cell  lines,  BALENTL  k   and  14,  gave  significant  suppression 
of  the  MLR  between  BALB/c  and  C57BL/6.   Furthermore,  BALENTL  14  also  inhi- 
bited the  generation  of  BALB/c  effector  cells  against  C57BL/6  spleen  cells. 
(Drs.  Kim,  Weinbaum  and  Asofsky) 


24-9 


Project  No.  Z01  Al  0013^-17  LM I 

Three  T  cell  lines,  BALENTL  k    (Ly  l"2+) ,  BALENTL  9  (Ly  l+2+)  and  BALENTL  13 
(Ly  l+2") ,  were  examined  for  their  sensitivity  to  glucocorticoids  and  their 
receptor  content.   All  these  lines  were  found  to  be  sensitive  to  dexametha- 
sone  and  to  contain  (high  glucocorticoid  receptors).   (Drs.  Schmidt,  Kim  and 
Thomas) 

D.  Hybri  domas 

Spleen  cells  from  BALB/c  mice  immunized  with  SSS-III  were  fused  with  drug 
marked  myeloma  cell  lines  (NS-1,  or  45.6TG  1.7  cell  line).   Several  stable 
hybrids  secreting  monoclonal  antibodies  specific  for  SSS-III  were  obtained. 
(Drs.  Kim  and  K.  Schroer) 

See  Project  No.  Z01  Al  001^9-09  LI  -  Hybridomas  Producing  Antibody  to  Insulin. 

E.  Other  Studies  With  Tumors 

The  possiblity  of  lymphotoxin  release  from  the  established  T- ,  B- ,  macrophage 
cell  lines,  were  determined  by  measuring  the  3H-thymidine  release  from  a 
fibroblast  cell  line,  P79^3.   Macrophage  lines  (J774.1,  P388D1,  PU5-1.8  and 
J77^.l)  secreted  the  cytotoxic  material,  however,  none  of  our  B  and  T  lymphoid 
lines  gave  any  positive  results. 

F.  Control  of  "Nonspecific"  Synthesis  of  Different  I sotypes  of  Immuno- 
globulin (Ig)  Following  Immunization 

Using  a  reverse  plaque-forming  cell  (PFC)  assay  which  detects  total  Ig  se- 
creting cells  or  those  secreting  Ig  of  one  particular  class,  regardless  of 
specificity,  in  vi  vo  experiments  have  been  done  to  study  mechanisms  which 
result  in  activation  of  B  cells.   It  is  shown  that  the  isotype  of  the  induced 
PFC  clearly  reflects  the  cellular  requirements  for  stimulation  of  B  cells  in 
the  different  cases.   For  example,  the  mitogen  LPS  which  requires  only  mini- 
mal signals  from  accesory  cells,  results  in  early  increases  in  PFC  restricted 
to  the  I gM  class.   In  addition,  polyclonal  B  cell  activation  can  occur  via 
two  different  T-cell  dependent  mechanisms.   First,  during  malaria  infections, 
a  marked  B  cell  activation  occurs.   Analysis  of  this  response  reveals  that 
all  PFC  classes  increase  in  a  parallel  fashion  suggesting  a  role  of  T  cell 
derived  lymphokines  which  act  non-speci f i cal ly  on  all  B  cell  precursors.   Re- 
infection of  mice  previously  given  malaria  however  results  in  preferential 
activation  of  I  gG  classes.   The  second  form  of  non-specific  B  cell  activation 
occurs  following  either  immunization  with  classical  T-dependent  antigens, 
e.g.  sheep  erythrocytes,  agg.  human  gamma  globulin,  or  heterologous  serum 
treatment  and  requires  the  participation  of  T  helper  cells  specific  for  in- 
ducing antigen.   In  this  case,  the  non-specific  increases  are   class  restricted, 
responses  occuring  predominantly  in  the  I gG  (particularly  IgGl)  class  and,  in 
one  case,  also  IgA. 


24-10 


Project  No.   Z01   Al   00134-17  LMI 

It  is  of  particular  interest  that  the  Ig  secreting  cell  populations  induced 
by  each  antigen  belong  to  overlapping  sets  of  cells.   The  finding  of  class- 
specific  increases  in  non-specific  PFC  is  not  limited,  and  has  been  shown 
using  hapten-carrier  conjugates  in  vivo  and  in  vi  tro,  as  well  as  the  above 
listed  antigens.   These  studies  are  now  aimed  at  the  demonstration  of  i sotype- 
specific  helper  cells,  and  the  possible  recognition  of  isotypes  and  idiotypes. 
Selective  recognition  may  cause  restricted  stimulation  of  B  cells.   (Drs. 
Rosenberg  and  Chiller  (not  N  I  H) ) 

G.   Immunoglobulin  Production  in  CBA/N  Mice. 

CBA/N  mice  bear  a  sex-linked  (X-l inked)  genetic  defect  which  precludes  anti- 
body responses  to  several  polysaccharide  antigens  and  results  in  decreased 
numbers  of  I gM  secreting  cells  with  a  parallel  decrease  in  levels  of  serum 
IgM.  This  genetic  defect  is  most  dramatic  in  (CBA/N  X  NZB)F.  male  mice, 
which  express  the  defect  fully,  despite  unusually  high  IgM  levels  seen  in  the 
paternal  NZB  strain.   Recent  work  has  shown  that  this  defect  can  also  regu- 
late respones  to  autologous  erythrocyte  antigens  as  assayed  on  bromalein- 
treated  isologous  RBC,  but  that  this  response  can  be  largely  regained  in 
older  mice.  The  results  suggest  that  the  defective  mice  suffer  from  low 
frequencies  of  B  cell  precursors  specific  for  certain  antigens  rather  than 
the  complete  absence  of  B-cell  population.   (Dr.  Rosenberg) 

H.   Immunoglobulin  and  Antibody  Synthesis  Following  Intratracheal  Sti- 
mulation with  Antigen 

Sheep  red  blood  cells  or  SSS-III  polysaccharide  were  administered  to  BALB/c 
mice  by  intratracheal  intubation.   Systemic  antibody  responses  were  almost 
absent  in  animals  immunized  by  this  means,  as  demonstrated  by  absent  or  very 
low  specific  plaque- forming  cells  (PFC)  in  spleens.   Draining  (bronchial) 
lymph  nodes  showed  substantial  PFC  responses  between  days  four  and  twelve  of 
immunization  similar  in  magnitude  to  PFC  responses  in  spleen  after  systemic 
immunization.   Antibody  belonging  to  all  classes  (isotypes)  was  seen,  without 
significant  preference  for  I gA  as  opposed  to  IgG.   (Drs.  Munoz  and  Asofsky) 


Publ i  cations : 

Kim,  K.  J.,  Weinbaum,  F.  I.,  and  Asofsky,  R. :   Functional  characteristics  of 
BALB/c  T  cell  lines:  Suppression  in  the  mixed  lymphocytes  response  and  the 
cell  mediated  lysis.   J.  Immunol.  121:  2299-2304 ,  1978. 

Kim,  K.  J.,  Kane  1 lopoulos-Langevi n ,  C. ,  Merwin,  R. ,  Sachs,  D.  ,  and  Asofsky, 
R. :   Establishment  and  characterization  of  BALB/c  lymphoma  lines  with  B  cell 
properties.   J.  Immunol.   122:  549-554,  1978. 


24-11 


Project  No.  Z01  Al  001 3^- 1 7  LM I 

Laskov,  R.,  Kim,K.  J.,  and  Asofsky,  R.:   Induction  of  amplified  synthesis 
and  secretion  of  IgM  by  fusion  of  murine  B-lymphoma  with  myeloma  cells. 
Proc  Natl  .  Acad.  Sci.  76:  915-919,  1978. 

Laskov,  R.,  Kim,  K.  J.,  and  Asofsky,  R. :   Induction  of  IgM  secretion  by  fusing 
murine  B-lymphoma  with  myeloma  cells.   In  Melchers,  F.,  Potter,  M.,  and  Warner, 
N.  L.  (Eds):  Current  Topics  in  Microbiology  and  Immunology.  Spr inger-Verlag, 
1978,  pp.  173-175. 

Aksamit,  R.  R. ,  and  Kim,  K.  J.:  Macrophage  cell  lines  produce  a  cytotoxin. 
J.  Immunol.  122:  1785-1788,  1979- 

Levy,  R.  B, ,  Shearer,  G.  M.,  Kim,  K.  J.,  and  Asofsky,  R. :   Xenogenic  serum- 
induced  murine  cytotoxic  cells:   I.  Importance  of  the  serum  source  in  the 
generation  of  primary  cytotoxic  effectors  without  the  addition  of  stimulation 
cells.   Cel lular  Immunol .   (In  press)  1 979 - 

Schmidt,  T.  M.,  Kim,  K.  J.  and  Thompson,  E.  G.:   Glucocorticoid  sensitivity 
and  receptors  in  BALB/c  T  cell  lymphoma  lines  expressing  restricted  patterns 
of  Ly  differentiation  antigens.   J.  Steroid  Biochemistry.   (in  press)  1979- 

McKeever,  P.  E. ,  Kim,  K.  J.,  Nero,  G.  B.,  Laskov,  R.,  Merwin,  R.  M.,  Logan, 
W.  J.,  and  Asofsky,  R. :   Two  spontaneous  BALB/c  lymphomas  synthesize  IgM; 
U9L„  and  uL  molecules  are  isolated  and  characterized  while  another  molecule 
resembles  IgD.   J.  Immunol.  122:  1261-1265,  1979- 

McKeever,  P.  E.,  Neiders,  M.E.,  Nero,  G.  B.,  and  Asofsky,  R. :  Murine  plasma 

cell  tumors  secreting  more  than  one  class  of  immunoglobulin.  VI.  Secretion  of 

completely  assembled  lgG2b  and  IgA  molecules  with  segregated  heavy  chains 

and  free  light  chains  by  spontaneous  myeloma  SAMM  368  in  culture.    J . 
Immunol .  122:  1972-1977,  1979- 

Rosenberg,  Y.  J.  and  Chiller,  J.  M.  :  The  ability  of  antigen-specific  helper 
cells  to  effect  a  class-specific  increase  in  total  Ig  secreting  cells  in 
spleens  after  immunization  with  the  antigens.  J .  Exp.  Med . .  (In  press)  1 979  - 


24-12 


SMITHSONIAN  SCIENCE 
PROJECT  NUMBER  (Do  NOT  us 


NFORMATION  EXCHANGE 
this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  Al  00135-05  LMI 


PERIOD    COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Properties  of  Immunoglobulin  Secreting  Cells 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Senior  Investigator  LMI/NIAID 

Laboratory  Chief         <  LMI/NIAID 

Biologist  LMI/NIAID 

Head,  I mmunochemi stry  Sect.  LCBGY/NCI 


Senior  Investigator  LI/NIAID 


PI  : 

H. 

C.  Morse  1 1 1 

Other: 

R. 

Asofsky 

J. 

H.  Goode 

M. 

Potter 

R. 

Riblet* 

M. 

Weigert 

0. 

Makela5-" 

R. 

Lieberman 

B. 

T. 

cooperating  units  (if  any)  *|nsti;tute  for  Cancer  Research,  Fox  Chase,  Philadelphia,  PA 
-"University  of  Helsinki,  Helsinki,  Finland 
"""Albert  Einstein  College  of  Medicine,  Bronx,  N.Y. 
'--University  of  Alberta.  Alberta,  Canada 


LAB/BRANCH 

Laboratory  of  Microbial  Immunity 


SECTION 

Experimental  Pathology  Section 


INSTITUTE  AND  LOCATION 

N (Al D,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

7/12 


PROFESSIONAL: 

3/12 


4/12 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   Q  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

A  large  series  of  NZB  plasmacytomas  was  studied  to  determine  the  classes  of 
immunoglobul in  produced.  The  time  course  for  plasmacytoma  development  and  the 
frequency  of  immunoglobulin  classes  produced  differed  significantly  from  tumors 
produced  in  BALB/c  mice.   Unique  immunoglobulins  carrying  markers  of  both 
lgG3  and  I gA  immunoglobulin  classes  were  detected. 

SAMM  368  is  a  BALB/c  plasmacytoma  secreting  two  classes  of  immunoglobulin  -  lgG2b 
and  I gA  -  which  do  not  share  idiotypes.   The  lgG2b  molecule  does  not  express  the 
appropriate  al lotype  markers  for  the  class.   Studies  of  this  protein  suggest  tha 
allotype  markers  for  lgG2b  are  expressed  by  the  CH,  domain. 


2*4-13 


PHS-6040 
(Rev.  10-76) 


Project  No.  Z01  Al  00135-05   LM I 


Project  Description: 


NZB  mice  were  induced  to  form  plasmacytomas  by  the  intraperitoneal  injection 
of  pristane.   Mice  were  followed  for  the  time  course  of  plasmacytoma  develop- 
ment, the  immunoglobulin  classes  produced  by  each  tumor,  and  the  capacity  of 
these  immunoglobulins  to  react  with  selected  haptens. 

NZB  plasmacytomas,  as  compared  to  BALB/c  tumors  were  1)  found  to  require 
longer  to  develop  after  pristane  injection;  2)  express  a  much  higher  frequency 
of  IgG's  (igGl,  lgG2a,  lgG2b,  I gG3)  and  a  lower  frequency  of  IgA's;  and 
3)  produced  unique  molecules  never  found  in  BALB/c,  which  have  characteristics 
of  both  I gA  and  I gG3  immunoglobulins.   Peptide  maps  of  the  heavy  chains  from 
these  lgA-lgG3  "doubles"  suggest  that  there  may  be  peptides  unique  to  this  set 
of  paraproteins  which  are  otherwise  very  much  like  IgA's. 

The  NZB  paraproteins  as  well  as  some  from  BALB/c  mice  were  tested  for  their 
capacity  to  bind  a  series  of  haptens.  A  large  number  of  new  hapten-bindi ng 
immunoglobulins  were  detected. 

A  BALB/c  plasmacytoma,  SAMM  368,  produces  an  I gG2b  molecule  which  lacks  appro- 
priate allotypic  markers  for  its  class  of  immunoglobulin.  Studies  of  other 
myeloma  variants  lacking  the  CH3  domain  of  lgG2b  or  with  a  substitution  of  the 
CHo  of  lgG2b  with  that  of  I gG2a  indicate  that  the  CH3  domain  carries  the  I gG2b 
allotype  markers.  This  portion  of  the  SAMM  368  heavy  chain  is  being  sequenced 
along  with  a  normal  I gG2b  heavy  chain  to  determine  the  structural  basis  for 
allotype  expression. 


Pub  1  i  cations : 

Morse  III,  H.  C.  ,  Riblet,  R.  ,  Asofsky,  R. ,  andWeigert,  M:  Plasmacytomas 
of   the   NZB   mouse.      J.     Immunol.,    121:    1969-1972,    1978. 

Makela,  0.,  Kaartinen,  M. ,  Karjalainen,  K. ,  Morse  111,  H.  C.,  Weigert,  M. 
and  Potter,  M. :  A  search  for  hapten-b inding  mouse  plasmacytoma  proteins. 
Eur.    J.    Immunol.,      3:    125-129,    1979- 


2k-]k 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl -Al -00136-07  LMI 


PERIOD  COVERED 

October  1.  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Functional  Activities  of  Subpopulat ions  of  Thymus-Der i ved  Cells 


NAMES,    LABORATORY    AND    INSTITUTE   AFFILIATIONS,    AND    TITLES   OF    PRINCIPAL    INVESTIGATORS 
PROFESSIONAL   g^NNE^AfJ^ON   THE   PROJECT  &taff    f&]  ^ 

OTHER:    R.  Asofsky  Laboratory  Chief 

S.O.  S harrow 
I .  Betel* 
M.  Mage 
K.  Bottomly** 

B.  J.  Fowlkes 

C.  Kanel lopoulos-Langevin 
M.  Potter 
M.  Timmons 
L .  Mo  r  row 
F-W  Shen*** 
E.  A.  Boyse*** 


Chemi  st 

Senior  Investigator 

Microbiolog  ist 

Vis  i  ting  Fel low 

Head,  Immunochemi stry  Seel 

Co-op  Student 

Student 


AND    ALL   OTHER 

LMI/NIAID 
LMI/NIAIO 
IB/NCI 

LB/NCI 

LMI/NIAID 
LMI/NIAID 
LCBGY/NCI 
LMI/NIAID 
LMI/NIAID 


COOPERATING  UNJTS   if  any   ,  ,  ,   _        .   _,.. 

»  REP- Inst i tutes  of  the  Organization  for  Health  Research  TNO  - 

The  Netherlands.  _, 

**  The  Institute  for  Cancer  Research,  Fox  Chase  Cancer  Center, 

***Memorial  Sloan  Kettering  Cancer  Center,  1275  York  AVe. ,  N.Y.  New  York  10021 


R  i  j  sw  i  j  k , 
Philadelphia, Pa. 


lab/branch 

Laboratory  of  Microbial  Immunity 


SECTION 

Experimental  Pathology  Section 


INSTITUTE  AND  LOCATION 

Nl AID,  NIH,  Bethesda,  Maryland   20205 


TOTAL  MANYEARS: 

23/12 


PROFESSIONAL: 

13/12 


10/12 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   fj  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

To  examine  subpopulat  ions  of  thymocytes ,  we  have  extended  the  use  of  flow  micro 
fluorometry  (FMF)  analysis  to  characterize  eel  1  surface  and  funct  ional  parameters 


of  peanut  lectin  agglutinated  cells.   In  addition  to  analysis  for  known  antigenic 
markers,  e.g.  the  Lyt  antigens,  we  have  extended  the  analysis  of  these  cells 
using  fluorescent  lectin  probes.   We  have  currently  undertaken  analyses  of  the 
functional  differentiation  of  these  thymocyte  subpopulat ions  for  eel  1 -mediated 
cytotoxicity  and  antigen,  mitogen,  or  monokine  induced  prol iferat ion ,  to 
assess  the  extent  of  functional  differentiation  determined  by  the  thymus. 

A  contaminant  antibody  detected  by  FMF  in  the  antisera  from  several  non-congenic 
Lyt  immunizations  has  been  examined  and  used  to  characterize  and  define  a  new 
lymphocyte  cell  surface  antigen  Ly  9  which  distinguishes  lymphocyte  populations 
from  all  other  tissues  and  cells.   Since  this  marker  is  expressed  on  lympho- 
cytic  precursors,  as  well  as  differentiated  lymphocytes,  and  not  on  erythrocytic 
precursors  or  differentiated  red  blood  cells,  it  may  prove  useful  for  the  study 
of  funct  ional  1 ineages  of  bone  marrow  precursors. 

PHS-6040  Zf=  I  5 

(Rev.  10-76) 


Project  No.  Z01 -Al -001 36-07  LMI 

Project  Description: 

I .   (A)   Expression  of  T-cell  Differentiation  Antigens  on  Normal  Thymocytes. 

Flow  microfluorometry  analysis  of  thymocyte  subpopulat ions  has  been 
extended  for  Lyt  antigens  and  other  cell  surface  differentiation  markers. 
Using  a  pre-enr ichment  of  thymocyte  subpopulat ions  with  peanut  lectin 
agglutination,  we  have  analyzed  the  possibility  that  cell  surface  differen- 
tiation seen  in  a  small  subset  of  cells  in  the  thymus  reflects  functional 
competence  of  the  cell  types  in  that  organ. 

Peanut  lectin  agglutination  allows  the  positive  enrichment  of  2  subsets 
of  thymocytes  with  different  Lyt  phenotypes.   Peanut  lectin  agglutinated 
(PNA_)  cells  are  nearly  all  Lyt  123  ,  high  Thy-1,  while  non-agglutinated 
(PNA  )  cells  are  highly  enriched  (to  about  60%)  for  low  Thy-1,  Lyt  1  23" 
thymocytes.   Cells  with  this  latter  phenotype  constitute  only  about  10% 
of  normal  thymocytes.  PNA  and  PNA  thymocytes  have  been  analyzed  co- 
ordinately  for  Lyt  phenotype  and  cytotoxic  T  cell  function.   More  recently 
an  additional  cell  surface  differentiation  marker,  CTL,  expressed  on 
cytotoxic  lymphocytes  and  their  precursors  has  been  used  in  these  analyses. 

(1)  The  analysis  of  cytotoxic  T  lymphocyte  precursors  (CTLp)  has 
demonstrated  that  following:  (a)  Although  Lyt  1  23   cells  are  enriched  in 
the  PNA  fraction,  the  Lyt  123  cells  in  that  fraction  are  both  necessary 
and  sufficient  for  generation  of  cytotoxic  function,  (b)  There  is  a  subset 
of  Lyt  123   cells  which  are  differentiated  for  the  CTL  marker.   This  subset 
corresponds  to  about  6%   of  the  PNA  cells  or  about  0.6%  of  the  starting 
normal  thymocyte  population.   Detection  of  this  population  is  only  possible 
with  the  pre-enr i chment  procedure  and  indicates  the  presence  of  a  differ- 
entiation sequence,  wherein  an  Lyt  123   thymocyte  acquires  the  CTL  marker  and 
functional  competence. 

(2)  Tn  col laborat Ton  with  Dr.  J.  Oppenheim's  laboratory,  NIDR,  using 
human  monocyte-der ived  lymphocyte  activation  factors,  (LAF),  we  have  analyzed 
the  differential  effect  of  isolated  factors  on  the  Lyt  phenotype  and  on  in- 
duced proliferative  activity  of  thymocytes,  cortisone  resistant  thymocytes 
and  PNA  and  PNA  thymocyte  subpopulat ions.   These  analyses  have  demon- 
strated that  the  conventional  lymphocyte  activating  factor  (LAF)  of  1 ^ ,000 
daltons,  which  is  highly  active  for  stimulation  of  thymocyte  proliferation, 
does  not  cause  a  phenotypic  shift  in  Lyt  antigens.   However,  a  higher  mole- 
cular weight  factor  (>60,000  daltons),  isolated  from  human  monocyte,  LPS 
stimulated  cultures,  not  only  induces  proliferation  in  thymocytes,  but  also 
causes  a  quantitative  increase  in  the  expression  of  Lyt  1  antigen  on  the  cell 
surface  of  thymocytes  exposed  to  the  factor  in  vi  tro.   This  increase  in  Lyt 

1  expression  is  seen  at  12-18  hr,  prior  to  the  blastogenic  increase  in  cell 
size.   This  phenotypic  shift  also  precedes  the  mitogenic  activation  by  the 
factor  which  is  measured  by  tritiated  thymidine  uptake  at  48  hr.  Both  PNA 
and  PNA  cells  display  this  increase  of  Lyt  1  antigen  in  response  to  the 
high  molecular  weight  LAF,  thus  indicating  no  correlative  parallel  selective 
activity  on  fractionated  thymocyte  subpopulat ions . 

24-16 


Project  No.  Z01 -Al -001 36-07  LM I 

(3)   As  an  alternative  to  antibody  probes  to  cell  antigens,  we  are  cur- 
rently examining  by  FMF  a  number  of  plant  and  animal  lectins  capable  of  re- 
cognizing complex  sugar  moieties  on  glycoprotein  or  glycol ipid  structures  of 
the  cell  surface.   Following  our  successful  application  of  peanut  lectin 
fractionation  to  enrich  minor  subpopulations,  we  intend  to  expand  to  the  use 
of  several  other  lectins.   We  are  attempting  to  further  identify  and  subdivide 
thymocyte  subpopulations,  for  which  there  is  evidence  by  physical  fractiona- 
tion, cell  surface  antigenic  characterization  or  functional  heterogeneity. 
These  several  aspects  are  being  investigated  in  collaboration  with  Dr.  M.  J. 
Waxdal  (See  project  ZOI -Al -00038-07  Ll). 

1  I .   (A)   Genetic  Analysis  of  Lymphocyte  Differentiation  Markers 

A  new  lymphocyte  cell  surface  alloantigen,  provisionally  designated 
Ly  3,    is  detected  as  an  extra  specificity  by  flow  microfluorometry  (FMF)  in 
sera  from  anti-Lyt  immunizations.   Ly  9-2,  one  of  the  apparently  allelic 
specificities  commonly  is  detected  in  anti-Lyt  3.1  immunizations;  (C3H/HeN  X 
SJL/J)F.  anti-C58  normal  thymocytes.   The  alternative  antigen  expression, 
Ly  9.1,  can  be  detected  routinely  in  sera  from  two  other  Lyt  immunizations; 
C57BL/6-H-2  anti-CE/J  normal  thymocytes  (anti-Lyt  2.1),  and  C58  anti-CE 
normal  thymocytes  (anti-Lyt  3.2).   This  thymocyte  alloantigen  has  both  a 
unique  strain  distribution  and  a  unique  cell/tissue  distribution,  differing 
from  previously  reported  cell  surface  antigens.   Ly  9  is  expressed  on  all 
thymocytes,  lymphocytes  and  apparently  on  lymphocyte  and/or  white  cell  pre- 
cursors in  the  bone  marrow.   This  antigen  is  not  expressed  to  any  significant 
degree  on  erythrocytes,  epidermal  cells,  sperm,  testis,  brain,  kidney,  liver 
or  lung.   FMF  analysis  and  absorption  typing  reveals  a  quantitative  difference 
between  the  level  of  antigen  expression  on  cells  from  thymus  versus  spleen  or 
lymph  node.   Cytotoxic  elimination  experiments  confirm  that  Ly  9  is  expressed 
on  both  T  and  B  cells  and  on  at  least  two  different  T  cell  functional  subsets. 

(B)  We  have  continued  to  analyze  the  genetic  inter-relationships  of 
the  (NZBXC58)  recombinant  inbred  lines  for  lymphocyte  cell  surface  antigen 
markers . 

Publ icat ions: 


Mathieson,  B.  J.,  Sharrow,  S.  0.,  Campbell,  P.  S.,  and  Asofsky,  R. :   An  Lyt 
differentiated  subpopulat ion  of  thymocytes  detected  by  flow  microfluorometry. 
Nature  277=  ^+78-480,  1979- 

Betel,  I.,  Mathieson,  B.  J.,  and  Sharrow,  S.  0.:   Differential  agglutination 
of  thymocytes  by  peanut  agglutinin:   Phenotypic  charcter izat ion  of  the  sub- 
populations  by  flow  microfluorometry.  In  Petters,  H.,  (Ed):  Protides  of  the 
Biological  Fluids.  Pergammon  Press,  Oxford.   In  Press,  1979- 

Mathieson,  B.  J.,  Mage,  M.,  Betel,  I.,  and  Sharrow,  S.  0.:   Cytotoxic  lympho- 
cyte precursors:   Phenotypic  analysis  and  functional  activity  of  peanut  lectin 
fractionated  thymocytes.   In  Peeters,  H.,  (Ed):  Protides  of  the  Biological 
Fluids.   Pergammon  Press,  Oxford.   In  Press,  1 979  - 

2^-17 


Project  No.  Z01 -Al -001 36-07  LMI 

Riblet,  R.,  Claflin,  L.  ,  Gibson,  D.  M.,  Mathieson,  B.  J.,  and  Weigert,  M. : 
Antibody  gene  linkage  studies  in  (NZBXC58)  recombinant  inbred  lines.  J . 
Immunol .   (In  press)  1 979  - 

Bottomly,  K.,  Mathieson,  B.  J.,  and  Mosier,  D.  E.:   Ant i -idiotype  induced 
regulation  of  helper  cell  function  for  the  response  to  phosphroy lchol i ne 
in  adult  BALB/c  mice.   J.  Exp.  Med..   1 48 :  1216-1227,  1978. 

Bottomly,  K. ,  Mathieson,  B.  J.  Cosenza,  H.,  and  Mosier,  D.  E.:   Idiotype 
specific  regulation  of  the  response  to  phosphoryl chol i ne  by  T  cells  from 
mice  with  high  and  low  levels  of  circulating  idiotype.   In  Cooper,  M.,  Mosier. 
D.,  Sher,  I.,  and  Vitteta,  E.,  (Eds.),  B  Lymphocytes  in  the  Immune  Response. 
Elsevier  North-Holland,  New  York.  (In  press)  1978. 


24-18 


SMITHSONIAN'  SCIENCE  INFORMATION  EXCHANGE]     U.S.  DEPARTMENT  OF 
PROJECT  NUMBER  (Do  NOT  use  this  space)   (HEALTH,  EDUCATION,  AMD  WELFARE 

PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJc-jT  number 
ZOl    Al    00137-13   LMI 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Biology  of  Graft-Versus-Host  Reactions 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI :   R.  Asofsky 


Laboratory  Chief 


LMI/NIAID 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Microbial  Immunity 


;ect i on 
Experimental  Pathology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

0/12 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (a!)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

No  progress  was  made  this  year.   Studies  of  the  "allogeneic  effect"  -  see 
ZOl  Al  00145-12. 


24-19 


PHS-6040 
(Rev.    10-76) 


M I THSOH I  AN  SCIENCE  INFORMATION  EXCHAflGEi     U.S.  DEPARTMENT  OF 
PROJECT  NUMBER  (Do  NOT  u:c  this  space)   i|!EALTH,  EDUCATION,  AND  WELFARE 

PUBLIC  HEALTH  SERVICE 
KCTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


T7o7 


RCJECT  NUMBER 


ZOl  Al  00138-05   LMI 


PERIOO    COVERED 

October  1,  1978  to  September  30,  1979 


TITLE   OF    PROJECT    (60   ch-iracters    or   less) 

Viruses  and  the  Immune  Response 


NAMES,  LABORATORY  AMD  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AMD  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


Senior  Investigator  LMI/NIAID 

Senior  Investigator  LMI/NIAID 

Senior  Investigator  LVD/NIAID 

Biologist  IB/NCI 

Visiting  Fellow  LMI/NIAID 

Biologist  LMI/NIAID 

Biologist  LVD/NIAID 

NIH  Postdoctoral  Fellow  LMI/NIAID 


PI  : 

He 

rbert  C.  Morse 

Other: 

T. 

M. 

Chused 

J. 

W. 

Hartley 

S. 

0. 

S  h  a  r  row 

W. 

F. 

Davi  dson 

J. 

H. 

Goode 

N. 

K. 

Wo  1  ford 

B. 

A. 

Taylor- 

J. 

D. 

Longstreth 

COOPERATING  UNITS  (if  any) 


The  Jackson  Laboratory,  Bar  Harbor,  Maine 


lab/branch 

Laboratory  of  Microbial  Immunity 


Experimental  Pathology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

36/12 


PROFESSIONAL: 

28/12 


8/12 


CHECK  APPROPRIATE  B0X(E3) 

□  (a)   HUMAN  SUBJECTS 

□  (at)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


(c)  NEITHER 


SUMMARY  CF  WORK  (200  words  or  less  -  underline  keywords) 

Genetic  analyses  of  xenotropic  murine  leukemia  viruses  in  inbred  mouse  strains 
have  shown  that  these  viruses  may  be  expressed  as  infectious  virus  or  as  virus- 
coded  antigens  (gp7_ls  termed  XenCSA)  on  the  surface  of  normal  lymphocytes. 
NZB  mice  have  two  independently  segregating  dominant  loci  for  infectious  virus 
expression  and  apparently  one  coding  for  XenCSA  in  the  absence  of  infectious 
virus.   The  levels  of  virus  expression  as  infectious  virus  and  XenCSA  in  NZB 
lymphocytes  is  governed  by  genes  affecting  lymphocyte  differentiation.   In  genetic 
crosses  of  mice  exhibiting  high  (DBA/2)  and  low  (C57BL/6)  levels  of  XenCSA,  a 
single  semidominant  gene  on  chromosome  k   at  or  near  Fv- 1  has  the  predominant 
effect  on  XenCSA  levels.  i 


24-20 


PHS-6040 
(Rev.    10-76) 


Project  No.  Z01  Al  00138-05   LM I 


Project  Description: 


Several  strains  of  mice  are  being  studied  to  determine  genetic,  developmental 
and  environmental  factors  affecting  the  expression  of  xenotropic  murine 
leukemia  viruses  (X-MuLV)  as  infectious  virus  or  as  virus-coded  antigen 
(gp70)  in  the  presence  or  absence  of  infectious  virus.   Strains  producing 
high  levels  of  infectious  virus  include  NZB,  F,  and  BXD-14,  a  recombinant 
inbred  derived  from  low-virus  strains  C57BL/6  and  DBA/2. 

In  crosses  with  NFS,  NZB  mice  were  shown  to  have  two  unlinked  dominant  loci 
governing  expression  of  infectious  X-MuLV  detected  by  focus  formation  in  mink 
cells.   Expression  of  infectious  virus  in  NZB's  differs  in  various  lymphoid 
tissues:  high  levels  of  X-MuLV  were  found  in  bone  marrow,  somewhat  lower 
levels  in  spleen  and  lymph  node,  and  the  lowest  levels  in  thymus.   Expression 
of  infectious  MuLV  in  T  cells  is  influenced  by  genes  governing  their  differ- 
entiation since  T  cells  purified  from  spleen  produce  10-fold  more  infectious 
X-MuLV  than  do  thymocytes.   Furthermore  splenic  B  and  T  cells  produce  equi- 
valent amounts  of  infectious  virus. 

Expression  of  X-MuLV-coded  gp70,  termed  XenCSA,  on  the  surface  of  lymphocytes 
was  detected  by  flow  microfluorometry  using  fluorescein- labeled  rabbit  anti- 
bodies specific  for  X-MuLV  gp70.   A  survey  of  over  100  inbred  strains  for 
expression  of  XenCSA  on  their  thymocytes  and  spleen  cells  revealed  marked 
variations  among  these  strains,  but  three  basic  phenotypes  were  observed: 
some  strains  have  high  antigen  expression  in  spleen  and  thymus;  others  have 
low  expression  in  thymus  with  high  expression  in  spleen;  and  the  great 
majority  exhibit  low  antigen  expression  in  both  tissues. 

Genetic  crosses  between  a  strain  with  high  XenCSA  levels  (DBA/2)  and  one  with 
low  levels  (C57BL/6)  showed  that  XenCSA  levels  are  determined  by  a  semi- 
dominant  gene  on  chromosome  k    in  close  proximity  to  Fv- 1 .   Although  this 
locus  has  the  predominant  effect  on  XenCSA  levels,  other,  as  yet  undetermined 
factors  act  to  modify  the  level  of  expression. 

In  crosses  between  NZB  and  NFS,  it  was  shown  that  high  XenCSA  levels  corre- 
late with  expression  of  only  one  of  the  two  loci  producing  infectious  X-MuLV 
and  that  a  third  locus  may  code  for  XenCSA  expression  in  the  absence  of 
i  nfect  ious  vi  rus . 

Studies  now  in  progress  indicate  that  the  patterns  for  X-MuLV  expression 
(as  infectious  virus  or  XenCSA)  in  F/St  mice  do  not  follow  the  patterns 
establ ished  for  NZB's. 


24-21 


Project   No.    Z01    Al    00138-05      LM I 


Publ i  cat  ions 


Chused,  T.  M. ,  and  Morse  III,  H.  C. :   Expression  of  XenCSA,  a  cell  surface 
antigen  related  to  the  major  glycoprotein  (gp70)  of  xenotropic  murine  leukemia 
virus,  by  lymphocytes  of  inbred  mouse  strains.   In  Morse  III,  H.C.  (Ed.): 
Origins  of  Inbred  Mice.   New  York,  Academic  Press,  1978,  pp.  297 

Morse  III,  H.  C. ,  Chused,  T.  M. ,  Boehm-Trui tt ,  M. ,  Mathieson,  B.  J.,  Sharrow, 
S.  0.,  and  Hartley,  J.  W. :   XenCSA:  Cell  surface  antigens  related  to  the  major 
glycoproteins  (gp70)  of  xenotropic  murine  leukemia  viruses.   J .  I mmunol . 
122:  kkl-k5k,    1979. 

Morse  III,  H.  C,  Chused,  T.  M.  ,  Sharrow,  S.  0.,  and  Hartley,  J.  W.  :   Vari- 
ations in  expression  of  xenotropic  murine  leukemia  virus  genomes  in  lymphoid 
tissues  of  NZB  mice.   J.  Immunol.   122:  23^5-23^8,  1979 • 

Morse  III,  H.  C. ,  Chused,  T.  M. ,  Hartley,  J.  W. ,  Mathieson,  B.  J.,  Sharrow, 
S.  0.,  and  Taylor,  B.  A.:   Expression  of  xenotropic  murine  leukemia  viruses 
as  cell-surface  gp70  in  genetic  crosses  between  strains  DBA/2  and  C57BL/6. 
J.  Exp.  Med.  \k3:    1183-1196,  1979- 

Morse  III,  H.  C,  and  Chused,  T.  M.  :   Polymorphisms:  Wild  Mouse.   Part  II. 
Relationship  to  other  retroviruses.   In  Altman,  P.  L.  and  Katz,  D.  D.  (Eds.): 
Inbred  and  Genetically  Defined  Strains  of  Laboratory  Animals.   Part  I.  Mouse 
and  Rat.   Maryland,  Federation  of  American  Societies  for  Experimental  Biology, 
1979,  PP.  229-232. 


24-22 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Oo  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl -Al -001 39-1 4  LMI 


PERIOD    COVERED 

nr.tnhp.r    1,     1978    to    Sppfgmhpr    J.Q  t     197? 


TITLE   OF    PROJECT    (80   characters    or   less) 

The  Immunologic  Response  of  Animals  to  Trypanosoma!  Antigens 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

LMI /N I Al D 

LPD/NIAID 

LMI /N I  AID 
LMI  /(Ml  Al  D 

DRS/NIH 


PI: 

J. 

F.  Finerty 

Research  Microbiolog 

st 

OTHER: 

D. 

M.  Dwyer 

Research  Microbiolog 

st 

L. 

P.  Gasbarre* 

Post-doctoral  Fellow 

L. 

Kendri  ck 

Biologi  st 

R. 

McKel  vi  n*;- 

Student 

Y. 

Rosenberg 

Vi  s  i  t ing  Scient i  st 

E. 

Graduate  Student 

C. 

T.Hansen 

Geneticist 

COOPERATING  UNITS  (if,  any)  ,'_.._  ,.  . 

*   WHO  Immunology  Research  &  Training  Center,  Lausanne,  Switzerland 

**     Howard  University 

"""  Laboratory  Clinical  Immunology,  Med.  Univ.  of  S.  Carolina,  Charleston,  S.  C 


LAB/BRANCH 

Laboratory  of  Microbial  Immunity 


SECTION 

Microbiology  and  Immunology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland   20205 


TOTAL  MANYEARS: 

18/12 


PROFESSIONAL: 

10/12 


8/1? 


CHECK  APPROPRIATE  BOX(ES) 
Q  (a)   HUMAN  SUBJECTS 

□  (a1  )  MINORS   Q  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


S  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords' 


Mice  infected  with  various  species  of  African  trypanosomes ,  succumb 
23-120  days  after  infection.   Survival  depends  on  the  presence  of  IgM 
antibody .   Mice  with  delayed  IgM  responses  survive  the  longest.   The 
data  indicate  a  poss  ible  rol e  of  I gM  ant  ibody  as  a  blocking  agent. 


24-23 


PHS-6040 
(Rev.    10-76) 


Project  No.  Z01 -Al -001 39-1 k   LMI 


Project  Description: 


The  purpose  of  this  study  was  to  assay  the  host  immune  response  in  laboratory 
animals  immunized  or  infected  with  African  trypanosomes ,  the  causative  agents 
of  African  Sleeping  Sickness.   Previous  studies  indicated  that  animals  could 
be  actively  immunized  against  Trypanosoma  rhodesiense  and  that  this  protection 
was  T  cell  dependent.   The  present  course  of  investigation  is  focused  on  (l) 
isolation  of  the  antigen(s)  responsible  for  immunization  and  (2)  the  use 
of  immuno-def icient  animals  to  assay  the  humoral  response. 

Recently,  methods  were  developed  that  can  isolate  and  separate  surface  coat 
antigen,  cytoplasmic  organelles  e.g.  kinetoplasts  and  cell  membranes  from 
T.  rhodes  iense.   A  lysing  solution,  consisting  of  low  ionic  strength  salts 
plus  a  chelating  agent  was  developed  that  allows  the  isolation  of  the 
parasite  into  the  components  mentioned  above.   A  gradient  solution  combined 
with  centr i fugat ion  allows  these  components  to  be  separated  and  isolated. 
These  techniques  will  allow  not  only  the  immune  response  to  these  components 
to  be  analyzed  but  also  the  area  of  the  cell  biology  of  these  parasites   to 
be  analyzed.   Using  SDS  acrylamide  electrophoresis,  the  cell  membranes  were 
resolved  into  major  components  that  are  similar  to  results  obtained  with 
other  hemof lagelates.   After  completion  of  electron  photomicrography,  these 
results  will  be  submitted  for  publication.   This  is  the  first  time  that  mem- 
branes from  the  human  pathogen,  T.  rhodesiense  have  been  isolated. 

The  second  aspect  of  this  study  concerned  the  humoral  response  to  African 
trypanosomes  utilizing  naturally  occurring  immuno-def icient  animals  e.g. 
CBA/N  and  congenital ly  athymic  (nude)  mice.   Recently,  we  found  that  "normal" 
mice  i.e.  normal  immune  responders  succumbed  to  T.  rhodesiense  infections 
in  20-28  days  after  infection,   whereas  CBA/N  mice  survive  longer  than  50  days. 

Since  trypanosomes  stimulate  the  host  to  synthesize  large  amounts  of  IgM 
and  this  was  suggested  to  interfere  with  a  protective  host  immune  response 
we  decided  to  study  the  effects  of  trypansomes  in  mice  with  a  naturally 
occurring  IgM  antibody  deficiency  (T- i ndependent)  to  polysaccharide  antigens, 
and  compare  them  to  normal  mice.   Thus,   CBA/N  (IgM  "deficient")  were  compared 
to  CBA/CaJ  (normal)  mice  upon  infection  with  T.  rhodesiense.   The  most  ob- 
vious result  was  that  CAB/CaJ  mice  and  a  mean  survival  of  23  ±  2  days,  whereas 
CBA/N  mice  had  a  mean  survival  time  of  62  ±  8  days  after  infection.   Immuno- 
globulin (Ig)  levels  were  quantitated  in  both  strains  of  mice  throughout 
infection.   A  characteristic  pattern  observed  in  CBA/CaJ  mice  was  a  rise  in 
all  Ig  isotype  levels,  followed  by  a  rapid  decrease  within  16  days  after 
infection.   CBA/N  mice  revealed  similar  Ig  responses  without  the  marked 
decrease  observed  in  CBA/CaJ  mice.   The  biggest  difference  was  observed  in 
the  IgM  antibody  responses.   IgM  antibody  was  detected  in  CBA/CaJ  mice  during 
the  2  weeks  following  infection,  and  then  became  undetectable.   In  contrast, 
IgM  antibody  was  undetectable  in  CBA/N  mice  for  ]k   days  after  infection,  and 
then  was  detected  on  day  16,  and  thereafter  .  Congen i c  B6:CBA/N  mice  revealed 
a  similar  finding.   The  data  suggest  that  a  delay  in  the  synthesis  of  IgM 
antibody  is  beneficial  to  hoast  survival.   Whether  this  reflects  a  delay  in 
IgM  blocking  antibody  to  the  trypansomes  or  a  delay  in  other  IgM  antibody 
responses  e.g.  "autoimmune"  antibody  is  under  investigation. 

Publications:  None  24-2^ 


(SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
iFROJECT  NUMBER  (Do  NOT  use  this  space) 


U.J.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl -A I -00 140-1 4  LMI 


PERIOD    COVERED 

|     October  1.  1978  to  September  30,  1979 


OF  PROJECT  (SO  characters  or  lessj 

Immunology  of  Malaria 


•JAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AMD  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI  : 

J. 

F.  Finerty 

OTHER: 

L. 

Kendr  ick 

R. 

McKel vin 

W. 

P.  Wei  dan z 

R. 

Rank*- 

D. 

Roberts-"'"* 

A. 

Tagl iabue 

Research  Microbiologist  LMI/NIAID 

Biologist  LMI/NIAID 

Student  LMI/NIAID 

Professor  LMI/NIAID 

Assistant  Professor  LMI/NIAID 

Research  Microbiologist  LMI/NIAID 

Visiting  Scientist  LIB/NCI 


COOPERATING  UNITS  (if  a;/; 

••The  Hahneman  Medical  College,  Phil.,  Pa. 
j  **Univers i ty  of  Arkansas  for  Medical  Sciences,  Little  Rock,  Arkansas 
'-•----National  Toxicologic  Research  Center,  Little  Rock,  Arkansas 
j lab/branch 

Laboratory  of  Microbial  Immunity 


SECTION 

Microbiology  and  Immunology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20014 


'TOTAL  MAN YEA 

8/12 


PROFESSIONAL: 

4/12 


4/12 


CHECK   APPROPRIATE   SOx(ES) 
JG  (a)      HUMAN    SUBJECTS 


n  (b)    HUMAN    TISSUES 


XjX(c)    NEITHER 


IG   (a1  j    .MINORS 


(a2)    INTERVIEWS 


I  JUMMAR"    OF    j/ORK    (200    *orae    or    less    -   underline    keywcr 


Malarial  antigen  was  used  to  both  induce  and  elicit  delayed  hypersensitivity 
(DH)  reactions  in  various  strains  of  mice.   Protection  was  correlated  with 


DH  in  the  early  aspect  of  the  immune  response. 
be  related  to  macrophage  activity. 


This  correlation  appears  to 


24-25 


phs-o040 

(Rev.  ! O-76 ) 


Project  No.  Z01 -Al-00140-14  LMI 
Project  Description: 

The  purpose  of  these  studies  is  to  assay  the  host  immune  response  to  rodent 
malarial  parasites  Plasmodium  berghei ,  P.  vinckei  and  P ■  yoel i  i .   Previous 
studies  demonstrated  that  T-cells  were  a  necessary  component  in  protection 
against  lethal  and  non-lethal  strains  of  rodent  malaria.   This  was  manifest 
in  vivo  by  delayed-hypersensi t ivi ty  reactions  (DH)  in  mice  by  the  footpad 
swelling  technique.   Swiss  mice  showed  greater  DH  to  malarial  antigens  than 
inbred  strains  of  mice.   Further,  P. yoel i  i  is  a  resolving  infection  in 
Swiss  mice,  but  most  inbred  strains  of  mice  become,  or  are  susceptible  to 
this  parasite.   This  is  particularly  evident  with  BALB/c  and  C,H  mice.   The 
latter  were  particularly  susceptible  to  the  non-lethal  P.  yoel i i ;  death 
occurred  J-]k   days  after  infection.   Since  the  parasitemia  reached  >80%, 
this  suggested  that  the  organism  changed  when  inoculated  into  C-H  mice. 
Parasitized  C,H  blood  was  subsequently  injected  into  Swiss  mice  and  para- 
sitemia was  monitored.   The  parasites  never  exceeded  15%,  same  as  the 
regular  strain  of  P.  yoel i  i ,  and  no  deaths  occurred.   This  data  suggested 
an  inherent  defect  in  the  C  H  mice  that  made  them  unable  to  clear  the 
parasites.   DH  responses  to  malarial  antigen  was  less  in  C,H  mice  than  in 
Swiss  mice.   Macrophages  are  a  necessary  component  in  the  host's  ability 
to  clear  malarial  parasites  plus  they  are  vital  in  DH  responses.   Pre- 
liminary results  indicated  that  the  responsiveness  of  C  H  macrophages  to 
malarial  parasites  plays  an  important  role  in  C,H  survival. 

Futher  studies  will  investigate  and  delineate  the  role  of  macrophages  in 
resistance  to  malaria. 

Publ icat ions : 


Finerty,  J.  F.,  Kendrick,  L.  P.  and  McKelvin,  R.:   Chemical  Enhancement  of 
Protective  Immunity.   In  Proceedings  of  a  Food  and  Drug  Administration  Con- 
ference, Inadvertant  Modification  of  the  Immune  Response,  Washington,  D.  C, 
in  press,  1 979 - 


24-26 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  ^Oo  NOT  use  this  space; 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJt'  T  NUMBER 


ZOl   Al  00141-05  LMI 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (60  characters  or  less) 

Immune   Responses   to  Malaria  and   Related    Intracellular  Protozoa 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI  : 

D. 

W.   Taylor 

R. 

Asofsky 

Other: 

Y. 

Rosenberg 

K. 

J.    Kim 

P. 

E.    McKeever 

C. 

B.    Evans 

J. 

Rodri  gues-Ramos 

Staff  Fel low 
Laboratory  Chief 
Vis  i  ting  Fel low 
Visiting  Associate 
Research  Associate 
Biological  Lab  Tech 
MBS  Student 


LMI /Nl  Al  D 
LMI /Nl  Al  D 

LMI/NIAID 
LMI /N I Al D 

LMI/NIAID 
LMI/NIAID 
LMI/NIAID 


COOPERATING    UNITS    (if   any; 

NONE 


LAB/ BRANCH 

Laboratory  of  Microbial  Immunity 


Microbiology  and  Immunology  Section  /  Experimental  Pathology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryalnd  20205 


TOTAL  MANYEARS: 

32/12 


PROFESSIONAL: 

24/12 


8/12 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 

□(al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

A  biochemical  and  functional  approach  is  being  used  to  analyze  the  antigenic 
similarities  and  differences  between  the  17XL  and  17XNL  strains  of  Plasmodi  urn 
yoel i  i .   Over  125  malarial  polypeptides  have  been  identified  using  isoelectri  c 
focusing  and  SDS-PAGE.   It  appears  that  both  quantitative  and  qualitative 
differences  exist  between  the  two  strains.  To  date,  twelve  hybri  doma  cell  lines, 
which  secrete  ant iplasmodi al  antibodies  (Ab) ,  have  been  established.   Monoclonal 
Ab  are  .being  used  to  identify  and  isolate  important  antigens  of  the  erythro- 
cytic stages  of  the  parasite.   In  addition,  polypeptides  of  P .  yoel i  i  are  being 
separated  by  preparatory  electrophoresis  and  fractions  which  contain  antigens 
that  react  with  hyperimmune  serum  and  induce  an  in  vi  tro  proliferation  response 
of  sensitized,  but  not  normal,  lymphocytes  have  been  identified. 


24-27 


PHS-6040 
(Rev.    10-76) 


Project  No.   Z01  Al  00141-05   LMI 


Project  Description: 

A  study  has  recently  been  initiated  to  evaluate  the  role  of  malarial  antigens 
in  immune  protection  and  pathogenesis.   Two  strains  of  the  rodent  malaria 
parasite  Plasmodium  yoe 1 i i  are  being  investigated.   The  1 7XL  strain  routinely 
produces  a  fatal  infection  in  BALB/c  mice;  whereas  the  17XNL  strain  causes  a 
self- 1 imi ting  infection.   Mice  which  have  recovered  from  infection  with  the 
1 7XNL  strain  are  completely  protected  from  challenge  with  the  lethal  form. 
Four  basic  research  approaches  are  being  used  to  isolate  and  identify  pro- 
tective and/or  suppressive  malarial  antigens. 

A.  Biochemical  Analysis  of  Plasmodial  Polypeptides 

A  procedure  has  been  developed  for  the  isolation  of  intact  erythrocytic  stages 
of  P.  yoel i  i  which  results  in  a  preparation  of  parasites  relatively  free  of 
host  material.   Blood,  collected  from  BALB/c  mice  with  patent  1 7XL  or  1 7XNL 
P .  yoe 1 i 1  infections,  is  washed  free  of  serum  proteins  and  passed  through  a 
Whatman  CF1 1  column  to  remove  host  leukocytes.   Normal  and  parasitized 
erythrocytes  are    incubated  in  0.65%  NaCl  for  ten  minutes  resulting  in  swelling, 
but  not  lysis,  of  red  blood  cells  (RBC).   Parasites  are  released  from  swollen 
erythrocytes  by  the  application  of  gentle  pressure  in  a  French  pressure  cell. 
Freed  parasites  and  RBC  ghosts  are  layered  onto  a  discontinuous  metrizamide 
gradient  and  centri fugated.   Four  distinct  bands  are  produced.   Fractions  con- 
taining rings  +  merozoites,  mature  trophozoites  and  schi zont-segmenters  are 
collected  washed  and  solubilized  in  the  non-ionic  detergent  NP40.   Such  pre- 
parations provide  the  starting  material  for  biochemical  and  immunologic 
(Section  C)  studies. 

The  technique  of  SDS-polyacry lamide  gel  electrophoresis  (SDS-PAGE)  was  used 
to  analyze  the  above  preparations  of  17SL  and  1 7XNL  polypeptides.   Gradient 
gels  stained  with  Coomassie  Blue  revealed  8-10  bands  (m.w.  25,000-300,000) 
with  both  quantitative  and  qualitative  differences  between  the  two  strains. 
A  new  protein  silver  staining  technique  (100  times  more  sensitive  than 
Coomassie  Blue)  has  been  used  on  reduced  and  non-reduced  samples  following 
SDS-PAGE  resulting  in  the  identification  of  30-35  distinct  bands.   When 
parasite  preparations  were  analyzed  using  2-dimensi onal  electrophoresis  (pro- 
teins separated  in  the  first  dimension  by  isoelectric  point  differences  using 
isoelectric  focusing  and  by  molecular  weight  differences  in  the  second 
dimension  using  SDS-PAGE)  at  least  125  polypeptides  were  identified.  Currently 
proteins  of  the  I7XL  and  1 7XNL  strains  and  various  stages  of  P.  yoel i  i  are 
being  analyzed  in  this  way.   (Drs.  Taylor,  McKeever,  Mr.  Evans,  and  Mr. 
Rodriques-Ramos  (student)). 

B.  Use  of  Monoclonal  Antibody  for  Identification,  Characterization 
and  Isolation  of  Malarial  Antigens 

During  the  last  five  months  a  number  of  hybridomas  have  been  produced  which 
secrete  ant i -malari al  antibody  (Ab) .   These  hybridomas  were  produced  by 
fusing  non-immunoglobul i n  secreting  BALB/c  myeloma  cells  (P3_X63_NS I /l )  with 

24-28 


Project  No.   Z01  Al  00141-05   LMI 

BALB/c  spleen  cells  removed  from  mice  infected  with  1 7XL  or  17  XNL  P.  yoel i  i 
four  days  prior  to  fusion.   Fusions  using  spleen  cells  from  hyperimmune  mice 
(completely  protected  from  challenge)  which  had  been  boosted  four  days  earlier 
wi  th  P.  yoel i  i  have  also  been  made.   Following  fusion,  cells  were  cultured 
in  microti ter  wells  and  culture  supernatants  were  screened  for  anti-plasmodial 
Ab  using  a  solid  phase  radioimmunoassay  (RIA).  The  assay  consisted  of  binding 
freed  parasites  plus  some  RBC  ghosts  (see  Section  A)  to  poly-L- lysine  coated 
polyvinyl  u-shaped  microtiter  wells,  blocking  of  excess  charges  with  bovine 
serum  albumin,  applying  test  culture  supernatants,  and  developing  with  an 
l2-M  -polyvalent  goat  anti-mouse  Ig.   The  isotype  of  the  monoclonal  anti-plasmo- 
dial Ab  was  determined  using  iodinated  monospecific  reagents.   Positive  hybrids 
were  cloned  and  injected  intraperi toneal ly  into  pri stane-pr imed  mice.  Currently 
12  hybridomas  (seven  from  17XL,  four  from  17XNL,  and  one  from  hyperimmune 
fusions)  are  being  maintained  and  it  is  hoped  they  will  prove  to  be  stable 
eel  1  1 ines. 

Monoclonal  Ab  produced  by  eight  of  the  hybridomas  have  been  studied  in  detail. 
Three  are  secreting  lgG„  and  the  other  five,  IgM.   One  of  the  Ab  (M4)  binds 
to  antigens  (Ags)  on  the  surface  of  freed  parasites  but  the  remainder  recog- 
nize internalized  Ags.   All  12  of  the  monoclonal  Abs  bind  to  Ags  common  to 
all  erythrocytic  stages  of  the  parasite  (i.e.  rings,  trophozoites  and  mero- 
zoites),  and  thus  do  not  demonstrate  the  presence  of  stage  specific  Ags.   One 
of  the  four  monoclonal  hybrids  (NLA10)  produced  by  fusion  with  17XNL  spleen 
cells  produces  Ab  which  seems  to  recognize  an  Ag  unique  to  the  17XNL  strain 
(determined  by  indirect  fluorescent  Ab  ( I  FA)  staining  and  RIA  analysis).   All 
other  hybridoma  Ab  appear  to  bind  equally  to  both  strains.   An  I  FA  study  showed 
that  some  of  the  hybridoma  Abs  recognized  only  P .  yoel i  i  Ags,  while  some  re- 
acted with  Ags  shared  among  P .  yoel i  i ,  P.  berghei  and  Babes  ia  mi  crot i . 

Monoclonal  Ab  are  currently  being  used  to  isolate  parasite  antigens  employing 
the  techniques  of  immune  precipitation  and  affinity  chromatography.  isolated 
antigens  will  be  biochemically  analyzed.   (Drs.  Kim,  Taylor,  and  Mr.  Evans). 

C.   Isolation  and  Identification  of  Immunologically  Relevant  Malarial 
Anti  gens 

Preparations  of  parasite  proteins  are  being  fractionated  by  agarose  prepara- 
tory electrophoresis  (PE)  and  DE52  chromatography.   Fractions  are  tested  for 
the  presence  of  malarial  Ags  by  Ouchterlony  analysis  using  hyperimmune  BALB/c 
serum  and  for  the  ability  to  transform  immune  lymphocytes  by  an  Ag- induced 
proliferation  assay.   PE  fractions  5"8  and  the  third  peak  eluted  from  DE52 
columns  (0.05M  buffer)  have  been  shown  to  contain  parasite  antigens.   Further 
biochemical  fractionation  of  malarial  polypeptides  will  be  undertaken  in  the 
coming  year  and  immunologically  relevant  Ags  will  be  analyzed  biochemically. 
(Dr.  Taylor  and  Mr.  Evans) 


24-29 


Project  No.   ZO 1  Al  00141-05   LMI 


D.   Identification  of  Antigens  Associated  with  Autoimmune  Hemolytic 
Anaemia 

Inoculation  of  irradiated  parasites  (17XL  strain)  into  BALB/c  mice  results  in 
the  induction  of  plasma  cells  secreting  Ab  against  bromelain-treated  normal 
mouse  red  blood  cells  (BrMRBC) ,  an  autoimmune  phenomena.   This  observation 
suggests  that  the  presence  of  parasite  Ags  or  antigenic  determinants  on  para- 
sitized RBC  is  sufficient  to  produce  an  autoimmune  response.   Active  infection 
is  not  required. 

The  anti-BrMRBC  response  also  appears  to  correlate  with  lethality  in  that 
1 7XL  infections  induce  a  specific  anti-BrMRBC  plaque  forming  cell  (PFC)  re- 
sponse peaking  at  day  four,  while  the  PFC  response  seen  during  the  17XNL 
infection  occurs  later  and  parallels  the  non-spcific  B  cell  activation  seen 
for  the  total  Ig  secreting  cell  populations.   Serial  passage  of  the  17XNL 
strain  in  BALB/c  mice  frequently  results  in  the  conversion  of  the  parasite  to 
the  lethal  form.   The  anti-BrMRBC  response  of  the  1 7XNL  strain  closely  approach- 
es the  1 7XL  kinetics  with  successive  passages,  thus  probably  reflecting  anti- 
genic changes  in  the  parasite  before  increases  in  parasitemia  and  lethality 
are  observed  in  the  host. 

Since  1 7XL  and  1 7XNL  strains  infect  mature  RBC  and  reticulocytes  respectively, 
it  was  investigated  whether  the  cell  type  involved  contributed  to  the 
different  anti-BrMRBC  response  kinetics  in  the  two  infections.   PFC  assays 
using  bromelain-treated  reticulocytes  or  mature  erythrocytes  showed  that  anti- 
BrMRBC  antibodies  are  predominantly  directed  to  antigens  on  mature  RBC  indi- 
cating that  only  neoantigens  expressed  on  the  mature  cells  induce  such  a 
response  and  partially  explain  why  specific  anti-BrMRBC  responses  are  not 
seen  during  stable  1 7XNL  infections.   Chemical  characterization  of  such  neo- 
antigens are  being  undertaken  and  may  yield  information  as  to  whether  anti- 
BrMRBC  Abs  contribute  to  the  anaemia  or  lethality  which  occurs  following 
infection  with  the  1 7XL  strain.   (Dr.  Rosenberg  and  Mr.  Evans) 

In  addition  to  the  above  studies,  several  other  areas  of  investigation  are 
currently  being  undertaken  to  elucidate  the  mechanisms  of  immunity  to  intra- 
cellular protozoans. 

Malarial  Induced  Immunosuppression 

Immunization  of  mice,  infected  8-11  days  previously  with  17XNL,  with  the 
antigens  SRBC  or  TNP-ficoll  induces  no  antibodies  specific  for  these  antigens. 
Such  suppression  has  been  shown  to  require  T  lymphocytes  and  macrophages. 
These  experiments  were  repeated  in  malaria  immune  mice  to  study  some  of  the 
conditions  required  to  generate  suppression.   In  such  immune  mice  reinfection 
with  malaria,  subsequent  immunization  with  SRBC  yielded  no  suppression  despite 
marked  activation  of  T  and  B  cells  showing  that  activation  of  macrophages  by 
large  numbers  of  parasites  is  important  in  generating  suppression  and  delin- 
eating the  mechanisms  responsible  for  polyclonal  B  cell  activation  from  those 
causing  suppression.   (Dr.  Rosenberg) 

24-30 


Project  No.   ZO 1  Al  00141-05   LM I 

Studies  on  Resistance  to  Babesia  Microti  Infections 

In  experiments  aimed  to  determine  the  role  of  T  and  B  lymphocytes  in  infec- 
tions with  B.  mi  croti ,  mice  suppressed  for  I gM  production  or  control  mice 
infected  with  this  parasite  and  parasi taemi as  were  followed  both  after  primary 
infection  and  challenge.   Such  suppressed  mice  showed  an  unexpected  resistance 
to  B.  microti  infections  compared  to  control  mice  and  in  addition  were  fully 
protected  to  reinfection.   These  studies  suggest  a  role  for  I gM  antibody  in 
the  invasion  of  erythrocytes  by  the  babesial  organism  and  may  yield  important 
information  on  mechanisms  of  invasion  by  intracellular  parasites  in  general. 
(Dr.  Rosenberg  and  Mr.  Evans) 

Publ i  cat  ions : 

Weinbaum,  F.  I.,  Weintraub,  J.,  Nkrumah,  F.  K. ,  Evans,  C.  B.,  Tigelaar,  R.  E. , 
and  Rosenberg,  Y.  J.:   Immunity  to  Plasmodium  berghei  yoelii  in  mice.   II. 
Specific  and  non-specific  cellular  and  humoral  responses  during  the  course 
of  infection.   J_.  Immunol.  ,  121:  629-636,  1978. 

Rosenberg,  Y.  J.,  and  Evans,  C.  B.   Resistance  of  mice  suppressed  for  I gM 
production  to  infection  with  Babesia  microti ■   Nature,  1979   (in  press). 


24-31 


fSM  1 THSOM  I  A.'J  SCIENCE  INFORMATION  EXCHANC 
PROJECT  NUMBER  (Oo  NOT  use  ih  i  u  apace) 


U.S.  DEPARTMENT  OF        I  PROJECT  NUMBER 
ALTH.  EDUCATION,  AND  WELFARE 


PUBLIC  HEALTH 
NOTICE  Or 
INTRAMURAL  RESEARCH  PROJECT 


ZOl  Al  001A2-06  LMI 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 


Development  of  Thymus-Der i ved  (T)  Suppressor  and  Amplifier  Cell  Function 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI  : 


P.  J.  Baker 


Head,  Microbiology  and  Immunology   LMI /N 1 Al D 
Sect  ion 


COOPERATING  UNITS  (if  any) 

NONE 


lab/branch  ,    , 

Laboratory  of   Microbial     Immunity 


SECTION 

Microbiology  and  Immunology  Section 


INSTITUTE  AND  LOCATION 

NIAID.  NIH.  Bethesda,  Maryland  20205 


TOTAL  MANYEARS 

0/12 


PROFESSIONAL: 


CHECK  APPROPRIATE  B0X(E3) 
□  (a)   HUMAN  SUBJECTS 

0  (al)  MINORS   □  (a2)  INTERVIEWS 


0  (t>)  HUMAN  TISSUES 


(c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  unaerline  keywords) 

Inactive  during  current  year. 


2^-32 


PHS-6040 
(Rev.  10-76) 


MITHSONIAN  SCIENCE  INFORMATION  EXCHANGE      U.S.  DEPARTMENT  OF 
PROJECT  NUMBER  (.Do  NOT  use  this  spacej   HEALTH,  EDUCATION,  AND  WELFARE 

PUBLIC  HEALTH  CERVlCE 


NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


"i?:j£  T  NUMBER 

ZOl  Al  00143-10  LMI 


PERIOD  COVERED 

October  1 ,  1978  to  September  30,  1979 


1 


TITLE  OF  PROJECT  (30  characters  or  less) 

Genetic  Control  of  the  Antibody  Response  to  Type 
Polysaccharide  (SSS-I  I  I) 


I  I  Pneumococcal 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI  : 

P. 

J.  Baker 

Head,  Microbio 

Other: 

B. 

Prescott- 

G. 

Caldes 

Chemi  st 

D. 

F.  Amsbaugh 

Biologi  st 

P. 

W.  Stashak 

Mi  crobiolgoist 

D. 

W.  Bailey** 

LMI /N I  AID 


LMI/NI 
LMI/NI 
LMI /n: 


AID 
AID 
AID 


COOPERATING  UNITS  (if  any) 

'-Biological  Research  Institute,  Rockville,  MD  20852 
**The  Jackson  Laboratory,  Bar  Harbor,  ME  04609 


LAB/ BRANCH 

Laboratory  of  Microbial  Immunity 


Microbiology  and  Immunology  Section 


INSTITUTE  AND  LOCATION 

IMIAID,    NIH,    Bethesda,    Maryl and      20205 


TOTAL  MANYEARS: 

20/12 


PROFESSIONAL: 

6/12 


14/12 


CHECK  APPROPRIATE  30X(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


(c)  NEITHER 


SUMMARY  OF  WORK  (200  *ords  or  less  -  underline  keywords) 

Congeni c-resi stant  strains  of  mice,  possessing  chromosomal  segments  from  a  high- 
responding  strain  on  a  low- respond i ng  background  strain,  were  used  to  isolate 
the  genes  involved  in  determining  the  capacity  of  bone  marrow-deri ved  precursors 
of  antibody-forming  cells  (B  ce  1 1 s)  to  make  an  ant i body  response  to  Type  I  I  I 
pneumococcal  polysaccharide  (SSS-I I  I)  and  to  assign  such  genes  to  well-defined 
genetic  linkage  groups  or  chromosomes.   The  results  obtained  show  that,  although 


at  least  five  genes  influence  the  capacity  of  B  cells  to  make  an  antibody 
response  to  SSS- III,  these  genes  are  located  on  different  (more  than  three) 
chromosomes . 


24-33 


PHS-6040 
(Rev.    10-76) 


Project  No.   Z01   Al   00 1 43" 10   LMI 


Project  Description: 


The  results  of  previous  studies  have  shown  that  the  capacity  of  bone  marrow- 
derived  precursors  of  ant i body- forming  cells  (B  cells)  to  make  an  antibody 
response  to  Type  III  pneumococcal  polysaccharide  (SSS-lll)  is  regulated  by 
the  activities  of  thymus-deri ved  (T)  suppressor  and  amplifier  lymphocytes. 
Several  autosomal  dominant  genes,  acting  in  an  independent  manner,  influence 
the  functional  activities  of  all  three  cell  types  and  thus  determine  the  magni- 
tude of  the  antibody  response  ultimately  produced  after  immunization.   None  of 
these  functional  activities  were  found  to  be  linked  to  genes  with  the  major 
histocompatibility  (H-2)  or  the  immunoglobulin  allotype  complex. 

C57BL/6By  mice  produce  an  extremely  low  antibody  response  to  SSS-lll,  a  1,3 
dextran  and  polyviny Ipyrol i dine  (PVP)  ,  whereas  BALB/cBy  mice  produce  a  high 
antibody  response  to  these  antigens.   The  availability  of  B6  congen i c-res is- 
tant  strains  having  chromosomal  segments  from  high- respondi ng  BALB/cBy  mice 
on  a  low- respondi ng  (C57Bl/6By)  background  provides  a  splendid  opportunity 
to  identify  and  isolate  genes  involved  in  determining  responsiveness  to  SSS-lll 
and  to  assign  such  genes  to  well-defined  genetic  linkage  groups  or  chromosomes. 
To  this  end  18  of  23  available  B6  congenic  strains  were  immunized  with  an 
optimally  immunogenic  dose  (0.5  yg)  of  SSS-lll  and  the  magnitude  of  the  anti- 
body response  produced  was  assessed.   The  responses  obtained  were  compared  to 
that  of  low-responding  C57BL/6By  mice  to  determine  whether  the  introduction 
of  known  chromosomal  segments  from  BALB/cBy  mice  (high  responders)  caused  a 
significant  increase  in  the  magnitude  of  the  antibody  response.   Although 
13  of  18  B6  congenic  strains  examined  gave  responses  like  those  of  low- 
responding  C57BL/6By  mice,  five  of  18  produced  responses  which  were  signifi- 
cantly greater  than,  but  not  equal  to,  that  of  hi gh- responding  BALB/cBy  mice. 
In  the  latter  case,  segments  from  chromosomes  17,  9  and  k   as  well  as  chromo- 
somal segments  containing  the  H-23  and  H-27  loci  appeared  to  contribute  to 
the  increased  responsiveness  noted.   These  findings  suggest  that  genes 
governing  the  capacity  of  B  cells  to  respond  to  SSS-lll  may  be  located  on 
different  chromosomes.   But,  it  remains  to  be  established  whether  the  activities 
of  these  isolated  genes  are  specific  for  the  antibody  response  to  SSS-lll  or 
influence  the  response  to  other  antigens  as  well.   Studies  are  in  progress  to 
resolve  this  issue  and  to  determine  if  such  genes  act  in  a  complementary 
(addi  ti  ve)  manner. 


Pub  1 i  cat  ions : 

Rudbach,  J.  A.  and  Baker,  P.  J.  (Eds.):   Immunology  of  Bacterial  Polysaccharides. 
Developments  in  Immunology,  Volume  2.   New  York,  Elsevier/North  Holland, 
1979,  157  pp.  """ 

Rudbach,  J.  A.,  and  Baker,  P.  J.:   Contributions  of  studies  with  bacterial 
polysaccharide  antigens.   In,  Rudbach,  J.  A.  and  Baker,  P.  J.  (Eds.): 
Immunology  of  Bacterial  Polysaccharides.   New  York,  Elsevier/North  Holland, 
1979,  PP.  1-19- 

2k-3k 


Project  No.   Z01  Al  00 1 ^3" 10   LMI 


Baker,  P.  J.,  and  Prescott,  B. :   Regulation  of  the  antibody  response  to 
pneumococcal  polysaccharides  by  thymus-deri ved  (T)  cells:   Mode  of  action  of 
suppressor  and  amplifier  T  cells.   In,  Rudbach,  J.  A.  and  Baker,  P.  J.  (Eds.) 
Immunology  of  Bacterial  Polysaccharides.   New  York,  Elsevier/North  Holland, 
1979,  PP.  67-105- 


24-35 


JHITHSOfilAN  SCIENCE  I  .'.'FORMAT  I  ON  EXCHANGE!     U.S.  DEPARTMENT  OF        I  PROJECT  NUMBER 
PROJECT  NUMBER  (Do  NOT  use  this  space)   IhEaLTH,  EDUCATION,  AMD  WELFARE  j 

PUBLIC  HEALTH  SERVICE 
NOTICE  Of 
INTRAMURAL  RESEARCH  PROJECT     ZOl  Al  00  1 44-  1  5   LM I 


PERIOD  COVERED 

October  1.  1978  to  September  30.  1979 


TITLE  CF  PROJECT  (30  characters  or  less) 

Regulation  of  the  Antibody  Response  to  Type  III  Pneumococcal  Polysaccharide 
(SSS-I  II). 


NAMES,  LA30RATORY  AMD  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:     P.  J.  Baker      Head,  Microbiology  &  Immunology  Sec.      LMI/N1A1D 
Other:   K.  B.  Schroer    Research  Associate  LMI/NIAID 

LMI/NIAID 
LMI/NIAID 
LMI/NIAID 
LMI/NIAID 


P. 

J.  Baker 

Head,  Microbiology 

K. 

B.  Schroer 

Research  Associate 

K. 

J.  Kim 

Visiting  Associate 

P. 

W.  Stashak 

Mi  crobiologi  st 

D. 

F.  Amsbaugh 

Bi  ologi  st 

G. 

Caldes 

Chemi  st 

B. 

P re scot t* 

COOPERATING  UNITS  (if  any) 

-:  Biological  Research  Institute,  Rockville,  MD  20852 


lab/branch 

Laboratory  of  Microbial  Immunity 


SECT  I  ON 

Microbiology  and  Immunology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS:  PROFESSIONAL:  OTHER: 

20/12  14/12  6/12 


CHECK  APPROPRIATE  BOX(ES) 
D  (a)   HUMAN  SUBJECTS  0  (b)  HUMAN  TISSUES  0  (c)  NEITHER 

□  (»0  MINORS   □  (a2)  INTERVIEWS 


SUMMARY  CF  WORK  (200  words  or  less  -  underline  keywords) 

Somatic  cell  (lymphocyte-myeloma)  hybri  ds  from  fusions  of  long-lived  cell  lines 


i th  antibody-forming  cells  from  mice  immunized  with  Type  I  1  I  pneumococcal 
polysaccharide  (SSS-I I l)  were  used  to  investigate  several  basic  issues  associated 


with  the  antibody  responseto  SSS-I  I  I.   The  results  obtained  showed  that  the 
plaque-forming  cell  (PFC)  repertoire  expressed  after  immuni  zat  i  on  wi  th  an 


optimally  immunogenic  dose  of  SSS-I I  I  was  reiterated  in  the  distribution  of 
i  sotypes  found  among  hybrids  making  antibody  specific  for  this  anti  gen.   Using 


the  monoclonal  products  of  hybridomas  as  probes  to  examine  the  diversity  of 

the  V-regions  of  antibody  specific  for  SSS-I  II,  it  was  found  that  the  CC4.6 

cross  reactive  i  di  otype  is  common  and  expressed  on  several  classes  of  immuno- 
globul  in  with  specificity  for  SSS-I  I  I. 


24-36 


PHS-6040 
(Rev.    10-76) 


Project  No.   Z01  A I  00144-15  LMI 


Project  Description: 

Plasmacytoma  cell  lines  which  are  drug  marked,  i.e.,  resistant  to  either  6- 
thioguanine  or  8-azaguanine  were  fused  in  the  presence  of  polyethylene  glycol 
to  spleen  cells  from  mice  immunized  with  Type  III  pneumococcal  polysaccharide 
(SSS-III);  here,  the  drug  marked  plasmacytoma  cell  line  dies  when  cultured 
in  medium  containing  hypoxanthine,  aminopterin  and  thymidine  (HAT).   Subse- 
quent cell  growth  is  a  feature  of  hybrid  cells  (hybri domas) ,  protected  from 
the  lethal  effects  of  HAT  by  the  HGPRT  enzyme  from  the  X-chromosome  of  spleen 
cells  from  immunized  mice.   Clones  secreting  antibody  specific  for  SSS-III 
can  then  be  selected  by  limiting  dilution  assays.   After  sub-cloning  i  n 
vi  tro,  desired  fusion  products  can  be  transferred  to  mice  to  elicit  ascites 
and  large  amounts  of  monoclonal  antibody  specific  for  SSS-III;  such  antibody 
can  be  used  to  prepare  ant i -i diotype  antibody  in  guinea  pigs  (or  other  animals) 
for  use  in  V-region  gene  analysis. 

For  the  antibody  response  to  SSS-III,  idiotypes  present  on  hybridomas  immuno- 
globulins were  sought  in  sera  from  immunized  mice.   The  first  such  screen  of 
hybrids  demonstrated  the  presence  in  sera  (interstrain  and  intrastrain)  of  an 
idiotypic  cross- react  ion  with  a  hybridoma  immunologul i n,  designated  CC4.6. 
This  CC4.6  cross-react i ve  idiotype  (CRl)  appears  to  be  present  on  a  large 
proportion  of  the  serum  antibody  specific  for  SSS-III  as  shown  by  similar 
inhibition  profiles  of  immune  serum  and  affinity  purified  CC4.6  immunoglobulin. 
No  other  hybrid  product  cross-reacted  with  the  CC4.6  idiotype.   Several  lines 
of  evidence  indicate  that  CRIs  elicited  in  hybridization  experiments  will 
reflect  the  repertoire  expressed  in  immunized  mice. 

Each  of  the  other  hybrids  making  antibody  specific  for  SSS-III  produced  unique 
idiotypes;  these  were  not  present  in  immune  serum  in  detectable  quantities. 
Since  the  plasmacytoma  cell  line  used  to  generate  these  hybrids  secreted 
I gG2b ,  k  immunoglobul in ,  one  explanation  for  the  absence  of  these  idiotypes 
in  immune  serum  is  that  the  antiidiotype  specificity  is  directed  against  a 
mixed  molecule  (parental  myeloma  k    chain  +  the  H  chain  from  immune  spleen 
cells);  but,  the  results  of  solid  phase  radioimmunoassays  failed  to  confirm 
this  possibility.   Although  antibody  made  by  these  clones  may  represent  <30% 
of  the  serum  antibody  produced  after  immunization,  subsequent  hybridization 
failed  to  yield  clones  bearing  these  idiotypes  which  may  occur  in  low  fre- 
quency.  Thus,  although  uncommon  clones  may  be  fused  randomly,  a  large 
number  of  clonotypes  must  be  examined  to  provide  an  accurate  measure  of  their 
precursor  frequency  and  diversity.   In  other  hybridization  experiments,  the 
CRl  was  found  to  occur  commonly  and  the  i sotype  distribution  of  hybridoma 
immunoglobulins  was  similar  to  that  for  the  antibody  response  of  mice  immunized 
with  SSS-III  (chiefly  I gM) .   The  CRl  was  common  and  expressed  on  several 
classes  of  immunoglobulin;  in  fact,  the  isotype  distribution  was  similar  to 
that  found  after  immunization  with  SSS-III. 

(CBA/N  X  BALB/c)F.  male  mice  (CB  mice)  bear  an  X- 1 i nked  defect  making  them 
unable  to  mount  an  antibody  response  to  SSS-III.   However,  somatic  cell 
hybrids  between  non-responding  CB  mice  and  plasmacytoma  cells  were  found 

24-37 


Project  No.   Z01   Al   00144-15   LMI 

to  secrete  1 gM  antibody  specific  for  SSS-III.   The  solid  phase  binding  of 
such  antibody  was  completely  inhibited  by  the  addition  of  free  antigen  (SSS- 
III).   The  solid  phase  binding  of  such  antibody  was  completely  inhibited  by 
the  addition  of  free  antigen  (SSS-III)  and  the  amount  of  antibody  detected 
in  culture  fluids  ranged  from  10  ng/ml  to  10  g/ml .   Eight  hybridoma  clones 
were  derived  from  such  a  fusion;  all  made  antibody  of  the  I gM  class.   These 
results  indicate  that  the  X- 1  inked  genetic  defect  does  not  result  from  the 
deletion  of  a  B  cell  subset,  capable  of  responding  to  certain  thymus-inde- 
pendent  antigens. 


Pub  1 i  cations : 

Schroer,    K.    R. ,    Kim,    J.    K. ,    Amsbaugh,    D.    F. ,    Stashak,    P.    W. ,    and   Baker,    P.J.: 
Lymphocyte   hybridomas   which   secrete   antibodies    to   the  Type    III    pneumococcal 
polysaccharide:    idiotypic   characterization.       In,    Sch lessi nger ,    D.     (Ed.): 
Microbiology    1980,   Washington,    D.C.,    American   Society    for  Microbiology,    1980, 
in   press. 

Schroer,    K.    R. ,    Kim,    J.    K. ,    Prescott,    B.,    and   Baker,    P.    J.:      Generation   of 
anti-Type    III    pneumococcal    hybridomas    from  mice  with   an    X-lined   B-lymphocyte 
defect.      J.    Exp.    Med. ,    in    press. 


24-38 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE     U.S.  DEPARTi.icnT  OF 
PROJECT  NUMBER  (Do  MOT  u;;e  this  space)   HEALTH,  EDUCATION,  AND  WELFAHE 

PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


ZOl  A!  00145-12   LMI 


PERIOD    COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (SO  characters  or  less) 

Mode  of  Action  of  Thymus-Deri ved  (T)  Suppressor  and  Amplifier  Cells 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


Head,  Microbiology  &  Immunology  Sec.  LMI/NIAID 

Research  Associate  LMI/NIAID 

Microbiologist  LMI/NIAID 

Biologist  LMI/NIAID 

Chemist  LMI/NIAID 


PI  : 

P. 

J.  Baker 

Other: 

K. 

B.  Schroer 

P. 

W.  Stashak 

D. 

F.  Amsbaugh 

G. 

Caldes 

B. 

P res cot t- 

COOPERATING  UNITS  (if  any) 

-Biological  Research  Institute,  Rockville,  Maryland  20852 


LAB/ BRANCH 

Laboratory  of  Microbial  Immunity 


Microbiology  and  Immunology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

10/12 


PROFESSIONAL: 

k/\2 


6/12 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 

□(al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


[3  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Sheep  erythrocytes  sensitized  with  a  hybridoma  immunoglobulin  capable  of  binding 
specifically  with  Type  III  pneumococcal  polysaccharide  (SSS-Tl  I )  were  used  as  in 
dicator  cells  for  the  detection  of  plaque- forming  cells  TPFQ  making  antjj^ 
idiotypic  antibody.   Significant  numbers  of  PFC  making  antibody  against  the 
CC4.6  idTotype,  a  common  idiotype  found  in  the  serum  of  mice  immunized  with 
SSS-III,  were  detected  during  the  late  stages  of  an  antibody  response  to  an 
optimally  immunogenic  dose  of  SSS-III;  larger  numbers  were  found  in  mice  given 
a  high  dose  (tolerogenic)  dose  of  SSS-III.   The  relationship  of  such  antibody 
to  the  mode  of  act  i  on  of  thymus-de rived  suppressor  and  ampl i  f ier  eel  Is  is  bei  ng 
investigated. 


24-39 


PHS-6040 
(Rev.    10-76) 


Project  No.   Z01  Al  00145-12  LMI 


Project  Description: 


There  is  ample  evidence  to  indicate  that  the  antibody  response  to  Type  III 
pneumococcal  polysaccharide  (SSS-III)  is  regulated  by  the  activities  of  two 
functionally  distinct  types  of  thymus-deri ved  (T)  cells  having  opposing 
functions;  these  cells  have  been  designated  suppressor  and  amplifier  T  cells. 
Also,  there  is  evidence  to  support  the  fact  that  the  antibody  response  is 
regulated  by  a  network  of  cellular  interactions  involving  the  formation  of 
antibody  and  anti- i diotypi c  antibody;  regulatory  T  cells  have  been  implicated 
in  such  a  network.   Therefore,  it  was  of  interest  to  determine  whether  anti- 
idiotypic  antibody  is  produced  during  the  course  of  an  antibody  response  to 
SSS-III  and  whether  the  development  of  such  antibody  is  associated  with  the 
expression  of  suppressor  T  cell  activity. 

The  development  of  somatic  cell  hybrids  secreting  large  amounts  of  antibody 
specific  for  SSS-III  and  bearing  a  common  cross-reactive  idiotype  (see  Z01 
Al  00144-15  LMI)  enables  one  to  examine  this  issue  at  the  cellular  level. 
Here,  erythrocytes  sensitized  with  SSS-I I l-binding  hybridoma  immunoglobulin 
can  be  used  as  indicator  cells  is  the  technique  of  localized  hemolys i s- in-gel 
for  the  detection  of  plaque-forming  cells  (PFC)  making  ant i -i diotypi c  antibody. 
The  results  of  preliminary  studies  show  that  significant  numbers  of  PFC  making 
anti-i diotype  antibody  were  found  in  mice  late  after  immunization  with  an 
optimally  immunogenic  dose  of  SSS-III;  however,  greater  numbers  were  detected 
in  mice  given  a  large  (tolerogenic)  dose  of  antigen.   Very  few  -  if  any  -  PFC 
making  ant i- i diotypi c  antibody  were  detected  after  priming  with  a  marginally 
immunogenic  dose  of  SSS-III,  a  procedure  known  to  activate  suppressor  T  cells. 
Although  it  would  be  tempting  to  speculate  that  suppressor  T  cells  are  in- 
volved in  the  formation  of  ant i -i diotypi c  antibody,  such  antibody  could  provide 
another  independent  mechanism  for  the  regulation  of  the  antibody  response.   In 
this  context,  suppressor  T  cells  have  been  shown  to  act  mainly  by  limiting  the 
extent  to  which  B  cells  proliferate  following  immunization;  but,  ant i -i di otypi c 
antibody  may  limit  the  magnitude  of  the  antibody  response  by  inhibiting  the 
secretion  of  antibody  by  antigen-stimulated  B  cells  as  has  been  suggested  by 
the  work  of  other  investigators.   These  issues  are  now  being  investigated. 


Pub! i  cat  ions : 

Pasanen,  V.  J.,  Asofsky,  R. ,  and  Baker,  P.  J.:   Synthesis  of  two  classes  of 

antibody,  yM  and  yG  or  yM  and  yA  by  identical  cells.   Amplification  of  the 

antibody  respone  to  pneumococcal  polysaccharide  Type  III.   J.  Exp.  Med. , 
149:  1227-1237,   1979- 


24-40 


SMITHSONIAN  3Cltj.CC  1 1,?  ORMATI  ON  EXCIiANG! 
PROJECT  NUMBER  (Do  NOT  Jse  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AMD  WELFARE 
PUHLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJE''  T  NUMBER 


201  Al  00146-06  LMI 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Immunological  Studies  on  Components  Isolated  from  Bacteria,  Parasites 
and  Plants. 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI  : 
Other: 


P.  J.  Baker 

B.  P res cot t* 

G.  Caldes 

D.  F.  Amsbaugh 

P.  W.  Stashak 

P.  R.  Beining-- 

G.  M.  F Tannery- 


Head,  Microbiology  5  Immunology  Sec.    LMI/NIAID 

Chemist  LMI/NIAID 

Biologist  LMI/NIAID 

Microbiolgoist  LMI/NIAID 


COOPERATING  UNITS  (if  any) 

"Biological  Research  Institute,  Rockville,  Maryland  20852 
--"Department  of  Biology,  University  of  Scranton,  Scranton,  Pennsylvania  18510 


lab/branch 

Laboratory  of  Microbial  Immunity 


Microbiology  and  Immunology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

12/12 


PROFESSIONAL: 

2/12 


10/12 


CHECK  APPROPRIATE  BOX(ES) 
D  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


(c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  IgM  antibody  response  to  the  1 ipotei  choi  c  aci  d  (LTA)  isolated  from  staphylo- 
coccus  aureus  ATCC  6538P  was  examined  by  a  newly  developed  procedure  in  which 


erythrocytes,  sensitized  with  peri odate-act i vated  LTA,  were  used  for  the  de- 
tection of  I gM-producing  plaque-forming  cells  (PFC) .   LTA-specific  PFC  were  f i rsl 
detected  two  days  after  immunization  with  heat-killed  bacteria  and  maximal  PFC 
were  attained  by  day  four;  specificity  of  such  PFC  was  affirmed  by  plaque- 
inhibifion  tests.   No  PFC  were  found  in  mice  given  isolated  LTA  over  a  10,000- 
fold  range  of  immunizing  doses.   Mice,  pretreated  with  a  carrier  known  to  activat 
thymus-deri ved  (T)  helper  lymphocytes,  produced  a  PFC  response  to  LTA  only  when 
immunized  with  LTA  bound  to  the  same  carrier.   This  suggests  that  carrier- 
specific  helper  T  ce 1  Is  are  needed  to  initiate  an  antibody  response  to  poorly- 
immunogenic  LTA.   Since  an  antibody  response  can  be  elicited  in  mice  given 
heat-killed  bacterial  cells,  bacterial  cell  wall  and/or  cell  membrane  components 
may  play  an  important  role  as  immunological  carriers  of  this  molecule. 


24-41 


PHS-6040 
(Rev.    10-76) 


Project  No.   Z01  Al  00146-06  LMI 


Project  Description: 


The  lipoteichoic  acid  (LTA)  of  gram  positive  bacteria  is  an  antigen  well- 
suited  for  use  in  basic  research  on  various  immune  phenomena.   It  is  composed 
of  a  glycerol  phosphate  polymer,  covalently  linked  to  a  lipid  moiety,  and 
contains  glycosyl  and  D-alanyl  side  groups.   LTA  is  known  to  interact  with  host- 
membrane  and  such  interactions  have  been  implicated  in  the  development  of 
arthritis.   LTA  may  also  serve  as  a  carrier  of  immunocytotoxi c  antigens  or, 
as  an  exposed  antigen,  may  play  a  decisive  role  in  the  binding  (colonization) 
of  bacteria  to  epithelial  cells  of  mucosal  surfaces.   Although  the  latter 
suggests  that  antibodies  specific  for  LTA  may  play  an  important  role  in 
limiting  the  pathogenesis  of  staphylococcal  infections,  this  has  been  difficult 
to  investigate;  isolated  LTA  is  considered  to  be  a  poor  immunogen  in  experimen- 
tal animals,  mainly  because  it  has  been  found  to  generate  extremely  low  serum 
antibody  and  antibody-producing  plaque-forming  cell  (PFC)  responses  after 
immunization.   Such  apparently  low  antibody  responses  may  be  due  to  the  i n- 
sensitivity  of  methods  commonly  used  for  the  detection  of  serum  antibody  and 
antibody-forming  cells. 

We  have  developed  a  method  for  the  sensitization  of  indicator  erythrocytes 
using  LTA  that  has  been  activated  by  treatment  with  periodate.   Erythrocytes 
sensitized  by  this  method  are  stable  and  can  be  used  effectively  not  only  in 
conventional  passive  immune  hemolysis  and  hemagglutination  tests  for  the 
detection  of  small  amounts  of  serum  antibody,  but  also  in  the  technique  of 
localized  hemolys i s-i n-gel  for  the  detection  of  cells  making  antibody  specific 
for  LTA.   The  results  obtained  using  such  indicator  erythrocytes  were  both 
reproducible  and  specific  for  LTA,  the  immunizing  antigen. 

Although  mice  were  found  to  give  a  reasonably  good  serum  antibody  and  PFC 
response  specific  for  LTA  after  immunization  with  heat-killed  Staphylococcus 
aureus ,  little  or  no  antibody  could  be  detected  in  mice  given  i  solated  (puri - 
f i  ed)  LTA  over  a  10,000-fold  range  of  immunizing  doses.   Prior  treatment  with 
a  relatively  large  dose  of  isolated  LTA  did  not  reduce  the  manitude  of  the 
antibody  response  produced  to  an  optimally  immunogenic  dose  of  heat-killed 
S.  aureus,  indicating  the  LTA  is  not  tolerogenic.   However,  mice  pretreated 
with  a  carrier  known  to  activate  thymus-der i ved  (T)  helper  lymphocytes,  en- 
abled mice  to  mount  an  antibody  response  to  LTA,  only  when  immunized  with  LTA 
bound  to  the  same  carrier;  the  magnitude  of  the  antibody  response  produced 
was  at  least  as  great  as  that  made  after  immunization  with  an  optimal  dose 
of  heat-killed  S.  aureus,  even  though  the  total  amount  of  carrier-bound  LTA 
used  was  extremely  small  (non- immunogen i c  in  mice  not  pretreated  with  carrier). 
These  findings  suggest  that  carrier-specific  helper  T  cells  are  needed  to 
elicit  an  antibody  response  to  poorly- immunogeni c  LTA;  since  an  antibody 
response  can  be  produced  in  mice  immunized  with  heat-killed  S.  aureus,  bacterial 
cell  wall  and/or  cell  membrane  constituents  must  play  an  important  role  in 
this  regard. 

The  availability  of  a  sensitive  and  reproducible  method  for  the  detection  of 
antibody  specific  for  LTA  now  permits  us  to  characterize  the  class  (isotype) 


24-42 


Project  No.   Z01   Al  00146-06   LMI 


of  antibody  produced  in  response  to  LTA  and  to  examine  the  role  of  such  anti- 
body in  the  development  of  protective  immunity  to  staphylococci.   Also,  studies 
with  carrier  molecules,  more  appropriate  for  use  in  man,  are  in  progress  to 
enhance  the  immunogen i ci ty  of  isolated  (purified)  LTA  for  potential  use  in 
vaccine  studies. 


Pub  1 i  cat  ions :    None 


24-43 


SMITHSONIAN    SCIENCE    INFORMATION    EXCHANG&I 
PROJECT    NUMBER    (Oo   NOT    use    this    space; 


.3.    DEPARTMENT   OF 

HEALTH,    EDUCATION      AND    WELFARE 

PUBLIC    HEALTH    SERVICE 

NOTICE   OF 

INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  Al  00153-03  LMI 


PERIOD    COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

In  Vi  tro  Response  of  Human  Peripheral  Lymphocytes  to  Infectious  Organisms 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI  :       Richard  Asofsky 


Laboratory  Chief 


LMI /N I Al D 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Microbial  Immunity 


SECTION 

Experimental  Pathology  Section 


INSTITUTE  AND  LOCATION 

Ml Al D.  NIH,  Bethesda,  Maryland   20205 


TOTAL  MANYEARS: 
0/12 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 

D  (al)  MINORS   n  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  CF  WORK  (200  ^ords  or  less  -  underline  keywords) 

No  work  was  done  on  this  project  this  year. 


2k-kk 


PHS-6040 
(Rev.    10-76) 


,'MITHSOHIAN  SCIENCE  INFORMATION  EXCHANGE     U.S.  DEPARTMENT  OF 
■RQJECT  NUMBER  (,Do  NOT  uie  this  space)   'HEALTH,  EDUCATION,  AfiD  WELFARE  | 

PUBLIC  HEALTH  SERVICE     | 
NOTICE  OF 
IHTRAHURAL  RESEARCH  PROJECT 


PROJECT  NUMCfcR 


ZOl  Al  00158-03  LMI 


PERIOD  COVERED 

October  1,  1978  to  September  30.  1979 


TITLE  OF  PROJECT  (60  characters  or  less) 

The  Immune  Response  to  Entamoeba  Antigens 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGACED  ON  THE  PROJECT 


PI 


J.  F.  Finerty 


Research  Microbiologist 


LMI /N I Al D 


COOPERATING    UNITS    (if    any) 

NONE 


LAB/ BRANCH 

Laboratory  of  Microbial  Immunity 


SECTION 

Microbiology  and  Immunology  Section 


INSTITUTE  AND  LOCATION 

NIAID.  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

0/12 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 
G  (a)   HUMAN  SUBJECTS 

Q  (al)  MINORS   jj  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□   (c)    NEITHER 


SUMMARY    OF    >/0RK    (200   words    or   less   -   underline    keywords) 

Inactive  during  current  year. 


2^-45 


PHS-6040 
(Rev.  10-76) 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AMD  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOI  Al  00186-06 


LMI 


Formerly   ZOI.   DE   0084-05 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (SO  characters  or  less) 

Pathogenesis  of  Autoimmunity  in  Inbred  Strains  of  Mice 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI  : 
OTHER: 


T.  M.  Chused 

H.  C.  Morse,  I 

W.  Davidson 

J.  Longstreth 

S.  0.  S harrow 

E.  Brown 


Medical  Officer  (Internal  Med)    LMI /Nl Al D 


Senior  Investigator 

Vi  s  i t  ing  Fel low 

NIH  Post-doctoral  Fellow 

Chemi  st 

Mi  crob  iolog  i  st 


LMI /N I  AID 
LMI /Nl Al D 
LMI /N I  Al  D 
IB/NCI 
LMI /Nl Al D 


COOPERATING  UNITS  (if  any) 


lab/branch 
Laboratory   of   Microbial     Immunity 


SECTION 

Experimental  Pathology  Section 


INSTITUTE  AND  LOCATION 

Ml Al D,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

26/12 


PROFESSIONAL: 

16/12 


10/12 


CHECK  APPROPRIATE  BOX(ES) 
Q   (a)   HUMAN  SUBJECTS 

(a1  )  MINORS   Q  (a2)  INTERVIEWS 


[j  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  purpose  of  this  project  is  to  understand  the  pathogenesis  of 
autoimmune  d  i  sease  by  studying  murine  modesl  such  as  New  Zealand 
Mice.   The  present  areas  of  investigation  are    the  early  activiation 
of  the  immune  system,  manifested  by  macrophage  activation  and  immuno- 
globulin production;  B  and  T  cell  membrane  receptors  and  differentiation 
antigen  abnormalities;  and  the  regulation  of  xenotropic  murine  leukemia 
virus  production  and  virus  coded  cell  surface  antigens.   The  genetic 
analys  is  of  these  parameters  has  identified  several  separate  genes 
which  presumably  contribute  to  the  development  of  autoimmune  disease. 


24-46 


PHS-6040 

(Rev.  10-76) 


Project  No.   Z01  Al  00186-06  LMI 
Formerly  Z01  DE  0084-05 


Project  Description: 


The  spontaneous  autoimmune  disease  of  New  Zealand  Black  (NZB)  mice  has  been 
under  investigation  for  more  than  15  years  without  a  definitive  explanation 
for  their  disorder.   We  have  previously  shown  that  NZB  splenic  B  cells 
spontaneously  produce  40  to  100  times  as  much  pentameric  IgM  as  non-auto- 
immune mice. 

Further  investigation  of  NZB  B  cells,  much  of  it  utilizing  the  fluorescence 
activated  cell  sorter,  has  shown: 

1.  NZB  spleen  contains  a  greatly  increased  number  of  IgD-dull  cells. 

2.  The  IgD-dull  cells  include  a  subpopul at  ion  of  large,  IgM  secreting 
plasmablasts. 

3.  These  plasmablasts  are  polyclonal ly  activated. 

4.  NZB  spleen  contains  ten  times  as  many  such  plasmablasts  as  normal 
mice. 

5.  Each  plasmablasts  contains  and  secretes  four  to  five  times  more  IgM 
than  those  of  normal  mice. 

6.  Ablation  of  T  cells  by  thymectomy,  irradiation  and  reconst i tut  ion 
with  anti  Thy  1  treated  bone  marrow  or  by  introducing  the  nude 
gene  onto  the  NZB  background  does  not  prevent  the  spontaneous  B 
eel  1  act  ivat ion. 

We  also  observed  that  NZB  T  cells  contained  pinocytozed  IgM,  presumably  anti- 
T  cell  autoantibody.   To  determine  whether  this  process  accounts  for  NZB  T- 
cel  1  abnormal  ities,  such  as  their  ability  to  give  a  primary  _i_n_  vi  tro  cytotoxic 
response  to  minor  h i stocompatabi 1 i ty  antigens  of  H-2  compatible  mice,   NZB 
B  cell  function  was  suppressed  with  anti-IgM  antibody.   Such  mu-suppressed 
mice  gave  the  same  level  cytotoxic  response  to  minor  histocompatibility  anti- 
gens, indicating  that  NZB  mice  also  have  a  primary  T  cell  defect.   A  macro- 
phage abnormality  that  could  produce  both  the  T  and  B  hyperfunct ion  has  not 
been  ruled  out. 

Genetic  analysis  of  both  the  T  and  B  abnormalities  is  in  progress. 

Dr.  Wendy  Davidson  has  examined  "motheaten"  mice,  a  spontaneous,  recessive 
mutation  on  chromosome  6  in  C57BL/6J  mice.   Motheaten  B  cell  express  charges 
similar  to,  but  more  extreme  than,  those  of  NZB  cells.   Their  T  cells  also 
appear  to  be  blast-like. 

Preliminary  study  of  BXSB  and  MRL  mice  indicates  that  they  have  patterns  of 
immunologic  activation  which  differ  from  those  of  NZB  and  motheaten.   Thus, 
we  now  have  several  distinct  models  of  murine  autoimmune  disease  whose  in- 
vestigation should  prove  fruitful  1. 

24-47 


Project  No.  Z01  Al  00186-06  LM I 

Formerly  Z01  DE  0084-05 

In  the  course  of  our  NZB  studies  we  have  shown  that  they  carry  at  least  two 
and  perhaps  three  separate  xenotropic  murine  leukemia  virus  genes.   These 
lines  are  now  in  the  seventh  to  ninth  backcross.   We  are  now  examining  the 
relationship  of  these  viruses  to  autoimmune  disease. 

To  facilitate  the  use  at  the  two-color  capability,  we  developed  in  collabora- 
tion with  Research  Organics,  Inc.  of  Cleveland,  Ohio,  a  modified  rhodamine- 
like  fluorophore,  XRITC,  which  has  absorption  and  emission  maxima  28  nm  higher 
than  those  of  tetramethyl rhodamine  i sothiocyanate . 


Publ icat  ions : 


Chused,  T.  M. ,  Moutsopoulos ,  H.  M.,  Sharrow,  S.  0.,  Hansen,  C.  T.  and 
Morse,  H.  C.  Ill:   Mechanism  of  autoimmune  disease  in  New  Zealand  Black  mice. 
In  Rose,  N.  R. ,  Bigazzi,  P.  E.,  and  Warner,  N.  L.,  (Eds.):  Genetic  Control 
of  Autoimmune  Disease.   Elsevier  North-Holland,  New  York.  ( I n  press) ,  1979- 

Davidson,  W.  F. ,  Morse,  H.C.  Ill,  Sharrow,  S.  0.,  and  Chused,  T.  M. :  Pheno- 
typic  and  functional  effects  of  the  motheaten  gene  on  murine  T  and  B  lympho- 
cytes.  J.  Immunol.,  122:  884-391,  1979- 

Chused,  T.  M. ,  Moutsopoulos,  H.  M.,  Sharrow,  S.  0.,  and  Hanson,  C.  T.  : 
Evidence  of  a  primary  B  lymphocyte  abnormality  in  NZB  mice.   In  Cooper,  M., 
Mosier,  D. ,  Scher,  F. ,  and  Vitetta,  E.,  (Eds.):  B  Lymphocytes  in  the  Immune 
Response,  Elsevier  North-Holland,  New  York,  (in  press) ,  1979- 


24-48 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  Al  00187-06  LMI 
Formerly  ZOl  DE  0085-05 


PERIOD    COVERED 


October  1,  1978  to  September  ^0,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Studies  in  Sjogren's  Syndrome 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI  : 
OTHER: 


M.  Chused 
M .  B  rown 
Moutsopoulos 
Law ley 
Mann 


Medical  Officer  LMI /N I  AID 

Biologist  LMI /NI Al D 

Visiting  Scientist  LMI /NI DR 

Senior  Investigator  D/NCI 

Senior  Investigator  l/NCI 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Microbial  Immunity 


SECTION 

Experimental  Pathology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

4/12 


PROFESSIONAL: 

2/12 


2/12 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   Q  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

It  is  the  purpose  of  this  project  to  determine  the  mechanism  and 
etiology  of  the  autoimmune  disease  Sjogren's  Syndrome.   Genetic 
factors,  such  as  HLA-A,  -B  and  -D  type  and  la- like  B  cell  auto- 
antigens  are  of  particular  interest.   In  addition,  clinical  para- 
meters such  as  circulating  immune  complexes  are  evaluated. 


PHS-6040 
(Rev.  10- 


24-49 


Project  No.  ZOI  Al  00187-06  LMI 
Formerly  Z01  DE  0085-05 


Project  Description: 


We  have  previously  shown  that  Sjogren's  syndrome  (SS)  is  associated  with  the 
serologically  defined  h istocompat ible  antigen  HLA-B8  (50%  +  in  SS ,  21%  +    in 
controls)  and,  to  a  greater  extent,  with  the  lymhpocyte  defined  antigen  HLA- 
Dw3   (84%  +  in  SS  alone,  lk%  +    in  controls). 

These  associations  were  studied  in  greater  detail  by  typing  a  series  of  2k   SS 
patients  with  a  panel  of  60  alloantisera  directed  against  human  la  antigens. 
Two  antisera,  la-172  and  la-AGS,  reacted  with  100%  of  SS  patients  but  25  and 
61%  of  normals,  respectively.   These  antigens  are  not  associated  in  the 
normal  population.   This  suggested  that  two  la  antigens  and  thus  possibly 
two  immune  response  genes  may  be  required  for  the  development  of  SS. 

We  recently  expanded  our  series  of  SS  patients  and  separated  them  into  those 
with  SS  alone  (primary)  and  SS  with  reheumatoid  arthritis  (secondary).   In- 
terestingly, we  observed  HLA  antigen  differences  between  the  two  groups. 
Patients  with  primary  SS  tended  to  have  three  independent  "families"  of 
HLA  antigens:   (1)  HLA-B8  and  HLA-DRw3   (2)  la-172,  la-350  and  (3)  la-715. 
Patients  with  secondary  SS  tend  to  carry  (1)  HLA-DRwA  (known  to  be  associated 
with  rheumatoid  arthritis)  and  (2)  la-172  and  la-350. 

From  this  we  conclude  that  the  la-172  complex  may  facilitate  the  development 
of  both  primary  and  secondary  SS;   that  primary  SS ,  in  fact,  requires  three 
immune  responses  genes;  and  that  the  immunost imulat ion  associated  with 
rheumatoid  arthritis  may  abrogate  the  requirement  for  HLA-DRw3  and  la-715 
observed  in  the  primary  disorder.   It  is  worth  noting  that  la-715  is  found 
in  both  primary  SS  and  systemic  lupus  erythematosus  two  diseases  which 
antinuclear  antibodies  are   frequently  found. 

The  genetic  differences  between  primary  and  secondary  SS  prompted  a  retro- 
spective chart  review  of  these  patients.   This  study  showed  that  salivary 
gland  enlargement,  splenomegaly  and  several  other  clinical  manifestations  are 
all  significantly  more  frequent  in  primary  SS . 

In  collaboration  with  Dr.  Thomas  Lawley  we  investigated  circulating  immune 
complexes  in  SS.   We  found  them  to  be  very  common  and  correlated  with 
clinical  activity  of  the  disease.   Because  serum  rheumatoid  factor  is  also 
very  frequent  in  SS  we  carefully  demonstrated  that  the  immune  complexes  are 
not   primarily  composed  of  rheumatoid  factor  and  immunoglobulin. 

Many  case  reports  have  indicated  that  lymphoma  occurs  relatively  often  in  SS. 
We  undertook  an  epidemiologic  study  to  qualify  this  risk,  finding  it  to  be 
kk   times  that  of  the  normal  population.   Waldenstom's  macroglobul in  is  also 
much  more  frequent  in  SS. 


24-50 


Project   No.    Z01    Al    00187-06    LMI 
Formerly    Z01    DE   0085-05 


Publ icat ions: 


Moutsopoulos,  H.  M. ,  Balow,  J.  E.,  Lawley,  T.  J.,  Stahl  ,  N.  I.,  Antonovych, 
T.  T.  and  Chused,  T.  M. :   Immune  complex  glomerulonephritis  in  sicca  syndrome. 
American  J.  Med.  64:955,  1978. 

Moutsopoulos,  H.  M. ,  Chused,  T.  M. ,  Johnson,  A.  H . ,  Knudsen,  B.,  Mann,  D.  L.: 
B  lymphocyte  antigen  in  sicca  syndrome.   Science  199: 1441-1 442 ,  1978. 

Chused,  T.  M. ,  Moutsopoulos,  H.  M.,  Johnson,  A.  H.,  and  Mann,  D.  L.:   la- 
antigens  in  Sjogren's  syndrome.   In  Rose,  N.  B.,  Bigazzi,  P.  E.  and  Warner, 
N.  L.  (Eds.),  Genetic  Control  of  Autoimmune  Diseases.   Elsevier  North-Holland, 
New  York,  (in  press) ,  1979- 

Kassan,  S.  S.,  Thomas,  T.  L.,  Moutsopoulos,  H.  M.,  Hoover,  R.,  Kimberly,  R. , 
Budman,  D.  R. ,  Costa,  J.,  Decker,  J.  L.,  and  Chused,  T.  M. :   Increased  risk 
of  lymphoma  in  sicca  syndrome.   Annals  of  Internal  Med.  89:888-892,  1978. 

Moutsopoulos,  H.  M. ,  Webber,  B.  L.,  Vlagopoulos ,  T.  P.,  Chused,  T.  M.,  and 
Decker,  J.  L.:   Differences  in  the  clinical  manifestations  of  sicca 
syndrome  in  the  presence  and  absence  of  rheumatoid  arthritis.  Amer.  Jour, 
of  Med.  66:733-736,  1979- 


24-51 


Z01   AI 
Project  Number 

SUMMARY 
00091-17 

00092-13 

00093-04 

00094-20 

00097-21 

00098-23 

00099-09 
00102-05 

00103-12 

00108-08 

00109-07 

00149-04 
00160-03 

00161-03 
00162-03 


LABORATORY  OF  PARASITIC  DISEASES 
1979  Annual  Report 
Table  of  Contents 


25-1 

The  Genetics,  Biology,  and  Control  of  Snail 
Intermediate  Hosts  of  Schistosomes  -  Richards  .  .  .  25-9 

The  pathogenesis  of  schistosome  infections  in 

mammalian  hosts  -  Cheever  25-14 

Studies  of  the  Immunologic  Response  to  Helminth 
Infections  -  Ottesen 25-18 

Studies  of  Entamoeba  histolytica  and  other 

Parasitic  Protozoa  -  Diamond 25-22 

Physiological  and  Cytochemical  Pathology  of 

Parasitic  Diseases  -  Mercado 25-27 

Biochemical  mechanisms  of  energy  metabolism  in 

mammalian  and  parasitic  organisms  -  Weinbach.  .  .  .  25-31 

Biophysical  Parasitology  -  Dvorak  25-35 

Pathogenesis  of  Disease  Caused  by  Infection  with 
Intracellular  Parasites  -  Neva 25-40 

Immunological  Studies  on  Toxoplasmosis  and  Other 
Parasitic  Diseases  -  Lunde 25-43 

Studies  on  the  Biology  and  Immunogenicity  of 

Malarial  Sporozoites  -  Gwadz 25-46 

Cellular  Immunology  of  Malaria  and  Other 

Parasitic  Diseases  -  Wyler 25-49 

Physiology  of  Anopheles  Mosquitoes  -  Beach 25-52 

Cell  Biology  of  Entamoeba,  Giardia  and  Other 

Parasitic  Protozoa  -  Gillin  25-56 

Immunochemistry  of  Parasitic  Diseases  -  Nash.  .  .  .  25-60 

Biochemical  Cytology  of  Host-Parasite  Interactions 

in  Parasitic  Protozoa  -  Dwyer  25-63 


00174-02         Culture,  physiology  and  antigenic  analysis  of 
sexual  and  asexual  erythrocytic  malaria 
parasites  -  Carter  and  Miller 25-69 

00184-01  Chemotherapy  of  Malaria:  The  Relationships 
Between  Gametocytocidal ,  Sporontocidal  and 
Hepatic  Schizontocidal  Drugs  -  Gwadz  25-75 

00185-01         Pathogenic  Protozoa:  Structure,  Division, 
Virulence  Factors,  and  Endogenous 
Viruses  -  Mattern 25-78 

Terminated  Projects,  Pacific  Research  Section,  LPD,NIAID 

00113-16         Studies  on  dengue  -  Rosen 25-82 

00163-02         Field  and  laboratory  studies  on  the  transovarial 

transmission  of  Japanese  and  St.  Louis  encephalitis 
viruses  by  mosquitoes  -  Rosen 25-83 

00164-02         Epidemiology  of  Infectious  Disease  in  the  Pacific 

Region  -  Dean 25-84 

00178-01         Studies  of  the  mechanism  of  transovarial 

transmission  of  arboviruses  by  mosquitoes  -  Tesh  .  .  25-85 


Laboratory  of  Parasitic  Diseases 

National  Institute  of  Allergy  and  Infectious  Diseases 

SUMMARY  -  October  1,  1978  through  September  30,  1979 

INTRODUCTION 

Over  the  past  year  many  changes  at  various  levels  of  professional 
personnel  occurred.   Carl  Mattern,  M.D. ,  who  had  previously  been  in  the 
Laboratory  of  Viral  Diseases,  was  transferred  to  LPD  because  of  administra- 
tive changes  in  LVD,  but  also  because  Dr.  Mattern 's  research  interests  have 
included  protozoology  and  viruses  of  amebae.   Gabriel  Schmunis,  M.D.  who 
was  a  Visiting  Fellow  with  us  some  years  ago  and  now  works  in  Brazil,  joined 
us  as  a  Visiting  Scientist.   Senior  Staff  Fellow  Charles  Oster,  M.D.  left 
us  to  take  a  senior  infectious  disease  position  at  Walter  Reed  Medical  Center. 
Phillip  Smith,  M.D.,  a  gastroenterologist  from  the  University  of  Colorado, 
is  due  to  join  Dr.  Nash  late  in  the  year  as  an  IPA  employee  for  work  on 
giardiasis.   Fouad  Boctor,  Ph.D.,  a  Visiting  Associate  from  Egypt,  will  be 
returning  there  late  this  fiscal  year,  and  T.  Takeuchi,  Ph.D.,  of  Japan, 
another  Visiting  Associate  who  worked  with  Dr.  Weinbach,  returned  to  Japan 
at  the  beginning  of  this  reporting  year.   Two  Research  Associates,  Brian 
Catto,  M.D.  and  Thomas  Quinn,  M.D.  finished  their  two  year  tours  with  us 
and  left  to  take  further  post-doctoral  work  in  Cleveland  and  Seattle, 
respectively,  while  one  new  Associate,  Mark  Hofstetter,  M.D.  just  arrived. 
Three  new  Visiting  Fellows  who  joined  us  were  Maria  do  Carmo  de  Souza,  Ph.D. 
of  Brazil,  D.C.  Kaushal,  Ph.D.  of  India  and  Nuzhat  Anwar,  Ph.D.  of  India. 
Renato  Gusmao,  M.D.  of  Brazil  who  had  been  with  us  as  a  Visiting  Fellow, 
started  graduate  work  at  Johns  Hopkins  School  of  Hygiene,  but  retains  an 
appointment  as  Guest  Worker  with  us  to  participate  in  field  research  on 
Chagas '  disease  in  Brazil.   Another  Guest  Worker  from  Brazil,  M.  Barral,  M.D. 
who  has  a  Kellogg  Foundation  Fellowship  arrived  to  work  with  Dr.  Cheever. 
Dr.  Martinez-Paloma  of  Mexico  spent  one  month  with  Dr.  Diamond  as  a  Guest 
Worker  in  July.   The  move  of  Dr.  Richards  and  Dr.  Sullivan  to  a  contract 
facility  in  Rockville  for  their  research  on  snail  vectors  of  schistosomiasis 
was  delayed  but  will  probably  take  place  in  late  summer  or  early  fall. 

Participation  in  field  research  abroad  continued  with  Dr.  Ottesen  and 
Ms.  Stanley  working  on  filariasis  for  several  weeks  in  Madras,  India  and 
Drs.  Neva  and  Gusmao,  along  with  Mr.  Gam  and  several  of  our  collaborators 
from  Duke  University  working  again  in  Brazil  on  Chagas'  disease.   Dr.  Neva 
returned  to  Brazil  for  follow-up  on  the  collaborative  project  in  Goiania, 
as  well  as  to  collect  triatome  vector  bugs  of  Chagas'  disease  in  Paulo 
Afonso.   Drs.  Gwadz  and  Beach  of  the  Malaria  Section  spent  several  weeks  in 
Egypt  as  advisors  on  a  PL-480  project  and  Dr.  Rosenberg  spent  several  weeks 
as  an  advisor  to  AID  in  Bangladesh,  where  he  had  worked  previously.   Drs. 
Ottesen  and  Nash  are  scheduled  to  visit  and  consult  with  CDC  personnel  in 
Puerto  Rico  about  possible  collaborative  work  on  schistosomiasis. 


25-1 


Laboratory  of  Parasitic  Diseases 

National  Institute  of  Allergy  and  Infectious  Diseases 

SUMMARY  -  October  1,  1978  through  September  30,  1979 

RESEARCH  ACCOMPLISHMENTS 


MALARIA  RESEARCH  PROGRAM     Search  for  antigens  with  hybridomas:   The  new 

technique  of  fusing  spleen  cells  from  immunized 
OF  THE  LPD,  NIAID  mice  with  myeloma  cells  is  being  used  to 

identify  specific  antigens  of  malaria  parasites. 
Polyethylene  glycol  is  used  to  promote  fusion  of  cells  and  the  resulting 
hybrid  cells  are  grown  in  a  selective  medium  which  permits  growth  only  of 
hybrid  cells.   When  the  resulting  hybrid  cells  are  cloned,  the  resulting 
progeny,  or  "hybridoma"  cells  produce  a  monoclonal  antibody  in  vitro  or  in 
vivo  as  an  ascites  tumor  in  mice,  which  is  characteristic  of  myeloma  cells. 
One  objective  of  this  technique  is  to  use  the  monoclonal  antibody  produced 
by  the  hybridoma  to  identify  specific  components  of  malaria  parasites.   Both 
internal  and  surface  antigens  of  gametes  of  P.  gallinaceum  have  been 
identified  with  this  method  (Rener,  Rosenberg  of  LMI ,  Carter  and  Miller). 
Hybridoma  cell  lines  have  also  been  developed  that  react  with  surface 
components  of  merozoites  of  P^  knowlesi,  as  determined  by  fluorescent  anti- 
body reactions  against  living  intact  merozoites.  (Epstein,  Miller  and  Carter) 

Gamete  physiology  and  immunology:   One  of  the  major  remaining  mysteries 
about  the  malarial  parasite  is  how  development  of  gametocytes  is  controlled. 
Are  some  of  the  asexual  forms  destined  to  become  gametocytes  or  are  there 
external  stimuli  which  initiates  their  development?  With  the  in  vitro 
culture  system  of  Trager  and  Jensen  the  conclusion  has  been  reached  that 
environmental  stimuli  control  gametocyte  production  of  P^  falciparum. 
(Carter  and  Miller).   Also,  the  addition  of  cyclic  AMP  to  the  culture  medium 
was  found  to  stimulate  conversion  of  asexual  forms  to  gametocytes  (Kaushal, 
Carter  and  Miller) . 

Surface  antigens  of  merozoites  of  P^  knowlesi  were  found  to  be  shared 
by  antigens  of  P.  falciparum.  (Miller,  Johnson  and  Carter).   The  common 
antigenic  specificity  between  these  two  species  of  malarial  parasites  might 
have  some  future  practical  application.   Further  work  has  been  done  to  try 
to  characterize  the  so-called  exf lagellation  factor,  a  material  in  the  gut 
of  mosquitoes  which  induces  gametogenesis  in  gametocytes  of  P.  gallinaceum. 
By  sephadex  chromatography  the  substance  appears  to  be  of  low  molecular 
weight  (only  200  to  300) ,  it  is  stable  to  heat  and  contains  several  amino 
acids.  (Beach). 

Merozoite  receptor:    Although  evidence  for  a  receptor  substance  on  red 
blood  cells  which  permitted  invasion  of  certain  species  of  malaria  parasites 
was  established  previously,  the  exact  immunochemical  nature  of  the  receptor 
has  not  yet  been  identified.   Another  aspect  of  the  merozoite-red  cell 
interaction  is   the  properties  of  the  merozoite  which  involve  attachment  to 


25-2 


red  cells.   Mild  treatment  of  merozoites  of  P^  knowlesi  with  trypsin  was 
found  to  destroy  ability  of  the  merozoite  to  invade  red  cells.   Such 
treatment  was  associated  with  loss  of  certain  high  molecular  weight  bands 
on  SDS  polyacrylamide  gel  electrophoresis,  suggesting  that  these  proteins 
may  be  involved  in  attachment  of  merozoites  to  red  cells.  (Johnson,  Epstein, 
Shiroishi  and  Miller. 

Sporozoite  antigens:   Collaborative  work  with  Dr.  Nussenzweig  at  NYU 
is  aimed  at  a  more  precise  understanding  of  immunity  to  sporozoites  of  P. 
knowlesi  in  the  rhesus  monkey.   Immunity  to  sporozoite  challenge  could  be 
transferred  passively  and  was  consistently  associated  with  demonstration  of 
anti-sporozoite  antibodies  by  various  in  vitro  tests.   Attempts  are  being 
made  to  identify  the  antigen(s)  responsible  for  protection  by  1"I  labelling 
and  by  hybridoma  production  of  specific  antibodies.  (Gwadz  and  Nussenzweig 
of  NYU) . 

Anti-malarial  drugs  affecting  gametocytes  and  liver  forms:   It  has~  been 
noted  that  anti-malarial  drugs  such  as  primaquine  that  affect  exo-erythrocytic 
liver  forms  generally  also  inhibit  gametocyte  development  in  the  mosquito. 
Since  a  system  for  testing  oocyst  development  in  mosquitoes  fed  upon  P. 
gallinaceum  infected  chickens  is  readily  available  and  the  same  effect  can 
also  be  checked  by  a  membrane  feeding  system,  these  two  types  of  tests  are 
being  used  to  check  drugs  for  activity  against  exo-erythrocytic  stages. 
Thus  far,  a  good  correlation  was  found  for  drugs  having  gametocytocidal 
effects  and  their  ability  to  act  as  hepatic  schizontocides.  (Gwadz,  Koontz, 
Miller  and  collaborators  at  WRAIR) . 

Anopheline  physiology:   The  steroid  hormone,  ecdysone,  until  recently 
believed  absent  in  adult  mosquitoes,  has  been  shown  to  play  a  major  role 
in  oogenesis.   Recent  studies  have  shown  that  ecdysone  also  modulated 
feeding  behavior  and  blood-meal  retention  in  female  mosquitoes  and  mating 
behavior  in  males.   Genetic  analysis  of  vector  competence  of  an  anopheline 
vector  in  Egypt,  A.  pharoensis,  has  been  initiated  in  an  effort  to  identify 
factors  determining  susceptibility.  (Beach  and  Rosenberg). 

Role  of  the  spleen  in  defense  against  malaria:   Last  year  it  was 
reported  that  the  role  of  the  spleen  in  host  defense  against  malaria  seemed 
to  depend  more  upon  rheologic  than  immunologic  factors.   This  conclusion 
was  confirmed  by  studies  of  clearance  in  the  rat  of  ->1Cr-labeled  red  cells 
parasitized  with  P^_  berghei.   Even  though  merozoite  invasion  of  RBCs  could 
be  prevented  in  vivo  by  antibody,  acute  resolution  of  malaria  was  spleen 
dependent  and  involved  decreased  deformability  of  infected  erythrocytes  and 
altered  splenic  micro-circulation.   (Quinn  and  Wyler) .   Congenitally  asplenic 
mice,  as  well  as  adult  splenectomized  mice,  could  not  overcome  an  acute 
malarial  infection.   Reconstitution  of  asplenic  mice  with  syngeneic  or 
autologous  spleen  cells  did  not  restore  protective  function.  (Wyler  and 
Oster) . 


25-3 


CELL  BIOLOGY  AND  PHYSIOLOGY    Amebae :   The  mechanism  of  action  and  partial 

characterization  of  a  "toxin"  from  extracts 
OF  PROTOZOAN  PARASITES         of  E.  histolytica  has  been  achieved.   Con  A, 

a  plant  lectin,  was  found  to  have  a  nearly 
identical  effect  as  the  amebal  toxin  on  cell  cultures.   The  effects  of  the 
amebal  toxin  can  be  inhibited  by  fetuin,  a  serum  glycoprotein,  and  the 
amebal  toxin  and  Con  A  are  inhibited  by  certain  hexosamines;  therefore, 
lectin-like  properties  seem  to  be  involved.   (Mattern) .   Amebae  of  greatly 
increased  virulence,  as  judged  by  the  ability  to  produce  liver  abscesses 
in  the  newborn  hamster,  were  selected  by  passage  in  the  newborn  hamster, 
whereas  virulence  is  usually  lost  by  passage  in  axenic  medium  only . (Mattern) . 
There  have  been  some  additional  improvements  in  the  axenic  cultivation 
technique  for  E_^   histolytica.   Yeast  extract  as  one  of  the  essential  com- 
ponents has  been  replaced  with  a  mixture  of  defined  ingredients  consisting 
of  purines,  pyrimidines,  nucleosides  and  B  complex  vitamins.   Use  of  butyric 
acid  has  been  found  to  be  a  key  factor  in  a  method  devised  to  permit  direct 
axenization  of  E.  histolytica  from  mixed  bacterial  cultures.  (Diamond). 
Investigation  of  the  sequence  of  electron  flow  in  respiration  of  axenized 
E.  histolytica  is  now  quite  complete.   It  goes  from  nicotinamide  adenine 
nucleotides,  f lavoproteins,  only  small  amounts  of  quinones  and  terminating 
in  iron-sulfur  centers.   Since  the  iron-sulfur  centers  are  one-electron 
transfer  carriers  the  end  product  of  their  reduction  is  H2O  rather  than  H2O2 
which  would  be  the  end  product  of  flavins  as  has  been  claimed  by  others. 
(Weinbach  and  Claggett). 

Giardia:   A  considerable  range  of  studies  has  been  initiated  with 
Giardia  lamblia.  Maximal  growth,  resistance  to  oxygen  and  attachment  to  glass 
surfaces  was  found  to  be  dependent  upon  the  presence  of  L-cysteine.   Serum 
or  plasma  fractions  also  promoted  attachment  of  giardia  trophozoites  to 
glass  surfaces.   In  contrast,  secretory  immunoglobulins  inhibited  attach- 
ment.  Factors  involved  in  excystation  of  cysts  are  also  under  study.   In 
spite  of  this  accumulation  of  knowledge  about  giardia  attempts  to  initiate 
new  cultures  from  patients  have  been  unsuccessful.  (Gillin) .   Biochemical 
studies  have  also  been  initiated  with  giardia,  since  it  can  be  grown 
axenically  in  medium  developed  for  amebae.   It  is  similar  to  E.  histolytica 
in  that  it  has  no  mitochondria,  cytochromes  or  a  tricarboxylic  cycle,  but 
in  contrast  its  respiratory  enzymes  are  particulate  and  has  a  different  type 
of  non-heme  iron  proteins.  (Weinbach,  Diamond  and  Keister). 

Leishmanial   The  time  spent  on  perfecting  techniques  to  isolate  membranes 
and  membrane  constituents  of  leishmanial  parasites  has  proven  to  be  rewarding. 
First  of  all,  good  yields  and  clean  preparations  by  electromicroscopy  have 
been  obtained  of  pellicular  membranes  and  microtubules  of  L.  donovani.   By 
use  of  lectin-fluorescein  conjugates,  components  of  the  pellicular  membrane 
could  be  "stained",  indicating  that  the  membranes  have  at  least  20  or  so 
glycoprotein  constituents  with  various  side  chain  carbohydrates,  as  well  as 
an  actin  or  actin-like  constituent.  (Dwyer) .   Antibodies  produced  in  rabbits 
against  these  pellicular  membranes  have  disclosed  evidence  of  polysaccharide 
antigens  common  to  6  different  trypanosomatid  species.  (Gottlieb  and  Dwyer). 
Also,  this  common  antigen  was  detected  as  an  exoantigen  in  the  sera  of 
L.  donovani  infected  hamsters.  (Dwyer).   Freeze-f racture  and-etching  studies 


25-4 


by  EM  have  demonstrated  many  details  of  supramolecular  structure  of  the 
pellicular  membranes.  (Da  Silva  and  Dwyer) .   Study  of  membrane  enzymes, 
especially  acid  phosphatase,  has  shown  that  the  flagellate  form  of  the 
organism  is  already  prepared  for  life  within  the  host  cell  phagolysosomes! 
(Gottlieb  and  Dwyer).   The  kinetoplast-mitochondrial  fraction  of  the  organisms 
is  being  studied  for  its  respiratory  metabolism,  a  point  of  interest  because 
most  biochemical  work  on  leishmanial  kinetoplasts  has  focused  mainly  on  their 
DNA.  (Weinbach  and  Dwyer).   The  techniques  used  for  study  of  leishmanial 
membranes  are  being  applied  to  isolation  and  characterization  of  membranes 
of  Trypanosoma  rhodesiense.  (Finerty  and  Dwyer) .   The  human  peripheral 
monocyte  which  becomes  a  macrophage  in  culture  is  being  used  as  a  host  cell 
for  L.  donovani  and  L.  tropica  to  investigate  the  leishmanial-host  cell 
interactions  that  permit  growth  of  the  parasite  within  phagolysosomes. 
(Berman,  Dwyer  and  Wyler) .   The  BALB/c  mouse  inoculated  in  the  foot-pad  so 
that  resulting  lesions  can  be  assessed  more  precisely  to  characterize 
virulence  has  given  quite  a  range  of  responses  with  different  strains  of 
leishmania  from  cutaneous  lesions.  (Neva). 

Trypanosoma  cruzi:   The  factor (s)  responsible  for  enhanced  attachment 
to  and  penetration  of  cells  by  trypomastigotes  that  is  present  in  the  serum 
of  chronically  infected  humans  or  animals  appear  to  be  in  the  >50,000 
molecular  weight  fraction  of  serum.   IgG  or  IgM  fractions  separately  were 
inactive  and  absorption  with  protein-A  decreased  the  activity.   (Schmunis 
and  Dvorak) .   DNA  synthesis  in  intracellular  T\_  cruzi  was  found  to  undergo 
a  synchronous  cycle  after  a  pre-replicative  lag  of  at  least  12  hours.   It 
was  concluded  that  T.  cruzi  trypomastigotes  are  in  theGg-Gj  phase  of  their 
cell  division  cycle  and  that  parasite  reproduction  occurs  independently  of 
events  controlling  the  host-cell  DNA  synthesis  cycle.   These  findings  of 
synchronous  growth  of  amastigotes  should  facilitate  biochemical  studies  on 
this  phase  of  the  parasite.  (Crane  and  Dvorak).   One  of  the  handicaps  to 
the  procedure  of  producing  hybrids  from  vertebrate  cells  X  T  cruzi  epimasti- 
gotes  has  been  a  more  efficient  method  of  selecting  definite  hybrid  clones. 
Use  of  a  different  vertebrate  cell,  one  which  lacks  certain  nutrients  and 
can  therefore  be  selected  for  by  using  selective  medium,  should  help  obviate 
this  problem.   The  P3-X63Ag8  cell  line  seems  to  serve  this  purpose.  (Crane 
and  Dvorak) . 

The  plaquing  technique  for  clonal  selection  of  intracellular  T^  cruzi 
has  now  been  tested  sufficiently  to  be  applicable  to  study  of  parasite 
strains  directly  from  infected  bugs.   Concomitant  titration  of  the  same 
trypomastigote  suspension  for  infectivity  at  serial  dilutions  and  for 
plaque  formation  indicates  that  the  minimal  infective  dose,  or  ID50,  is  not 
necessarily  equivalent  to  plaque  forming  units.   Therefore,  the  property 
of  producing  plaques  requires  more  than  the  ability  to  simply  initiate 
infection  of  cells.   Ability  of  strains  to  produce  plaques  is  better  at  33°c, 
in  fact,  very  few  strains  have  been  found  to  produce  plaques  at  elevated 
temperature.   In  this  respect,  growth  curves  and  plaque  formation  give 
similar  results.  (Neva).   An  interesting  phenomenon  of  bacterial-parasite 
interaction  which  came  to  notice  accidentally  has  been  investigated. 
Filtrates  of  Pseudomonas  f luorescens  were  found  to  produce  morphologic 
changes  in  trypomastigotes  of  J_^_  cruzi,  including  lysis.   The  main  effect 
of  the  bacterial  product  appears  to  be  on  the  cell  membrane,  and  the  lytic 


25-5 


factor  is  resistant  to  heat,  is  not  inhibited  by  trypsin  and  has  a  molecular 
weight  c 10,000  daltons.  (Mercado) . 

HELMINTHIC  INFECTIONS      Immune  response  in  human  schistosomiasis: 

Antibodies  to  a  glycoprotein  fraction  from  adult 
schistosome  worms  when  measured  by  the  ELISA 
procedure  were  found  to  correlate  with  intensity  of  infection.  (Nash  and 
Lunde) .   The  levels  of  antibody  in  patients  at  various  time  stages  of  their 
infection  were  related  to  antigens  from  appropriate  stages  of  the  parasite  - 
e.g.  higher  levels  to  cercarial  antigen  in  acute  disease  and  higher  levels 
to  adult  worm  antigen  in  chronic  cases.   Although  IgE  to  schistosome  antigens 
can  be  measured  by  the  ELISA  technique,  it  is  not  as  sensitive  as  conven- 
tional procedures.  (Lunde  and  Ottesen) . 

Schistosomal  antigens  and  neurotransmitters:   After  labelling  _in  vitro 
cultured  schistosomes  with  radioactive  amino  sugars  excretory-secretory  (E-S) 
materials  have  been  studied.   Addition  of  various  metabolic  inhibitors  to 
the  system  can  either  increase  or  decrease  the  release  of  large  and  small 
molecular  weight  substances  into  the  medium.   This  approach  is  being  used 
to  study  synthesis  and  turnover  of  schistosome  tegument  as  well  as  antigenic 
E-S  components  of  the  parasite.  (Nash).   The  kinetics  of  uptake  and  metabol- 
ic pathways  leading  to  synthesis  of  serotonin,  a  major  neurotransmitter  of 
schistosomes,  have  been  defined  in  the  schistosomule  which  is  the  early 
developmental  stage.   Previous  work  on  serotonin  was  with  adult  worms.  (Catto) , 

Experimental  pathogenesis  of  schistosomiasis:   Collagen  content  of  the 
liver  in  rabbits  with  Symmers '  fibrosis  due  to  S.  japonicum  infection 
increased  greatly  up  to  30  weeks  after  infection.   The  liver  pathology  did 
not  continue  to  progress  beyond  this  time  because  of  death  of  worms  and 
decreased  oviposition  of  remaining  worms.   Markedly  different  intestinal 
lesions  in  rabbits  were  found  to  be  produced  by  a  Japanese  as  contrasted  to 
a  Phillipine  strain  of  S_;_  japonicum.   The  Phillipine  strain  produced  serosal 
tumor-like  masses  and  hence  egg  excretion  did  not  reflect  intensity  of 
infection.   It  will  be  important  to  determine  whether  such  differences  in 
oviposition  exist  in  human  infections.  (Cheever) .   The  substance  from  schisto- 
somal egg  granulomas  which  was  found  to  stimulate  collagen  synthesis  and 
fibroblast  proliferation  of  mammalian  cells  was  found  to  be  distinct  from 
soluble  egg  antigen,  and  was  found  to  also  increase  c-AMP  and  prostaglandin 
E  2  synthesis  in  cell  cultures.   (Wyler,  Wahl,  Cheever  and  Wahl). 

Snail  vectors  of  schistosomiasis:   Now  a  total  of  as  many  as  12 
genetically  different  susceptibility  types  of  Biomphalaria  glabrata  snails 
to  S^  mans on i  have  been  demonstrated.   Another  mechanism  for  insusceptibile 
snails  has  been  found  in  which  the  miracidia  penetrate  but  fail  to'  develop 
despite  an  apparent  absence  of  host  tissue  reaction.   This  type  of  insus- 
ceptibility is  being  referred  to  as  "insuitability"  of  the  vector  snail, 
in  contrast  to  the  "resistant"  snails  in  which  miracidia  are  encapsulated 
and  destroyed  by  amebocytes.   This  "resistant"  type  of  insusceptibility  can 
be  reversed  by  preinfection  of  the  snail  with  a  heterologous  trematode 
parasite,  such  as  Echinostoma  paraensei,  but  the  "unsuitable"  type  of 
insusceptibility  is  not  altered  by  heterologous  preinfection.  (Richards). 


25-6 


Three  different  genetic  stocks  of  B^  glabrata  are  being  tested  for  their 
ability  to  develop  resistance  of  the  4  most  commonly  used  molluscicides. 
Successive  generations  of  survivors  of  exposures  to  calculated  90  percent 
lethal  doses  are  being  reared  for  tests  of  possible  molluscidide  resistance. 
(Sullivan) . 

Host  response  to  filarial  infection:   Patients  with  tropical  pulmonary 
eosinophilia  who  have  no  circulating  microf ilarial  (mf)  because  they  are 
efficiently  trapped  in  the  lungs  represent  one  extreme  of  the  human  response 
to  filarial  infection.   At  the  other  extreme  are  those  who  are  asymptomatic 
and  circulate  abundant  mf .   This  hypo-responsiveness  appears  to  involve 
serum  inhibitory  factors.   These  latter  patients  may  also  have  specific 
cellular  immune  hypo-responsiveness  which  appears  related  to  both  serum  and 
suppressor  cell  factors.  (Ottesen) .   Complement-enhanced  leukocyte 
adherence  has  been  demonstrated  with  mf  and  immune  serum.   Chromium- labelled 
mf  of  Ih_  immitis  are  being  studied  for  more  precise  information  of  their 
clearance  in  immune  animals.  (Weil  and  Ottesen). 


25-7 


Laboratory  of  Parasitic  Diseases 

National  Institute  of  Allergy  and  Infectious  Diseases 

SUMMARY  -  October  1,  1978  through  September  30,  1979 

HONORS  AND  AWARDS 

Dr.  Louis  H.  Miller  continues  to  serve  on  the  editorial  boards  of 
Experimental  Parasitology,  Journal  of  Parasitology  and  Journal  of  Molecular 
Medicine.   He  is  a  member  of  the  Steering  Committee  for  Immunology  of  Malaria 
of  the  TDR  of  WHO,  and  was  elected  to  membership  in  the  American  Society  for 
Clinical  Investigation. 

Dr.  Louis  S.  Diamond  was  appointed  a  member  of  the  Executive  Committee 
of  the  Society  of  Protozoologists  and  serves  on  the  editorial  board  of 
Experimental  Parasitology. 

Dr.  Mattern  serves  on  the  editorial  board  of  the  Journal  of  Protozoology. 

Dr.  Cheever  is  a  member  of  the  editorial  board  of  the  American  Journal 
of  Tropical  Medicine  and  Hygiene. 

Dr.  Dwyer  serves  on  the  editorial  board  of  the  Journal  of  Protozoology 
and  was  re-appointed  as  adjunct  Associate  Professor  at  Rockefeller  University 
and  at  the  University  of  Massachusetts. 

Dr.  Neva  completed  several  years  of  service  on  the  editorial  board  of 
the  Journal  of  Infectious  Diseases  and  continues  as  a  member  of  the  editorial 
board  of  the  American  Journal  of  Tropical  Medicine  and  Hygiene.   He  was 
re-appointed  Visiting  Lecturer  at  the  Harvard  School  of  Public  Health  and 
the  Johns  Hopkins  School  of  Hygiene  and  Public  Health.   He  is  a  member  of 
the  Steering  Committee  of  the  NAS-Institute  of  Medicine's  study  on  Clinical 
Investigation  in  Developing  Countries. 

Dr.  Ottesen  served  as  Chairman  of  the  NIAID  Clinical  Research  Committee 
and  began  to  serve  on  the  editorial  board  of  Experimental  Parasitology.   He 
was  an  invited  participant  in  a  WHO  Immunology  Course  given  in  Lausanne, 
Switzerland  and  has  been  asked  to  serve  as  a  member  of  the  WHO  TDR  Scientific 
Working  Group  on  Immunology  of  Filar iasis.   He  was  also  elected  to  the 
Infectious  Disease  Society  of  America. 


25-8 


SMITHSONIAN   SCIENCE    INFORMATION   EXCHANGi 
PROJECT  NUMBER    (Do   NOT   use   this   space) 


U.S.   DEPARTMENT   OF  I   PROJECT   NUMBER 

HEALTH.    EDUCATION.    AND   WELFARE  nnnQ1     ,7    T  __ 

PUBLIC   HEALTH   SERVICE  ZOl   AI    00091-17    LPD 

NOTICE  OF 

INTRAMURAL  RESEARCH   PROJECT 


PERIOD   COVERED 
I  October   1,    1978    to  September   30,    1979 


TITLE  OF   PROJECT   (30  characters   or  less,) 
The  Genetics,    Biology,    and   Control   of   Snail  Intermediate  Hosts   of   Schistosomes 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:     C.  S.  Richards  Parasitologist,  LPD,  NIAID 

Other:   J.  T.  Sullivan  Visiting  Fellow,  LPD,  NIAID 


COOPERATING  UNITS  (if  any)  Dr.  K.  J.  Lie  and  Dr.  Donald  Heyneman,  The  George  Williams 
Hooper  Foundation,  University  of  CA. ,  Dr.  Ernesto  Ruiz,  San  Juan  Laboratories, 
Bureau  of  Laboratories,  CDC,  San  Juan,  P.R.   Dr.  David  Woodruff,  Dr.  Phil 
LoVerde,  Ms.  Madeleine  Fletcher,  and  Mr.  Dennis  Minchella,  Dept.  of  Biology, 


LAB/8RANCH 
Laboratory  of  Parasitic  Diseases 


SECTION 

Host-Parasite  Relations  Section 


INSTITUTE  AND  LOCATION 

NIAID,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

39/12 


PROFESSIONAL: 

21/12 


18/12 


CHECK  APPROPRIATE  BOX(ES) 
C  (a)  HUMAN  SUBJECTS 

□  (a!)  MINORS   Q  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


(2  (c)  NEITHER 


SUMMARY  OF  WORK  (200  *ords  or  less  -  underline  keywords) 

Genetic  variations  occur  in  susceptibility  to  infection  in  Biomphalaria 
glabrata  and  inf ectivity  in  Schistosoma  mansoni.   Studies  indicate  at  least  12 
genetically  different  susceptibility  types  in  jJ.  glabrata.   Thirteen  strains 
or  substrains  of  S_.   mansoni  apparently  differing  genetically  in  snail  infectivity 
are  being  compared.   Ten  of  these  are  of  Puerto  Rican  origin. 

Two  different  mechanisms  of  insusceptibility  to  S_.  mansoni  in  various  genetic 
stocks  of  B.    glabrata  are  being  compared:   active  resistance  with  amebocytic 
encapsulation  and  destruction,  and  unsuitability  for  parasite  development  in 
the  absence  of  observed  host  tissue  reaction.   Genetic  tendency  in  B.  glabrata 
for  amebocytic  accumulations,  and  relationship  between  this  and  insusceptibility 
to  infection  with  S_.   mansoni  are  being  studied. 

B_.  glabrata,  when  infected  with  x-irradiated  miracidia  of  certain  species  of 
trematodes,  acquires  specific  resistance  to  a  subsequent  challenge  with 
nonirradiated  miracidia  of  the  same  species.   In  addition,  preinfection  with 
25-9 

P-S-6040 
(Rev.  10-76) 


Serial  No-  Z01  AI  00091-17  LPD 
Cooperating  Units  (continued): 

Purdue  Univ.,  Dr.  M.  R.  Kasschau,  Dept.  of  Physics,  M.D.  Anderson  Hospital 
and  Tumor  Inst.,  University  of  Texas. 

Summary  (continued) : 

nonschistosome  miracidia  interfers  with  the  active  resistance  of  the 
snails  to  S_.  mansoni,  rendering  them  temporarily  susceptible.   These 
phenomena  are  being  studied  with  the  trematodes  Echinostoma  paraensei 
and  Ribieroa  marini. 

The  ability  of  three  stocks  of  B_.  glabrata  to  develop  resistance  to  the 
4  most  commonly  used  molluscicides  is  being  compared.   Successive  genera- 
tions of  survivors  of  molluscicide  exposures  are  being  reared  and  tested 
for  molluscicide  resistance. 

Project  Description: 

The  objective  is  to  study  molluscan  and  trematode  genetics  with 
particular  attention  to  applications  to  control  measures.   To  study  genetic 
variations  in  snail  susceptibility  and  parasite  infectivity.   To  study 
host-parasite  interaction,  including  molluscan  cellular  defence  mechanisms 
and  unsuitability  for  parasite  development.   To  study  snail  amebocytes 
particularly  as  related  to  defence  against  parasites.   To  study  prospective 
agents  for  biological  control  of  intermediate  hosts  of  schistosomes.   To 
study  causes  of  tumors  in  mollusks.   To  study  the  biology  of  the  snails  with 
the  aim  of  improving  the  efficiency  of  control  measures.   To  study  the 
genetics  of  molluscicide  resistance  in  snails.   To  study  the  contributions 
and  complications  of  molluscan  diseases  and  molluscan  genetics  to  bio- 
medical research. 

Occurrence  in  15.  glabrata  of  two  different  pigmentation  genes  on 
different  chromosomes  has  facilitated  formal  genetic  studies.   Each  gene  has 
three  alleles  (wildtype,  blackeye,  and  albino;  coalesced  mantle  pigment, 
discrete  mantle  spots,  and  absence  of  black  mantle  pigment)  with  combina- 
tions resulting  in  nine  phenotypes.   Six  morphological  single  gene 
characters  in  B_.  glabrata  have  now  been  described,  apparently  all  on 
different  chromosomes.   Crosses  will  be  performed  to  determine  if  linkage 
occurs  between  any  of  these  morphological  characters  and  factors 
regulating  susceptibility  to  S_.  mansoni  infection.   Attempted  matings 
between  each  of  three  stocks  of  B.  glabrata  from  different  localities  in 
Puerto  Rico  and  ten  genetically  different  laboratory  stocks  of  J3.  glabrata, 
have  indicated  general  incompatibility.   Hybrid  F]_s  have  resulted  with 
only  two  of  the  laboratory  stocks. 

Need  for  evaluation  of  the  role  of  genetics  in  snail  control  continues. 
Resistant  B_.  glabrata  may  prove  useful  in  biological  control  of  schisto- 
somiasis by  dilution  or  replacement  of  populations  of  susceptible  snails  in 
endemic  areas.   Resistant  B.  straminea  may  be  effective  in  interspecific 


25-10 


Serial  No.  Z01  AI  00091-17  LPD 

competition.   Genetic  resistant  and  susceptible  stocks  of  B.  glabrata 
have  been  provided  Mr.  Dennis  Minchella,  Purdue  University,  for  laboratory 
competition  studies.   Progress  in  understanding  causes  of  susceptibility  or 
insusceptibility  of  j3.  glabrata  to  infection  with  S_.  mansoni  accelerates 
the  prospect  of  safe  field  testing  of  resistant  snail  stocks  for  control. 

Individual  snails  of  various  J3.  glabrata  stocks  are  exposed  as 
juveniles,  young  adults,  and  old  adults  to  miracidia  of  a  series  of  S. 
mansoni  strains.   Selection  is  carried  out  until  snail  lines  demonstrating 
consistent  susceptibility  or  insusceptibility  patterns  are  established. 
Snails  of  genetically  characterized  stocks  are  exposed  to  miracidia  of 
various  S_.  mansoni  strains  and  substrains,  with  selection  until  consistent 
infectivity  patterns  are  characterized.   Results  indicate  about  12 
genetically  different  susceptibility  types  in  15.  glabrata;  about  13  geneti- 
cally different  infectivity  strains  in  _S.  mansoni.   Ten  of  the  S_.  mansoni 
strains  or  substrains  are  of  Puerto  Rican  origin,  involving  collaboration 
with  the  San  Juan  Laboratories,  CDC,  P.R.  (Dr.  Ernesto  Ruiz).   Snail 
crosses  and  parasite  crosses  are  in  progress  or  planned  to  determine  the 
genetic  factors  involved.   Establishing  genetic  B_.  glabrata  and  S_.   mansoni 
lines  provides  material  for  biochemical,  biological  and  cytogenetic 
intraspecif ic  analyses.   Such  genetic  lines  have  been  provided  on  request 
to  many  other  investigators.   In  collaborative  studies  at  Purdue  University, 
Dr.  David  Woodruff  and  Ms.  Madeleine  Fletcher  are  pursuing  allozyme  studies 
on  our  genetic  lines  of  13.  glabrata  and  S.  mansoni. 

Studies  on  the  mechanisms  involved  in  snail  trematode  interactions 
are  continuing.   When  a  free  swimming  j5_.  mansoni  miracidium  in  freshwater 
penetrates  a  potential  host  snail,  it  encounters  a  drastic  osmotic  change  in 
the  snail  hemolymph.   Ability  to  adapt  to  this  change  may  involve  the  free 
amino  acids  of  parasite  and  host.   A  former  LPD  guest  worker,  Dr.  Margaret 
Kasschau  (Univ.  of  Texas)  studied  the  free  amino  acids  of  cercariae  and 
adults  of  S_.  mansoni.   Collaborative  studies  are  planned  to  compare  the  free 
amino  acids  of  j3.  glabrata  hemolymph  and  S_.  mansoni  of  genetically 
characterized  lines. 

Two  basically  different  mechanisms  of  insusceptibility  to  S_.  mansoni 
occur  in  host-parasite  combinations  involving  various  genetic  lines  of 
_B.  glabrata  and  S_.  mansoni.   Active  resistance  involves  host  recognition 
with  rapid  amebocytic  encapsulation  and  destruction.   Unsuitability 
involves  delayed  or  aborted  parasite  development  in  the  absence  of  observed 
host  tissue  reaction.   The  occurrence  of  these  phenomena  is  being  surveyed 
in  various  _B.  glabrata-S.  mansoni  combinations,  and  snail  crosses  are 
in  progress  to  study  the  genetics  of  these  different  host-parasite  relations. 

Two  nonschistosome  trematodes  that  infect  _B.  glabrata  are  now  cultured 
in  our  laboratory:   Echinostoma  paraensei  and  Ribieroa  marini.   With 
these  parasites  we  are  extending  the  studies  of  Lie  and  his  coworkers,  who 
have  demonstrated  acquired  resistance  to  trematodes  in  B_.  glabrata. 
Snails  sensitized  by  infection  with  x- irradiated  R.  marini  miracidia 
acquire  resistance  to  a  challenge  with  nonirradiated  R.  marini  miracidia 

25-11 


Serial  No.  Z01  AI  00091-17  LPD 

given  7  to  10  days  subsequently.   This  resistance  is  acquired  as  soon 
as  3  days,  and  persists  for  at  least  3  weeks  post-exposure  to  irradiated 
miracidia.   This  marks  the  first  independent  confirmation  of  acquired 
resistance  in  molluscans  and  is  the  first  demonstration  of  this  phenomenon 
outside  of  the  Echinostomatidae.   Initial  attempts  to  interfere  with 
insusceptibility  to  S_.  mansoni  (by  first  infecting  insusceptible  snails 
with  irradiated  E.  paraensei  and  then  challenging  with  nonirradiated 
S_.  mansoni)  have  indicated  that  this  procedure  will  block  insusceptibility 
that  is  due  to  active  resistance,  but  not  that  due  to  unsuitability. 

Amebocytes  play  an  important  role  in  defence  of  _B_.  glabrata  against 
infection  by  S_J  mansoni.   Studies  on  occurrence  of  genetic  amebocytic 
accumulations  are  continuing.   In  some  genetic  lines  of  B_.  glabrata 
transitory  amebocytic  accumulations  occur  in  the  atrium  in  young  adults, 
during  the  period  of  maximum  reproductive  activity.   When  exposed  to  S_. 
mansoni,  these  snails  test  susceptible  as  juveniles,  insusceptible  as 
young  adults  with  atrial  amebocytic  accumulations,  and  revert  to  suscepti- 
bility in  old  age.   Ultramicroscopic  studies  on  these  amebocytic  accumula- 
tions are  planned  in  collaboration  with  Purdue  University  (Dr.  Phil  LoVerde) . 

Microsporidia,  amebae,  and  bacteria  cause  extensive  damage  in  some 
snail  hosts.   Search  for  viral  or  fungal  infections  are  also  in  progress. 
These  and  other  snail  diseases  need  to  be  examined  for  potential  human 
infection,  and  prospective  use  as  agents  in  biological  control  of  snails. 
It  is  essential  to  be  aware  of  the  occurrence  of  these  infections  and 
their  relations  with  the  snails  and  other  parasites  to  plan,  conduct,  and 
interpret  experimental  research  on  snails  with  validity. 

Studies  on  a  variety  of  tumors  observed  in  _B.  glabrata,  many  of  which 
are  genetic,  will  be  continued. 

An  inbred  laboratory  stock  of  _B.  glabrata,  the  M  line  albino,  and  two 
stocks  recently  sent  to  us  from  field  collections,  one  from  Puerto  Rico 
and  the  other  from  St.  Lucia,  are  being  tested  for  resistance  to  the 
molluscicides,  Bayluscide,  Frescon,  potassium  pentachlorophenate,  and 
copper  sulfate.   Successive  isolation  of  and  rearing  of  offspring  from 
survivors  of  molluscicide  exposures  over  several  generations  should 
result  in  some  degree  of  resistance.   This  resistance  will  be  characterized 
statistically  with  the  use  of  a  potency  probit  analysis  computer  program, 
and  the  genetics  of  its  transmission  will  be  studied.   Currently,  dosage- 
mortality  regression  plots,  e.e.,  LC^q  determinations,  are  being  established 
for  the  4  molluscicides  in  each  of  the  3  nonselected  parental  stocks. 

Publications: 

1.  Kassim,  0.  0.  and  Richards,  C.  S.:  Schistosoma  mansoni:  Lysozyme 
activity  in  Biomphalaria  glabrata  during  infection  with  two  strains  of 
the  parasite.   Exp.  Parasit.,  46:   213-217,  1978. 


25-12 


Serial  No.  Z01  AI  00091-17  LPD 

2.  Kassim,  0.  0.  and  Richards,  C.  S.:   Biomphalaria  glabrata:   Lysozyme 
activities  in  the  hemolymph,  digestive  gland  and  headfoot  of  the  inter- 
mediate host  of  Schistosoma  mansoni.   Exp.  Parasit.,  4_6:   218-224,  1978. 

3.  Kassim,  0.  0.  and  Richards,  C.  S.:  Radioisotope  labelling  of 
Schistosoma  mansoni  miracidia  for  in  vivo  studies  in  Biomphalaria 
glabrata.   J.  Invert.  Pathol.,  (in  press,  1979). 

4.  Kassim,  0.  0.  and  Richards,  C.  S.:   Host  reactions  in  Biomphalaria 
glabrata  to  Schistosoma  mansoni  miracidia,  involving  variations  in 
parasite  strains,  numbers  and  sequence  of  exposures.   Internat.  Jour,  for 
Parasit.  (in  press,  1979). 

5.  Lie,  K.  J.,  Heyneman,  D.,  and  Richards,  C.  S.:   Specificity  of 
natural  resistance  to  trematode  infections  in  Biomphalaria  glabrata. 
Internat.  Jour,  for  Parasit.  (in  press,  1979). 


25-13 


SMITHSONIAN    SCIENCE    INFORMATION    EXCHANGE|  U.S.    DEPARTMENT   OF  PROJECT   NUM8ER 

PROJECT   NUMBER    (Do   NOT    use    this    sDace)      jHEALTH,    EDUCATION,    AND    WELFARE 

P&eUS0T^TcVSERV,C£  ZOl  AI    00092-13    LPD 

I    INTRAMURAL   RESEARCH    PROJECT 


PERIOD    COVERED 

October  1,  1978  to  September  30,  1979 


i TITLE  OF  PROJECT  (30  characters  or  less) 

The  pathogenesis  of  schistosome  infections  in  mammalian  hosts. 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


Other: 


A.  W.  Cheever  Assistant  Chief,  LPD,  NIAID 

T.  E.  Nash  Medical  Officer,  LPD,  NIAID 

E.  A.  Ottesen  Medical  Officer,  LPD,  NIAID 

D.  J.  Wyler  Medical  Officer,  LPD,  NIAID 

M.  N.  Lunde  Research  Zoologist,  LPD,  NIAID 


cooperating  units  (if  any)   Southwest  Foundation  for  Research  &  Education,  San  Antonio, 
TX  (Dr.  Kuntz) ;  Dept.  Gastroenterol.  WRAIR  (Dr.  Dunn);  Lab.  Statistical  and 
Math.  Methodology,  DCRT,  NIH  (Dr.  Mosimann,  Mrs.  Minker) ;  Div.  Immuno- 
parasitology,  NMRI  (Dr.  Dean) 


LAB/8RANCH 

Laboratory  of  Parasitic  Diseases 


SECTION 

Host  Parasite  Relations 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Md.  20205 


TOTAL  MANYEARS: 

2.5 


PROFESSIONAL: 
1.0 


1.5 


CHECK  APPROPRIATE  BOX(ES) 
G  (a)   HUMAN  3U8JECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


3  (b)  HUMAN  Tl 


U  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Schistosome  infections  are  studied  quantitatively  in  man  and  experimental 
animals.   Schistosoma  japonicum  infections  in  rabbits  regularly  produced 
Symmers'  clay  pipestem  fibrosis  of  the  liver,  but  this  was  often  accompanied 
by  a  mixed  macronodular-micronodular  cirrhsis.   Morphologic  fibrosis  and 
collagen  content  decreased  between  30  and  50  weeks  of  infection.   The  size  of 
hepatic  granulomas  around  eggs  remained  nearly  constant,  but  several  factors 
combined  to  drastically  reduce  the  number  of  eggs  reaching  the  liver.   Hepatic 
fibrosis  was  similar  in  rabbits  infected  with  Japanese  and  Philippine  strains 
of  the  parasite.   The  passage  of  eggs  in  the  feces  parallelled  the  intensity 
of  infection  in  rabbits  infected  with  the  Japanese,  but  not  the  Philippine, 
worm  strain,  apparently  because  most  eggs  were  not  deposited  near  mucosal 
surfaces  by  the  latter. 


25-14 


PHS-6040 

[Rev.    10-76) 


Serial  No.  Z01  AI  00092-13  LPD 

The  nature  and  pathogenesis  of  lesions  produced  in  mammalian  hosts 
by  schistosomes  of  medical  importance  are  studied  in  man  and  experimental 
animals.   Schistosome  infections  are  quantitated  by  measurement  of  worm 
numbers,  passage  of  eggs  in  excreta  and  accumulation  of  eggs  in  host 
tissues. 

Symmers'  fibrosis  of  the  liver  was  produced  in  rabbits  with  heavy 
S.  japonicum  infection.   Hepatic  fibrosis,  evaluated  histologically  and  by 
measurement  of  hydroxyproline  content  of  the  liver,  decreased  markedly 
between  30  and  50  weeks  after  infection,  confirming  earlier  microscopic 
findings  of  Japanese  investigators.   Several  factors  combine  to  decrease 
the  insult  to  the  liver  during  this  time:  1)  worm  numbers  decrease 
slightly  2)  the  number  of  eggs  laid  by  each  worm  pair  decreased  by  about 
50%  and  3)  the  proportion  of  eggs  laid  which  reach  the  liver  decreased 
markedly.   Modulation  of  granuloma  size  was  minimal,  although  this  is 
an  important  factor  in  moderating  pathology  in  several  host  species 
infected  with  _S.  mansoni.   Cirrhosis  in  infected  rabbits  appears  to  be 
caused  by  the  S_.  japonicum  infection,  a  situation  without  a  counterpart 
in  man  or  other  experimental  animals.   This  is  a  major  drawback  to  this 
model;  but  it  remains  the  only  experimental  model,  other  than  the  chimpanzee, 
of  Symmers1  fibrosis  of  the  liver. 

Passage  of  eggs  in  the  feces  of  rabbits  infected  with  a  Japanese  strain 
of  S.  japonicum  is  proportion  to  worm  numbers,  but  this  is  not  the  case 
with  a  Philippine  strain,  in  which  many  less  eggs  are  passed.   This 
appears  to  be  explained  by  focal  deposition  of  eggs  in  the  intestinal 
serosa  by  the  Philippine  strain.   A  few  rabbits  infected  with  the 
Japanese  strain  and  showing  similar  anatomic  lesions  had  similar  patterns 
of  egg  passage.   Passage  of  eggs  in  the  feces  is  the  only  currently 
available  technique  for  measuring  intensity  of  schistosome  infection  in 
man,  so  that  if  generally  valid  this  finding  has  clinical  importance. 
We  are  therefore  comparing  the  two  worm  strains  in  capuchin  monkeys  to  see 
if  this  pattern  holds  for  primates.   Collagen  synthesis  in  the  liver  and 
cell  mediated  immune  responses  are  also  being  examined  in  the  monkeys  in 
collaboration  with  Dr.  Ottesen  (LPD)  and  Dr.  Dunn  (WRAIR) . 

Schistosome  infections  appear  to  produce  much  more  fibrosis  in  the 
liver  than  in  the  gut,  although  egg  deposition  is  similar  in  the  two 
organs.   We  are  pursuing  this  at  the  descriptive  level  and  quantitating 
intestinal  fibrosis  through  measurement  of  hydroxyproline  levels. 

The  study  of  bladder  "cancers"  produced  by  S_.  haematobium  in  capuchin 
monkeys  is  nearly  terminated.   The  papillary  lesions  of  the  bladder 
epithelium  regressed  with  time  (2  to  5  years  after  infection)  and  are 
probably  inflammatory  rather  than  neoplastic  in  nature.   Regression  of  the 
lesions  occurred  as  the  schistosome  infection  disappeared.   The  monkeys 
had  considerable  resistance  to  reinfection  and  active  infections  of  high 
intensity  could  not  be  maintained. 


25-15 


Serial  No.  Z01  AI  00092-13  LPD 

During  the  next  year  the  reversibility  of  hepatic  fibrosis  in  S. 
japonicum  infected  rabbits  will  be  examined  following  chemotherapy,  with 
or  without  subsequent  colchicine  treatment  (Dr.  Dunn).   The  biology, 
pathology  and  immunology  of  S_.  japonicum  and  S.  mansoni  infections 
in  capuchin  monkeys  will  be  studied.   Pathology  and  collagen  metabolism 
in  inbred  strains  of  mice  will  be  looked  at  in  relation  to  their  resistance 
to  schistosome  infection  (Dr.  Dean,  NMRI  and  Dr.  Dunn). 

Understanding  of  the  lesions  caused  by  schistosomiasis  in  man,  the 
associated  morbidity  and  their  relation  to  intensity  of  infection  are 
basic  to  evaluating  the  importance  of  the  disease  and  to  some  aspects  of 
control  and  treatment.   The  detailed  study  of  animal  models  contributes 
to  our  understanding  of  the  parasite  and  its  possible  interactions  with 
the  human  host. 

1.  Ottesen,  E.  A.,  Hiatt,  R.  A.,  Cheever,  A.  W. ,  Sotomayor,  Z.  R.  and 
Neva,  F.  A.:   The  acquisition  and  loss  of  antigen-specific  cellular  immune 
responsiveness  in  human  schistosomiasis.   Clin.  Exp.  Immunol.  33:  38-47, 
1978. 

2.  Mosimann,  J.  E. ,  Malley,  J.  D.,  Cheever,  A.  W.  and  Clark,  C.  B.: 
Size  and  shape  analysis  of  schistosome  egg-counts  in  Egyptian  autopsy 
data.   Biometrics  34:   341-356,  1978. 

3.  Kamel,  I.  A.,  Elwi,  A.  M. ,  Cheever,  A.  W. ,  Mosimann,  J.  E.  and  Danner, 
R.:   Schistosoma  mansoni  and  ^_.  haematobium  infections  in  Egypt.   IV. 
Hepatic  lesions.   Am.  J.  Trop.  Med.  Hyg.  27:   931-938,  1978. 

4.  Cheever,  A.  W.:   Schistosomiasis  and  neoplasia.   J.  Nat.  Cancer  Inst. 
61:   13-18,  1978. 

5.  Nash,  T.  E.,  Ottesen,  E.  A.  and  Cheever,  A.  W.:   Antibody  response  to 
a  polysaccharide  antigen  in  schistosomiasis.   II  Modulation  of  antibody 
response.   Am.  J.  Trop.  Med.  Hyg.  27:   944-950,  1978. 

6.  Kuntz,  R.  E.,  Cheever,  A.  W. ,  Bryan,  G.  T.,  Moore,  J.  A.  and  Huang, 
T.  C:   Schistosomiasis:   Natural  history  of  papillary  lesions  in 

the  urinary  bladder  in  schistosomiasis.   Cancer  Research  38:   3836-3839, 
1978. 

7.  Boctor,  F.  N.,  Nash,  T.  E.  and  Cheever,  A.  W. :   Isolation  of  a  poly- 
saccharide antigen  from  Schistosoma  mansoni  eggs.   J.  Immunol.  122: 
39-43,  1979. 

8.  Lunde,  M.  N. ,  Ottesen,  E.  A.  and  Cheever,  A.  W.:   Serological  differ- 
ences between  acute  and  chronic  schistosomiasis  mansoni  detected  by 
enzyme-linked  immunosorbent  assay.  Am.  J.  Trop.  Med.  Hyg.  28:   87-91,  1979. 


25-16 


Serial  No.  Z01  AI  00092-13  LPD 

9.   Kassim,  0.  0.,  Cheever,  A.  W.  and  Richards,  C.  S.   Schistosoma 
mansoni:   comparison  of  infections  in  mice  with  different  strains  of 
worms.  Exp.  Parasitol.   (in  press) . 


25-17 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Oo  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  ANO  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  Of 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  AI  00093-04  LPD 


PERIOD   COVERED 
October    1,    1978    to    September    30,    1979 


(TITLE   OF    PROJECT   (30   characters   or   less) 

Studies   of    the   Immunologic   Response    to   Helminth   Infections 


NAMES,  LABORATORY  ANO  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


Other: 


E.  A.  Ottesen  Senior  Investigator,  LPD,  NIAID 

B.  A.  Catto  Research  Associate,  LPD,  NIAID 

G.  J.  Weil  Research  Associate,  LPD,  NIAID 

M.  N.  Lunde  Research  Zoologist,  LPD,  NIAID 

A.  W.  Cheever  Assistant  Chief,  LPD,  NIAID 

F.  A.  Neva  Chief,  LPD,  NIAID 

T.  J.  Lawley  Medical  Investigator,  NCI 


COOPERATING  UNITS  (if  any)  Tuberculosis  Chemotherapy  Center,  Madras,  India  (Dr.  S.P. 
Tripathy) ;  Medical  College  of  Madras,  India  (Prof.  K.V.  Thiruvengadam) .   Bio- 
medical Research  Institute,  Rockville,  Md.  (Dr.  F.A.  Lewis),  Bureau  of  Veterinary 
Research,  FDA,  Beltsville,  Md.  (Dr.  K.G.  Powers). 


LAB/3RANCH 

Laboratory   of   Parasitic   Digeases 


SECTION 

Host-Parasite  Relations 


INSTITUTE  AND  LOCATION 

NIAID,  Bethesda,  Md. 


TOTAL  MANYEARS: 

62/12 


PROFESSIONAL: 

38/12 


24/12 


CHECK  APPROPRIATE  30X(,£3) 
|3  (a)   HUMAN  SUBJECTS 

3  (a!)  MINORS   □  (a2)  INTERVIEWS 


(b)  HUMAN  TISSUES 


(c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  major  goals  of  this  project  are  to  characterize  the  host's  immune  response  to 
helminth  infections  and  to  relate  the  findings  to  the  pathogenesis  of  clinical 


disease.   Chronic  filariasis  and  schistosomiasis  are  both  characterized  by 
cellular  immune  hyporesponsiveness  to  parasite  antigens  which  may  play  a  role 
in  the  persistence  of  the  parasite  within  the  host.   The  mechanisms  involved  in 
this  hyporesponsiveness  include  both  serum  inhibitory  factors  and  mononuclear 
suppressor  cell  populations.   Profound  immunologic  hyperresponsiveness  of  the 
immediate  type  (IgE)  characterizes  the  asthma-like  tropical  eosinophilia 
syndrome  of  human  filariasis.   The  most  important  antigens  are  those  derived  from 
microfilariae.   By  contrast  patients  with  patent  microfilaremia  are  hyporespon- 
sive.   Clearance  of  these  microfilariae  depends  on  anti-surface  antibodies.   In 
acute  schistosomiasis  not  only  is  there  marked  cellular  and  humoral  immune 
activity  but  also  the  presence  of  large  numbers  of  circulating  immune  complexes 
which  may  be  important  in  the  syndrome's  pathogenesis. 


Larval  schistosomes (schistosomules)  contain  the  biogenic  amine,  serotonin. 
i uptake  mechanisms  and  metabolic  precursors  have  been  defined. 


Its 


PHS-6040 
(Rev.  IO-76) 


25-18 


Serial  No.  Z01  AI  00093-04  LPD 

Project  Description: 

The  character  and  evolution  of  the  immune  response  in  man  and  experi- 
mental animals  has  been  studied  during  and  following  infection  with  parasitic 
helminths . 

Filariasis .   The  questions  of  why  some  individuals  in  an  endemic  area 
become  infected  with  filaria  (bancrofti)  while  others  appear  to  resist  it 
and  of  why  even  among  infected  individuals  the  clinical  manifestations  of 
disease  can  be  so  varied  have  led  to  a  search  for  the  relevant  immunologic 
parameters  both  in  field  studies  of  human  filariasis  and  in  work  on  labor- 
atory models  of  the  disease.   Field  studies  in  the  Cook  Islands  (Project 
//J0233-I-74)  and  in  India  (Project  #76-1-231)  have  shown  that  in  endemic 
areas  the  greatest  cellular  immune  reactivity  (lymphocyte  transformation)  and 
the  highest  production  of  antibodies  to  filarial  antigens  (determined  with 
ELISA  techniques)  occur  not  in  infected  patients  but  in  those  equally  exposed 
to  infective  parasites  but  who  have  not  acquired  the  infection.   In  fact, 
there  exists  in  infected  individuals  a  state  of  specific  cellular  immune 
unresponsiveness  to  filarial  antigens  which  appears  to  be  determined  both  by 
serum  inhibitory  factors  and  by  suppressor  cells  which  can  be  identified 
among  the  adherent  subpopulation  of  mononuclear  peripheral  blood  cells .   The 
nature  of  these  suppressive  elements  is  currently  being  explored.  (Ottesen) 
Also  being  studied  is  the  pathogenesis  of  the  recurrent  attacks  of  lymph- 
angitis (i.e.,  filarial  fevers)  and  the  possible  role  of  immune  complexes  in 
initiating  them  (Ottesen,  Lawley) . 

Tropical  eosinophilia  (TE)  is  a  form  of  filariasis  characterized  by 
chronic  lung  disease  with  episodes  of  paroxysmal  nocturnal  asthma,  profound 
blood  eosinophilia,  markedly  elevated  IgE  levels  and  high  filarial  antibody 
titers.   Using  the  in  vitro  correlate  of  IgE-mediated  allergic  responses 
(i.e. ,  the  histamine  release  reaction  of  IgE-coated  basophils  challenged  in 
vitro  with  the  sensitizing  allergen)  and  a  radioenzymatic  assay  for  the 
detection  of  histamine,  we  have  documented  and  quantified  the  high  degree  of 
allergic  sensitization  to  filaria  in  patients  with  TE  (compared  to  those  with 
other  forms  of  filarial  disease) .   The  especial  hypersensitization  to  micro- 
filariae and  their  products  may  be  most  important  in  determining  the  symptoms 
and  pathology  associated  with  this  syndrome  (Ottesen,  Neva) .   At  the  other 
extreme,  individuals  with  circulating  microfilariae  who  might  be  expected  also 
to  be  allergically  sensitized  were  paradoxically  hyporesponsive  to  antigens 
derived  from  microfilariae.   The  mechanisms  underlying  this  hyporesponsiveness 
appear  to  involve  serum  inhibitory  factors  whose  nature  is  under  study 
(Ottesen) . 

Microfilaria  (ME)  have  been  used  as  a  model  for  the  study  of  immunity  to 
tissue  helminths.   In  the  canine  and  human  systems  most  infected  individuals 
have  antibody  directed  against  soluble  microfilarial  antigens.   Individuals 
who  are  immune  to  the  microfilarial  stage  (infected  but  amicrofilaremic)  have 
circulating  antibody  directed  against  microfilarial  surface  structures.   In 
the  presence  of  immune  serum  normal  peripheral  blood  leukocytes  adhere  to  MF 


25-19 


Serial  No.  Z01  AI  00093-04  LPD 

in  vitro .   Cell  adherance  is  enhanced  by  the  presence  of  complement  and 
accompanied  by  lysosomal  enzyme  release  onto  the  surface  of  the  MF  (Weil, 
Ottesen) . 

Experiments  involving  the  infusion  of  radiolabeled  MF  of  Dirof ilaria 
immitis  into  dogs  have  shown  that  injected  MF  circulate  freely  in  normal 
and  microf ilaremic  infected  dogs,  with  relative  concentration  in  the 
microvasculature.   MF-immune  dogs  clear  injected  MF  from  the  blood  within 
fifteen  minutes.   The  MF  are  primarily  trapped  in  the  lung  (Weil,  Powers, 
Ottesen) . 

Schistosomiasis .   Previous  studies  in  our  laboratory  have  defined  the 
progressive  loss  of  parasite  antigen-specific  cellular  immune  responsiveness 
(lymphocyte  transformation)  in  patients  whose  schistosome  infections  evolve 
from  the  acute  to  chronic  stages.   As  we  have  explored  the  mechanisms 
involved  in  this  modulation  of  antigen  responsiveness,  it  has  become  clear 
that  just  as  in  the  patients  with  filariasis,  individuals  with  chronic  S^. 
mansoni  infections  have  inhibitory  elements  both  in  their  serum  and  in  their 
adherent  mononuclear  cell  populations.   The  serum  and  cell  suppressive  effects 
may  be  additive  and  their  natures  are  being  investigated  (Ottesen) . 

Serum  samples  from  patients  followed  closely  for  almost  two  years  after 
developing  acute  schistosomiasis  have  been  studied  for  the  presence  of  CI 
binding  immune  complexes.   All  patients  with  symptomatic  acute  schisto- 
somiasis had  circulating  immune  complexes  which  either  declined  gradually  as 
the  infection  became  chronic  or  declined  rapidly  after  treatment.   The 
finding  of  circulating  complexes  in  patients  with  chronic  infection  was  much 
less  common  (2  of  11  patients)  than  it  was  in  acutely  infected  individuals. 
Studies  to  determine  the  antigenic  make-up  of  these  complexes  and  their 
potential  role  in  the  pathogenesis  of  the  acute  syndrome  are  in  progress 
(Lawley,  Ottesen) . 

Biochemical  studies  on  the  major  neurotransmitter  of  schistosomes  (i.e., 
serotonin)  have  defined  the  presence  of  this  amine  in  the  schistosomule  stage 
of  the  organism.   The  kinetics  of  uptake  and  matabolic  pathways  leading  to  its 
synthesis  have  been  defined  (Catto) .   Cercariae  of  _S.  mansoni  upon  penetration 
of  the  skin  release  a  factor  causing  discharge  of  amine-rich  granules  from 
tissue  mast  cells.   This  factor  which  is  not  found  in  the  post-penetration 
schistosomules  is  likely  responsible  for  the  schistosome  dermatitis  which 
develops  after  initial  exposure  to  infective  schistosomes  (Catto,  Lewis, 
Ottesen) . 

Publications: 

1.  Ottesen,  E.  A.,  Hiatt,  R.  A.,  Cheever,  A.  W. ,  Sotomayor,  and  Neva,  F.  A.: 
The  Acquisition  and  Loss  of  Antigen-Specific  Cellular  Immune  Responsiveness 
in  Acute  and  Chronic  Schistosomiasis  in  Man.   Clin.  Exp.  Immunol.   33: 
38-47,  1978. 

2.  Ottesen,  E.  A.  and  Weller,  P.  F. :   Eosinophilia  Following  Treatment  of 
Patients  with  Schistosomiasis  mansoni  and  Bancroft's  filariasis.   J.  Inf. 
Pis.   139:   343-347,  1979. 

25-20 


Serial  No.  Z01  AI  00093-04  LPD 

3.  Ottesen,  E.  A.,  Neva,  F.  A.,  Paranjape,  R.  S.,  Tripathy,  S.  P., 
Thiruvengadam,  K.  V.  and  Beaven,  M.  A. :   Specific  Allergic  Sensitization  to 
Filarial  Antigens  in  Tropical  Eosinophilia  Syndrome.   Lancet  I_:   1158-1161, 
1979. 

4.  Nash,  T.  E.,  Ottesen,  E.  A.,  and  Cheever,  A.  W. :   Antibody  Response  to  a 
Polysaccharide  Antigen  Present  in  the  Schistosome  Gut.   II.   Modulation  of 
Antibody  Response.   Am.  J.  Trop.  Med.  Hyg.   27:   944-950,  1978. 

5.  Neva,  F.  A.  and  Ottesen,  E.  A.:   Tropical  (Filarial)  Eosinophilia.   New 
Eng.  J.  Med.   298:   1129-1131,  1979. 

6.  MacQueen,  J.  M. ,  Ottesen,  E.  A.,  Ottesen,  C. ,  Amos,  D.  B.,  and  Ward, 
F.  E. :   HLA  Histocompatibility  Antigens  in  a  Polynesian  Population  -  Cook 
Islanders  of  Mauke.   Tissue  Antigens  13:   121-128,  1979. 

7.  Lunde,  M.  N. ,  Ottesen,  E.  A.,  and  Cheever,  A.  W. :   Serological  Differences 
Between  Acute  and  Chronic  Schistosomiasis  mansoni  Detected  by  Enzyme  Linked 
Immunosorbant  Assay  (ELISA) .   Am.  J.  Trop.  Med.  Hyg.   28:   87-91,  1979. 

8.  Coolidge,  C. ,  Weller,  P.  F. ,  Ramsey,  P.  G. ,  Ottesen,  E.  A.,  Beaver,  P.  C. 
and  von  Lichtenberg,  F.  C. :  Zoonotic  Brugia  Filariasis  in  New  England.  Ann. 
Int.  Med.   90:   341-343,  1979. 

9.  Ottesen,  E.  A.   Modulation  of  the  Host  Response  in  Human  Schistosomiasis 
II.   Adherent  Suppressor  Cells  which  Inhibit  Lymphocyte  Proliferative 
Responses  to  Parasite  Antigens.   J.  Immunol.   (In  press). 

10.  Ottesen,  E.  A. :   Filarial  Infection  and  the  Host  Response  in  Man. 
Paradoxes  and  Insights.   In  Escape  from  Immune  Surveillance:   The  Interface 
Between  Immune  Mechanisms  and  Disease.   D.  B.  Amos,  R.  S.  Schwartz  and  B.  W. 
Janicki,  eds.   Academic  Press  (In  press). 

11.  Ottesen,  E.  A. :   Visceral  Larva  Migrans  and  Other  Migratory  Helminths 
of  Man.   In  Principles  and  Practice  of  Infectious  Disease,  Mandell,  G.  L. , 
Douglas,  R.  G. ,  and  Bennett,  J.  E.  eds.  J.  Wiley  and  Sons,  New  York  (In 
press) . 

12.  Catto,  B.  A.  and  Ottesen,  E.  A.:   Serotonin  Uptake  in  Schistosomules  of 
Schistosoma  mansoni.   Comp.  Biochem.  Physiol.  (In  press) . 

13.  Lawley,  T.  J.,  Ottesen,  E.  A.,  Hiatt,  R.  A.  and  Gazze,  L.  A.:  Circulat- 
ing Immune  Complexes  in  Acute  Schistosomiasis.   Clin.  Exp.  Immunol.  (In  press). 

14.  Lunde,  M.  N. and  Ottesen,  E.  A.:   Enzyme-linked  immunosorbent  assay  (ELISA) 
for  detecting  IgM  and  IgE  antibodies  in  human  schistosomiasis.   Am.  J.  Trop. 
Med .  Hyg .   (In  press) . 

15.  Cohen,  S.  G.  and  Ottesen,  E.  A.:   "Eosinophils  in  immune  function"  in 
Oppenheim,  J.,  Rosenstreich,  D.  and  Potter,  M.  (eds).   Cell  Biology  of 
Immunity  and  Inflammation,  Harvard  Elsevier-North  Holland,  Inc.   (In  press). 

25-21 


SMITHSONIAN    SCIENCE    INFORMATION    EXCHANGE! 
PROJECT   NUMBER    (Do   NOT    use    this    soacej 


J. 3.    DEPARTMENT   OF 
HEALTH,    EDUCATION,    AND    WELFARE 
PUBLIC   HEALTH   SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH   PROJECT 


PROJECT  NUMBER 

ZOl  AI   00094-20   LPD 


PERIOD    COVERED 

S ep t ember    30,    1978   to   October    1,    1979 


TITLE  OF   PROJECT   (30  characters   or  less) 
Studies   of   Entamoeba  histolytica   and  other   Parasitic   Protozoa 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


L.  S.  Diamond 


Head,  Parasite  Growth  and  Differentiation  Section, 
LPD,  NIAID 


Other:   F.  D.  Gillin  Senior  Staff  Fellow,  LPD,  NIAID 

D.  B.  Keister  Biologist,  LPD,  NIAID 

E.  C.  Weinbach  Head,  Physiology  and  Biochemistry  Section,  LPD,  NIAID 
C.  F.  T.  Mattern  Medical  Officer,  LPD,  NIAID 


COOPERATING  UNITS  (if  any)   --._  .-.. 

American  Type  Culture  Collection,  Rockville,  MD;  Hospital  General,  Centro 
1  edico  Nacional,  I.M.S.S.,  Mexico  City. 


LAB/ BRANCH 

Laboratory  of  Parasitic  Diseases 


SECTION 

Parasite  Growth  and  Differentiation 


NST1TUTE  ANO  LOCATION 

NIAID,  Bethesda,  MD   20205 


TOTAL  MANYEARS: 
4.0 


PROFESSIONAL: 
1.3 


2.7 


CHECK  APPROPRIATE  BOX(ES) 
S  (a)   HUMAN  SUBJECTS 

D  (a!)  MINORS  "J   (*2)  INTERVIEWS 


H  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Yeast  extract  an  ingredient  of  TYI-S-33  medium  devised  for  axenic  cultivation  of 
Entamoeba  histolytica  has  been  replaced  with  a  mixture  of  defined  nutrients 
consisting  of  adenine,  guanine ,  cytosine,  uracil,  AMP,  ADP,  ATP,  and  NH^Cl  for- 
tified with  B_  vitamins.   A  method  has  been  devised  enabling  rapid  direct 
axenization  of  E.  histolytica  from  cultures  in  which  the  parasite  is  grown  with 
a  mixed  bacterial  flora  and  thus  circumvents  the  intermediate  step  of  growing  ^ 
the  amebae  monoxenically  prior  to  axenization.   Butyric  acid  added  to  the  medium 
used  for  axenization  is  a  key  factor  in  success  of  this  technique.   An  improved 
medium  for  axenic  cultivation  of  Giardia  lamblia  has  been  achieved  by  means  of 
supplementing  TYI-S-33  medium  with  crude  bovine  bile  preparations  containing  free 
and  conjugated  bile  acids.   Growth  is  superior  to  that  attained  with  TP-S-1 
currently  the  medium  of  choice  for  this  parasite.   Modification  of  a   technique 
devised  in  this  laboratory  has  enabled  cyropreservation  of  refractory  strains 
of  E.  histolytica.   Success  is  attributed  to  the  substitution  of  sucrose  for 
glucose  in  the  cryopreservant ,  the  use  of  TYI-S-33  for  cultivation  of  the  amabae 


before  and  after  freezing  and  the  use  of  plastic  vials  in  the  place  of  glass. 
25-22 

3HS-6040 
(Rev.  IO-76) 


Serial  No.  Z01  AI  00094-20  LPD 

Project  Description: 

The  objectives  of  this  project  are  5  fold:   1)  to  develop  and  refine 
techniques  for  the  axenic  cultivation  of  Entamoeba  histolytica,  related 
Entamoeba  and  other  parasitic  protozoa;  2)  to  determine  their  nutritional 
requirements  in  vitro  and  to  define  the  physical  and  chemical  conditions  for 
optimal  growth;  3)  to  study  the  mechanisms  of  their  pathogenicity;  4)  to  study 
and  characterize  the  viruses  of  E.  histolytica;  5)  to  devise  or  improve 
existing  methods  for  the  freeze-preservation  of  these  parasites  at  cryogenic 
temperatures  and  to  study  mechanisms  involved  in  resistance  to  freeze-preser- 
vation. 

1.  Development  of  a  Defined  Medium  for  Axenic  Cultivation  of  E.  histoly- 
tica  -  There  are  3  undefined  components  of  TYI-S-33  the  medium  currently  used 
in  our  laboratory  for  axenic  cultivation:   Trypticase  (casein  digest),  bovine 
serum  and  yeast  extract  (YE) .   Current  investigations  are  aimed  at  replacing 
YE  with  defined  nutrients.   A  highly  purified  (98%)  commerical  preparation  of 
yeast  mucleic  acids  (YNA)  was  found  to  support  amebic  growth  provided  it  was 
supplemented  with  the  equivalent  concentrations  of  the  B  vitamins  present  in 
yeast.   A  mixture  of  adenine,  guanine,  cytosine,  uracil,  AMP,  ADP  and  ATP  in 
the  proportions  present  in  yeast  supported  serial  subculture  (25  to  date) 
when  substituted  for  YNA.   The  yields  of  amebae  although  considerably  lower 
than  those  obtained  with  YNA  were  equal  to  the  best  yields  obtained  with 

the  older  TP-S-1  medium.   Glutamic  acid,  glutamine  and  NH.C1  when  added 
separately  to  the  above  mixture  of  purines,  pyrimidines  and  nucleosides 
further  enhanced  growth  increasingly  in  the  order  presented.   Addition  of 
cholesterol  and  lecithin  alone  or  in  combination,  or  a  standard  mixture  of 
trace  metals  did  not  enhance  growth.   Subtle  effects  could  have  been  masked 
by  the  bovine  serum  and  Trypticase  which  are  rich  sources  of  these  nutrients. 

Projected  future  studies  entail  determination  of  the  optimal  concen- 
trations of  the  defined  nutrients  shown  to  be  of  value  in  replacement  of  YNA, 
testing  of  additional  defined  components  of  yeast,  and  attempts  to  replace 
trypticase  and  bovine  serum  with  defined  nutrients  (Diamond  and  Cunnick) . 

2.  Iron  and  Nutrition  of  E.  histolytica  -  Diamond  (1978)  postulated  that 
the  protozoa,  none  of  which  have  been  shown  to  produce  siderophores,  could 
conceivably  use  preformed  siderophores  synthesized  by  other  microorganisms  in 
the  environment  as  do  certain  bacteria  and  algae.   In  TYI-S-33  there  are  two 
sources  of  iron,  that  which  occurs  in  the  three  biological  ingredients  and 
had  been  designated  intrinsic  iron,  and  that  which  is  present  in  the  ferric 
ammonium  citrate  (FAC)  supplement,  extrinsic  iron.   When  established  cultures 
of  E.  histolytica  growing  in  TYI-S-33  were  deprived  of  extrinsic  iron  through 
deletion  of  FAC,  the  rate  of  growth  decreased  and  stabilized  after  a  few 
subcultures  at  a  lower  rate.   If  in  addition  the  intrinsic  iron  was  chelated 
with  Desferal,  a  potent  microbial  siderophore,  growth  was  marginal.   However, 
if  FAC  was  combined  with  sufficient  Desferal  to  bind  all  iron  and  was  added 

to  the  medium  in  which  the  intrinsic  iron  was  previously  bound  with  Desferal, 
growth  of  the  amebae  was  significantly  increased.   This  increase  was  below  that 
obtained  with  medium  containing  intrinsic  iron  alone,  but  well  above  that  ob- 

25-23 


Serial  No.  Z01  AI  00094-20  LPD 

tained  with  the  Desferal  bound  intrinsic  iron.   This  suggests  that  the  ameba 
can  remove  some  iron  from  the  Desferal  complexes  in  vitro  and  could  possibly 
meet  some  of  its  iron  requirements  in  vivo  through  use  of  preformed  microbial 
siderophores  (Diamond  and  Keister) . 

Direct  axenization  of  E.  histolytica  from  Mixed  Bacterial  Cultures  -  A 
method  has  been  developed  enabling  rapid  direct  axenization  of  E.  histolytica 
from  cultures  in  which  the  parasite  in  grown  in  the  presence  of  a  mixed 
intestinal  flora  (the  customary  culture  technique  for  isolating  the  organism 
from  the  host)  and  circumvents  the  laborious  intermediate  step  of  growing  the 
amebae  monoxenically  is  association  with  a  trypanosomatid  or  Fusobacterium 
symbiosus.   Key  factors  in  this  technique  are  as  follows:   1)  Use  of  a  newly 
devised  liquid  medium  for  growing  the  amebae  with  bacteria  before  axenization. 
The  medium  is  specially  designed  to  match  TYI-S-33  ingredients  and  tonicity 
in  order  to  facilitate  transfer  of  the  amebae  from  xenic  to  axenic  conditions 
of  growth.   2)  Supplementation  of  TYI-S-33  with  butyric  and  isobutyric  acids 
during  the  axenization  phase.   We  have  noted  that  bacterial  flora  which  produce 
butyric  acid  significantly  enhance  growth  of  several  species  of  Entamoeba. 
Recently  Scheff  (1978)  reported  that  the  ability  of  Fusobacterium  (see  above) 
and  allied  species  to  produce  butyric  acid  was  related  to  their  ability  to 
support  monoxenic  growth  of  the  E.  histolytica- like  Laredo  strain  of  ameba. 
Attempts  will  be  made  to  axenize  other  entamoebae,  specifically  E.  coli  and 
E.  gingivalis .   Neither  of  these  species  from  man  have  been  axenized  (Diamond 
and  Keister) . 

Improved  Medium  for  Axenic  Cultivation  of  Giardia  lamblia  -  Studies 
originating  in  this  laboratory  have  shown  that  although  TYI-S-33  medium  is 
far  superior  to  TP-S-1  for  axenic  cultivation  of  E.  histolytica,  the  reverse 
is  true  for  G.  lamblia.   Except  for  the  incorporation  of  yeast  extract  in  the 
former  medium  and  Panmede,  an  ox-liver  digest,  in  the  latter,  the  two  media  are 
essentially  similar  in  respect  to  the  other  ingredients.   The  association  of 
G_.  lamblia  with  the  gall  bladder  in  human  giardiasis,  the  presence  of  bile 
constituents  in  Panmede,  and  the  observation  that  partial  substitution  of 
Panmede  for  the  yeast  extract  in  TYI-S-33  improved  its  ability  to  support 
growth  of  G.  lamblia  led  to  exploration  of  the  use  of  bile  in  the  cultivation 
of  this  parasite.   TYI-S-33  was  supplemented  singly  with  the  following 
commercial  preparations:   1)  curde  bile  containing  taurocholic  acid  (40%), 
glyocholic,  cholic  and  deoxycholic  acids  in  lower  condentrations ;  2)  dehydrated 
unfractionated  bovine  bile,  and  3)  bacteriological  grade  of  bovine  bile 
consisting  of  a  mixture  of  free  and  conjugated  bile  acids.   Growth  of  G. 
lamblia  in  the  presence  of  each  of  these  supplements  was  markedly  superior  to 
that  obtained  with  TP-S-1.   The  fact  that  a  synthetic  taurocholic  acid  of 
high  purity  (98%)  failed  to  stimulate  growth  suggests  that  enhancement  of 
growth  by  the  crude  preparations  was  not  due  alone  to  this  particular  compound. 
Addition  of  a  quantity  of  Panmede  to  TYI-S-33  equal  in  weight  to  each  of  the 
above  supplements  was  not  sufficient  to  stimulate  growth.   This  suggested 
that  multiple  factors  in  Panmede  were  responsible  for  its  growth  stimulating 
effect,  or  that  the  growth  enhancing  factors  were  present  in  low  concentrations. 
Future  studies  will  attempt  to  define  the  factors  in  bile  which  are  responsible 
for  stimulation  of  growth.   Specific  fractions  commercially  available  or 

25-24 


Serial  No.  Z01  AI  00094-20  LPD 

prepared  in  the  laboratory  will  be  studied.   Combinations  of  conjugated  and 
free  bile  acids  in  varying  proportions  will  be  tested  (Keister  and  Diamond) . 

Cryopreservation  of  E.  histolytica  -  Modification  of  a  technique  originated 
in  this  laboratory  has  led  to  the  cryopreservation  of  amebal  strains  which 
have  been  refractory  in  previous  repeated  trials.   Success  is  attributed 
principally  to  having  1)  substituted  sucrose  for  glucose  as  one  of  the  con- 
stituents of  the  cryopreservant  employed,  2)  the  use  of  TYI-S-33  instead  of 
TP-S-1  for  cultivation  of  the  amebae  prior  to  freeze-preservation,  and  as  the 
basic  freezing  and  recovery  vehicle,  3)  the  use  of  plastic  vials  in  place  of 
glass.   The  following  specific  attributes  of  TYI-S-33  have  been  found  to  play 
vital  roles  in  the  success  of  freeze-preservation.   Cells  in  late  logarithmic 
or  stationery  phase  are  generally  most  suitable  for  cryopreservation.   The 
relatively  long  logarithmic  and  stationery  phases  which  are  characteristic 
of  TYI-S-33  and  the  smooth  transition  between  the  two  enables  one  to  select- 
ively and  unhurriedly  choose  and  process  vigorous  viable  organisms.   Under  the 
most  ideal  conditions  devised  to  date,  the  recovery  of  viable  E_.  histolytica 
immediately  after  thawing  is  in  the  order  of  a  few  percent.   The  ability  to 
routinely  initiate  cultures  of  E.  histolytica  with  a  few  hundred  amebae  has 
enabled  us  to  obtain  vigorous  growing  cultures  in  100%  of  the  samples  thawed. 
Future  projected  investigations:   Improvement  in  the  numbers  of  amebae  recovered 
can  be  made  by  refinement  of  the  cooling  rates  and  procedures  for  thawing. 
Attention  will  be  given  to  optimizing  these  aspects  of  the  technique.   A  major 
effort  will  be  put  into  exploration  of  the  use  of  membrane  stabilizers,  such 
as  spermine  and  spermidine,  in  an  attempt  to  minimize  the  loss  of  cell  membrane 
integrity  which  is  a  common  result  of  freezing  injury  leading  to  death  of  the 
amebae  (Diamond,  Claggett,  Gillin,  Keister  and  Cunnick) . 

Role  of  Iron  and  Nutritional  Immunity  in  Human  Amebic  Disease  -  A 
clinical  and  laboratory  study  aimed  at  identifying  the  role  of  iron  in  the 
pathogenesis  of  human  amebic  disease  which  was  initiated  in  1977  in 
collaboration  with  staff  members  of  the  Hospital  General,  Centro  Medico 
Nacional,  I.M.S.S.,  Mexico  City  has  been  halted  temporarily  due  to  changes  in 
the  status  of  key  hospital  personnel.   However,  collating  of  acquired  data 
in  in  progress  and  should  be  ready  for  analysis  in  the  near  future. 

For  results  of  collaborative  studies  with  investigators  in  other  sections 
within  the  laboratory  see  the  following  annual  reports:   Project  No.  Z01  AI 
00098-23-LPD,  PI  E.  C.  Weinbach,  Project  No.  Z01  AI  00185-01-LPD,  PI  C.  F.  T. 
Mattern. 

Publications: 


1.  Diamond,  L.  S. ,  Harlow,  D.  R. ,  and  Cunnick,  C.  C:  A  new  medium  for  the 
axenic  cultivation  of  Entamoeba  histolytica  and  other  Entamoeba.  Trans .  Roy . 
Soc.  Trop.  Med.  &  Hyg.  72_:   431-432,  1978. 

2.  Diamond,  L.  S.,  Harlow,  D.  R. ,  Phillips,  B.  P.,  and  Keister,  D.  B.: 
Entamoeba  histolytica,  iron  and  nutritional  immunity.   Arch.  Invest.  Med.  (MEX) 
9  (Suppl.  1):   329-338,  1978.  '    ~  

25-25 


Serial  No.  Z01  AI  00094-20  LPD 

3.  Diamond,  L.  S.,  Tanimoto-Weki,  M. ,  and  Martinez-Palomo ,  A.:   Production  of 
cecal  lesions  in  new  born  guinea  pigs  with  axenically  cultivated  Entamoeba 
histolytica.   Arch.  Invest.  Med.  (MEX)  9_   (Suppl.  1):   223-228,  1978. 

4.  Mattern,  C.  F.  T.,  Keister,  D.  B.,  and  Diamond,  L.  S.:   Experimental 
Amebiasis  IV:   Amebal  viruses  and  the  virulence  of  Entamoeba  histolytica. 
Amer.  J.  Trop.  Med.  &  Hyg.   28:   653-657,  1979. 

5.  Gillin,  F.  D. ,  and  Diamond,  L.  S.:   Clonal  growth  of  Entamoeba 
histolytica  and  other  Entamoeba  in  agar.   J.  Protozool.   25:   539-543,  1978. 

6.  Gillin,  F.  D. ,  and  Diamond,  L.  S.:   Clonal  growth  of  Entamoeba  in  agar: 
Some  applications  of  this  technique  to  the  study  of  their  cell  biology. 
Arch.  Invest.  Med.   (MEX)  _9  (Suppl.  1):   237-246,  1978. 

7.  Weinbach,  E.  C. ,  Claggett,  C.  E.,  Takeuchi,  T.,  and  Diamond,  L.  S.: 
Biological  oxidations  and  flavoprotein  catalysis  in  Entamoeba  histolytica. 
Arch.  Invest.  Med.   (MEX)  9_   (Suppl.  1):   89-98,  1978. 

8.  Takeuchi,  1.,  Weinbach,  E.  C,  Gottlieb,  M.  and  Diamond,  L.  S.: 
Mechanism  of  L-serine  metabolism  in  Entamoeba  histolytica.  Comp.  Biochem. 
Physiol.  B.  Comp.  Biochem.  621B:   281-285,  1979. 

9.  Gillin  G.  D.,  and  Diamond,  L.  S.:   Entamoeba  histolytica  and  Entamoeba 
invadens :   Effects  of  temperature  and  oxygen  tension  on  axenic  growth. 
Experimental  Parasitology  (In  press). 

10.  Gillin,  F.  D.  and  Diamond,  L.  S.:   Clonal  growth  of  Giardia  lamblia 
trophozoites  in  a  semi-solid  Agarose  medium.   Journal  of  Parasitology 
(In  press) . 


25-26 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  sDace) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  AI   00097-21  LPD 


PERIOD   COVERED  ,  „„       ,.__ 

October    1,    1978    to   September   30,    1979 


TITLE  OF    PROJECT   (30   characters   or   less) 

Physiological  and   Cytochemical  Pathology   of   Parasitic  Diseases 


NAMES,   LABORATORY   AND    INSTITUTE  AFFILIATIONS,    AND   TITLES   OF    PRINCIPAL    INVESTIGATORS   AND   ALL   OTHER 
PROFESSIONAL   PERSONNEL   ENGAGED   ON   THE  PROJECT 

PI:      Teresa   I.   Mercado     Research  Physiologist,    LPD,    NIAID 


COOPERATING  units  (if  any)    Ms.   Alba  Colon-Whitt   and  Dr.    Theodore   S.    Theodore, 
Laboratory   of    Streptococcal  Diseases,    NIAID,    Clinical  Pathology  Dept.,    NIH 


LAB/BRANCH 

Laboratory  of  Parasitic  Diseases 


SECTION 

Physiology  and  Biochemistry 


INSTITUTE  AND  LOCATION 

NIAID,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

12/12 


PROFESSIONAL: 

12/12 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (a1 )  MINORS   □  (a2)  INTERVIEWS 


0  (b)  HUMAN  TISSUES 


□  (c)   NEITHER 


SUMMARY   OF   WORK   (200   words   or  less   -   underline   keywords) 

During    the   course   of    studies   related    to   the   structure   and    function   of   parasite 
(Trypanosoma   cruzi)    membranes,    contamination  of    a   parasite   suspension  with 
a  motile,    rod-like   organism,    identified   as   Pseud omonas    f luorescens,    occurred 
which   caused    lysis    of    the   flagellates.      In  view  of    the   potential   significance 
of    this   observation  not   only    in   terms    of    cellular   biology   and    physiology 
in  general,    but  more   specifically,    because   of    its    impact   on  parasite   chemo- 
therapy  and/or    immunology   it  was   deemed    advisable    to   study   this    interaction 
more   extensively.      Our   findings   are  described    in   this   report. 


25-27 


3HS-6040 
(Rev.    10-76) 


Serial  No.  Z01  AI  0097-21  LPD 

Project  Description: 

The  Tulahuen  strain  of  Trypanosoma  cruzi  was  used.   Trypomastigotes 
were  isolated  from  blood  employing  column  chromatography  (Mercado,  T.  and 
Katusha,  K.  1979.   Prep.  Biochem. ,  9_:    97-106).   Pseudomonas  fluorescens  was 
cultured  in  nutrient  broth  and  resuspended  in  phosphate  buffer-saline 
(pH  7.4)  to  an  0D  of  0.1  at  570  nm.   A  chemically-defined  medium  containing 
sodium  glutamate,  mannitol,  Na  ,  K  ,  Ca"1^",  and  Mg"^"  was  also  used.   The 
cultures  were  incubated  for  3,  18,  24,  48,  and  72  hours.   They  were  centri- 
fuged  at  3,000  rpm  (RC3  Sorvall)  for  10  min. ,  and  the  supernatants  were 
filtered  through  Millipore  (0.45  urn).   The  filtrates  were  checked  further 
for  sterility  by  incubation  in  blood  agar  plates  and  nutrient  broth.   To 
examine  the  trypanosome-bacterium  (TB)  interaction  we  mixed  0.15  and  0.30 
ml  respectively  of  a  parasite-buffer  suspension  (e.g.,  25  X  10°/ml)  with 
0.30  ml  of  the  Pseudomonas  filtrates.   They  were  examined  immediately  after 
mixing,  after  10  minutes,  and  after  12  and  24  hours.   The  mixtures  were 
maintained  at  4°  C.   In  another  series  the  bacteria  were  sonicated  in 
buffer-saline  glucose  and  this  material  as  well  as  the  supernatant 
fractions,  unfiltered  and  filtered,  were  used  in  the  assays.   Our  assays 
were  based  on  examination  of  a  drop  of  the  TB  mixtures  for  not  longer  than 
a  minute  because  of  the  possibility  that  drying  of  the  cover-slipped 
preparations  would  interfere  with  an  accurate  appraisal  of  the  parasites. 
They  were  assessed  mainly  on  the  basis  of  shape  and  motility.   Light  and 
phase  microscopy  were  used.   For  permanent  preparations  smears  were  air 
dried,  fixed  in  methanol,  and  stained  with  Giemsa.   In  order  to  ascertain 
further  the  occurence  of  a  bacterial  exof actor,  we  concentrated  the  active 
fractions  following  filtration  of  the  supernatant  samples.   This  was  done 
in  3  ways:   (1)  filtrate  was  concentrated  10  times,  (2)  filtrate  was 
dialyzed  against  distilled  water  (three,  250  ml  changes)  and  the  dialyzate 
concentrated  as  described  in  (1),  (3)  filtrate  dialyzed  as  in  (2)  and  the 
dialyzate  lyophilized  and  reconstituted  in  saline-glucose  buffer.   The 
trypomastigotes  were  isolated  immediately  after  bleeding  and  the  TB  mixtures 
assayed  on  the  same  day.   All  buffers  and  the  DEAE  cellulose  column  were 
treated  with  gentamicin  at  a  final  concentration  of  50  micrograms /ml.   The 
anti-trypanosomal  activity  of  the  filtrates  was  also  assessed  following 
incubation  at  56°  C  for  30  minutes,  boiling  (5  min.),  freezing  at  -20°  C  and 
following  treatment  with  trypsin  (1  mg/ml)  for  30  min.   Approximation  of 
the  molecular  size  of  the  exofactor  was  made  by  filtration  employing  filters 
of  different  porosities.   An  important  consideration  from  these  observations 
was  whether  or  not  the  modified  parasites  would  induce  infection  and/or 
be  protective  against  a  challenge  with  parasitized  blood.   Four  animals 
were  inoculated  intraperitoneally  with  8  X  10"  trypomastigotes  of  which 
only  5%  were  typically  motile,  lanceolate  organisms.   In  another  experiment, 
2  animals  were  inoculated  with  0.4  and  0.5  ml  respectively  of  a  suspension 
of  sonicated  cells.   The  animals  were  challenged  after  one  month  with 
freshly-drawn  blood  containing  1  and  2  X  10"  trypomastigotes. 

In  the  presence  of  P.  fluorescens,  trypomastigotes  of  T_.  cruzi  are  con- 
siderably immobilized.  HJsually  one  bacterium  approaches  a  single  flagellate 
and  with  persistent  to  and  fro  movements  directed  to  the  posterior  end 


25-28 


Serial  No.  Z01  AI  00097-21  LPD 

(kinetoplast-f lagellar  site)  it  quickly  subdues  the  flagellate.   Giemsa- 
stained  preparations  revealed  a  rounding-up  of  the  parasite  following  the 
initial  encounter;  the  flagellum  appeared  wrapped  around  the  cell  body  and 
as  the  incubation  time  increased  disintegration  of  this  structure  and  of 
the  nucleus  occurred.   The  cell  bodies  later  became  considerably  reduced 
in  size  and  assumed  bizarre  triangular  and  rounded  shapes.   Filtrates  of 
supernatant  fractions  of  the  centrifuged  bacterial  cultures  caused 
immobilization  and  accompanying  cellular  disintegration  within  12  hours; 
however,  an  almost  instantaneous  paralysis  followed  by  lysis  occurred  when 
the  filtrates  were  concentrated.   Cell  membranes  were  scattered  about 
and  some  parasites  though  still  intact,  were  noticeably  swollen  and  their 
protoplasm  became  transparent  with  an  apparent  loss  of  organelle  boundaries. 
Cultures  grown  in  nutrient  broth  exhibited  an  optimal  immobilizing  effect 
after  incubation  for  48  hours;  the  immobilization  response  however,  was 
also  observed  following  incubation  for  18  and  24  hours.   In  contrast  to 
nutrient  broth,  media  36,  though  a  good  medium  for  the  culture  of  P. 
f luorescens,  was  poor  for  the  production  of  the  lytic  factor.   Only  in  media 
36  inoculated  from  broth  cultures  was  the  lytic  factor  produced  in  sufficient 
amounts  to  elicit  the  immobilization  response  and  lysis  of  the  parasites. 
Preliminary  experiments  on  the  characterization  of  the  lytic  factor  dis- 
closed that  it  is  resistant  to  heat,  freezing,  is  not  inhibited  by  trypsin, 
and  has  a  molecular  weight  lower  than  10,000  daltons.   We  did  not  observe 
typical  Tulahuen  strain  parasitemias  in  animals  inoculated  with  the  T_. 
cruzi  bizarre  forms.   Three  of  4  animals  became  infected,  but  the  highest 
parasitemia  (observed  after  10  days)  was  29  per  30  microscopic  fields 
examined.   The  fourth  animal  did  not  become  infected.   No  alteration  of 
the  chronic  infection  was  elicited  following  a  challenge  with  1  X  10 
flagellates,  based  on  the  examination  of  a  drop  of  blood,  the  animals  had 
become  negative  after  33  days.   Similarly,  animals  inoculated  with  a 
suspension  of  P_.  f luorescens  sonicated  cells  did  not  develop  lethal 
infections  following  a  challenge,  after  one  month,  with  1  and  2  X  10"  tryp- 
omastigotes  respectively;  very  mild  parasitemias  were  observed  (e.g.,  6 
parasites  per  30  microscopic  fields) . 

Our  studies  on  the  characterization  of  the  lytic  factor  are  being 
continued.   However,  based  on  the  microscopic  observations  of  the  flagellate- 
bacterium  interaction,  in  the  apparent  absence  of  cell  lysis,  and  the 
significant  lysis  produced  by  concentrated  dialyzed  filtrates,  we  believe 
the  active  factor  is  secreted  extracellularly  and  the  observed  effects  are 
not  a  consequence  of  bacterial  cell  lysis.   This  assumption  is  substantiated 
further  by  the  fact  that  supernatant  fractions  of  sonicated  cells 
elicited  essentially  a  rounding-up  of  the  parasite  with  the  production 
of  bizarre  forms  and  not  a  lytic  effect.   The  extracellular  factor  have 
so  far  disclosed  certain  properties  which  are  comparable  to  some  _P. 
f luorescens  exoenzymes  reported  by  other  investigators. 

Although  our  animal  inoculation  results  are  only  preliminary,  they 
are  interesting  particularly  in  view  of  a  report  (Bomford,  R.  and  McHardy,  N. 
1979.   Parasitology  78:  77-87)  in  which  they  described  an  enhancement  of 


25-29 


Serial  No.  Z01  AI  00097-21  LPD 

the  protective  effect  of  a  T_.  cruzi  epimastigote  vaccine  in  the  presence 
of  the  bacterial  species,  Corynebacterium  parvum.   The  apparent  protective 
effect  resulting  in  our  study  of  the  P_.  f luorescens-T.  cruzi  interaction 
is  currently  being  examined  more  extensively.   The  influence  of  other 
bacterial  species  such  as  Escherichia  coli  and  P_.  aeruginosa  as  well  as 
species  of  African  trypanosomes  which  multiply  in  the  blood  stream,  not 
in  tissues,  will  also  be  studied.   Changes  produced  on  the  parasite  membrane 
will  also  be  studied  biochemically  and  cytochemically . 

The  present  study  though  not  projected  as  part  of  the  main  program 
related  to  the  physiological  and  cytochemical  pathology  of  parasitic 
disease  has  served  to  emphasize  the  importance  of  research  on  parasite 
interactions  and  their  potential  significance  leading  to  a  chemotherapeutic 
or  immunologic  control  of  parasitic  infections. 

Publications: 


1.  Mercado,  T.  I.  and  Katusha,  K.:   1979.   Isolation  of  Trypansoma  cruzi 
from  the  blood  of  infected  mice  by  column  chromatography.   Prep.  Biochem., 
9^:97-106. 

2.  Mercado,  T.  I.  and  Garbus,  J.:   Creatine  phosphokinase  isoenzymes  and 
Trypanosoma  cruzi  infections.   Comp,  Biochem.  Physiol.  (In  press). 

3.  Mercado,  T.  I.:   1979.   Observations  on  lactate  dehydrogenase  isozymes 
in  the  plasma  of  mice  infected  with  Trypanosoma  cruzi.  Isozyme  Bull. 
12:39. 


25-30 


SMITHSONIAN    SCIENCE    INFORMATION    EXCHANGE!  U.S.    DEPARTMENT   OF  |   PROJECT   NUMBER 

PROJECT  NUMBER    (Do   NOT   use   this   space)       HEALTH,    EDUCATION,    AND   WELFARE 

PUBLIC   HEALTH   SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH   PROJECT 


ZOl  AI    00098-23 


PERIOD   COVERED 

October    1,    1978    to   September    30,    1979 


TITLE  OF    PROJECT   [3Q   characters   or   less) 

Biochemical  mechanisms   of    energy  metabolism    in  mammalian  and   parasitic 

organisms. 


NAMES,    LABORATORY    AND    INSTITUTE   AFFILIATIONS,    AND    TITLES   OF    PRINCIPAL    INVESTIGATORS   AND    ALL   OTHER 

PROFESSIONAL   PERSONNEL   ENGAGED  ON   THE  PROJECT 

PI:     E.  C.  Weinbach   Head,  Physiology  and  Biochemistry  Section,  LPD,  NIAID 

Other:   L.  S.  Diamond    Research  Zoologist,  LPD,  NIAID 

C.  E.  Claggett   Biological  Laboratory  Technician  (Biochem.),  LPD,  NIAID 

D.  B.  Keister    Biologist,  LPD,  NIAID 

D.  M.  Dwyer      Research  Microbiologist,  LPD,  NIAID 


COOPERATING  UNITS  (if  any) 

Laboratory  of  Chemical  Physics,  NIAMDD  (H.  Kon  and  F.  Inoue) ;  Metabolism 
Branch,  NCI  (D.  Tschudy  and  P.  Ebert);  University  of  Stockholm,  Sweden 
(T.  Barnard) 


lab/branch 

Laboratory  of  Parasitic  Diseases 


section 

Physiology  and  Biochemistry 


INSTITUTE  AND  LOCATION 

NIAID,    Bethesda,   Maryland    20205 


TOTAL  MANYEARS: 

36/12 


PROFESSIONAL:  OTHEfi: 

22/12  14/12 


CHECK  APPROPRIATE  30X(ES) 
G  (a)   HUMAN  SUBJECTS  ~\   (b)  HUMAN  TISSUES  3  (c)  NEITHER 

D  (al)  MINORS   H  (a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  worts  or  less  -  underline  keywords) 

The  study  of  aerobic  energy  metabolism  in  parasites  and  mammals  was  continued 
with  efforts  devoted  primarily  to  elucidating  the  sequence  of  electron 
transfer  in  the  respiratory  chain  of  Entamoeba  histolytica.   Unique  bio- 
chemical properties  of  the  amebae  are  their  absence  of  heme  proteins,  large 
content  of  non-heme  iron  and  acid-labile  sulfide,  minute  amounts  of  quinones, 
and  absence  of  covalently  bound  flavins.   Hydrogen  peroxide  is  formed  only 
when  artificial  electron  carriers  mediate  electron  transfer  from  substrates 
to  molecular  oxygen,  indicating  that  iron-sulfur  centers  and  not  flavin  is  the 
final  electron  carrier.   Biochemical  studies  of  Giardia  lamblia  were  initiated 
this  year.   Respiratory  metabolism  of  G.  lamblia  resembles  that  of  E_.  histolytica 
by  its  lack  of  a  functional  tricarboxylic  acid  cycle  and  cytochromes; 
mediating  electron  transfer  by  f lavoproteins  and  non-heme  iron  proteins.   Only 
a  small  amount  of  acid-labile  sulfide  was  detected  in  G.  lamblia.   Differences 
in  the  locus  of  the  respiratory  enzymes  and  substrate  specificities  are 
clearly  evident  in  the  aerobic  metabolism  of  these  two  enteric  protozoa. 
Another  study  initiaed  this  year  is  the  examination  of  respiratory  metabolism 
25-31 

PHS-6040 
(Rev.  10-76) 


Summary  (continued):  Serial  No.  Z01  AI  00098-23  LPD 

of  kinetoplasts  from  Leishmania  donovani.   Respiration  was  supported  by 
Kreb's  cycle  substrates,  particularly  succinate,  and  the  presence  of 
functional  NADH  oxidoreductose  was  demonstrated.   Mammalian  studies 
centered  on  the  assessment  of  whether  protein  synthesis  or  respiration 
was  first  affected  during  cellular  heme  depletion  in  cultured  mur ine 
erythroleukemia  cells. 

Project  Description: 

The  object  of  this  project  is  to  conduct  fundamental  studies  on  the 
mechanisms  of  aerobic  energy  metabolism  in  mammalian  and  parasitic 
organisms.   Currently  these  studies  center  primarily  on  identification  and 
characterization  of  components  of  the  respiratory  chains  of  pathogenic 
protozoa,  and  elucidation  of  the  sequence  of  electron  flow  to  molecular 
oxygen. 

Tissues  and  cells  are  disrupted  mechanically  and  subcellular  fractions 
are  isolated  by  differential  and  gradient  centrifugations.   Cellular  and 
subcellular  constituents,  enzyme  activities,  and  metabolic  pathways  are 
determined,  characterized  and  elucidated  by  enzymatic,  chemical,  radio- 
chemical and  physical  methods.   Changes  in  oxygen  concentration  are 
determined  polarographically  with  the  Clark  electrode.   Electron  probe 
microanalysis  is  used  to  localize  elements  in  cestode  calcareous  corpuscles. 

Parasite  studies:   Continued  investigation  of  the  respiratory  chain 
in  Entamoeba  histolytica,  strain  HK-9,  cultivated  axenically  in  Diamond's 
liquid  medium,  was  focused  primarily  on  elucidating  the  sequence  of 
electron  flow  to  molecular  oxygen.   We  now  have  identified  the  following 
electron  carriers  in  E_.  histolytica:   nicotinamide  adenine  nucleotides, 
f lavoproteins,  minute  amounts  of  quinones,  and  iron-sulfur  centers. 
Combined  use  of  inhibitors  and  spectrophometric  analyses  have  shown  that 
the  sequence  of  electron  flow  is  from  reduced  nicotinamide  adenine 
nucleotides  (NAD(P)H)  to  flavins  to  iron-sulfur  centers  (Weinbach,  Takeuchi 
and  Claggett) .   Electron  paramagnetic  resonance  (EPR)  studies  and  direct 
chemical  analyses  have  revealed  the  presence  of  multiple  iron-sulfur 
centers  in  E.  histolytica.   These  centers  are  one-electron  tranfer  proteins 
which  lead  to  the  production  of  H2O.   Flavins,  in  contrast,  are  two- 
electron  transfer  carriers  and  lead  to  the  production  of  H2O2.   We  did  not 
detect  H2O2  as  an  end  product  of  aerobic  metabolism  in  E_.  histolytica; 
therefore,  we  conclude  that  the  terminal  acceptor  in  this  parasite  is  an 
iron-sulfur  center,  rather  than  flavin  as  claimed  by  others  (Weinbach  and 
Claggett).   Our  studies  of  the  enteric  protozoa  were  extended  to  another 
human  pathogen,  Giardia  lamblia.   In  collaboration  with  Diamond  and  Keister, 
the  axenically-cultivated  trophozoites  were  found  to  have  active  endogenous 
respiration  (32  nanomoles  02/min/mg  protein)  and  a  high  affinity  for  oxygen. 
Substrate  specificity  and  inhibitor  sensitivity  indicated  that  this 
eukaryote,  which,  like  E_.  histolytica,  also  lacks  mitochondria,  is  devoid 
of  tricarboxylic  acid  cycle  enzymes  and  cytochromes.   Heme  iron  could  not 
be  detected  by  chemical  or  EPR  analysis.   G.  lamblia  exhibits  unusual 


25-32 


Serial  No.  Z01  AI  00098-23  LPD 

EPR  signals  and  has  a  low  content  of  acid-labile  sulfide.   This  parasite 
may  contain  non-heme  proteins  of  the  rubredoxin-type  and  only  small 
amounts  of  the  f erred ox in- type  (Weinbach  and  Kon) .   Alcohol  dehydrogenase, 
which  is  exceedingly  active  in  _E.  histolytica,  is  absent  in  G.  lamblia. 
Sonicated  trophozoites  were  equally  effective  in  oxidizing  NADH  or 
NADPH,  demonstrating  the  presence  of  a  functional  NAD(P)H  oxidoreductase. 
Separation  of  the  particulate  and  soluble  fraction  of  G.  lamblia  disclosed 
that  the  particulate  fraction  contained  most  of  the  reductase  activity 
and  required  no  exogenous  carrier  to  mediate  electron  transfer  to  molecular 
oxygen.   Spectrof luorometric  analysis  showed  that  virtually  all  of  the 
flavin  was  acid-extractable  and  not  covalently  bound  (Claggett) .   Flavo- 
antagonists  inhibited  the  respiration  of  intact  trophozoites,  and 
NAD(P)H  oxidoreductase  activity.  Another  study  that  initiated  this  year 
resulted  from  a  "spin-off"  from  Dr.  Dwyer's  work  on  the  cell  membranes 
of  Leishmania  donovani.   During  the  course  of  cell  fraction  of  this 
parasite,  suspension  of  clean  but  fragmented  kinetoplasts  were  obtained. 
Our  preliminary  studies  of  these  preparations  reveals  their  capacity  to 
oxidize  a  number  of  tricarboxylic  acid  cycle  substrates.   Oxidation  of 
succinate  was  particularly  vigorous  (Vmax  =  200  nanoatoms/min)  ,  and  is  ex- 
tremely sensitive  to  malonate.   Unlike  its  mammalian  counterpart,  the 
malonate  inhibition  was  not  overcome  by  addition  of  excess  substrate. 
Electron  transfer  to  molecular  oxygen,  unlike  that  of  the  enteric  amebae, 
is  through  the  cytochromes.   Respiratory  control,  indicative  of  oxidative 
phosphorylation,  is  absent  in  these  fragmented  kinetoplasts  (Weinbach 
and  Dwyer ) . 

Mammalian  studies:   The  previous  collaboration  with  Dr.  Tschudy,  NCI, 
on  the  relation  of  heme  biosynthesis  and  biological  oxidations  was  resumed 
with  a  study  of  respiratory  phenomena  in  cultured  murine  erythroleukemia 
(MEL)  cells.   The  synthesis  of  hemoglobin  in  these  cells  is  strongly 
inhibited  by  succinylacetone.   Examination  of  MEL  cells'  respiratory 
capacity  showed  an  active  endogenous  respiration  and  the  presence  of  an 
NADH:   (quinone  acceptor)  oxidoreductase.   There  was  little  difference 
in  these  activities  in  cells  treated  with  succinylacetone.   Our  preliminary 
data  show  that  although  cell  growth  and  hemoglobin  synthesis  was  markedly 
diminished  by  the  succinylacetone,  respiration  was  unaffected  (Weinbach, 
Ebert  and  Tschudy) . 

This  project,  which  attempts  to  elucidate  vital  biochemical  mechanisms 
common  to  all  aerobic  cells,  has  obvious  implications  for  biomedical 
research  in  general  (illustrated  by  the  numerous  collaborative  projects), 
and  provides  a  rational  basis  for  understanding  bioenergetic  mechanisms 
associated  with  parasites  and  parastism.   The  parasite  studies  will  be 
continued  with  emphasis  on  identifying  the  ultimate  electron  carrier  in 
E_.  histolytica  and  G_.  lamblia  that  replaces  cytochromes  in  higher 
eukaryotic  organisms.   We  hope  to  purify  the  amebal  NAD(P)H  (quinone 
acceptor)  oxidoreductase  by  affinity  chromatography  utilizing  a  new 
technique  of  menadone  (the  acceptor)  bound  to  sephorose0   This  technique 


25-33 


Serial  No.  Z01  AI  00098-23  LPD 

also  should  enable  us  to  separate  the  oxidoreductase  from  other  amebal 
enzymes,  particularly  glutathione  reductase.   We  have  begun  preliminary 
experiments  with  this  enzyme  which  appears  to  be  of  physiological  importance 
in  both  IS.  histolytica  and  G.  lamblia,  based  on  the  studies  of  Dr.  F.  Gillin, 
LPD.  (see  her  report).   A  study  of  iron-sulfur  electron  transfer  proteins 
in  G_.  lamblia  appears  to  be  particularly  promising  in  view  of  the  marked 
differences  from  those  of  E.  histolytica.   The  rewarding  preliminary  experi- 
ments with  kinetoplasts  from  L.  donovani  will  be  expanded  with  the  objective 
of  elucidating  the  respiratory  mechanisms  of  this  organelle.   The  mammalian 
work  on  the  effects  of  succinylacetone  on  cultured  murine  erythroleukemia 
cells  will  be  expanded  to  other  aspects  of  hemoglobin  biosynthesis.   A 
report  of  our  initial  findings  is  in  preparation.   Because  of  shortage  of 
manpower  the  expected  collaboration  on  the  study  of  energy  metabolism  in 
human  blood  platelets  was  not  resummed  this  year.   We  hope  that  this  can  be 
done  during  the  ensuing  year.   Unfortunately,  Dr.  Takeuchi  had  to  return 
to  Japan  earlier  than  expected  during  the  past  year  and  he  could  not 
finish  his  work  on  isopropanol  reductase  in  E.  histolytica.   We  now  have 
done  this  and  a  paper  is  in  preparation.   The  microprobe  analysis  of  metal 
distribution  in  cestode  calcareous  corpuscles  was  done  in  Dr.  Barnard's 
laboratory  in  Sweden  and  a  few  samples  were  analyzed  by  the  Tousimis 
Research  Corporation  in  Bethesda.   Although  difficulty  was  experienced  by 
both  groups  in  sectioning  the  samples,  differences  were  observed  in  the 
distribution  of  metals  between  the  matrix  and  outer  shell  of  the  corpuscles. 
The  untimely  death  of  Dr.  T.  von  Brand  temporarily  suspended  this  work. 
The  data  must  be  collated  and  analyzed  to  ascertain  if  sufficient  information 
is  available  for  publication. 

Publications: 


1.   Weinbach,  E„  C,  Claggett,  C.  E. ,  Takeuchi,  T.  and  Diamond,  L.  S.: 
Biological  oxidations  and  f lavoprotein  catalysis  in  Entamoeba  histolytica. 
Arch.  Invest.  Med,  9:   89-98  (1978). 

2o   Takeuchi,  T„,  Weinbach,  E.  C,  Gottlieb,  M.  and  Diamond,  L.  S„: 
Mechanism  of  L-serine  metabolism  in  Entamoeba  histolytica.   Comp.  Biochem. 
Physiol.  B.  Biochem.  62B:   281-285  (1979).  ~ 

3.  Weinbach,  E.  C.  and  Bueding,  E.:   Theodor  von  Brand:   A  Tribute.  J. 
Parasitol.  65:   182-184  (1979). 

4.  Pazoles,  C.J.,  Claggett,  C.E.,  Creutz,  C.E.,  Pollard,  H.B,  and  Weinbach, 
E.C.:   Identification  and  subcellular  localization  of  catalase  activity  in 
bovine  adrenal  medulla  and  cortex.   Arch.  Biochem.  Biophys.  (in  press) . 


25-34 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  OEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  AI  00099-09  LPD 


PERIOD   COVERED 

October  1,    1978   to   September   30,    1979 


TITLE   CF    PROJECT    (.30   characters 
Biophysical  Parasitology 


less) 


NAMES,  LABORATORY  ANO  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Dvorak      Research  Microbiologist,  LPD,  NIAID 


PI 


Other: 


J.  A. 


M.  S.  Crane 
G.  A.  Schmunis 


Research  Associate,  LPD,  NIAID 
Visiting  Scientist,  UFRJ,  Brazil 


(JUUHtKAIING  UNITo  UT  any)  „  „  nn 

Applied  Clinical  Engineering  Section,  DRS;  Television  Engineering  Section,  CC; 
Instituto  de  Microbiologia,  UFRJ,  Brasil 


LAB/BRANCH 

Laboratory  of  Parasitic  Diseases 


SECTION 

Physiology  and  Biochemistry 


INSTITUTE  AND  LOCATION  „„„„, 

NIAID,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 


42/12 


PROFESSIONAL: 

30/12 


12/12 


CHECK  APPROPRIATE  80X(ES) 
G  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


S3  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

A  multiparametric  approach  utilized  to  study  the  interaction  of  Trypanosoma  cruzi 
with  vertebrate  cells  indicates  that  the  infection  of  vertebrate  cells  by  T.  cruzi 
is  an  active  process.   Cytochalasin  does  not  inhibit  the  infection  of  non-phago- 
cytic  vertebrate  cells  by  trypomastigotes.   Chronic  chagasic  serum  enhances  the 
ability  of  trypomastigotes  to  penetrate  vertebrate  cells.   The  phenomenon  is  not 
specific  to  parasite  strain,  host  cell  type  or  serum  source.   Total  immunoglobins 
are  capable  of  enhancing  penetration.   However,  purified  specific  immunoglobulin 
fractions  are  inactive.   Activity  is  restored  to  the  IgG  fraction  with  an  as  yet 
undefined  factor  present  in  normal  human  serum.   The  first  round  of  DNA  synthesis 
by  a  population  of  intracellular  amastigotes  is  synchronous.   Synchrony  decays 
during  subsequent  parasite  divisions.   The  confirmation  of  a  lag  period  prior  to   } 
the  initial  round  of  DNA  synthesis  indicates  that  trypomastigotes  are  in  an  extend-j- 
ed  G   [G  ]  phase  prior  to  infection  of  a  vertebrate  cell.   A  group  of  vertebrate 
cell  X  T?  cruzi  hybrids  have  been  produced  utilizing  P3-X63Ag8  vertebrate  cells  and 
T.  cruzi  epimastigotes.   The  vertebrate  cell  hybrids  express  T.  cruzi  antigen  as 
demonstrated  by  IFA.   Antigen  expression  has  been  stable  for  14  weeks. 
25-35 j 

?HS-o040 
(Rev.  10-76) 


Serial  No.  Z01  AI  00099-09  LPD 
Project  Description: 

A  multiparametric  approach  is  being  utilized  to  study  the  interaction  of 
Trypanosoma  cruzi  with  vertebrate  cells.   Five  major  topics  are  presently 
being  studied:   1.  Elucidation  of  the  mechanism  of  entry  of  a  trypomastigote 
into  a  non-phagocytic  cell;  2.  The  effect  of  immune  serum  on  the  ability  of 
a  trypomastigote  to  attach  to  and,  subsequently,  penetrate  a  vertebrate  cell; 

3.  The  patterns  of  macromolecular  synthesis  and  utilization  of  precursors  by 
host  cells  and  parasites  during  the  intracellular  phase  of  the  cycle; 

4.  Elucidation  of  the  mechanism  responsible  for  the  transformation  of 
epimastigotes  to  trypomastigotes  and;  5.  The  production  of  T.  cruzi  X 
vertebrate  cell  hybrids  that  express  T.  cruzi  antigens. 

1.  Mechanism  of  entry  of  trypomastigotes  into  non-phagocytic  vertebrate  cells 

It  has  been  postulated  that  phagocytosis  is  the  sole  mechanism  of  infec- 
tion of  vertebrate  cells  by  trypomastigotes.   Data  to  support  the  postulate 
were  collected  from  studies  of  macrophages  which  are  normally  phagocytic. 
The  entry  of  trypomastigotes  into  non-phagocytic  cells  is  postulated  as  being 
due  to  the  induction  of  vertebrate  cell  phagocytosis  by  the  parasite.   This 
concept  is  at  variance  with  data  collected  in  this  laboratory.   For  example, 
as  reported  previously,  protease  inhibitors  inhibit  the  penetration  of  fibro- 
blasts by  trypomastigotes  to  the  same  extent  as  protease  +  inhibitor.   Con- 
sequently, it  would  appear  that  inactivation  of  labile  enzymes  present  on 
host  cell  or  parasite  rather  than  removal  of  receptors  is  a  better  inter- 
pretation of  the  data.   In  addition,  it  has  been  reported  that  Cytochalasin 
inhibits  the  penetration  of  macrophages  by  trypomastigotes.   A  recent  study 
in  this  laboratory  has  demonstrated  that  Cytochalasin  does  not  inhibit  the 
penetration  of  fibroblasts  by  trypomastigotes.   Furthermore,  when  tested 
together  as  a  mixed  system,  the  ability  of  trypomastigotes  to  penetrate 
fibroblasts  is  many-fold  greater  than  macrophages.   Under  identical 
conditions,  macrophages  rapidly  phagocytosed  sheep  red  blood  cells  whereas 
the  fibroblasts  did  not.   Although  still  indirect,  these  data  lend  further 
support  to  the  hypothesis  that  trypomastigotes  are  capable  of  actively 
penetrating  vertebrate  cells. 

2 .  Influence  of  immune  serum  on  the  ability  of  T.  cruzi  trypomastigotes  to 
penetrate  vertebrate  cells 

T_.    cruzi  antiserum  has  a  lytic,  agglutinogenic  and  opsonizing  effect  upon 
trypomastigotes  and  may  induce  redistribution  and  loss  of  surface  antigens. 
The  fact  that  immune  serum  may  partially  protect  against  lethal  infection  of 
mice  when  passively  transferred  could  indicate  that  T.  cruzi  antibodies  may 
have  an  inhibitory  effect  upon  infection  of  vertebrate  cells.   To  test  this 
possibility,  trypomastigotes  were  incubated  with  human  fibroblasts  (WI-38) 
in  the  presence  of  normal  or  chagasic  human  serum  and  the  subsequent  infec- 
tion quantified.   It  was  observed  that:   1.  human  chagasic  sera  increase  the 
attachment  and  penetration  of  blood  trypomastigotes  from  3  different  strains 
of  parasite.   2.   Dialysis  of  chronic  chagasic  serum  slightly  decreases 


25-36 


Serial  No.  Z01  AI  00099-09  LPD 

attachment  and  penetration  compared  to  non-dialyzed  serum.   However,  penetra- 
tion is  still  greater  than  observed  with  normal  serum.   3.  the  factor (s) 
responsible  for  increasing  attachment  and  penetration  are  in  the  _>  50,000 
MW  fraction  of  the  serum.   4.  The  total  immunoglobulins  have  essentially  the 
same  activity  as  whole  serum.   However,  the  IgM  and  IgG  fractions  are  in- 
active.  Activity  was  restored  when  the  IgG  (but  not  IgM)  fraction  was  mixed 
with  fresh  human  normal  serum.   5.   Absorption  of  chagasic  serum  was 
Protein-A  or  epimastigotes  decreased  attachment  and  penetration.   Absorption 
with  WI-38  cells  or  sheep  red  blood  cells  had  no  effect.   6.   Fixed  trypo- 
mastigotes  incubated  with  chagasic  serum  do  not  attach  to  WI-38  cells. 
Serum  from  chronically  infected  rabbit,  mouse  or  monkey  increases  the 
penetration  of  WI-38  cells  by  homologous  or  heterologous  trypomastigotes. 
7.   Human  chagasic  and  chronic  rabbit  sera  increase  the  penetration  of 
BESM  cells  by  trypomastigotes. 

3.  Studies  of  the  pattern  of  DNA  synthesis  of  host  cells  and  parasites 
during  the  intracellular  phase  of  the  cycle 

DNA  synthesis  of  intracellular  T_.  cruzi  amastigotes  following  the 
infection  of  BESM  cells,  was  studied  by  autoradiography.   After  penetration, 
there  was  a  pre-replicative  lag  period  (a.  12  hr)  followed  by  a  synchronous 
round  of  DNA  synthesis  which  was  found  to  be  independent  of  parasite 
number/BESM  cell  and  the  host  cell  DNA  synthesis  cycle.   Parasite  reproduc- 
tion occurred,  for  the  first  time,  at  ^  21  hr  post  infection.   It  was 
concluded  that  T_.  cruzi  trypomastogotes  are  in  the  G0/G^  phase  of  their 
cell  division  cycle  and,  after  penetration,  parasite  reproduction  occurs 
independent  of  events  controlling  the  host  cell  DNA  synthesis-growth  cycle. 
The  early  synchronous  growth  of  intracellular  amastigotes  should  facilitate 
further  studies  on  the  biochemical  events  controlling  trypomastigote-to- 
amastigote  reproduction.   A  further  application  is  envisaged  for  studies 
involved  with  the  mode  of  action  of  drugs  with  trypanocidal  activity. 

4.  Elucidation  of  the  mechanism  responsible  for  the  transf oriaation  of 
epimastigotes  to  trypomastigotes. 

The  transformation  of  T_.  cruzi  from  one  morphologic  form  to  another 
occurs  naturally  at  several  points  in  the  life  history  of  the  parasite.   One 
of  these  transformations,  epimastigote  to  trypomastigote,  was  chosen  for 
study  in  an  attempt  to  elucidate  the  conditions  required  for  transformation 
to  occur.   Thus  far,  numerous  substances  known  to  induce  transformations  in 
vertebrate  cells  have  been  tried.   These  include  0-20  mM  thymidine  and  0-20 
mM  dibutryl-cyclic  AMP,  0-5  mM  adenosine  and  0-5  mM  monobutryl-cyclic  GMP 
which  have  no  effect  on  growth  or  morphology.   Adenosine  (10-20  mM) ,  10  mM 
monobutryl-cyclic  GMP  and  5  mM  theophylline  inhibit  growth  of  epimastigotes 
but  no  transformation   to  trypomastigotes  occurs.   Although  all  of  these 
data  represent  negative  experiments  with  respect  to  transformation,  they 
point  out  clearly  that  the  biochemistry  of  transformation  of  these  parasites 
is  obviously  different  from  that  found  in  vertebrate  cells. 


25-37 


Serial  No.  Z01  AI  00099-09  LPD 

5.   The  production  of  vertebrate  cell  hybrids  that  express  T.  cruzi  antigens 

The  production  of  vertebrate  cell  X  T.  cruzi  hybrids  initiated  last  year 
has  been  continued.   The  technique  for  selection  of  hybrid  clones  expressing 
T.  cruzi  antigen  is  the  only  major  problem  remaining  that  requires  development 
to  make  the  entire  system  practical.   This  problem  can  probably  be  overcome 
using  affinity  columns  or  a  cell  sorter.   However,  three  clones  have  been 
established  utilizing  a  P3-X63Ag8  vertebrate  cell  line  and  epimastigotes  as 
starting  material.   The  P3-X63Ag8  cell  line  is  HAT  sensitive  which  allows  for 
selection  of  hybrids  along  more  classical  lines.   Thus  far,  all  three  hybrid 
clones  express  T_.  cruzi  antigen  as  demonstrated  by  IFA. 

Aside  from  the  obvious  usefulness  of  this  system  to  basic  cell  biological 
questions,  there  are  several  major  Chagas'  disease-related  problems  that  can 
now  be  studied.   The  parasite  antigens  can  be  isolated,  characterized  and  used 
as  specific  immunogens.   It  is  not  yet  known  if  all  hybrid  cell  lines  express 
the  same  antigens.   If  not,  the  cell  hybrids  could  provide  a  model  for  the 
study  of  possible  immunopathologic  lesions  resulting  from  ^T.  cruzi  infection. 

Significance  and  Proposed  Course  of  Program: 

(1)  The  study  of  host-parasite  interactions  at  the  cellular  and  sub- 
cellular level  is  a  topic  of  critical  importance.   The  outcome  of  this 
interaction  directly  influences  the  physiologic  state  and  survival  potential 
of  both  the  host  and  the  parasite. 

(2)  The  methodology  and  research  philosophy  being  developed  are  directly 
applicable  to  other  areas  of  biomedical  research.   For  example,  the  controlled- 
environment  cluture  system  developed  with  this  program  is  now  in  routine  use 

in  laboratories  with  such  diverse  Interests  as  oncology,  immunology,  neuro- 
physiology and  morphogenesis.   The  video  systems  developed  with  this  program 
are  being  used  for  analysis  of  macroscopic  as  well  as  microscopic  images  in 
basic  research  and  diagnostic  clinical-medical  fields. 

Research  will  continue  in  an  attempt  to  further  our  understanding  of  the 
interaction  of  T.  cruzi  with  vertebrate  cells  in  vitro  and  relate  this 
information  to  the  course  of  Chagas'  disease  in  nature.   Basic  information 
obtained  with  the  controlled-environment  culture  system  and  video  systems 
will  be  utilized  in  more  complex  in  vitro  model  systems. 

The  basic  methodology  and  research  philosophy  developed  with  this 
program  will  be  used  for  the  analysis  of  other  host-parasite  interactions. 

Publications : 

1.   Dvorak,  J.  A.  and  Howe,  C.  L:   Toxoplasma  gondii-vertebrate  cell 
interactions:   II.   The  intracellular  reproductive  phase.   J.  Protozool. 
26:   114-117,  1979. 


25-38 


Serial  No.  Z01  AI  00099-09  LPD 

2.  Weller,  P.  F. ,  Dvorak,  J.  A.  and  Whitehouse,  W.  C:   Human  eosinophil 
stimulation  promoter  lymphokine:   Production  by  antigen  stimulated 
lymphocytes  and  assay  with  a  new  electro-optical  technique.   Cellular  Immunol, 
40:   91-102,  1978. 

3.  Dvorak,  J.  A.  and  Schmunis,  G.  A.:   The  influence  of  acute  and  chronic 
human  chagasic  sera  on  the  ability  of  Trypanosoma  cruzi  to  attach  to  and 
penetrate  human  diploid  fibroblasts  (abs,  4th  Int.  Congress  of  Parasitol. , 
1978,  Vol.  E,  pp  26-27. 

4.  Dvorak,  J.  A.:   Letter  to  the  Editor.   J.  Parasitol.   26:   158,  1979. 

5.  Dvorak,  J.  A.:   Trends  in  the  use  of  in  vitro  cell  cultures  for 
Trypanosoma  cruzi  research.   in  I_n  Vitro  Cultivation  of  Pathogens  of  Tropical 
Diseases.   (In  press). 

6.  Dvorak,  J.  A. :   I_n  vitro  studies  of  the  interaction  of  Trypansoma  cruzi 
with  vertebrate  cells.   International  Congress  on  Chagas  Disease,  Rio  de 
Janeiro.   (In  press). 

7.  Crane,  J.  St.  J.  and  Dvorak,  J.  A.:  Studies  on  DNA  synthesis  during  the 
intracellular  cycle  of  Trypanosoma  cruzi:  Host-parasite  inter-relationhsip. 
International  Congress  on  Chagas  Disease,  Rio  de  Janeiro.   (In  press). 


25-39 


JMITHSClilAN  SCIENCE  INFORMATION  EXCHANGE!     U.S.  DEPARTMENT  OF 
PROJECT  NUMBER  (Co  NOT  use  this  space)   HEALTH,  EDUCATION,  AND  WELFARE 

PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


;CT  NUMBER 


ZOl   AI   00102-05      LPD 


I PERICD    COVERED 


October    1,    1978    to    September    30,    1979 


fITLE  OF   PROJECT   (SO  characters   or  less) 
Pathogenesis   of   Disease   Caused  by    Infection  with   Intracellular   Parasites 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  ANO  TITLES  OF  PRINCIPAL  INVESTIGATORS  ANO  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


F.A.  Neva 


Others:    Renato  Gusmao 
Albert  Gam 


Chief,  Laboratory  of  Parasitic  Diseases,  NIAID 

Visiting  Fellow,  LPD, NIAID 

Biological  Laboratory  Technician  (Micro.),  LPD, NIAID 


COOPERATING  UNITS  (if  any )  Prof  essors  Joffre  Rezende  and  Anis  Rassi,  University  of  Goias, 
Goiania,  Brazil.   Professor  Aluizio  Prata,  University  of  Brasilia,  Brasilia, 
Brazil.   Dr.  Frances  Ward,  Duke  University  Medical  Center,  Durham,  N.C. 
Dr.  Euripides  Ferreria,  University  of  Parana,  Curtiba,  Brazil. 


LAB/BRANCH 
Laboratory  of  Parasitic  Diseases 


SECTION 
Cell  Biology  and  Immunology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  Bethesda,  Maryland   20205 


TOTAL  MANYEAfiS: 

27/12 


PROFESSIONAL: 
16/12 


11/12 


CHECK  APPROPRIATE  30X(ES) 
3(a)   HUMAN  SUBJECTS 

0  (al)  MINORS   n  (a2)  INTERVIEWS 


3  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  association  of  certain  HLA-tissue  types  with  chronic  Chagas '  disease 
noted  previously  could  not  be  confirmed  in  a  much  expanded  study.  B-cell  typing 
remains  to  be  checked  and  basic  immuno genetic  information  concerning  the 
Brazilian  population  has  been  a  useful  by-product  of  the  study. 


The  plaguing  technique  for  intracellular  phase  T^  cruzi  shows  promise  as 
a  tool' for  clonal  selection  and  analysis  of  isolates  of  the  parasite.   Other 
applications  of  cell  culture  for  characterization  of  strains  of  T.  cruzi  include 
temperature  tolerance  and  growth  curves. 


Use  of  the  foot-pad  site  for  inoculation  of  BALB/c  mice  and  subsequent 
measurements  of  the  lesion  has  proved  to  be  a  useful  model  for  experimental  work 
with  strains  of  cutaneous  leishmaniasis. 


25-40 

(Rev.  !0-7S) 


Project  No.   Z01  AI  00102-05  LPD 

Project  Description: 

The  variable  geographic  distribution  of  the  late  sequelae  that 
constitute  chronic  Chagas '  disease  suggest  that  genetic  factors  may  be 
involved  in  development  of  the  chronic  disease.   Therefore,  it  was  considered 
important  to  thoroughly  explore  possible  association  of  HLA-tissue  types 
with  chronic  Chagas'  disease.   A  preliminary  study  last  year  disclosed 
correlations  between  certain  forms  of  chronic  Chagas'  disease  and  presence 
as  well  as  absence  of  certain  HLA  types.   However,  the  study  groups  were 
relatively  small  (12  to  18  cases)  so  it  was  necessary  to  expand  the  numbers 
of  patients  studied,  and  family  members  were  also  included.   Again  such  a 
study  required  rather  complicated  logistics  and  collaboration  of  other  groups 
for  patient  selection  and  the  HLA-typing  at  A,  B  and  C  loci.   A  controlled 
freezing  apparatus  was  also  transported  to  Brazil  so  lymphocytes  could  be 
harvested  from  patients,  frozen  and  brought  back  to  the  U.S.  for  D-locus 
typing.   After  review  of  clinical  and  laboratory  findings  between  36  and  53 
patients  were  available  for  analysis.   In  this  expanded  study  the  apparent 
correlations  of  certain  HLA  types  with  chronic  Chagas'  disease  in  last  year's 
study  could  not  be  confirmed.   B-cell  typing  (DRw)  on  the  frozen  cells  has 
not  yet  been  performed,  but  will  be  done.   Quite  apart  from  possible  HLA 
relationships  with  a  parasitic  infection,  this  study  has  disclosed  some 
findings  of  intrinsic  interest  to  immuno gene tics.   For  example,  the  genetic 
diversity  of  this  Brazilian  population  is  unique  and  several  new  HLA 
specificities  will  probably  emerge  from  the  family  studies.   The  fact  that 
the  HLA  background  and  status  of  Chagas'  disease  has  been  established  in  a 
population  that  can  be  followed  may  make  it  worthwhile  to  monitor  the  study 
groups  for  the  next  several  years  for  evolution  of  their  disease  state. 
(Neva,  Gusmao,  Gam  and  collaborators). 

The  assessment  of  different  isolates  of  Trypanosoma  cruzi,  especially 
activity  of  the  intracellular  phase  of  the  organism,  using  cell  cultures 
as  a  host  system  has  continued.   Technical  features  of  the  intracellular 
plaquing  procedure  have  been  standardized.   The  ability  of  isolates  or 
strains  to  form  plaques  is  correlated  with  minimal  infective  dose  for  cell 
cultures  as  titrated  in  serial  dilutions,  and  with  growth  curves  over  3 
cycles  of  parasite  development.   The  minimal  infective  dose  does  not 
necessarily  correlate  with  the  ability  of  a  strain  to  produce  plaques.   There 
is  evidence  that  the  plaquing  technique  can  be  used  for  cloning  isolates  of 
T.  cruzi  because  successive  plaque  isolation  appears  to  enhance  a  property 
of  the  isolate,  such  as  ability  to  produce  plaques  at  an  elevated  temperature 
of  38°C.   Although  most  isolates  of  T.  cruzi  have  been  found  to  grow 
optimally  at  33°C,  a  recent  isolate  from  a  Brazilian  patient  with  acute 
Chagas'  disease  was  found  to  grow  better  at  38°C.   All  observations  lend 
support  to  the  concept  that  assessment  of  biologic  activity  of  T.  cruzi  by 
cell  culture  techniques  is  a  valid  approach  and  that  the  plaquing  procedure 
will  be  useful  for  clonal  selection  and  analysis  of  populations  of  1\_  cruzi. 
(Neva  and  Gam) . 

Further  work  has  been  done  on  the  foot-pad  infection  in  the  genetic 
in-bred  BALB/c  mouse  with  leishmania  causing  cutaneous  leishmaniasis.   Use 


25-41 


Project  No.   Z01  AI  00102-05  LPD 

of  this  inoculation  site  and  subsequent  measurement  of  width  and  thickness 
of  lesions  at  weekly  intervals  for  quantitative  assessment  of  lesion  size 
has  proved  to  be  a  useful  model  for  experimental  work.   A  previous  experi- 
ment with  resistant  C57  Bl  and  DBA  mice,  their  Fl  progency  with  BALB/c  and 
back-cross  with  BALB/c  gave  results  suggesting  a  single  gene  inheritance 
pattern  for  susceptibility.   A  similar  experiment  repeated  with  DBA  and 
BALB/c  mice  was  still  equivocal  although  similar  to  what  would  be  expected 
of  a  single  gene  effect.   A  distinctly  reduced  foot-pad  size  in  several 
experiments  with  a  standard  strain  of  L^_   tropica  was  initially  thought  to 
represent  loss  of  virulence  of  the  strain.   However,  the  decreased  response 
was  found  due  to  use  of  a  smaller  inoculum,  and  this  dose-response  relation- 
ship was  clearly  demonstrated  with  another  strain  of  L^  tropica  as  well. 
Reproducibility  of  the  foot-pad  response  was  shown  with  strains  of  L.  tropica 
after  storage  in  liquid  nitrogen.   Although  factors  such  as  inoculum  size 
can  influence  response  with  this  system,  the  foot-pad  size  seems  basically  to 
reflect  intrinsic  virulence  of  the  strain  of  leishmania.  (Neva) . 

Publications: 


1.  Neva,  F.A.,  Wyler,  D.J.,  and  Nash,  T.E.:   Cutaneous  leishmaniasis  -  A 
case  with  persistent  organisms  after  treatment  in  presence  of  normal  immune 
response.   Am.  J.  Trop .  Med.  Hyg.  28:   467-471,  1979. 

2.  Bjorvatn,  B. ,  and  Neva,  F.A. :   A  model  in  mice  for  experimental 
leishmaniasis  with  a  West  African  strain  of  Leishmania  tropica.   Am.  J.  Trop. 
Med.  Hyg.  28:   472-479,  1979. 

3.  Bjorvatn,  B. ,  and  Neva,  F.A.:  Experimental  therapy  of  mice  infected  with 
Leishmania  tropica.   Am.  J.  Trop.  Med.  Hyg.   28:   480-485,  1979. 

4.  Ottesen,  E.A.,  Neva,  F.A.,  Paranjape,  R.A. ,  Tripathy,  S .P . ,  Thiruvengadam, 
K.V.,  and  Beaven,  M.A. :  Specific  allergic  sensitisation  to  filarial  antigens 
in  tropical  eosinophilia  syndrome.   Lancet :  1158-1161,  June  2,  1979. 

5.  Ottesen,  E.A. ,  Hiatt,  R.A. ,  Cheever ,  A.W. ,  Sotomayor,  Z.R.  and  Neva,  F.A. : 
The  acquisition  and  loss  of  antigen-specific  cellular  immune  responsiveness 

in  acute  and  chronic  schistosomiasis  in  man.   Clin,  exp .  Immunol.   33: 
38-47,  1978. 


25-42 


(SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
iPROJECT  NUMBER  [Do  NOT  use  this  space) 


U.S.  OEPARTMENT  OF 
HEALTH,  EDUCATION,  ANO  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00103-12  LPD 


I  PERIOD   COVERED 

{October  1.  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less] 

Immunological  Studies  on  Toxoplasmosis  and  Other  Parasitic  Diseases 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


Other: 


M.  N.  Lunde  Research  Zoologist 

A.  W.  Cheever  Assistant  Chief,  LPD,  NIAID 

E.  A.  Ottesen  Senior  Investigator,  LPD,  NIAID 

T.  E.  Nash  Senior  Investigator,  LPD,  NIAID 


COOPERATING  UNITS  (if  an> 


LAB/BRANCH 

Laboratorv  of  Parasitic  Diseases 


SECTION 

Host-Parasite  Relations 


INSTITUTE  AND  LOCATION 

NIAID.  Bethesda,  Md, 


TOTAL   MANYEARS: 
18/12 


PROFESSIONAL: 
13/12 


5/12 


CHECK  APPROPRIATE  BOX(ES) 
C  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   H  (a2)  INTERVIEWS 


g]  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Cercarial/adult  (c/a)  antibody  ratios  from  acutely  infected  patients  were  hi 
than  from  individuals  with  chronic  schistosomiasis.   Specific  IgM  titers  to 
soluble  egg  antigen  (SEA)  were  found  in  all  patients  with  acute  or  early 
schistosomiasis  but  from  only  a  few  patients  with  chronic  schistosomiasis, 
antibody  in  the  chronic  patients  correlated  with  the  presence  of  circulating 
immune  complexes .   Specific  IgE  antibody  was  found  in  most  of  the  sera  from 
acute  patients  and  in  only  a  few  of  the  ones  with  chronic  schistosomiasis, 
and  toxoplasma  antigen  added  to  serum  have  been  detected  by  ELISA  at  levels 
10  and  100  ng/ml  respectively. 


gher 


IgM 


SEA 
of 


25-43 


-HS-6040 

(Rev.  10-76) 


Serial  No.  Z01  AI  00103-12  LPD 

Project  Description: 

Emphasis  has  been  focused  on  improving  the  enzyme- linked  immunosorbent 
assay  (ELISA)  for  serodiagnosis  and  study  of  host  parasite  immune  responses 
in  parasitic  diseases,  especially  schistosomiasis  and  toxoplasmosis.   The 
ELISA  is  used  to  measure  immunoglobulin  class  specific  antibodies  to  antigens 
prepared  from  different  stages  of  the  life  cycle  of  Schistosoma  mansoni  and 
also  to  quantitate  antigens  from  these  life  cycle  stages.   The  following 
findings  relative  to  schistosomiasis  were  established: 

1.  Sera  from  patients  with  acute  schistosomiasis  were  found  to  have 

higher  ELISA  titers  with  cercarial  antigen  than  with  adult  antigen.   In 

contrast  sera  from  patients  with  chronic  schistosomiasis  were  found  to  have 

higher  titers  with  adult  worm  antigen.   This  difference  could  be  quantified 

and  expressed  by  determining  the  ratio  of  cercaria/adult  (c/a)  antigen 

0D.nn   extinction  values  at  a  1:16  dilution  of  serum. 
490nm 

2.  Acute  patients,  those  with  high  c/a  ratios  had  schistosome  specific 
IgM  antibody  but  only  2  out  of  11  patients  with  chronic  schistosomiasis  had 
positive  IgM  titers.   Interestingly  these  two  patients  were  also  the  only 
ones  of  the  group  found  to  have  circulating  immune  complexes.   (Lawley  et  al. , 
to  be  published) .   These  observations  suggest  a  possible  link  between  the 
determinants  which  lead  to  chronic  IgM  production  and  those  responsible  for 
the  persistence  of  circulating  immune  complexes. 

3.  IgE  antibody  was  demonstrated  in  11  of  13  acutely  infected  patients 
with  titers  ranging  from  1:8  to  1:64  while  sera  from  all  chronically  infected 
persons  except  one  were  negative  for  IgE  antibodies  in  the  ELISA.   Schisto- 
some IgE  was  demonstrated  almost  exclusively  with  egg  antigen  as  cercarial 
and  adult  worm  antigens  elicited  only  negative  or  weakly  positive  responses. 
One  interpretation  of  these  observations  is  that  the  schistosome  egg  antigens 
play  a  predominant  role  in  eliciting  hypersensitivity  responses  in  the 
infected  host. 

4.  Fab'  fragments  of  IgG  from  a  monkey  infected  with  S_.  mansoni  have 
been  labeled  with  horseradish  peroxidase  and  used  in  an  ELISA  procedure  to 
detect  antigen.   Using  this  procedure  soluble  egg  antigen  (SEA)  can  be 
detected  as  low  as  10  ng/ml  when  added  to  10%  serum  in  PBS.   This  is  being 
done  as  a  prelude  to  hopefully  being  able  to  detect  circulating  antigen  and 
antigen  moities  of  immune  complexes. 

The  diagnosis  of  toxoplasmosis  is  usually  based  on  demonstration  of 
antibodies  by  different  serological  methods.   However,  for  confirmation  of 
active  toxoplasmosis  it  would  be  helpful  if  a  method  could  be  obtained  for 
the  detection  of  circulating  antigen.   Using  Fab'  fragments  of  IgG  from  a 
rabbit  infected  with  toxoplasms  which  have  been  conjugated  with  peroxidase 
toxoplasma  antigen  has  been  detected  at  the  100  ng/ml  level  when  added  to 
10%  serum. 


25-44 


Serial  No.  Z01  AI  00103-12  LPD 
Proposed  Course: 

1.  As  sera  becomes  available  from  patients  post  treatment  for  schisto- 
somiasis we  plan  to  use  ELISA  to  study  immune  response  after  treatment.   If 
changes  can  be  found  in  levels  of  specific  immunoglobulins  with  antigens  from 
different  stages  of  the  life  cycle  then  we  have  a  handle  for  evaluating 
chemo  therapy . 

2.  Methods  currently  available  for  the  detection  of  circulating  antigen 
in  schistosomiasis  have  used  radiolabeled  antibody  or  counter  current  Immuno- 
electrophoresis.  Perhaps  ELISA  methodology  offers  a  means  of  detecting  such 
antigen  in  human  subjects.   In  the  presence  of  circulating  antibody  these 
antigens  can  form  complexes  which,  indeed,  have  been  demonstrated  in  schisto- 
somiasis.  If  ELISA  proves  successful  in  measuring  circulating  immune  com- 
plexes, then  complexes  of  other  parasitic  diseases  will  be  studied.   Along 
these  lines  we  hope  to  eventually  separate  and  identify  the  antigen  and  anti- 
body moities  of  these  complexes  since  they  can  be  considered  a  pathogenic 
mechanism  in  parasitic  diseases. 

3.  Improvements  in  the  ELISA  techniques  to  detect  circulating  antigen 
in  tomoplasmosis  will  be  continued. 

Significance  to  Biomedical  Research: 

Application  of  ELISA  for  multipurpose  seroepidemiological  and  clinical 
studies  is  being  contemplated  in  areas  endemic  for  malaria,  trypanosomiasis 
and  schistosomiasis  by  the  World  Health  Organization.   We  have  shown  how 
ELISA  can  be  a  useful  tool  in  distinguishing  acute  and  chronic  schisto- 
somiasis thus  making  it  useful  for  seroepidemiological  and  clinical  studies. 
Further  investigations  employing  ELISA  to  detect  circulating  antigens  and 
circulating  immune  complexes  should  be  valuable  from  a  diagnostic  standpoint 
and  also  as  a  means  of  identifying  the  role  of  complexes  in  the  disease 
process. 

Publications : 

1.  Lunde,  M.  N. ,  Ottesen,  E.  A.  and  Cheever,  A.  W. :   Serological  differences 
between  acute  and  chronic  Schistosomiasis  mansoni  using  enzyme-linked 
immunosorbent  assay  (ELISA).   Am.  J.  Trop.  Med.  Hyg.   28:   87-91,  1979. 

2.  Lunde,  M.  N.  and  Ottesen,  E.  A. :   Enzyme-linked  immunosorbent  assay  (ELISA) 
for  detecting  IgM  and  IgE  antibodies  in  human  schistosomiasis.   Am.  J.  Trop. 
Med.  Hyg.   (In  press) . 


25-45 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE] 


PROJECT  NUMBER  (Do  NOT  use  this  space]   HEALTH,  EDUCATION,  AND  »ELFARE 

PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


U.S.  DEPARTMENT  OF 


PROJECT  NUMBER 


ZOl  AI  00108-08  LPD 


PERIOD    COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (30  characters  or  less) 

Studies  on  the  Biology  and  Immunogenicity  of  Malarial  Sporozoites, 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  ANO  TITLES  OF  PRINCIPAL  INVESTIGATORS  ANO  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


R.  W.  Gwadz 


Research  Entomologist,  LPD,  NIAID 


Other:   R.  S.  Nussenzweig  Professor  and  Head,  Division  of  Parasitology,  NYU 

School  of  Medicine 


COOPERATING  UNITS  (if  any; 

New  York  University,  School  of  Medicine 


LAB/BRANCH 

Laboratory  of  Parasitic  Diseases 


SECTION 

Malaria  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Md. 


TOTAL  MANYEARS: 


9/12 


PROFESSIONAL: 
3/12 


6/11 


CHECK  APPROPRIATE  BOX(ES) 
J   (a)   HUMAN  SUBJECTS 


□  (b)  HUMAN  TISSUES 


*  (c)  NEITHER 


□  (a! )  MINORS 


INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  purpose  of  this  project  is  to  study  the  immune  mechanisms  involved  in 
sporozoite-induced  infections,  and  to  develop  protocols  for  immunization 
with  sporozoite  antigens.   Studies  underway  are  concerned  with  development 
of  methods  for  immunizing  rhesus  monkeys  with  Plasmodium  knowlesi  sporozoites, 
and  characterization  of  the  structure,  function  and  immunogenicity  of  surface 
determinants  of  sporozoites. 


25-46 


,:HS-6040 

i  Rev.  10-761 


Serial  No.  Z01  AI  00108-08  LPD 

Sporozoite  immunization 

Rhesus  monkeys  have  been  immunized  by  intravenous  injection  of  sporo- 
zoites  of  the  H  strain  (Malaysian)  of  P_.  knowlesi.   Animals  resistant  to 
challenge  by  bite  of  _P.  knowlesi  infected  mosquitoes  showed  consistently 
high  levels  of  anti-sporozoites  antibodies  as  determined  by  circum-sporozoite 
precipitin  (CSP)  reactions  and  immunof luorescense  (IFA) .   In  addition,  serum 
from  protected  monkeys  inactivate   sporozoites  after  a  1  hour  incubation  in 
a  test  for  sporozoite  neutralization  activity  (SNA) . 

Monkeys  that  received  similar  quantities  of  sporozoite  antigen  but  failed 
to  develop  antibodies  as  determined  by  CSP,  IFA  or  SNA  tests  were  not  resis- 
tant to  challenge  by  bite  of  infected  mosquitoes.   We  can  demonstrate  a 
positive  correlation  between  these  serological  tests  and  the  presence  of  pro- 
tective immunity  against  sporozoites. 

Passive  transfer  of  serum  from  immune  donor  monkeys  to  non-immune 
recepients  confers  complete  protection  in  the  recepient  against  sporozoite 
challenge,  demonstrating  the  critical  role  of  antibody  in  the  anti-sporozoite 
reaction. 

Characterization  of  sporozoite  surface  antigens 

P_.  knowlesi  sporozoites  were  radiolabelled  by  lactoperoxidase  mediated 
iodination  and  disrupted  by  French  pressure  cell.   SeS — polyacrylamide  gel 
electrophoresis,  followed  by  autoradiography  revealed  the  presence  of  a  small 
number  of  labelled  proteins  in  the  extract.   Immunoprecipitation  with  specific 
antisera  to  P_.  knowlesi  detected  primarily  one  of  these  membrane  components, 
with  molecular  weight  of  approximately  100,000  daltons. 

Proposed  course: 

The  following  investigations  will  be  pursued  in  the  coming  year  with  the 
eventual  goal  of  immunization  against  sporozoites  of  the  human  malarias. 

1)  improvement  of  the  P_.  knowlesi/monkey  model  with  respect  to  immunization 
schemes . 

2)  characterization  of  the  sporozoite  surface  antigens  most  responsible  for 
immunity.   The  techniques  of  metabolic  labelling  and  hybridoma  production  of 
specific  antibodies  are  being  used  to  facilitate  this  characterization. 

3)  non-response  of  some  monkeys  to  sporozoite  antigens  and  resulting  non- 
protection  has  stimulated  an  examination  of  histocompatability  types  in 
rhesus  monkeys. 

Significance: 

A  malaria  vaccine  effective  against  mosquito-injected  sporozoites  would 
be  of  great  importance  to  human  populations  at  risk  in  much  of  the  tropical 

25-47 


Serial  No.  Z01  AI  00108-08  LPD 

world.  The  ready  application  of  this  type  of  immunization  has  met  with  a 
number  of  practical  difficulties.  The  development  of  a  Simian  model  for 
studies  of  the  mechanisms  and  methodology  for  inducing  anti-sporozoite 
immunity  would  be  very  useful  for  the  solution  of  some  of  these  problems. 
In  addition,  characterization  of  the  functional  sporozoite  antigens  could 
lead  to  synthesis  and  elimination  of  unreasonable  dependence  on  mosquito- 
produced  sporozoite  antigens  for  immunization. 

Publications : 


1.   Gwadz,  R.  W. ,  Cochrane,  A.  H. ,  Nussenzweig,  V.  and  Nussenzweig,  R.  S.: 
Preliminary  studies  on  vaccination  of  rhesus  monkeys  with  irradiated 
sporozoites  of  Plasmodium  knowlesi  and  characterization  of  surface  antigens 
of  these  parasites.   Bull.  WHO   (in  press). 


25-48 


SMITHSONIAN    SCIENCE    INFORMATION    EXCHANGE 
PROJECT  NUMBER    (Do   NOT   use    this   space) 


U.S.    DEPARTMENT   OF 
HEALTH,    EDUCATION,    ANO    WELFARE 
PUBLIC    HEALTH    SERVICE 
NOTICE  Of 
INTRAMURAL  RESEARCH   PROJECT 


PROJECT   NUMBER 


ZOl  AI   00109-07   LPD 


PER  I  00    COVERED 

October    1,    1978    to   September   30,    1979 


TITLE   OF   PROJECT    [30   characters   or  less) 

Cellular   Immunology   of  Malaria   and   Other   Parasitic  Diseases 


NAMES,  LABORATORY  ANO  INSTITUTE  AFFILIATIONS,  ANO  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:     D.  J.  Wyler    Senior  Investigator,  LPD,  NIAID 

Other:   C.  N.  Oster    Senior  Staff  Fellow,  LPD,  NIAID 
Research  Associate,  LPD,  NIAID 
Clinical  Associate,  LCI,  NIAID 
Senior  Staff  Fellow,  LMI,  NIDR 
Research  Microbiologist,  LMI,  NIDR 
Cheever  Assistant  Chief,  LPD,  NIAID 

Research  Microbiologist,  LPD,  NIAID 


C. 

N. 

Oster 

T. 

C. 

Quinn 

J. 

D. 

Berman 

L. 

M. 

Wahl 

S. 

M. 

Wahl 

A. 

W. 

Cheeve: 

D. 

M. 

Dwyer 

COOPERATING  UNITS  (if  any)  Laboratory  of  Microbiology  and  Immunology,  NIDR,  Dept, 
Anatomy  and  Cell  Biology,  Johns  Hopkins  School  of  Medicine  (Dr.  Li  Chen); 
Dept.  of  Medicine,  V.  A.  Hospital,  Memphis,  TN.  (Dr.  A.  Postlethwaite) 


of 


LAB/BRANCH 

Laboratory   of   Parasitic  Diseases 


SECTION 
Malaria 


INSTITUTE   AND    LOCATION 

NIAID,    Bethesda,   Md . 


TOTAL  MANYEARS: 
24/12 


PROFESSIONAL: 
11/12 


13/12 


CHECK  APPROPRIATE  90X(ES) 
%   (a)   HUMAN  SUBJECTS 

D  (al)  MINORS   □  (a2)  INTERVIEWS 


30  (b)  HUMAN  TISSUES 


2  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Investigations  are  directed  at  host  defense  mechanisms  and  immunoregulation  in 
malaria  and  leishmaniasis ,  and  the  possible  role  of  the  schistosomal  egg 
granuloma  in  the  immunopathogenesis  of  hepatic  fibrosis.   Presently,  research 
is  aimed  at  (1)  spleen  function  in  malaria  immunity  (2)  regulation  of  intra- 
cellular growth  of  leishmania  in  human  macrophages,  and  (3)  characterization 
of  a  fibroblast  stimulating  factor  produced  in  vitro  by  schistosomal  egg 
granulomas.   In  addition,  clinical  studies  are  covered  by  project  ZOl  AI 
00141-03  FY  1979. 


25-49 


=HS-6040 
(Rev.  10- 


Serial  No.  Z01  AI  00109-07  LPD 


Project  Description: 


This  project  investigates  host  defense  in  malaria  and  leishmaniasis,  and 
immunopathogenesis  of  hepatic  fibrosis  in  schistosomiasis.   The  studies 
include  continuation  of  the  program  investigating  the  role  of  the  spleen  in 
malaria,  as  well  as  clinical  immunology  of  leishmaniasis.   In  both  cases, 
the  motivation  for  this  research  is  the  belief  that  through  gaining  greater 
insights  into  basic  defense  mechanisms,  new  stratagiesmay  emerge  for  pre- 
vention of  these  important  protozoal  diseases.  Schistosomiasis  research, 
on  the  other  hand,  investigates  a  model  we  described  which  provides  the 
first  molecular  link,  between  chronic  inflammation  (schistosomal  egg 
granuloma)  and  fibroblast  activation  which  may  lead  to  hepatic  fibrosis. 
The  practical  goal  will  be  to  consider  in  what  pharmacologic  or  immunologic 
manner  the  fibrotic  process  could  be  inhibited. 

Studies  of  splenic  host  defense  mechanisms  in  rodent  malaria  have  re- 
vealed that  clearance  of  infected  erythrocytes  by  the  spleen  is  not  anti- 
body-dependent but  rather  determined  by  factors  involving  decreased 
def ormability  of  infected  erythrocytes  (Wyler  and  Quinn)  and  altered  splenic 
microcirculation  (Wyler  and  Quinn;  Wyler  and  Chen) .   Antibody  protects  in 
vivo  in  malaria  by  preventing  merozoite  invasion  of  RBC  (Quinn  and  Wyler). 
Acute  resolution  of  malaria  is  spleen-dependent.   Congenitally  asplenic 
mice  as  well  as  adult-splenectomized  mice  cannot  overcome  an  acute  malaria 
infection,  but  are  protected  from  a  challenge  with  the  homologous  parasite 
strain  following  rescue  with  chloroquine  (Wyler  &  Oster) .   Reconstitution 
of  asplenic  mice  with  syngeneic  or  autologous  spleen  cells  do  not 
restore  the  protective  function.   These  findings,  taken  together,  clearly 
indicate  that  splenic  architecture  -  not  merely  spleen  cell  populations  - 
are  critical  in  malarial  host  defense. 

Leishmania  species  which  are  human  pathogens  have  been  sucessfully  grown 
in  human  monocyte-derived  macrophages  (Wyler,  Berman  &  Dwyer)  and  the 
intracellular  parasite  has  been  shown  to  multiply  within  the  phagolysosomes. 
This  model  is  being  employed  for  studying  the  leishmanicidal  effects  of 
drugs  (Wyler  and  Berman),  as  well  as  host  factors  which  regulate  intracellular 
growth  of  the  parasite  (Wyler) . 

Schistosomal  egg  granulomas,  when  isolated  from  infected  mice  and 
cultured  in  vitro,  elaborated  soluble  substances  which  stimulated  fibro- 
blasts in  vitro  (Wyler,  Wahl,  Cheever,  Wahl) .   This  substance  is  distinct 
from  soluble  egg  antigens  (SEA) ,  although  SEA  can  also  directly  stimulate 
fibroblasts.   The  stimulating  substance  also  can  increase  PGE2,  CAMP,  and 
collagen  synthesis  in  fibroblast  cultures. 

All  projects  are  being  discontinued  at  N.I.H.  since  the  P.I.  is  leaving 
N.I.H.  in  November  1979,  but  will  be  continued  in  his  new  laboratory. 


25-50 


Serial  No.  Z01  AI  00109-07  LPD 
Publications : 

1.  Wyler,  D.  J.,  Wahl,  S.  M.  ,  Wahl,  L.  M. :   Hepatic  Fibrosis  in  schisto- 
somiasis.  Egg  granuloma  secrete  fibroblast  stimulating  factor  ui  vitro . 
Science,  202:435-440,  1978. 

2.  Wyler,  D.  J.,  Wasserman,  S.  I.,  Karchmer,  A.  W:   Substances  which 
modulate  leukocyte  migration  are  present  in  CSF  during  meningitis.   Annals 
of  Neurology,  5:322-326,  1979. 

3.  Wyler,  D.  J.:   Leishmaniasis.   In  Current  Therapy  (Conn,  H. ,  Ed.)  1979. 
W.  B.  Saunders  Co.,  Philadelphia,  Pa. 

4.  Wyler,  D.  J.  and  Miller,  L.  H. :   Plasmodium  species  (Malaria)  (Chapter 
227).   In  Principles  and  Practices  of  Infectious  Diseases  (Mandell,  G. 

L. ,  Douglas,  R.  G. ,  and  Bennett,  J.  E. ,  eds . )  (in  press). 

5.  Wyler,  D.  J.,  Oster,  C.  N.  ,  and  Quinn,  T.  C:   The  role  of  the  spleen  in 
malaria  infections.   In  The  Role  of  the  Spleen  in  the  Immunology  of 
Parasitic  Diseases.   (in  press) 

6.  Wyler,  D.  J.:   Cellular  aspects  of  immune  regulation  in  malaria.   Bull. 
W.H.O.   (in  press). 

7.  Wyler,  D.  J.  Herrod,  H.  and  Weinbaum,  F.  I.:   Response  of  sensitized 
and  unsensitized  human  lymphocyte  subpopulations  to  Plasmodium  falciparum 
antigens.   Infect,  and  Immun.  ^4_:106,  1979. 

8.  Wyler,  D.  J.  Oppenheim,  J.  J.  and  Koontz,  L.  C. :   The  influence  of 
malaria  infection  on  the  elaboration  in  vitro  of  soluble  mediators  by 
adherent  mononuclear  cells.   Infect,  and  Immun.  _24:151,  1979. 

9.  Quinn,  T.  C.  and  Wyler,  D.  J.:   Intravascular  clearance  of  parasitized 
erythrocytes  in  rodent  malaria.   J.  Clin.  Invert.  6_3:1187,  1979. 

10.  Neva,  F.  A.,  Wyler,  D.  J.,  and  Nash,  T.   Curaneous  leishmaniasis  -  a 
case  with  persistent  organisms  after  treatment  in  presence  of  normal  immune 
response.   Am.  J.  Trop.  Med.  Hyg.  2_8:467,  1979. 

11.  Wyler,  D.  J.,  Weinbaum,  F.  I.  and  Herrod,  H. :   Characterization  of  the 
in  vitro  proliferative  responses  of  human  lymphocytes  to  leishmanial  anti- 
gens .   J.  Infect.  Pis. ,  1979  (in  press). 

12.  Quinn,  T.  C.  and  Wyler,  D.  J.   Resolution  of  acute  malaria  (Plasmodium 
berghei  in  the  rat):   Reversibility  and  spleen  dependence.   Am.  J.  Trop. 
Med.  Hyg. ,  1980  (in  press) . 

13.  Quinn,  T.  C.  and  Wyler,  D.  J.   Mechanisms  of  action  of  hyperimmune 
serum  in  mediating  protective  immunity  to  rodent  malaria  (Plasmodium 
berghei) .   J .  Immuno 1 .  (in  press) , 


25-51 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  ANO  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  AI  00149-04  LPD 


PERIOD  COVERED 

October    1,    1978   to    September    30,    1979 


TITLE  OF   PROJECT   1,30  characters   or  less) 
Physiology   of   Anopheles  Mosquitoes 


NAMES,    LABORATORY    ANO    INSTITUTE   AFFILIATIONS,    AND    TITLES   OF    PRINCIPAL    INVESTIGATORS   AND    ALL    OTHER 
PROFESSIONAL   PERSONNEL   ENGAGED   ON   THE   PROJECT 


PI: 


Other: 


R.  F.  Beach         Staff  Fellow,  LPD,  NIAID 
R.  M.  Rosenberg     Staff  Fellow,  LPD,  NIAID 

R.  W.  Gwadz         Research  Entomologist,  LPD,  NIAID 


COOPERATING  UNITS  (it  any) 

None 


LAB/BRANCH 

Laboratory  of  Parasitic  'Diseases 


SECTION 

Malaria 


INSTITUTE    ANO    LOCATION 

NIAID,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 


39/12 


PROFESSIONAL:  OTHER: 

24/12  15/12 


CHECK  APPROPRIATE  BOX(ES) 
2   (a)   HUMAN  SUBJECTS 

<Z   (a1  )  MINORS  Q   (a2)  INTERVIEWS 


(b)  HUMAN  TISSUES 


2  (=)  NEITHER 


SUMMARY  OF  WORK  (,200  words  or  less  -  unaerline  keywords) 

This  project  consists  of  a  series  of  studies  on  the  physiology,  behavior ,  genetics 
and  vector  competence  of  anopheline  mosquitoes  which  serve  as  vectors  of  malaria. 
Special  emphasis  has  been  placed  on  the  neuro-hormonal  regulation  of  feeding 
behavior ,  oogenesis  and  blood  meal  retention  in  female  mosquitoes  and  the  onset  of 
mating  behavior  in  male  mosquitoes.  The  genetic  and  physiological  basis  for  vecto 
competence  is  being  investigated  in  Anopheles  pharoensis .  The  concept  of  antibody 
as  a  systemic  insecticide  is  being  explored  using  various  mosquito  metabolic 
products  and  hormones  as  antigens. 


25-52 


?HS-o040 
(Rev.    10-76 


Serial  No.  Z01  AI  00149-04  LPD 

Project  Description: 

The  object  of  this  project  is  to  investigate  a  wide  range  of  physiological, 
behavioral  and  genetic  parameters  which  influence  the  capacity  of  anopheline 
mosquitoes  to  transmit  malaria.   The  following  studies  are  currently  underway. 

1)  Neuro-hormonal  control  of  blood  feeding  behavior. 

The  demonstration  in  our  laboratory  that  the  biting  behavior  of  female 
mosquitoes  is  modulated  by  the  ovarian  hormone,  ecdysone,  has  raised  the  possi- 
bility that  blood  feeding  (and  consequent  disease  transmission)  could  be 
inhibited  by  providing  exogenous  sources  of  this  hormone.   Indeed,  when 
ecdysone  analogues  are  fed  to  female  mosquitoes  in  their  sugar  meals,  blood- 
feeding  behavior  is  inhibited. 

2)  Initiation  of  Oogenesis. 

Ovarion  follicles  do  not  develop  until  triggered  by  a  blood  meal. 
Although  the  midgut  and  ovaries  are  apposed,  at  least  some  of  the  stimuli  from 
the  engorged  gut  to  the  ovary  are  relayed  by  the  brain.   Using  the  techniques 
of  decapitation  and  blood  enemas,  evidence  now  indicates  that  two  signals  are 
needed  for  the  ovary  to  produce  mature  eggs.   The  first  stimulus  triggered 
by  the  ingestion  of  even  small  amounts  of  blood  activates  oocyte  pinocytosis; 
the  volume  of  the  blood  meal  stimulates  the  brain  to  release  a  hormone  which 
insures  oocyte  maturation. 

3)  Relationships  between  oogenesis  and  blood-meal  retention. 

Retention  of  the  blood  meal,  as  measured  by  the  time  of  defecation,  is  a 
function  of  ovarian  development.   Females  decapitated  soon  after  a  blood  meal 
do  not  mature  eggs  and  defecate  earlier  than  normal.   Ovariectomized  females 
resemble  decapitated  females  by  defecating  earlier  than  normal,  but  when  treated 
with  the  ovarian  hormone,  ecdysone,  these  females  retain  blood.   These  and  other 
results  suggest  that  the  blood  meal  initiates  oogenesis,  the  developing  oocysts 
produces  ecdysone,  and  ecdysone  prolongs  retention  of  gut  contents. 

4)  Neuro-hormal  regulation  of  male  mating  behavior. 

Newly  emerged  male  mosquitoes  do  not  mate  because  they  can  not  perceive 
the  stimuli  which  elicits  mating  behavior,  the  flight  sounds  of  the  female 
mosquito.   Males  detect  this  sound  via  erect  hairs  on  their  antennae.   However, 
at  emergence  and  for  some  time  thereafter  these  hairs  are  recumbent  on  the 
antennal  shift  and  do  not  respond  to  female  sounds. 

Antennal  hair  erection  is  triggered  by  the  release  of  neurotransmitters 
from  the  nerves  which  ennervate  the  antennae.   In  vitro-  and  in  vivo  hair 
erection  can  be  stimulated  by  certain  a-adrenergic  compounds  such  as  L- 
epinephrine.   However,  these  drugs  cannot  stimulate  hair  erection  in  young 
males. 


15-53 


Serial  No.  Z01  AI  00149-04  LPD 

Hemolymph  transfer  experiments  have  shown  that  a  humoral  factor  is 
involved  in  the  inhibition  of  antennal  hair  erection  in  newly  emerged  males. 
One  of  the  candidates  for  this  inhibitory  factor  is  ecdysone,  the  growth 
hormone  present  at  the  time  of  adult  emergence  but  absent  soon  thereafter. 
When  young  males  are  injected  with  ecdysone,  the  length  of  the  non-erect 
period  can  be  extended,  there  is  no  response  to  an  epinephrine  stimulus  and 
the  males  will  not  mate.   It  appears  that  high  ecdysone  titres  at  emergence 
prevent  antenna  response. 

5)  Antibody  as  systemic  insecticides. 

Ideally,  host  antibodies  to  mosquito  internal  organs  and  secretions  could 
enter  the  mosquito  with  the  blood  meal  and  disrupt  normal  function.   In  nature, 
the  mosquito's  host  has  the  opportunity  to  form  antibody  only  to  mosquito 
salivary  fluid.   We  are  examining  the  possibility  of  immunizing  vertebrates 
with  antigens  that  could  cause  premature  death  of  the  biting  mosquito. 
Testing  of  the  antigenic  potential  of  mosquito  ovaries  and  midgut  has  already 
begun. 

6)  Genetics  of  vector  competence  of  Anopheles  pharoensis. 

Factors  which  determine  vector  competence,  particularly  those  which  deter- 
mine the  susceptibility  or  refractoriness  of  mosquitoes  to  the  pathogens  they 
transmit,  have  been  shown  to  be  genetically  determined.   This  study  is  designed 
to  establish  patterns  of  susceptibility  within  populations  of  the  Egyptian 
malaria  vector  An.  pharoensis .   Mosquito  lines  susceptible  or  refractory  to 
Plasmodium  cynomolgi  (a  simian  parasite  closely  related  to  the  human  parasite, 
P_.  vivax)  are  being  established  and  maintained  by  single  pair  and  mass  matings. 
These  lines  will  be  used  to  determine  the  mode  of  inheritance  of  susceptibility, 
and  once  genetically  defined  these  lines  will  be  used  to  study  some  of  the 
factors  which  determine  susceptibility. 

Proposed  Course: 

Investigations  will  be  continued  on  the  role  of  ecdysone  in  regulating  a 
number  of  physiological  and  behavioral  events  in  adult  mosquitoes.   A  radio 
immunoassay  will  be  employed  to  determine  ecdysone  titers  in  male  mosquitoes. 
Particular  emphasis  will  be  placed  on  a  comprehensive  study  of  the  laboratory 
biology  and  characteristics  of  An.  pharoensis  in  support  of  on-going  studies 
of  field  populations  of  this  species  in  Egypt.   Sera  from  animals  immunized 
with  ecdysone  or  juvenile  hormone  will  be  tested  as  systemic  insecticides. 
These  hormones  are  critical  physiological  modulators  in  mosquitoes  and  inter- 
ference in  their  activity  could  have  dramatic  results. 

Significance  to  Biomedical  Research: 

AnoDheline  mosquitoes  are  solely  responsible  for  the  transmission  of 
malaria  from  man  to  man.   However,  factors  which  regulate  their  behavior  and 
physiology  are  poorly  understood.   Our  investigations  are  aimed  at  developing 
an  understanding  of  certain  critical  and  possibly  vulnerable  stages  in  the 


25-54 


Serial  No.  Z01  AI  00149-04  LPD 

life  cycle  of  these  disease  vectors.   Disruption  of  one  of  these  events  or 
interference  in  the  capacity  to  transmit  malaria  could  prove  to  have  a  major 
impact  on  the  health  on  man  in  the  tropics. 

Publications : 

1.  Beach,  R.  F.:   Mosquitoes:   Biting  behavior  inhibited  by  ecdysone. 
Science  (in  press) 

2.  Nijhout,  H.  F.  and  Carrow,  G.  M. :   Diuresis  after  a  bloodmeal  in  female 
Anopheles  freeborni.   J.  Insect.  Physiol.  24:    293-298,  1978. 

Nijhout,  H.  F.  and  Martin,  S.  K. :   Alpha-adrenergic  activity  of  isoproterenal 
in  mosquito  antennae.    Experientia  34:   758-759,  1978. 


25-55 


SMI  THSONI  AN    SCIENCE    INFORMATION    EXCHANGE 
iPROJECT  NUMBER    (.Do   NOT   use   this   space) 


U.S.    DEPARTMENT   OF  I   PROJECT   NUMBER 

HEALTH,    EDUCATION,    AND    WELFARE 

p5BUMOTIC£0FS£RVICE  ZOl   AI    00160-03   LPD 

INTRAMURAL  RESEARCH   PROJECT 


[ PERIOD    COVERED 

;October    1,    1978    to    September    30,    1979 


TITLE   OF    PROJECT   (30   characters   or  less) 

Cell   Biology    of     Entamoeba,    Giardia  and  Other   Parasitic   Protozoa 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:      F.  D.  Gillin       Senior  Staff  Fellow,  LPD,  NIAID 

Other:   L.  S.  Diamond      Head,  Parasite  Growth  &  Differentiation  Section, 

LPD,  NIAID 
T.  E.  Nash         Medical  Officer,  LPD,  NIAID 


COOPERATING  UNITS  (if  any) 

•Dr.  Andrew  Plaut,  Gastroenterology  Div.,  Tufts  New  England  Medical  Center. 
Dr.  Pierre  Daggett,  American  Type  Culture  Collection. 


LAB/8RANCH 

Laboratory  of  Parasitic  Diseases 


SECTION 

Parasite  Growth  &  Differentiation  Section 


INSTITUTE  AND  LOCATION 

NIAID,  Bethesda,  Md.   20205 


TOTAL  MANYEARS: 

21/12 


PROFESSIONAL:  jOTHEfl: 

12/12  9/12 


CHECK  APPROPRIATE  30X(E3) 

j  (a)   HUMAN  SUBJECTS  Xj  (b)  HUMAN  TISSUES  ~  (c)  NEITHER 

E  (a1 )  MINORS   □  (a2)  INTERVIEWS     


SUMMARY  OF  WORK  (200  worts  or  less  -  underline  keywords) 

The  current  work  is  part  of  a  study  of  the  cell  biology  and  cell  physiology  of 
human  intestinal  protozoan  pathogens.  Previous  reports  were  f ocussed  predomi- 
nantly upon  E.  histolytica.   The  present  report  describes  studies  with  Giardia 


lamblia  in  the  following  areas: 


1)  The  role  of  reducing  agents  in  promoting  attachment,  growth  and  resis- 
tance to  killing  by  oxygen. 

2)  The  kinetics  of  and  requirements  for  attachment  in  a  model  system  and 
the  effects  of  host  factors  and  chemotherapeutic  agents  upon  attachment. 

3)  Interactions  of  G_.  lamblia  trophozoites  with  complement. 

4)  Permeability  and  surface  properties  of  cysts ■ 

5)  Isolation  of  new  strains. 

6)  Antibody  response  in  serum  and  secretions . 
25-56 

=hS-6040 
(Rev.  10-76) 


Serial  No.  Z01  AI  00160-03  LPD 

Project  Description; 

Our  interests  are  in  the  cell  biology  and  cell  physiology  of  two  impor- 
tant human  intestinal  protozoan  pathogens,  as  well  as  in  the  biochemical  and 
immunological  aspects  of  the  interactions  of  these  parasites  with  their  hosts. 
While  previous  reports  were  focussed  predominantly  on  Entamoeba  histolytica, 
the  current  report  describes  new  studies  with  Giardia  lamblia,  which  is 
increasingly  recognized  as  a  problem  within  the  United  States. 

I.  Growth,  Oxygen-Sensitivity  and  the  role  of  L-cysteine. 

Maximal  growth  of  G_.  lamblia  depended  upon  the  presence  of  L- 
cysteine  (6-12  mM) ,  even  under  N„  atmosphere.   L-cysteine  appeared  to  be  a 
specific  growth  factor.   Of  many  compounds  tested,  only  glutathione,  N-acetyl- 
L-cysteine  or  L-cystine  supported  slight  growth.   Both  L-cysteine  and  D- 
ascorbic  acid  also  functioned'  as  reducing  agents  as  they  delayed  the  killing 
of  G.  lamblia  trophozoites  by  increased  0„  tension:   ^140  mm  Hg,  compared 
with  30  mm  or  less  under  normal  growth  conditions. 

II.  Attachment  to  glass 

A.  Giardia  lamblia  trophozoites  show  a  striking  tendency  to  attach 
to  the  walls  of  their  culture  vessel.   We  have  observed  this  attachment  in 
axenic  cultures  as  well  as  with  trophozoites  from  duodenal  aspirates  and  from 
in  vitro  excystation. 

A  convenient  new  assay  for  attachment  in  axenic  cultures  was  developed. 
It  took  advantage  of  the  fact  that  after  removal  of  the  free  cells,  attached 
Giardia  trophozoites  can  be  detached  by  chilling  and  then  enumerated  in  a 
Coulter  Counter.   Free  swimming  and  attached  cell  populations  were  separated 
and  used  to  initiate  cultures.   The  doubling  time  of  the  cultures  initiated 
with  attached  cells  was  8.6  hours  compared  with  11.5  hours  for  cultures 
initiated  with  free  cells;  however  the  final  yields  were  the  same.   Attached 
cells  also  had  2-3  fold  higher  cloning  efficiencies.   Thus,  although  tropho- 
zoites could  detach  and  re-attach,  the  attached  population  as  a  whole,  appeared 
"healthier". 

B.  As  a  pre-requisite  to  studying  the  process  of  parasite  attach- 
ment in  mammalian  cell  and  organ  cultures,  the  kinetics  and  requirements  for 
the  attachment  of  the  trophozoites  to  glass  were  determined  and  a  simple 
maintenance  medium  (MM-2)  was  devised. 

Attachment  was  rapid;   ^  75%  of  the  maximum  values  were  attained  in 
1  hour  or  less.   Both  attachment  and  survival  were  dependent  upon  cysteine; 
other  thiol  compounds  were  30-80%  as  active,  while  ascorbic  acid,  a  non-thiol 
reducing  agent,  was  inactive.   Serum  (bovine  or  human) ,  which  also  promoted 
attachment  and  survival,  could  be  replaced  by  Cohn  serum  fraction  III  which 
consists  mainly  of  8-globulins.   Other  bovine  serum  fractions  such  as  Cohn  II, 
IV,  or  V  (albumin) ,  as  well  as  transferrin,  B-lipoprotein,  f etuin  and  gastric 
mucin  were  inactive.   This  activity  was  in  the  >  10,000  molecular  weight 


25-57 


Serial  No.  Z01  AI  00160-03  LPD 

fraction  of  the  Cohn  III  preparation.   Both  attachment  and  survival  were  very 
sensitive  to  pH  and  ionic  strength:   pH  6.9-7  and  200-300  milliosmoles/kg 
were  optimal.   The  maintenance  medium  (MM-2)  derived  from  these  studies  has 
been  employed  to  study  the  effects  of  the  chemotherapeutic  agent  Atabrine 
(quinacrine  HC1)  upon  attachment  and  survival. 

Secretory  factors,  including  immunoglobulins  are  likely  to  be  impor- 
tant in  host  defense  against  giardiasis.   In  preliminary  studies,  low  concen- 
trations (6  ug/ml)  of  a  partially  purified  preparation  of  secretory  IgA  from 
pooled  human  colostrum  (C/S-IgA)  inhibited  attachment  of  trophozoites.   At 
higher  C/S-IgA  concentrations  or  with  longer  incubations,  trophozoites  were 
killed.   These  effects  were  observed  in  growth  or  maintenance  medium  in  the 
presence  of  Cohn  serum  fraction  III,  but  were  totally  prevented  by  1%  whole 
serum. 

Highly  purified  S-IgA  from  another  human  source  (p/S-IgA)  inhibited 
attachment  to  a  lesser  extent,  without  killing  the  parasites.   Heating  the 
C/S-IgA  (56  C,  20  min)  reduced  the  attachment-inhibiting  activity  to  the  level 
observed  with  p/S-IgA.   It  is  not  known  whether  the  S-IgA  molecules  have  anti- 
giardial  specificity,  however  binding  of  IgA  from  the  C/S-IgA  to  trophozoites 
was  revealed  by  indirect  fluorescence. 

III.  Host  Defences  (complement) 

Others  have  shown  that  the  alternate  complement  pathway  is  activated 
by  _E.  histolytica,  resulting  in  lysis  of  the  parasite.   In  contrast.,  we  have 
shown  that  G_.    lamblia  trophozoites  do  not  activate  complement.   Killing  of  the 
parasites  by  rabbit  hyper-immune  serum,  however,  is  strictly  complement 
dependent. 

IV.  Excystation 

We  have  confirmed  the  studies  from  Meyer's  laboratory  showing  that: 
1)  excystation  is  promoted  by  a  period  of  cyst  maturation  at  low  temperature 
followed  by  mild  acid  treatment  and  2)  cyst  preparations  vary  greatly  in  their 
ability  to  excyst.   We  have  shown  that  regardless  of  these  factors,  cyst  popu- 
lations are  heterogeneous:   most  cysts  are  able  to  exclude  fluorescent  com- 
pounds which  penetrate  trophozoites  e.g.  quinacrine.   Secondly,  a  small  pro- 
portion of  cysts  have  exposed  sugars  similar  or  identical  to  N-acetylgluco- 
samine,  as  shown  by  binding  of  fluorescent  lectins.   In  contrast,  to  date, 
we  have  not  observed  binding  of  any  lectin  to  the  trophozoite.   Acid  treat- 
ment of  the  cysts  increased  both  their  permeability  to  fluorescent  probes 
and  surface  labelling  with  lectins. 

V.  Isolation  of  G^.  lamblia  from  patients 

Only  one  strain  of  G_.  lamblia  is  currently  established  in  axenic 
culture.   Although  our  attempts  to  establish  new  cultures  from  duodenal 
aspirates  have  not  been  successful,  we  have  made  several  useful  observations: 


25-58 


Serial  No.  Z01  AI  00160-03  LPD 

1)  Of  the  six  media  tested,  TP-S-1  was  the  best. 

2)  Human  serum  was  superior  to  bovine  serum. 

3)  Trophozoites  died  within  1  hour  if  left  in  the  duodenal  fluid. 

4)  Only  cultures  from  which  the  mucus  and  duodenal  fluid  were  removed, 
by  changing  the  medium,  survived  longer  than  24  hours. 

VI.   Sera,  parotid  secretions  and  duodenal  fluid  from  patients  and  con- 
trols (normal  and  hypogammaglobinemic)  are  being  gathered  (in  collaboration 
with  Dr.  T.  Nash).   An  Elisa  method  for  determining  antibody  response  to  G_. 
lamblia  is  being  developed.   The  effects  of  host  defenses  upon  (1)  tropho- 
zoite attachment  and  survival  and  (2)  upon  excystation  (as  described  in 
previous  sections)  will  be  determined. 

Publications: 


Gillin,  F.D.,  and  Diamond,  L.S.:   Clonal  growth  of  Entamoeba  histolytica  and 
other  Entamoeba  in  agar.   J.  Protozool.  25:   539-543,  1979. 

Gillin,  F.D.,  and  Diamond,  L.S.:   Clonal  growth  of  Entamoeba  in  agar:   some 
applications  of  this  technique  to  the  study  of  their  cell  biology. 
Arch.  Invest.  Med.  9_(1)  :   237-246,  1979. 

Gillin,  F.D.,  and  Diamond,  L.S.:   Entamoeba  histolytica  and  Entamoeba  invadens : 
Effects  of  temperature  and  oxygen  tension  on  axenic  growth.   Exp.  Parasitol. 
(in  press) 

Gillin,  F.D.,  and  Diamond,  L.S.:   Clonal  growth  of  Giardia  lamblia  trophozoites 
in  a  semi-solid  agarose  medium.   J.  Parasitol.   (in  press) . 


15-59 


SMITHSONIAN    SCIENCE    INFORMATION    EXCHANGE  U.S.    DEPARTMENT   OF  |   PROJECT   NUMBER 

PROJECT   NUMBER    (Oo    NOT    use    this    soace)       HEALTH,    EDUCATION,    AND    aELFARE    | 

I  PUBLIC    HEALTH    SERVICE 

NOTICE  Of 
INTRAMURAL   RESEARCH   PROJECT 


01   AI   00161-03   LPD 


PERIOD    COVERED 

October    1,    1978    to    September    30,    1979 


TITLE  OF   PROJECT   ^30  characters   or  less) 
Immunochemistry   of   Parasitic   Diseases 


NAMES,    LABORATORY    AND    INSTITUTE   AFFILIATIONS,    AND    TITLES   OF    PRINCIPAL    INVESTIGATORS    AND    ALL   OTHER 
PROFESSIONAL   PERSONNEL   ENGAGED   ON   THE  PROJECT 

PI:       T.  E.  Nash,  Senior  Investigator,  LPD,  NIAID 

Others:    Fouad  Boctor,  Fogarty  Fellow,  LPD,  NIAID 

A.  W.  Cheever,  Assistant  Chief,  LPD,  NIAID 
David  Taylor,  Fellow,  Naval  Medical  Research  Institute 
Eric  A.  Ottesen,  Senior  Investigator,  LPD,  NIAID 
Nasir-ud-Din,  Associate  in  Biological  Chemistry,  Laboratory  of 
Carbohydrate  Chemistry,  Harvard  Medical  School 
Carl  F.   T.  Mattern,  Senior  Investigator,  LPD,  NIAID 
Wendell  A.  Daniel,  Biologist,  LPD,  NIAID 
Frances  D.  Gillin,  Senior  Staff  Fellow,  LPD,  NIAID 
Louis  S.  Diamond,  Head,  Parasite  Growth  &  Differentiation,  LPD, 

Milford  N.  Lunde.  Research  Zoologist,  LPD,  NIAID 


NIAID 


COOPERATING  UNITS  (if  any) 

Laboratory  of  Clinical  Investigation,  NIAID,  Naval  Medical  Research  Institute, 
Harvard  University 


LAB/BRANCH 

Laboratory  of  Parasitic  Diseases 


SECTION 

Host-Parasite  Relations  Section 


INSTITUTE  ANO  LOCATION 

NIAID,  Bethesda,  Md. 


TOTAL  MANYEARS: 


22/12 


PROFESSIONAL: 
17/12 


5/12 


CHECK  APPROPRIATE  30x(ES) 
[X  (a)   HUMAN  SUBJECTS 

Q  (a1  )  MINORS   Q  (a2)  INTERVIEWS 


Xj  (b)  HUMAN  TISSUES 


(c)  NEITHER 


SUMMARY  OF  «0RK  (200  words  or  less  -  underline  keywords) 

A  number  of  studies  centering  around  the  identification  and  composition  of 
schistosome  antigens  as  well  as  the  response  of  the  host  to  parasite  antigens 
are  in  progress.   A  series  of  experiments  were  performed  in  order  to  study  the 
control  of  release  of  schistosome  excretory-secretory  (E-S)  substances  in 
vitro  following  incorporation  of  labelled  N-acetyl-glucosamine .   The  processes 
involved  are  complex  and  multiple,  inhibited  by  Na  flouride,  colchcine,  carbachol 
and  secretions  from  other  schistosomes.   Damage  to  the  schistosome  tegument  was 
associated  with  increased  release  of  materials.   The  schistosome  secretory 
processes  are  most  likely  dependent  on  energy  metabolism,  microtubular  function 
and  worm  movement.   Identification  of  large  and  small  molecular  weight  E-S 
products  of  adult  S_.    mansoni  and  j$.  malayi  microfilaria  continue.   IgG  ELISA 
antibody  to  a  glycoprotein  fraction  of  adult  worms  in  infected  patients  was  found 
to  correlate  with  intensity  of  infection.   Clinical  studies  involving  G.  lamblia 
trophozoites  obtained  from  duodenal  aspirates  of  patients  as  well  as  the  specific 
antibody  responses  of  the  host  to  the  parasite  have  been  initiated. 


25-60 


PHS-6040 

(Rev.  10-/6) 


Serial  No.  ZOL  AI  00161-03  LPD 

Project  Description: 

A  major  effort  of  this  laboratory  continues  to  be  the  study  of  schistosome 
antigens.   Recent  investigations  have  concentrated  on  the  control  of  release  of 
schistosome  excretory-secretory  (E-S)  antigens.   After  labelling  adult 
schistosomes  with  tritiated  N-acetylglucosamine,  attempts  were  made  to  inhib- 
it E-S  processes  by  certain  inhibitory  procedures  or  substances.   The  metabolic 
inhibitors  Na  azide  and  Na  arsenite  resulted  in  the  release  of  both  large  and 
small  molecular  weight  products  into  the  media.   This  release  corresponded 
with  sloughing  of  the  worm's  outer  surface,  the  tegument.   On  the  other  hand, 
Na  flouride  treated  worms  showed  decreased  release  of  E-S  materials  and  no 
tegumental  loss.   Worms  held  at  0  degrees  C  showed  a  transitory  early  in- 
creased release  of  materials.   These  worms  were  not  irreversibly  damaged  since 
they  were  viable  after  rewarming.   It  is  postulated  that  procedures  which 
functionally  or  mechanically  disrupt  the  tegnment  result  in  increase  loss  of 
material.   Both  carboachol,  an  acetylcholine  like  substance,  and  colchcine 
inhibited  secretory  processes  at  certain  time  periods.   Cycloheximide  failed 
to  inhibit  secretory  processes  over  the  time  period  studied.   Electronmicro- 
scopy  confirmed  that  Na  aresenite  caused  tegumental  sloughing  at  the  level 
of  the  plasma  membrane.   Additionally,  it  was  found  that  large  molecular 
weight  E-S  substances  inhibited  the  release  of  secretions  from  other  schisto- 
somes (Nash,  Mattern,  Daniel). 

Antibodies  to  a  glycoprotein  fraction  derived  from  adult  schistosomes 
were  measured  in  infected  patients'  sera  by  an  ELISA  test.   The  levels  of  IgG 
antibodies  found  correlated  with  the  worm  burden  of  the  host.   In  contrast, 
antibodies  to  purified  gut  associated  proteoglycan  as  measured  by  a  similar 
ELISA  test,  correlated  with  IFA  IgG  antibodies  to  schistosome  gut  epitielial 
cells.   Levels  were  highest  in  acutely  infected  patients  and  fell  in  the 
chronically  infected  individuals  despite  high  levels  of  egg  excretion.   There- 
fore antibodies  to  purified  fractions  of  worms  have  correlated  with  important 
and  different  clinical  parameters  (Nash  and  Lunde) .   This  is  distinct  from 
the  failure  of  clinical  parameters  to  correlate  with  antibodies  to  crude 
fractions. 

Studies  are  underway  to  investigate  the  synthesis  and  turnover  of 
schistosome  tegument  using  radioactively  labelled  amino  sugars  (Nash  and 
Taylor) . 

The  analysis  of  small  and  large  molecular  weight  E-S  material  derived 
from  adult  S_.  mansoni  cultured  with  carbohydrate  precursor  materials 
continues.   Presently,  emphasis  is  being  placed  on  identification  of  small 
molecular  weight  glucosamine  containing  compounds. 

Analysis  of  small  and  large  molecular  weight  E-S  products  of  microfilaria 
of  B_.  malayi  has  begun  (Nash  and  Ottesen)  . 


25-61 


Serial  No.  Z01  AI  00161-03  LPD 

Clinical  studies  involving  patients  infected  with  G.  lamblia  have  been 
initiated.   Headway  is  being  made  in  attempts  to  culture  the  parasite  from 
duodenal  secretions.   Specific  antibody  responses  to  G.  lamblia  are  being 
studied  in  sera  and  other  body  secretions. 

Publications : 

1.  Nash,  T.  E. :   Antibody  response  to  a  polysaccharide  antigen  in  schistoso- 
miasis.  I.  Sensitivity  and  specificity.   Am.  J.  Trop.  Med.  Hyg.   27 :   939, 
1978.  --—   - 

2.  Nash,  T.  E.,  Ottesen,  E.  A.,  Cheever,  A.  W. :   Antibody  response  to  a 
polysaccharide  antigen  present  in  schistosome  gut.   II.   Modulation  of 
antibody  response.   Am.  J.  Trop.  Med,  and  Hyg.   27 :   944-950,  1978. 

3.  Amyx,  H.  L. ,  Asher,  D.  M. ,  Nash,  T.  E. ,  Gibbs,  C.  J.,  Gagdusek,  D.  C.: 
Hepatic  amebiasis  in  spider  monkeys.   Am.  J.  Trop.  Med.  Hyg.   27 :   888,  1978. 

4.  Boctor,  F.  M. ,  Nash,  T.  E. ,  Cheever,  A.  W. :   Isolation  of  a  polysaccharide 
antigen  from  Schistosoma  mansoni  eggs.   J.  Immunol.   122 :   39-43,  1979. 

5.  Nash,  T.  E. :   Schistosoma  mansoni:   Pattern  of  release  of  secretory 
products.   Experimental  Parasitology.   (In  press). 

6.  Nasir-ud-din,  Nash,  T.  E.,  Jeanloz,  R.  W. ,  McArthur,  J.  W.  and  Gminskin, 
D.  M. :  Immunologically  induced  alteration  in  the  morphology  of  the  cervical 
mucus  of  Macaca  radiata.   Fertility  and  sterility.   (In  press) . 

7.  Neva,  F.  A.,  Wyler ,  D.  J.  and  Nash,  T.  E. :   Cutaneous  leishmaniasis  -  A 
case  with  persistent  organism  after  treatment  in  presence  of  normal  immune 
response.   Am.  J.  Trop.  Med,  and  Hyg.   28.:   467-471,  1979. 


25-62 


SMITHSONIAN   SCIENCE    INFORMATION    EXCHANGE!  U.S.   DEPARTMENT   OF 

PROJECT   NUMBER    (Do    NOT    use    this    saace)      IHEALTH,    EDUCATION,    AND    WELFARE 

PUBLIC   HEALTH    SERVICE 
NOTICE  Of 
|    INTRAMURAL  RESEARCH   PROJECT 


PROJECT   NUMBER 


ZOl  AI  00162-03  LPD 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (30  characters  or  less) 

Biochemical  Cytology  of  Host-Parasite  Interactions  in  Parasitic  Protozoa 

{NAMES,    LABORATORY    AND    INSTITUTE   AFFILIATIONS,    AND    TITLES   OF    PRINCIPAL    INVESTIGATORS   AND    ALL    OTHER 
PROFESSIONAL   PERSONNEL   ENGAGED   ON   THE  PROJECT 


PI:     D.  Mo  Dwyer 

Other:   J.  Berman 

J.  Finerty 

M.  Gottlieb 

P.  Pinto  DaSilva 

E.  C.  Weinbach 

D.  J.  Wyler 

T.  B.  Fioretti 


Research  Microbiologist,  LPD,  NIAID 

Clinical  Associate,  LPD,  NIAID 

Research  Microbiologist,  LMI,  NIAID 

Guest  Investigator* 

Head,  Membrane  Biology  Section,  LPP ,  NCI 

Head,  Physiology  &  Biochemistry  Section,  LPD,  NIAID 

Senior  Investigator,  LPD,  NIAID 

Chemist,  LPD,  NIAID 


COOPERATING  UNITS  (if  any) 

*   Dept.  Pathobiology,  School  of  Hygiene  &  Public  Health,  Johns  Hopkins 
University,  Baltimore,  Md. 


LA8/.BRANCH 

Laboratory  of  Parasitic  Diseases 


SECTION 

Cell  Biology  and  Immunology 


INSTITUTE  AND  LOCATION 

NIAID,  Bethesda,  Md, 


TOTAL  MANYEARS: 

48/12 


PROFESSIONAL: 

36/12 


12/12 


CHECK  APPROPRIATE  30X(E3) 
□  (a)   HUMAN  SUBJECTS 


3  (b)  HUMAN  TISSUES 


(c)  NEITHER 


£  (a1  )  MINORS 


(a2)  INTERVIEWS 


SUMMARY  OF  «0RK  (200  words  or  less  -  underline  keywords) 

Aspects  of  the  cell  biology  and  immunology  of  host-parasite  interactions 
among  several  intra-  and  extracellular  parasitic  protozoa  are  being  investi- 
gated.  Leishmania  sp.  and  Trypanosoma  sp.  are  being  used  as  models  of 
intracellular  and  extracellular  parasitism,  respectively.   Emphasis  is 
placed  upon:  1)  determining  some  of  the  basic  chemical  and  antigenic  properties 
of  parasite  surface  membranes;  2)  ascertaining  the  nature  and  extent  of  the 
interactions  of  parasite  surfaces  with  specific  host  cell  types;  3)  defining 
the  basic  mechanisms  involved  in  the  intracellular  survival  and  multiplication 
of  parasites  within  host  cells;  and  4)  attempts  to  determine  the  means  by 
which  parasites  circumvent  host  immuno-def ense  systems.   Techniques  employed 
in  these  studies  include:  subcellular  fractionation,  ultracyto-  and  immuno- 
chemistry,  electron  microscopy,  affinity  chromatography,  polyacrylamide  gel 
electrophoresis,  lectin  assays,  radio  isotope  labeling,  gel  immunoassays,  and 
in  vitro  cell  culture. 


25-63 


PHS-6040 
(Rev.  IO-76) 


Serial  No.  Z01  AI  00162-03  LPD 


Project  Description  and  Objectives: 


How  parasites  evade  host  immune  responses,  are  nourished,  multiply,  and 
eventually  destroy  the  host  are  questions  being  asked  in  this  investigation. 
In  as  much  as  all  initial  interactions  between  host  and  parasite  occur  at 
the  level  of  their  respective  cell  membranes,  and  understanding  of  the 
nature  and  physiology  of  these  membranes  is  essential.   It  is  to  this  end 
that  parasite  surface  membrane  structure,  chemical  composition,  antigenic 
nature,  and  physiology  are  being  investigated  specifically  with  regard  to 
leishmaniasis  and  trypanosomiasis.   Further,  parasite  interactions  with 
specific  host  cell  types  and  immune  systems  are  being  studied  to  ascertain 
the  role  of  parasite  surface  membranes  in  infectious  disease  processes. 

Methods  Employed: 

Techniques  used  include:  1)  fine  structure  (transmission  and  scanning 
electron  microscopy,  ultrastructural  cyto-  and  immunochemistry,  freeze 
fracture  and  -etching  methods,  and  autoradiography);  2)  subcellular 
fractionation;  3)  analytical  and  preparative  polyacrylamide  slab  gel 
electrophoresis  and  isoelectric  focusing;  4)  antibody  and  lectin  binding 
assays;  5)  radio  isotope  labeling  and  autoradiography;  6)  chromatography 
(gel  permeation  and  affinity);  7)  qualitative  and  quantitative  gel  immuno- 
precipitin  assays;  and  8)  in  vitro  cell  culture. 

Major  Findings: 

I.   Isolation  and  characterization  of  subcellular  components  from  Leishmania 
and  Trypanosoma. 

1.   Isolation,  structure,  and  carbohydrate-and  antigenic  composition 
of  Leishmania  donovani  surface  membranes  (Dwyer) . 

Utracentrif uge  bouyant  density  floatation  methods  were  devised  to  in- 
crease the  yield  of  isolated  L_.  donovani  promastigote  pellicular  membranes 
(PM) .   Using  these  methods,  the  PM  yield  (>98%  purity)  was  increased  by  °^4 
fold  (i.e.  >15  mg  protein/isolation  from  5X10-1-0  cells)  over  our  preceeding 
method.   These  membranes  had  a  density  equivalent  to  ^1.19  g/cm-*  and  dis- 
played a  characteristic  structural  asymmetry  (i.e.  subpellicular  micro- 
tubules remained  attached  to  the  PM  inner  lamina).   Dissolution  of  the  PM 
bilayer  occurred  to  varying  degrees  following  treatment  with  7  different 
nonionic  and  fitter  ionic  detergents.   The  subpellicular  microtubules 
remained  insoluble  to  varying  degrees  in  these  detergents.   Triton  X-100 
treatment,  although  only  ^50%  effective  with  regard  to  its  solubilizing 
activity,  rendered  most  PM  peptide  constituents  (^40)  soluble.   The  PM 
Triton  extract  (PM-TE)  was  used  for  subsequent  antibody  and  lectin  studies. 
X-ray  microprobe  analyses  indicated  the  presence  of  relatively  large 
quantities  of  sulfur  and  phosphorous  in  the  isolated  PM  presumably  repre- 
senting phospholipids  and  sulfur  containing  amino  acid  residues. 

The  isolated  L.    donovani  PM  were  specifically  "stained"  with  fluorescein 
conjugated  heavy  meromyosin.   These  results  indicate  the  presence  of 

25-64 


Serial  No.  Z01  AI  00162-03  LPD 

actin  or  an  actin-like  constituent  in  the  PM-microtubule  complex. 

Isolated  PM  were  specifically  "stained"  for  the  sugars  _-D-mannose 
(Man),   -  and  _S-linked  galactose  (_-,  3-gal) ,  N-acetyl-galactosamine 
(NAc  GaT) ,  -glucosamine  (NAcGlu) ,  and  _-L-fucose  (Fuc)  using  eight  different 
lectin-fluorescein  conjugates.   Fine  structure  localization  results  with 
ferritin  conjugates  of  these  lectins  indicated  that  most,  if  not  all, 
glycosylated  ligands  were  asymmetrically  oriented  in  the  PM  outer  surface 
lamina.   Detergent  solubilized  PM  separated  by  sodium  dodecylsulf ate- 
polyacrylamide  gel  electrophoresis  (SDS-PAGE)  and  stained  with  Periodic 
acid-Schiffs  reagent  (PAS)  had  ^20  major  presumptive  carbohydrate 
containing  constituents  ranging  in  apparent  molecular  weight  from  <1.2 
to  >    28  x  104  daltons.   Similar  SDS-PAGE  PM  preparations  were  "stained" 
with  various  lectin-fluorescein  conjugates,  photographed,  and  subsequently 
stained  with  Coomassie  Blue  to  determine  both  the  characteristic  specific 
lectin  -  "stained"  carbohydrate  -  and  peptide  banding  patterns.   With 
the  exception  of  a  presumptive  glycolipid  band,  all  lectin  -  "stained 
PM  bands  were  coincidently  stained  for  protein  suggesting  that  most  PM 
carbohydrates  were  side  chain  ligands  on  membrane  glycoproteins.   The 
lectin  -  "stained"  PM  bands  had  relative  mobilities  coincident  with  those 
obtained  by  the  PAS  method.   Many  PM  glycoproteins  were  "stained"  by  2 
or  more  of  the  various  lectins  indicating  compositional  heterogeneity 
in  their  carbohydrate  side  chains.   Cumulative  results  suggest  that  PM 
have  a  minimum  of  20  glycoprotein  constituents  some  of  which  contain 
Man,  _-  and  g-Gal,  NAcGal,  NAcGlu  and  Fuc,  and  these  are  presumably 
oriented  on  the  PM  external  surface  lamina. 

Antibodies  were  raised  in  rabbits  against  the  isolated  PM.   These  have 
been,  and  currently  are  being  used  in  a  variety  of  gel  immunoprecipitin 
assays  to  ascertain  the  antigenic  complexity  of  the  PM.   Precipitin  and 
SDS-PAGE  analyses  have  demonstrated  the  polyvalancies  of  these  sera  against 
the  PM-TE.   These  anti-membrane  sera  have  also  given  positive  precipitin 
results  against  7  PM  mannose  containing  glycopeptides  which  were  isolated 
from  PM  via  lectin-af f inity  chromatography.   Polysaccharide  antigens 
isolated  from  6  different  trypanosomatid  species  (by  Gottlieb)  were  speci- 
fically precipitated  by  anti-_L.  donovani  PM  sera.   These  results  suggest 
that  all  of  these  species  possess  a  common  or  cross-reacting  surface 
membrane  antigen.   Further,  this  "common"  antigen  was  also  detected  as  an 
"exoantigen"  in  the  sera  of  L.  donovani  infected  hamsters.   Sera  from  such 
animals  concomitantly  gave  a  single  positive  precipitin  against  the 
PM-TE  indicating  the  presence  of  antibodies  against  some  PM  antigens  other 
than  the  "common"  polysaccharide. 

2.   Freeze-fracture  and  -etching  of  L.  donovani  PM  (Pinto  DaSilva  and 
Dwyer ) . 

Freeze— fracture  and  -etching  studies  were  performed  in  order  to 
ascertain  further  information  concerning  the  supramolecular  structure  of 
isolated  PM.   Fine  structure  observations  of  such  preparations  demonstrated 
the  intramembranous  particle  (IMP)  distribution  in  PM.   Greater  numbers  of 

25-65 


Serial  No.  Z01  AI  00162-03  LPD 

IMP  were  associated  with  the  "P-"  rather  than  the  "E-face"  of  PM.   The 
subpellicular  microtubules  (MT)  remained  attached  to  the  fractured  PM 
inner  lamina.   The  "P-Face"  IMP  appeared  to  be  aligned  with  respect  to  the 
attached  MT;  however,  no  structural  bridge  was  discerned  between  the  MT 
and  PM  inner  lamina.   The  PM  "E-face"  IMP  were  randomly  distributed. 
These  results  suggest  that  the  MT  might  be  attached  to  the  PM  via  a  inner 
lamina  peripherally  oriented  membrane  protein. 

3.  Enzyme  characterization  of  L.    donovani  PM  (Gottlieb  and  Dwyer) . 

In  order  to  establish  functional  biochemical  and  physiological 
markers  for  isolated  L_.  donovani  PM  a  series  of  enzymes  were  investigated 
using  standard  colorimetric  and  radioisotopic  procedures.   Some  of  these 
have  also  been  localized  in  the  PM  using  ultrastructure  cytochemical 
techniques.   The  enzymes  activities  were  characterized  with  regard  to  pH 
and  temperature  optima,  cation  requirement,  and  the  time  course  of  the 
reaction.   All  enzymes  examined  to  date  have  temperature  optima  above  that 
used  for  optimum  growth  (i.e.  26°C).   Acid  phosphatase  had  a  pH  optimum 
of  5,  was  fluoride  sensitive,  and  was  as  active  in  whole  cells  as  in 
homogenates  suggesting  its  external  surface  orientation.   In  isolated 
membrane  fractions,  this  activity  was  enhanced  5-7  fold  over  that  of  the 
homogenate.   No  activity  was  present  in  the  alkaline  pH  range.   These 
results  in  conjunction  with  cytochemical  localization  results  have  demon- 
strated that  this  enzyme  is  indeed  localized  on  the  outer  surface  of  the 
PM.   This  finding  is  of  considerable  significance  as  acid  phosphatase  is 
a  lysosomal  marker  enzyme!   These  results  suggest  that  the  organism's 
plasma  membrane  is  pre-adapted  for  life  in  a  hydrolytic  environment. 
Hexokinase  appears  not  to  be  associated  directly  or  at  least  firmly  bound 
to  the  PM.   5 '-nucleotidase  was  fluoride  sensitive  and  was  distinguished 
from  the  acid  phosphatase  by  pH  optimum  and  substrate  specificity.   Its 
activity  was  enhanced  >5  fold  over  the  initial  homogenate.   Cytochemical 
results  indicate  that  this  enzyme  is  PM  bound.   ATPase  activity  was  not 
significantly  inhibited  by  fluoride  and  had  a  pH  optimum  of  %7.0.   The 
activity  was  stimulated  by  Mg++,  slightly  inhibited  (<20%)  by  ouabain, 
and  was  ^2  fold  enriched  in  the  PM  fraction  over  the  initial  homogenate. 
Preliminary  cytochemical  data  indicate  that  this  enzyme  is  localized  on 
the  PM  inner  lamina. 

4.  Isolation  and  characterization  of  L.  donovani  kinetoplast-mito- 
chondria  (Weinbach  and  Dwyer) . 

A  highly  enriched  kinetoplast-mitochondrian  (K-M)  fraction  was  obtained 
from  homogenates  of  L.  donovani  using  various  differential  and  sucrose 
gradient  centrifugation  techniques.   The  isolated  K-M  had  a  bouyant 
density  of  ^1.22  g/Cm35  and  they  characteristically  possessed  inner  and 
outer  membranes,  cristae,  and  some  amount  of  supercoiled  K-DNA.   The 
terminal  respiratory  metabolism  of  these  organelles  is  being  investigated 
currently  with  regard  to  characterization  of  their  constituent  enzymes 
and  terminal  electron  acceptors.   For  details  see  Dr.  Weinbach 's  report 
(i.e.  Z01  AI  00098-23). 


15-66 


Serial  No.  Z01  AI  00162-03  LPD 

5.   Isolation  of  surface  membranes  from  Trypanosoma  rhodesiense 
(Finer ty  and  Dwyer). 

Methods  were  devised  for  the  successful  isolation  of  pellicular 
membranes  (PM)  from  T_.    rhodesiense  bloodstream  forms.   These  membranes 
had  a  bouyant  density  equivalent  to  %1. 176g/cnH.   They  retained  their 
structural  and  spacial  asymmetric  association  with  subpellicular  micro- 
tubules, however,  they  lacked  the  surface  coat  characteristic  of  the 
intact  parasite  plasma  membrane.   Results  of  SDS-PAGE  analyses  indicated 
that  the  isolated  PM  contained  -v3  2  major  peptides  ranging  in  apparent 
molecular  weight  from  <1.4  -  30  X  10  daltons.   These  membranes  also 
contained  a  characteristic  major  peptide  band  at  V5.3  X  10^  daltons  which 
was  presumed  as  tubulin. 

II.  Host-parasite  interactions  _in  v i tr o . 

1.   Human  monocyte  derived  macrophages  (Berman,  Dwyer,  and  Wyler). 

The  intracellular  fate  of  _L.  donovani  and  L_.  tropica  amastigotes  was 
investigated  _in  vitro  using  a  human  peripheral  monocyte  derived  macro- 
phage culture  system.   Results  obtained  using  fine  structure  labeling 
methods  indicated  that  the  intracellular  parasites  reside  and  multiply 
within  the  phagolysosomal  system  of  this  host  cell  type.   These  results 
suggest  that  parasite  survival  is  based  upon  resistance  to  host  lysosomal 
enzyme  digestion.   For  details  see  Wyler 's  report  (i.e.  #Z01  AI  00109-07 
LPD). 

Proposed  Course: 

1.  Various  monospecific  antibody,  lectin,  and  radiochemical  probes 
will  be  used  further  to  qualitatively  and  quantitatively  elucidate  the 
nature  of  the  intact  Leishmania  sp.  and  Trypanosoma  sp.  PM. 

2.  Specific  antibody  and  lectin  affinity  chromatography  and  radio- 
labeling  techniques  will  be  used  to  preparatively  isolate  specific 
parasite  PM  constituents  for  their  subsequent  detailed  chemical  analyses 
(i.e.,  amino  acid  and  carbohydrate  composition). 

3.  Monospecific  antibodies  against  Leishmania  PM  antigens  will  be  used 
as  probes  to  ascertain  the  presence  and  identity  of  parasite  antigens  on 
the  surface  of  infected  macrophages  in  vitro.   Such  antibodies  might  also 
be  tested  for  their  efficacy  to  suppress  parasite  growth  and  multipli- 
cation in  cultured  macrophages  In  vitro. 

4.  Lipid  compositional  analyses  of  the  isolated  parasite  PM  are 
planned  in  collaboration  with  a  tentative  postdoctoral  candidate  and 
Dr.  Edgar  Ribi  (Rocky  Mt.  Lab.,  NIAID) . 

5.  Studies  will  be  pursued  concerning  the  nature  and  cellular  origin 
of  parasite  exoantigens  present  in  host  circulation,  and  their  possible 


15-67 


Serial  No.  Z01  AI  00162-03  LPD 

effects  on  the  state  of  host  immune  responsiveness. 

Significance: 

Basic  research  concerning  the  molecular  and  structural  composition  of 
specific  protozoan  parasite  surface  membranes  and  their  interactions 
with  specific  host  cell  types  might  provide  a  basis  for  future  immuno- 
prophylaxis  programs  and  regimens  and,  or,  a  more  rational  approach 
toward  the  development  of  effective  delivery  systems  for  chemotherapeutic 
treatment  of  these  diseases  of  man. 

Publications: 

1.  Dwyer ,  D.  M.:   Membrane  interactions  between  Leishmania  and  host 
cells.   In  Schlessinger,  D.  (Ed.):   Microbiology-1979.   Washington,  D.  C, 
American  Society  of  Microbiology,  130-134. 

2.  Dwyer,  D.  M.:   Externally  disposed  surface  membrane  constituents  of 
Leishmania  donovani  promastigotes.   Proc.  IV  Int.  Cong.  Parasitol., 
Sect.  F:   37-38,  1978. 

3.  Capron,  A.  and  Dwyer,  D.  M. :   The  surface  antigens  of  parasitic 
protozoa.   In  Hutner,  S.  (Ed.):   Protozoological_Actualities.  Allen  Press, 
Lawrence,  Kansas.   (In  press) . 

4.  Dwyer,  D.  M.:   The  recent  advances  in  in  vitro  cultivation  methods  of 
trypanosomatids.   In  Hutner,  S.  (Ed.):   Protozoological  Actualities. 
Lawrence,  Kansas,  Allen  Press.   (In  presiT! 

5.  Dwyer,  D.  M. :   Isolation  and  partial  characterization  of  surface 
membranes  from  the  human  protozoan  pathogen,  Leishmania  donovani.   Proc. 
Nat.  Acad.  Sci.  USA.   (In  press) . 

6.  Dwyer,  D.  M.  and  D'Alesandro,  P.  A.:   Isolation  of  surface  membranes 
from  Trypanosoma  lewis i  bloodstream  forms.   J.  Parasitol.   (In  press). 

7.  Berman,  J.  D.,  Dwyer,  D.  M. ,  and  Wyler,  D.  J.:   Growth  of  Leishmania 
in  human  macrophages  _in.  vitro.   Infect.  Immun.   (In  press)  . 


25-68 


(SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE!     U.S.  DEPARTMENT  OF 
PROJECT  NUMBER  (Do  NOT  use  this  space)   IHEALTH,  EDUCATION,  AND  WELFARE 

PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  AI   00174-02   LPD 


PERIOD   COVERED 

October    1,    1978    to   September    30.    1979 


! TITLE   OF   PROJECT   (80   characters   or  less 
Culture,    physiology   and   antigenic   analysis   of    sexual   and    asexual   erythrocytic 


malaria   parasites 


NAMES,   LABORATORY   ANO    INSTITUTE   AFFILIATIONS,    AND   TITLES  OF    PRINCIPAL    INVESTIGATORS   AND   ALL   OTHER 

PROFESSIONAL   PERSONNEL   ENGAGED  ON   THE   PROJECT 

PI:     R.  Carter  Visiting  Scientist,  LPD,  NIAID 

L.  H.  Miller  Head,  Malaria  Section,  LPD,  NIAID 

Other:   R.  Gwadz  Res.  Entomologist,  LPD,  NIAID 

J.  Johnson  Visiting  Associate,  LPD,  NIAID 

R.  F.  Beach  Staff  Fellow,  LPD,  NIAID 

J.  Rener  Staff  Fellow,  LPD,  NIAID 

N.  Epstein  Visiting  Associate,  LPD,  NIAID 

D.  C.  Kaushel  Visiting  Fellow,  LPD,  NIAID 

T.  Shiroishi  Zoologist,  LPD,  NIAID 

Y.  Rosenberg  Visiting  Fellow,  LMI ,  NIAID 

I.  Green  Senior  Investigator,  LI,  NIAID 


cooperating  units  (if  any)  M#  Aikawa,  Case  Western  Reserve  University,  Cleveland; 
J.  D.  Haynes,  Dept.  of  Immunology,  WRAIR;  R.  Schmidt-Ullrich,  Tufts 
University  Medical  School,  Boston. 


LAB/BRANCH 

Laboratory  of  Parasitic  Diseases 


SECTION 
Malaria 


INSTITUTE   AND    LOCATION 

NIAID,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

130/12 


PROFESSIONAL: 

79/12 


51/12 


CHECK  APPROPRIATE  30X(ES) 
JXi  (a)   HUMAN  SUBJECTS 

D  (aQ  MINORS   Q  (a2)  INTERVIEWS 


3  (b)  HUMAN  TISSUES 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Animals  have  been  successfully  vaccinated  against  sexual  and  asexual  malaria 
parasites.   In  addition,  Plasmodium  falciparum,  the  major  malaria  of  man, 
has  been  grown  in  culture  for  the  production  of  asexual  stages  and  gametocytes. 
The  present  study  will  1)  Improve  culture  conditions  for  the  production  of 
large  numbers  of  gametocytes,  gametes  and  merozoites  of  J?,  falciparum;  2) 
Study  of  the  physiology  of  exf lagellation,  fertilization  and  invasion  of  red 
cells  by  malaria  merozoites;  3)  Characterize  structure,  function  and  immuno- 
genic ity  of  surface  determinants  on  gametes  and  merozoites;  4)  Evaluate  gamete 
vaccines  in  model  systems  for  identification  of  the  best  antigens  and  adjuvants. 


25-69 


PHS-6040 
(Rev.  10-76) 


Serial  No.  Z01  AI  00174-02  LPD 
Project  Description: 
Objectives: 

1.  Improvement  of  culture  conditions  for  sexual  and  asexual  parasites  of 
Plasmodium  falciparum.   This  will  include  methods  for  fractionation  of 
asexual  and  sexual  parasites  and  attempts  to  understand  the  controls  on 
gametocytogenesis  and  development  of  gametocytes  in  culture. 

2.  Study  of  the  physiology  of  gamete  formation,  fertilization  and  merozoite 
invasion  of  RBCs. 

3.  Study  of  structure,  function  and  immunogenicity  of  antigenic  determinants 
on  the  surface  of  extracellular  sexual  and  asexual  parasites.   This  will 
include  the  use  of  monospecific  antibodies  produced  by  hybridomas. 

4.  Study  of  immunogenicity  of  crude  antigenic  preparations  of  sexual  and 
asexual  stages  in  model  systems. 

Each  objective  is  interrelated  with  the  others:   Culture  will  be  needed 
for  production  of  antigenic  material  for  analysis  of  both  sexual  and 
asexual  erythrocytic  parasites.   Structure  and  function  of  parasite  deter- 
minants will  be  studied  in  the  framework  of  parasite  physiology.   Successful 
immunization  with  whole  parasite  preparations  will  identify  sources  of  more 
purified  immunogenic  materials. 

Methods: 


1.  Continuous  culture  of  red  cell  stages  of  Plasmodium  falciparum;  isolation 
of  sexual  and  asexual  stages;  purification  of  merozoites  and  gametes. 

2.  Use  of  model  systems:  P_.  gallinaceum-chicken  for  study  of  gametes  and 
gamete  immunity;  _P.  knowlesi-rhesus  for  study  of  mechanism  of  invasion  and 
gamete  immunity. 

3.  Use  of  polyspecific  antibody  to  parasite  antigens  for  immune  precipita- 
tion; high-resolution  gel  electrophoresis  and  electrof ocussing  separation 
techniques;  i-25j  lactoperoxidase  surface  labelling;  general  metabolic 
labelling  of  all  parasite  components. 

4.  Monospecific  antibody  produced  in  hybridomas  for  analysis  of  parasite 
surfaces  and  isolation  of  surface  determinants. 

Major  Findings: 

1.   The  conversion  of  asexual  parasites  of  P_.  falciparum  to  gametocytes  in 
culture  was  shown  to  be  controlled  by  environmental  stimuli.   On  subculture 
gametocyte  production  ceases  during  a  period  of  rapid  asexual  proliferation 
coincident  with  the  deterioration  of  the  health  of  the  asexual  parasites. 
At  parasitemias  above  2%,  asexual  parasites  begin  to  convert  to  gametocytes. 


25-70 


Serial  No.  Z01  AI  00174-02  LPD 
This  was  uninfluenced  by  the  age  of  the  red  cells  in  culture. 

2.  Using  a  method  for  measuring  conversion  to  gametocytes,  it  was  shown 
that  1  mM  cAMP  added  to  the  culture  medium  at  the  end  of  the  rapid  phase 
of  asexual  growth  stimulated  the  almost  complete  conversion  of  asexual 
parasites  to  gametocytes.  Metabolities  of  cAMP  had  no  influence  on  con- 
version.  Theophylline,  a  phosphodiesterase  inhibitor,  stimulated  gameto- 
cytogenesis  in  the  presence  of  limiting  concentrations  of  cAMP. 

3.  Following  immunization  of  Balb/c  mice  with  gametes  of  P_.  gallinaceum, 
spleen  cells  from  the  immunized  mice  have  been  fused  with  myeloma  cells 
using  polyethylene  glycol.   Hybrid  cells  grown  in  selective  medium  (HAT) 
have  been  tested  for  the  production  of  antigamete  antibodies  using  immuno- 
fluorescent  antibody  test  against  gamete  preparations  as  an  assay.   Cloned 
lines  of  "hybridoma"  cells  producing  antigamete  antibodies  have  been 
selected;  high  levels  of  monoclonal  antibodies  were  produced  by  growing 
the  cloned  cell  lines  as  ascites  tumors  in  Balb/c  mice.  Monoclonal  anti- 
bodies have  a  variety  of  specificities  for  gamete  antigens  including  internal 
and  surface  antigens  of  macrogametes  and  antigens  involved  in  immobilization 
and  agglutination  reactions  of  microgametes.   Using  1^25  surface  labelled 
macrogametes  certain  monoclonal  antibodies  have  been  shown  to  precipitate 

a  single  major  protein. 

4.  Hybridoma  cell  lines  have  been  developed  against  merozoites  of  P_.  know- 
lesi. Most  such  hybridoma  will  produce  antibodies  which  are  specific  for 
surface  components  of  the  merozoites  as  determined  by  fluorescent  antibody 
reactions  against  living  intact  merozoites. 

5.  Mild  digestion  of  merozoites  of  P_.  knowlesi  with  trypsin  destroys  the 
ability  of  the  merozoites  to  penetrate  erythrocytes.   Such  digestion  is 
associated  with  the  loss  of  certain  high  molecular  weight  bands  on  SDS 
polyacrylamide  gel  electrophoresis.   These  proteins  may  be  involved  in  the 
attachment  of  merozoites  to  red  cells. 

6.  Human  serum  from  individuals  with  a  high  degree  of  naturally-acquired 
immunity  to  P_.  falciparum  precipitates  surface  antigens  of  merozoites  of 
P_.  knowlesi  following  I-"-*  surface  labelling.   The  precipitation  of  these 
bands  is  specifically  inhibited  by  antigens  of  P_.  falciparum.   This  indicates 
that  a  number  of  antigens  of  P_.  falciparum  share  common  specificities  with 
the  surface  antigens  of  merozoites  of  P_.  knowlesi. 

7.  a)   It  is  regularly  noted  that  active  malarial  infections  exert  a  non- 
specific suppressive  effect  on  the  host's  immune  system  and  may  affect 
vaccination.   We  have  found  that  monkeys  with  chronic  P_.  knowlesi  infections 
showing  a  rising  parasitemia  at  the  time  of  immunization  develop  little 
immunity  to  gametes  after  immunization  with  an  antigen  preparation  con- 
taining microgametes,  macrogametes  and  trophozoites  in  Freund's  complete 
adjuvant  (FCA) .   Chronically  infected  monkeys  without  patent  parasitemia 
developed  Immunity  to  the  sexual  stages  of  the  parasite. 


25-71 


Serial  No.  Z01  AI  00174-02  LPD 

b)   It  has  been  suggested  that  the  merozoite  is  the  most  effective  anti- 
gen for  immunization  against  the  asexual  stages  of  the  malaria  parasite. 
When  monkeys  were  immunized  with  _P.  knowlesi  trophozoites  (12  hours  old) 
in  FCA,  they  developed  Immunity  against  the  asexual  stages  of  the  parasite 
as  evidenced  by  low  grade  infections  when  challenged. 

8.   A  factor  has  been  identified  in  the  gut  of  mosquitoes  which  induces 
gametogenesis  in  gametocytes  of  P_.  gallinaceum.   Preparations  containing 
this  factor  may  be  obtained  by  feeding  Anopheles  stephensi  on  saline 
solutions  and  collecting  the  fluid  excreted  through  the  mosquito  anus 
during  the  feeding  process.   Preliminary  chemical  characterization  of  the 
active  factor  has  shown  it  to  be  a  molecule (s)  of  molecular  weight  200-300 
following  fractionation  on  a  sephadex  P400  column.   The  factor  is  stable 
to  boiling  for  10  minutes  but  is  destroyed  by  heating  to  300°C.   In 
chlorof orm/methanol  extraction  the  factor  is  recovered  only  from  the 
methanol  fraction.   The  only  amino  acids  identified  in  the  active  fraction 
from  sephadex  P400  are  lysine,  serine,  threonine  and  glycine.   The  factor 
is  on  present  evidence, a  low  molecular  weight  organic  molecule  without 
lipid  properties. 

Proposed  Course: 

1.  Determine  the  role  of  cAMP  in  gamecytogenesis  of  P_.  falciparum  in 
culture  at  the  metabolic  level  and  in  relation  to  the  phase  of  growth  of 
the  parasites  in  culture. 

2.  Use  the  methods  worked  out  in  the  studies  described  here  to  obtain 
cultures  of  P.  falciparum  producing  large  numbers  of  gametocytes  free  from 
asexual  parasites  in  order  to  conduct  studies  on  the  biology  and  immunology 
of  antigens  of  the  sexual  stages  of  this  parasite. 

3.  Using  metabolic  labels,  determine  the  nature  of  the  gamete  proteins 
synthesized  during  gametogenesis  in  terms  of  their  location  in  the  gametes, 
their  biological  function  and  their  role  in  antigamete  immunity. 

4.  Further  characterize  the  monoclonal  antigamete  antibodies  produced 
from  hybridomas  and  especially  i)  determine  their  ability  to  prevent  gamete 
fertilization  in  vitro  and  in  the  mosquito  and  ii)  isolate  the  target 
gamete  antigens  and  determine  their  locations,  biological  functions  and  role 
in  antigamete  immunity. 

5.  Determine  the  chemical  nature  of  mosquito  exf lagellation  factor  (MEF) 
using  sephadex  column  fractionation,  mass  spectroscopy  and  gas  liquid 
chromatography. 

6.  Use  incorporation  of  labelled  amino  acids  into  mature  schizonts  of 

P.  knowlesi  to  determine  whether  merozoites  contain  antigens  formed  during 
the  final  stage  of  schizont  development  preceeding  rupture. 


25-72 


Serial  No.  Z01  AI  00174-02  LPD 

7.  Analyze  surface  antigens  of  merozoites  by  immune  precipitation  of 
surface  labelled  and  metabolic  labelled  antigens  and  characterize  such 
antigens  in  relation  to  their  role  in  red  cell  invasion  and  protective 
immunity. 

8.  Further  characterize  the  monoclonal  anti-merozoite  antibodiesproduced 
from  hybridoma  cell  lines  and  especially  i)  determine  their  ability  to 
prevent  merozoite  invasion  of  red  cells  _in  vitro  and  ii)  isolate  the 
target  merozoite  antigens  and  determine  their  location,  biological  function 
and  role  in  protective  immunity. 

9.  Develop  the  Babesia  bovis  culture  system  for  analagous  studies  on 
babesia  merozoites.   These  babesial  merozoites  invade  red  cells  in  a 
similar  manner  to  malarial  merozoites  and  offer  certain  advantages  over 
malarial  merozoites  that  include  long  stability  and  availability  in  large 
numbers  from  culture.   In  addition,  the  probable  receptor,  a  complement 
component,  has  been  identified.   We  will  ask  the  question:   Will  isolated, 
purified  merozoite  proteins  induce  protective  immunity? 

Publications: 


1.  Carter,  R. ,  Gwadz,  R.  W.  and  McAuliffe,  F.  M.:   Plasmodium  gallinaceum: 
Transmission  blocking  immunity  in  chickens  I.   Comparative  Immunogenicity 
of  Gametocyte  and  Gamete-containing  Preparations.   Expl.  Parasitol. ,  47, 
185-193  (1979).  ~"'"~  """~ 

2.  Carter,  R.,  Gwadz,  R.  W.  and  Green,  I.:  Plasmodium  gallinaceum: 
Transmission  blocking  immunity  in  chickens  II.   The  effect  of  antigamete 
antibodies  in  vitro  and  in  vivo  and  their  elaboration  during  infection. 
Expl.  Parasitol.,  47,  194-208  (1979). 

3.  Carter,  R.  and  Miller,  L.  H. :  A  Method  for  the  study  of  gametocyto- 
genesis  by  Plasmodium  falciparum  in  culture:  Evidence  for  environmental 
modulation  of  gametocytogenesis.   Bull.  Wld.  Hlth.  Org,  (in  press). 

4.  Carter,  R. ,  Gwadz,  R.  W.  and  Green,  I.:   Naturally  acquired  immunity 
and  antimalarial  antibodies  in  relation  to  infectivity  to  mosquitoes  in 
endemic  Plasmodium  falciparum.   Report  of  Scientific  Working  Group  on 
Immunology  of  Malaria,  Panama,  June  1979  (in  press) . 

5.  Carter,  R.  and  Gwadz,  R.  W.:   Infectiousness  and  Gamete  Immunization 
in  Malaria.   Chapt.  17  in  Malaria  in  Man  and  Experimental  Animals.  Ed. 
J.  P.  Krier,  Academic  Press  (in  press). 

6.  Gwadz,  R.  W. ,  Carter,  R.  and  Green,  I.:   Gamete  Vaccines  and  Transmis- 
sion-Blocking Immunity  in  Malaria.   Bull.  Wld.  Hlth.  Org,  (in  press). 


-5-73 


Serial  No.  Z01  AI  00174-02  LPD 

7.  Miller,  L.  H.,  Aikawa,  M. ,  Johnson,  J.  G.,  and  Shiroishi,  T.:   Inter- 
action between  cytochalasin  B-treated  malarial  parasites  and  erythrocytes, 
Attachment  and  function  formation.   J.  Exp.  Med.,  149,  172-184,  (1979). 

8.  Martin,  S.  K.  ,  Miller,  L.  H. ,  Ailing,  D.,  Okoye,  V.  C,  Osan,  G,  J.  F., 
Osunkoye,  B.  0.,  Deane,  M. :   Severe  malaria  and  glucose-6-phosphate- 
dehydrogenase  deficiency:   A  reappraisal  of  the  malaria/G-6-PD  hypothesis. 
Lancet  I:   524-526  (1979). 

9.  Spencer,  H.  C.,  Miller,  L.  H. ,  Collins,  W.  E.  ,  Knud-Hansen,  C, 
McGinnis,  M. ,  Lobos,  R0  Ao  ,  Feldman,  R.  A.:   The  resistance  factor  to 
Plasmodium  vivax  in  Honduras.   The  Duffy  blood  group  negative  phenotype. 
Amer.  J.  Trop.  Med.  Hyg.  27_,  664-670  (1978). 

10.  Miller,  L.  H.,  McGinnis,  M.  H. ,  Holland,  P.  V.,  Sigmon,  P.:  The  Duffy 
blood  group  phenotype  in  American  blacks  infected  with  Plasmodium  vivax 
in  Vietnam.   Amer0  J.  Trop.  Med.  Hyg.,  27,  1069-1072  (1978). 

11.  Gwadz,  R.  W.  and  Green,  I.:   Malaria  immunization  in  rhesus  monkeys: 

A  vaccine  effective  against  both  the  sexual  and  asexual  stages  of  Plasmodium 
knowlesi.   J.  Exp,  Med.,  148,  1311-1323  (1978). 

12.  Miller,  L.  H. :   Editorial  Note.   Continuous  in  vitro  cultivation  of  the 
human  malaria  parasite.   Annals  of  Internal  Medicine.,  89_,  419  (1978). 

13.  Martin,  S.  K. ,  Miller,  L.  H.,  Hicks,  C.  U.,  David  West,  A.,  Ugbode,  C, 
Deane,  M.:   Frequency  of  Blood  Group  Antigens  In  Nigerian  Children  with 
Falciparum  Malaria.   Trans.  Roy.  Soc.  Trop.  Med.  &  Hyg.,  73,  216-218. 


25-^ 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
[PROJECT  MUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF         PROJECT  NUMBER 
HEALTH,  EDUCATION,  AND  WELFARE  j 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


ZOl  AI  00184-01  LPD 


PERIOD    COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Chemotherapy   of  Malaria:      The  Relationships   Between  Gametocytocidal, 
Sporontocidal   and   Hepatic   Schizontocidal  Drugs. 


NAMES,    LABORATORY   ANO    INSTITUTE  AFFILIATIONS,    AND   TITLES  OF    PRINCIPAL    INVESTIGATORS   AND   ALL   OTHER 
PROFESSIONAL   PERSONNEL   ENGAGED   ON   THE   PROJECT 


PI: 


R.  W.  Gwadz    Research  Entomologist,  LPD,  NIAID 


Other:   L.  C.  Koontz   Biologist,  LPD,  NIAID 

L.  H.  Miller   Head,  Malaria  Section,  LPD,  NIAID 


COOPERATING  UNITS  (If  any) 

I  Cols.  C.  J.  Canfield  and  D.  Davidson,  Division  of  Experimental  Therapeutics, 
WRAIR,  Walter  Reed  Army  Medical  Center 


LAB/ BRANCH 

Laboratory  of  Parasitic  Diseases 


SECTION 

Malaria 


INSTITUTE   AMD    LOCATION 

NIAID,    Bethesda,   Maryland    20205 


TOTAL  MANYEARS: 

14/12 


3SI0NAL: 
2/12 


12/12 


CHECK  APPROPRIATE  BOX(Eo) 
□  (a)   HUMAN  SUBJECTS 

(al )  MINORS   H  (a2)  INTERVIEWS 


(b)  HUMAN  TISSUES 


(c)  NEITHER 


SUMMARY  OF  WORK  (,200  words  or  less  -  underline  keywords) 

We  are  attempting  to  develop  a  simple  rapid,  inexpensive  method  for  screening 
for  anti-malarial  drugs  with  activity  against  hepatic  schizonts.   We  are 
determining  if  drugs  with  known  activity  against  hepatic  schizonts  i.e.  drugs 
which  block  relapses  of  certain  human  and  simian  malarias,  will  have 
gametocytocidal  activity  in  the  _P.  gallinaceum/chicken/Aedes  aegypti  system. 


25-75 


?US-6QA0 
(Rev.  10- 


Serial  No.  ZC1  AI  00184-01  LPD 

Project  Description: 

Gametocytes  share  certain  similarities  with  latent  exoerthrocytic 
hepatic  schizonts  in  that  both  stages  remain  dormant  until  stimulated  to 
undergo  further  development.   The  stimulus  for  gametocytes  to  complete 
development  to  the  gamete  stage  is  provided  in  the  gut  of  a  feeding  mosquito. 
The  stimulus  for  the  exoerythrocytic  parasite  to  complete  development  in  the 
liver  is  unknown.   The  two  stages  also  share  a  common  sensitivity  to 

-aminoquinoline  drugs,  particularly  primaquin.   Primaquin  is  the  drug  of 
choice  for  radical  cures  of  relapsing  malarias  such  as  P_.  vivax.   Therefore, 
drugs  exhibiting  gametocytocidal  effects  might  produce  a  radical  cure  of 
a  relapsing  malaria  by  eliminating  latent  parasites  from  the  liver. 
Alternatives  to  primaquine  are  desireable  because  of  the  drug's  long  course 
of  therapy  (14  consecutive  days) ,  because  of  unacceptable  side  effects  in 
some  individuals,  and  because  of  the  high  rate  of  drug  failure  against 
some  strains  of  P_.  vivax. 

Methods: 

We  have  developed  a  simple  screen  using  P_.  gallinaceum,  in  chickens 
which  can  detect  drugs  with  gametocytocidal  or  sporontocidal  properties  by 
their  effects  on  the  subsequent  development  of  the  sexual  stages  of  the 
parasite  in  Aedes  aegypti  mosquitoes.   Gametocytocidal  drugs  act  directly 
on  gametocytes  circulating  within  erythrocytes  in  the  vertebrate  hosts. 
Effects  are  irreversible  and  present  as  non-inf ectivity  of  the  parasite 
to  mosquitoes.   Sporontocidal  drugs  affect  the  parasite  after  the  parasite 
because  extracellular  and  after  initiation  of  gametogenesis  within  the 
gut  of  the  mosquito  vector.   In  this  screen  a  candidate  drug  is 
administered  directly  to  a  chicken  showing  a  patent  parasitemia.   One  batch 
of  mosquitoes  is  fed  on  the  chicken  just  prior  to  drugging  and  another 
batch  five  hours  later.   The  two  groups  of  mosquitoes  are  then  held  6-7 
days,  dissected,  and  their  guts  examined  for  the  presence  and  number  of 
developing  oocysts.   Drugs  with  gametocytocidal  or  sporontocidal  properties 
effect   parasite  development  in  two  ways  1)  Oocyst   numbeis  are  significantly 
reduced,  usually  by  over  95%;  2)  Oocysts  which  do  develop  are  less  than  1/3 
normal  size  at  7  days  post-feeding  and  do  not  complete  development. 
Drugs  with  blood  schizontocidal  properties  have  no  effect  on  the  development 
of  the  parasite  in  the  mosquito. 

Gametocytocidal  and  sporontocidal  drugs  can  be  further  differentiated 
with  a  2  step   technique  using  membrane  feeders.   1)  Blood  from  a 
previously  drug-treated  chicken  is  washed,  resuspended  in  normal  plasma 
and  fed  to  mosquitoes  through  a  membrane.   2)  Plasma  from  a  drug-treated 
chicken  is  mixed  with  parasitized  blood  from  an  untreated  bird  and  fed  to 
mosquitoes  through  a  membrane.   Drugs  with  gametocytocidal  properties 
affect  the  parasite  in  the  circulation;  washing  (technique  #1)  does  not 
restore  gametocyte  infectivity.   Conversely,  plasma  from  a  drug  treated 
chicken  has  no  inhibitory  properties  (technique  #2).   Sporontocidal  drugs 
affect  the  parasite  only  within  the  gut  of  the  mosquito.   Parasites 


25-76 


Serial  No.  Z01  AI  00184-01  LPD 

washed,  resuspended  in  normal  plasma  and  fed  to  mosquitoes  are  infectious. 
Plasma  from  drug-treated  chickens  mixed  with  parasites  from  untreated 
chickens  inhibits  oocyst  growth. 

Results: 

A  series  of  coded  compounds  provided  by  the  Division  of  Experimental 
Therapeutics,  WRAIR,   as   tested  with  the  P.  gallinaceum /mosquito  screen. 
After  the  code  was  broken  we  found  that  drugs  effective  in  producing 
radical  cures  of  P_.  cynomolgi  in  rhesus  monkeys,  i.e.  drugs  which  act 
against  latent  hepatic  schizonts,  had  clearly  demonstrable  gametocytocidal 
properties  in  our  screen. 

Drugs  with  sporontocidal  properties  were  less  clearly  differentiated. 
Some  of  these,  such  as  pyrimethamine,  appeared  to  have  both  gametocytocidal 
and  sporontocidal  effects,  although  pyrimethamine  does  affect  hepatic 
schizonts. 

Proposed  Course  of  the  Project: 

This  study  will  continue  and  be  expanded  to  evaluate  a  wide  variety 
of  compounds  provided  through  WRAIR.   Particular  emphasis  will  be  placed 
on  development  of  methods  for  differentiating  gametocytocidal  drugs  from 
sporontocidal  drugs.   Protocols  will  be  developed  for  the  screening  of 
newly  synthesized  compounds  with  the  aim  of  developing  new  classes  of 
hepatic  schizontocides. 

Significance  to  Biomedical  Research: 

A  substitute  for  primaquine,  the  only  available  hepatic  schizontocide, 
is  considered  one  of  the  major  needs  for  malaria  therapy.   However, 
development  of  new  drugs  has  been  hampered  by  the  lack  of  a  simple, 
inexpensive  screen  capable  of  evaluating  a  wide  range  of  candidate  compounds. 
We  feel  that  the  P_.  gallinaceum/Ae.  aegypti  system  may  function  as  a 
primary  screen  for  hepatic  schizontocides,  with  a  capacity  for  evaluating 
large  numbers  of  compounds  including  classes  of  drugs  not  yet  tested 
against  the  latent  tissue  phases  of  the  malaria  parasite. 

Publications: 

1.   Koontz,  L.  C.  Jacobs,  R.  L.,  Lummis,  W.  L. ,  and  Miller,  L.  H. : 
Plasmodium  berghei:   Uptake  of  clindamycin  and  its  metabolities  by  mouse 
erythrocytes  with  clindamycin-sensitive  and  clindamycin-resistant  parasites. 
Exp.  Parasitol.  (in  press). 


25-77 


[DEPARTMENT   Or 
BUCATION,    AND    WELFARE 


SMITHSONIAN    SCIENCE    INFORMATION    EXCHANGEl  'J 

PROJECT   NUMBER    (Do    NOT    use    this    space)       HEALTH,    cuuwuun,    «nu    icu 

PUBLIC   HEALTH   SERVICE 
NOTICE  Of 
!    INTRAMURAL  RESEARCH   PROJECT 


PROJECT   NUMBER 

ZOl  AI  00185-01  LPD 


PER  I  00    COVERED 

October    1,    1978    to   September    30,    1979 


TITLE  OF    PROJECT   (80   characters   or  less; 

Pathogenic  Protozoa:      Structure,    Division,    Virulence  Factors,    and 

Endogenous   Viruses. 


NAMES,  LABORATORY  ANO  INSTITUTE  AFFILIATIONS,  ANO  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:  C.  F.  T.  Mattern   Senior  Investigator,  LPD,  NIAID 


COOPERATING  UNITS  (if  any)  Dr.  L.  S.  Diamond,  LPD;  Dr.  B.  M.  Honigberg,  University  of 
Massachusetts;  Drs.  Michael  R.  Spence  and  John  K.  Frost,  Johns  Hopkins  University; 
Dr.  Shigeko  Nomura,  NCI;  Dr.  Lawrence  Abramson,  NNMC,  Bethesda. 


LAB/BRANCH 

Laboratory  of  Parasitic  Diseases,  NIAID 


SECTION 

Cell  Biology  and  Immunology  Section 


NSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

31/4 


PROFESSIONAL: 

1 


21/4 


CHECK  APPROPRIATE  30X(ES) 
G  (a)   HUMAN  SUBJECTS 

Q  (a!)  MINORS   □  (a2)  INTERVIEWS 


b   HUMAN  TISSUES 


□  (c)  NEITHER 


UMMARY  OF  «I0RK  (200  *oras  or  less  -  underline  keyworas) 

The  goal  of  this  project  (recently  transferred  from  LVD  to  LPD  with  three 
personnel)  is  to  evaluate  a  variety  of  factors  which  influence  the  virulence 
of  pathogenic  protozoa,  especially  Entamoeba  histolytica  and  Trichomonas 
vaginalis. 


Ongoing  studies  continue  evaluating  amebal  viruses,  selection  pressures  by 
repeated  animal  passage  at  limiting  dilution,  and  an  amebal  "toxin"  in  the 


virulence  of  several  strains  of  E.  histolytica.   Amebal  extracts  are  toxic 
for  mammalian  cells,  producing  rounding  and  aggregation  of  cells  in  tissue 
culture,  closely  resembling  the  cytotoxicity  of  the  plant  lectin,  concanavalin 
A..   The  former  effect  is  inhibited  by  normal  serum  of  many  species.   We  have 
identified  the  serum  component  as  the  glycoprotein,  fetuin.   Fetuin  and  the 
hexosamine,  N-acetyl-D-galactosamine  inhibit  and  reverse  the  cytotoxicity  of 
amebal  extracts  as  does<=<-methyl-mannoside  the  cytotoxicity  of  concanavalin 
ft..   We  believe  the  amebal  toxin  to  have  lectin-like  properties.  We  are  employing 
affinity  chromatography  to  purify  the  "toxin"  for  further  studv. 
!        25-78 

PH 3-6040 
(Rev.  IO-76) 


Serial  No.    Z01  AI    00185-01  LPD 
Summary    (continued): 

Studies   continue  on  alternate  passage  of  relatively  avirulent  amebal   strains 
in  newborn  hamster    liver   and    axenic   culture.      We  have   successfully 
increased  virulence   about   1,000-fold    twice  with   each   of    two   strains,    one 
having   remained   highly  virulent   in   axenic   culture   alone   for    20  months. 

Project  Description: 

Entamoeba  histolytica  "toxin". 

The  major  emphasis  of  this  project  in  the  past  year  has  been  in  the 
identification  of  the  factor  in  normal  serum  which  neutralizes  the  cyto- 
toxicity of  crude  amebal  extracts  for  baby  hamster  kidney  cells.   The 
cytotoxic  effect,  seen  only  in  the  absence  of  serum,  was  first  described 
by  Lushbaugh  et  al. ,  Medical  University  of  South  Carolina. 

Amebal  extracts  produce  rounding  and  aggregation  of  cells  with  dose 
dependent  kinetics.   The  serum  neutralizing  factor  has  been  found  to  be, 
in  all  probability,  the  serum  glycoprotein,  fetuin.   At  concentrations 
of  50  to  100  ug/ml,  three  of  four  commercially  available  fetuin  preparations 
completely  inhibit  10-5  amebal  equivalents  of  "toxin" /ml.   The  purest 
available  fetuin  (GIBCO,  prepared  by  the  Spiro  method,  99%  pure)  is  the 
most  active  preparation  tested,  showing  partial  protection  against 
the  above  "toxin"  at  12.5  ug/ml.   Other  purified  glycoproteins  similar 
to  fetuin,  including  asialof etuin,  oi—1   acid  glycoprotein,  and  carcino- 
embryonal  antigen  at  100  ug/ml  were  ineffective  in  neutralizing  amebal 
"toxin". 

A  number  of  purified  IgG  fractions  of  normal  sera  (mouse,  rabbit, 
goat,  human)  were  ineffective  at  1  to  16  mg/ml  against  the  "toxin". 

Serum  (>0.5%)  or  fetuin  (50-100  ug/ml)  not  only  completely  inhibit 
the  toxin,  they  also  reverse  the  rounding  and  aggregation  after  the  cells 
have  been  in  that  state  for  as  many  as  5  days.   Such  cells,  so  treated, 
reattach  to  the  tissue  culture  well  and  divide. 

Concanavalin  A,  a  plant  lectin,  produces  a  virtually  identical 
cytopathology  with  baby  hamster  kidney  cells  which  toxicity  is  inhibited 
and  reversed  by  0.1  Mof-methylmannoside  and  less  efficiently  by  mannose. 
The  amebal  "toxin",  however  is  also  inhibited  by  a  hexose,  the  hexosamine, 
N-acetyl-D-galactosamine . 

The  similarity  in  cytopathological  effects  of  amebal  extract  and 
concanavalin  A,  their  inhibition  and  reversal  by  N-acetyl-D-galactosamine 
and  °C-methyl-mannoside,  respectively,  and  the  evidence  provided  by 
Lushbaugh  et  al.  that  the  amebal  "toxin"  is  a  protein  of  25,000-35,000 
daltons  suggests  to  us  that  the  "toxin"  has  lectin-like  properties.   This 
is  supported  by  a  known  lectin-like,  mannose  inhibited,  carbohydrate- 
binding  surface  protein  of  the  free-living,  but  sometimes  pathogenic 


25-79 


Serial  No.  Z01  AI  00185-01  LPD 

ameba,  Acanthamoeba  castellani.   (Mattern,  Keister,  Natovitz) . 

Selection  and  maintenance  of  virulent  subpopulations  of  E.  histolytica. 

The  Rahman  strain  of  E.  histolytica  has  been  increased  3  orders  of 
magnitude  in  virulence  by  alternate  passages  for  1  week  in  newborn  hamster 
liver  and  1  week  in  axenic  culture.   For  passage  in  medium,  liver  lesions 
were  selected  from  among  the  largest  lesions  produced  at  limiting 
dilution.   The  first  isolate,  after  three  such  passages,  has  remained 
highly  virulent  for  the  newborn  hamster  liver  for  a  period  of  20  months 
of  biweekly  culture  in  axenic  medium.   A  repeat  experiment  has  resulted 
in  a  second  virulent  Rahman  line  which  has  remained  virulent  for  5 
months  of  continuous  axenic  culture. 

Strain  H-302:NIH  E.  histolytica  has  been  similarly  increased  100-to 
1,000-fold  in  virulence  by  alternate  liver  passage  and  axenic  culture. 
After  up  to  8  liver-culture  passages,  all  such  lines  have  rapidly  declined 
in  virulence  in  axenic  culture  alone.   We  are  continuing  the  liver 
passages,  repeating  the  experiment,  and  evaluating  virulence  stability 
of  clonal  lines  of  this  strain.   (Mattern,  Keister) . 

Trichomonas  vaginalis. 


A  collaborative  project  on  T.  vaginalis  (B.  M.  Honigberg,  Univ.  of 
Mass.;  Michael  R.  Spence  and  John  K.  Frost,  Johns  Hopkins  Univ.)  is 
pending  approval  of  their  grant  in  order  to  obtain  and  thoroughly 
characterize  fresh  clinical  isolates  of  trichomonads. 

Ultrastructural  study  of  microfilaria. 

An  electron  microscope  study,  paralleling  light  microscope  observa- 
tions, was  undertaken  on  the  phenomenon  of  antibody  dependent  cell 
adhesion  between  human  leukocytes  and  Brugia  maylai  microfilaria. 
Deposition  of  eosinophile  lysosomal  material  onto  the  microf ilarial 
surface  was  demonstrated.   (Weil,  Daniel) 

Ultrastructural  study  of  schistosomes. 

In  conjunction  with  studies  of  the  release  of  schistosome  antigens 
by  metabolic  inhibitors  (Na  azide,  Na  fluoride,  and  Na  arsenite)  the 
kinetics  of  tegumental  damage  was  followed  by  electron  microscope  study 
of  Schistosoma.   (Nash,  Daniel) 


25-80 


Serial  No.  Z01  AI  00185-01  LPD 
Publications. 

1.  Mattern,  C.  F.  T. ,  Keister,  D.  B.  and  Caspar,  P.  A.:   Experimental 
Amebiasis  III.   A  rapid  in  vitro  assay  for  virulence  of  Entamoeba 
histolytica.   Am.  J.  Trop.  Med.  Hyg.,  27.:   88  2-887,  1978. 

2.  Mattern,  C.  F.  T. ,  Keister,  D.  B.  and  Diamond,  L.  S .:  Experimental 
Amebiasis  IV.   Amebal  viruses  and  the  virulence  of  Entamoeba 
histolytica.   Am„  J.  Trop.  Med.  Hyg.,  28,  653-657,  1979. 

3.  Mattern,  C.  F.  T. ,  Keister,  D.  B.  and  Caspar-Natovitz,  P.:  Entamoeba 
histolytica  "toxin":   fetuin  neutralizable  and  lectin-like.   Am.  J. 
Trop.  Med.  Hyg.  (in  press). 

4.  Mattern,  C.  F.  T.:   Structure  of  viruses.   in  "Medical  Microbiology: 
Principles  and  Concepts",  Samuel  Baron,  editor.   Addison-Wesley ,  San 
Francisco  (in  press). 


25-81 


SMITHSONIAN   SCIENCE    INFORMATION    EXCHANGE  u7s.   DEPARTMENT  OF  j   PROJECT  NUMBER 

PROJECT   NUMBER    (Oo   NOT   use   this    saace)       HEALTH,    EDUCATION,    AND   WELFARE 

PUBLIC   HEALTH   SERVICE 
NOTICE  Of 
INTRAMURAL   RESEARCH    PROJECT 


ZOl      AI      00113-16      LPD 


PERIOD   COVERED 
October    1,    1978    to    September    30,    1979 


TITLE   OF    PROJECT    (80   characters    or   less) 

Studies   on   dengue 


!  NAMES,    LABORATORY    AND    INSTITUTE   AFFILIATIONS,    AND    TITLES   OF    PRINCIPAL    INVESTIGATORS   AflO    ALL   OTHER 
PROFESSIONAL    PERSONNEL    ENGAGED    ON    THE   PROJECT 


PI: 


Leon  Rosen,  Head,  Pacific  Research  Section,  LPD.NIAID 


COOPERATING  UNITS  (if  any) 


LAB/ BRANCH 

Laboratory    of    Parasitic   Diseases 


SECTION 

Pacific  Research  Section 


INSTITUTE  AND  -OCATION 

NIAID,  NIH,  Honolulu,  Hawaii 


TOTAL  MANYEARS- 


PROFESSIONAL: 


CHECK  APPROPRIATE  30X(E3) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   Q  (a2)  INTERVIEWS 


T  (b)  HUMAN  TISSUES 


□   (c)    NEITHER 


SUMMARY    OF    *ORK    (200    words   or   Less   -   underline    keywords) 

This   project   was    terminated   October    1,    1978 


I 


25-82 


?H 5-6040 
(Rev.    10-75) 


SMITHSONIAN    SCIENCE    INFORMATION    EXCHANGE 
PROJECT  NUMBER    (Do   NOT   use   this   space) 


U.S.    DEPARTMENT   OF 
HEALTH,    EDUCATION,    AND   WELFARE 
PUBLIC   HEALTH   SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH   PROJECT 


PROJECT  NUMBER 
ZOl     AI      00163-02     LPD 


PERIOD   COVERED 
October   1,    1978   to   September   30,    1979 


TITLE  OF   PROJECT    (30   characters   or   less) 
Field  and  laboratory   studies  on   the  transovarial   transmission  of   Japanese 
and  St.    Louis   encephalitis  viruses  by  mosquitoes. 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


Leon  Rosen,  Head,  Pacific  Research  Section,  LPD,NIAID 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Parasitic  Diseases 


SECTION 

Pacific   Research   Section 


INSTITUTE   AND   LOCATION 

NIAID,    NIH,    Honolulu,    Hawaii 


TOTAL  MANYEARS: 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 

G  (a!)  MINORS   0  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


(c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

This  project  was  terminated  October  1,  1978. 


25-83 


?^S-6040 
(Rev.  10-76) 


SMITHSONIAN    SCIENCE    INFORMATION    EXCHANGE 
PROJECT  NUMBER    ^Oo   NOT   use    this   space) 


U.S.    DEPARTMENT   OF  I   PROJECT  NUMBER 

HEALTH,    EDUCATION,    ANO    WELFARE 

PUBLIC   HEALTH    SERVICE  I      ZOl       AI       00164-02       LPD 

NOTICE  Of 
INTRAMURAL  RESEARCH   PROJECT 


PERIOD    COVERED 

October   1,    1978    to    September    30,    1979 


TITLE  OF  PROJECT  (SO  characters  or  less) 

Epidemiology  of  Infectious  Disease  in  the  Pacific  Region 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  ANO  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


A.  Dean     Pacific  Center  for  Geographic  Disease  Research 
Honolulu  under  Contract  to  LPD.NIAID 
(Contract  No.   No  1-A1-02228) 


COOPERATING  UNITS  (if  an> 


LAB/BRANCH 

Laboratory  of  Parasitic  Diseases 


SECTION 

Pacific  Research  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Honolulu,  Hawaii 


I  TOTAL  MANYEARS: 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 

Q  (a!)  MINORS  Q   (*2J  INTERVIEWS 


Q  (b)  HUMAN  TISSUES 


(c)  NEiTHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

This   project  was    terminated   October    1,    1975 


25-84 


PHS-6040 
(Rev.    10-76) 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl  AI   00178-01  LPD 


PERIOD  COVERED 

October   1,    1978    to   September   30,    1979 


TITLE  OF    PROJECT   (30   characters   or   less) 

Studies   of    the   mechanism  of    transovarial   transmission   of   arboviruses   by 
mosquitoes. 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


Robert  B.  Tesh,  Staff  Member,  Pacific  Research  Section,  LPD 


COOPERATING  UNITS  (if  any) 


LAB/8RANCH 

Laboratory  of  Parasitic  Diseases 


SECTION 

Pacific  Research  Section 


INSTITUTE  AND  LOCATION 

NIAID,    NIH,    Honolulu,    Hawaii 


TOTAL  MANYEARS: 


PROFESSIONAL: 


CHECK   APPROPRIATE   BOX(ES) 
£   (a)      HUMAN    SUBJECTS 

q  (al)   MINORS       □  (a2)    INTERVIEWS 


(b)    HUMAN    TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  *ords  or  less  -  underline  keywords) 

This   project   was    terminated   October   1,    1978 


25-85 


PHS-6040 
(Rev.    IO-76) 


LABORATORY  OF  STREPTOCOCCAL  DISEASES 

197  9  Annual  Report 

Table  of  Contents 


Z01  AI 


Project  Number  page 

Summary        26-1 

00001-18       Biochemical  Studies  on  Staphylococcal 
and  Streptococcal  Membranes,  Cell 
Walls,  and  Mesosomal  Vesicles  —  Huff    26-11 

00002-14  Studies  on  the  Nutrition,  Physiology 
and  Ultrastructure  of  Staphylococcus 
aureus  —  Theodore  "        26-15 

00004-19  Electron  Microscopy  of  Bacteria  in 
Relating  Structure  and  Function  — 
Cole  26-19 

00005-14       Studies  on  Bacteriophages  and  Genetics 

of  Streptococci  —  Colon-Whitt  26-24 

00006-08       Competence  Development  and  Genetic 

Exchange  Mechanisms  in  Streptococci  — 
Ranhand  26-28 

00007-04       Viruses  of  Spiroplasmas  and  Myco- 
plasmas —  Cole  26-33 

00181-01       Isolation,  Purification,  and 

Characterization  of  Toxins  and 
Inflammatory  Factors  of  Streptococci  — 
Calandra  26-37 


PHS-NIH 

Summary  Statement 

Laboratory  of  Streptococcal  Diseases 

October  1,  1978  to  September  30,  1979 

Research  Progress 

Bacterial  Lipoteichoic  Acid 

A.  Lipoteichoic  acids  (TA)  are  ubiquitous  glycerol-phosphate 
polymers  of  Gram-positive  bacteria  that  are  primarily  associated, 
in  cell  fractions,  with  the  mesosomes.   Previous  studies  showed 
large  changes  in  total  extractable  LTA  occurring  with  stages  of 
culture  growth,  so  we  examined  cells  (of  Staphylococcus  aureus) 
at  different  culture  ages  by  electron  microscopy  to  determine 
possible  changes  in  the  number  or  sizes  of  mesosomes.   No 
differences  were  found,  despite  10-fold  changes  in  LTA  content. 
Extraction  procedures  were  then  changed  from  use  of  whole  cells 
(old  method)  to  use  of  mechanically-disrupted  cells.   Subsequent 
comparisons  showed  that,  whereas  exponentially-growing  cells 
extracted  by  either  method  gave  comparable  results,  whole 
stationary  cells  yielded  only  10%  of  the  LTA  extractable  if  the 
cells  were  first  disrupted.   Use  of  the  latter  method  (applied 
also  to  Streptococcus  faecium  and  Bacillus  licheniformis)  demon- 
strated that  the  amount  of  total  LTA  was  actually  constant  (at  1- 
3%  dry  weight  of  cell)  throughout  the  cell  cycle.   The  finding 
emphasizes  the  previously  unrecognized  need  for  complete  cellular 
disruption  prior  to  application  of  other  procedures  (i.e., 
delipidization  with  chloroform-methanol,  which  seems  to  fix  LTA 
in  an  unextractable  form) ,  if  total  LTA  is  to  be  successfully 
extracted.   The  result  also  raises  questions,  as  yet  unanswered, 
concerning  reasons  for  variation  in  LTA  extractability  according 

to  cell  age.   It  is  possible  that  changes  in  envelope  permeability, 
in  age-associated  cell  wall  fragility,  or  in  dissociation  of  LTA 
from  unextractable  complexes  with  protein,  are  responsible,  and 
all  these  possibilities  will  require  exploration.   It  may  also  be 
that  the  variability  is  related  to  other  observations  on  the 
partition  of  LTA  among  the  various  cellular  fractions.   It  was 
found  that  if  cells  were  frozen  before  protoplasting  (an  essential 
first  step  in  fractionation) ,  LTA  shifted  to  the  periplasm  and 
the  shift  was  greater  in  exponential  than  in  stationary  cells. 
Mechanical  disruption  alone  caused  a  shift  of  LTA  to  the  soluble 
fraction;  this  was  enhanced  by  the  absence  of  added  magnesium 
ion,  particularly  in  exponential  cells.   Continuing  studies  will 
utilize  standard  procedures  (unfrozen  cells,  added  magnesium, 
Braun  mechanical  disruption)  now  shown  to  be  optimal,  in  exploring 
LTA  turnover,  reactions  of  LTA  with  mesosomal  proteins  and 
lipids,  and  intracellular  enzymes  involved  in  LTA  degradation. 
(Huff,  Theodore,  Popkin) 

B.  By  use  of  a  hot  phenol  extraction  instead  of  previous 
methods,  purified  LTA  without  protein,  nucleic  acids,  hexoamines, 
or  cell  wall  contaminants,  was  obtained  form  Staphylococcus 

26-1 


aureus .   The  material  contained  only  glycerol  and  phosphate 
(1:1),  d-alanine,  glucose,  and  5  fatty  acids,  of  which  the  C,s 
species  was  predominant.   Immunization  of  rabbits  with  this  LTA 
coupled  to  methylated  bovine  serum  albumen  produced  antiserum 
that  was  specific  for  the  glycerol-phosphate  backbone  moiety  of 
the  LTA;  antisera  raised  previously  against  whole  cells,  mesosomes, 
membranes,  or  hot  water  and  cold  phenol  extracts,  were  cross- 
reacting  and  did  not  detect  LTA  because  of  specificities  directed 
primarily  against  contaminating  proteins.   The  specific  antiserum 
was  of  high  activity  (2.5  mg  antibody/ml)  and  was  employed  to 
verify  chemical  data  indicating  the  presence  of  LTA  primarily  in 
the  mesosomal  fraction  of  cells  and  its  absence  in  culture 
fluids.   It  will  now  be  used  (as  IgG)  to  localize  LTA  by  immuno- 
electron  microscopy  in  thin  sections  of  intact  cells,  which  was 
not  possible  with  previous  antisera.   In  addition,  the  sensitive 
serologic  detection  of  LTA  has  been  made  quantitative  by  develop- 
ment of  a  radioimmunoassay  method  for  use  in  ongoing  studies  of 
LTA  structure,  biosynthesis,  and  functional  role  in  the  bacterial 
cell.   (Theodore;  Greenberg  and  Kalica,  LID,  NIAID) 

Legionnaires'  Disease  Organism 

The  morphology  and  ultrastructure  of  the  cultured  bacillus 
responsible  for  Legionnaires'  Disease,  first  investigated  by  us 
last  year,  was  reaffirmed  by  subsequent  and  repeated  electron 
microsopic  examinations.   The  cultured  organism,  as  well  as  that 
first  correctly  reported  in  human  lung  sections  in  conjunction 
with  our  CDC  collaborators  (Am.  J.  Clin.  Pathol.  71_,  43-50,  1979) 
is  a  simple  double-membrane-enveloped  rod  ultrastructurally 
characteristic  of  Gram-negative  bacteria.   No  organelles  such  as 
pili  or  flagella  are  present,  and  no  layer  representing  pepti- 
doglycan  between  outer  and  inner  (cytoplasmic)  membrane  was 
visualized.   Except  for  large  lucent  granules  or  inclusions 
ultrastructurally  typical  of  polyhydroxybutyrate,  no  unusual 
intracytoplasmic  features  were  found.   The  furrowing  type  of  cell 
division  seen  is  that  of  Gram-negative  bacteria.   These  features, 
which  were  reported  at  the  International  Symposium  of  Legion- 
naires' Disease  (CDC,  Atlanta,  November  1978)  and  subsequently 
published  in  its  proceedings  (Ann.  Int.  Med.  90_,  642-647,  1979) 
were  confirmed,  for  the  most  part  by  findings  of  other  investi- 
gators, that  were  also  presented  at  the  meeting.   No  new  or 
different  ultrastructural  information  has  been  reported  since. 
The  ultrastructural  features  do  not  contribute  to  an  explanation 
of  the  pathogenicity  or  virulence  of  this  bacterium.   (Cole, 
Popkin,  LSD;  Chandler  et  al,  CDC) 

New  Erythrocyte-Adherent  Bacterium  Infecting  Man 

A  middle-aged  white  male,  splenectomized  25  years  earlier, 
developed  arthralgias,  anorexia,  weight  loss,  and  episodes  of 
chills  and  fever  associated  with  petechiae  and  tender  nodular 
purpuric  lesions  of  the  feet.   The  history,  especially  in 


26-2 


relation  to  contact  with  animals,  arthropods,  or  other  similar 
illnesses,  was  non-contributory.   Blood  cultures,  serologic  tests 
for  infectious  agents,  and  other  laboratory  examinations,  were 
negative.   However,  direct  examination  of  blood  smears  revealed 
Wright-  or  Giemsa-staining,  Gram-positive,  bacteria-like  bodies 
adherent  to  red  cells;  similar  bodies,  better  revealed  by  a 
silver  stain,  were  found  in  large  numbers  extracellularly  in 
biopsies  of  the  cutaneous  lesions  and  in  the  bone  marrow. 
Inoculation  of  embryonated  eggs,  tissue  cultures,  and  5  species 
of  normal  and  splenectomized  animals  produced  no  results. 
Samples  of  peripheral  blood,  furnished  to  us  in  subsequent 
consultation  with  the  clinician  (Dr.  G.  L.  Archer,  Medical 
College  of  Virginia) ,  were  examined  by  electron  microscopy  in 
negative  stains  and  after  sectioning.   We  also  cultured  samples 
aerobically  and  anaerobically  in  several  media.   All  cultures 
remained  sterile,  but  electron  microscopy  showed  short  rod-shaped 
bacteria  on  red  cell  surfaces,  but  not  within.   No  destruction  or 
alteration  of  the  cells  was  evident.   The  bacteria  were  typically 
of  Gram-positive  nature  by  internal  features  (mesosomes) ,  septate 
division,  and  structure  of  the  cell  wall — with  the  exception  of 
an  additional  unique  feature  of  an  outer  membrane  7.5-8.0  nm  wide 
surmounting  the  wall.   After  several  episodes  of  the  clinical 
features  described,  each  relieved  temporarily  by  vancomycin  and 
cephalosporins  but  recurring  with  reappearance  of  the  red  cell- 
associated  bacteria,  the  patient  was  cured  by  chloramphenicol  and 
the  bacteria  disappeared.   Negative  serologies,  lack  of  culture 
in  artifical  media  or  animals,  and  the  Gram-positive  ultra- 
structure,  indicated  that  the  bacterium  was  not  Bartonella 
bacilliformis  or  any  other  previously  known  red  cell-associated 
prokaryote.  Gram-positive  bacteria  adherent  to  red  cells  and 
causing  disease  in  humans  have  not  been  reported,  and  this  case 
(New  Eng.  J.  Med. ,  in  press)  represents  the  first  instance  of 
what  must  be  a  rare  condition  caused  by  a  bacterium  that  has  yet 
to  be  cultivated,  classified,  and  examined  for  mode  of  patho- 
genicity.  (Cole,  Popkin,  LSD;  Archer,  Medical  College  of 
Virginia) 

Streptococcal  Erythrogenic  Toxins 

Our  prior  studies  established  that  a  non-lysogenized  strain  (64 
x  4  02)  of  a  Group  A  streptococcus,  contrary  to  extant  dogma, 
could  produce  an  erythrogenic  toxin  (ET) .   Four  antigenically 
distinct  ETs  (A,  B,  C,  and  D)  are  known,  but  only  ETB  (identified 
by  monospecific  antisera  and  standard  reference  strains)  was 
produced  by  64  x  4  02.   The  question  raised  was  the  location  of 
the  genetic  coding  for  the  production  of  ETB.   No  evidence  of 
plasmids  (either  by  isolation  procedures  or  by  loss  of  ETB 
production  after  exposure  to  "curing"  acridine  dyes)  was  found  in 
64  x  402.   Its  DNA  (as  well  as  that  from  a  strain  producing  both 
ETs  A  and  B)  failed  to  transform  a  competent  Group  H  streptococcus 
to  ETB  ,  although  a  streptomycin  resistance  marker  was  transferred, 
These  results  appeared  to  rule  out  either  extrachromosomal  or 
chromosomal  DNA  as  the  site  of  genetic  information  for  ETB 
production.   Another  possibility  was  the  presence  in  64  x  4  02  of 

26-3 


a  defective,  and  therefore  undetected,  phage;  but  recent  attempts 
to  prove  this  by  recombination  after  infection  of  64  x  402  with  a 
virulent  mutant  phage  and  examination  of  progeny,  were  unsuccessful. 
The  location  of  the  ET  coding  thus  remained  obscure — not  only  for 
ETB,  but  also  for  the  other  toxins.   In  continued  exploration, 
strain  64  x  4  02  was  lysogenized  by  a  phage  from  a  strain  producing 
ETs  A  and  C-with  resultant  production  (in  addition  to  ETB  already 
present)  of  ETC  but  not  A.   We  then  attempted  to  lysogenize  52 
other  Group  A  strains  that  were  ETB-  with  4  phages  from  ETB 
strains.   Only  2  6  strains  were  sensitive  to  the  phages,  only  8 
were  lysogenized,  and  none  of  these  now  produced  ETB.   Present 
information  thus  suggests  that  production  of  ETs  A  and  B  is  not 
related  to  lysogenizing  phages,  but  production  of  ETC  may  be; 
technical  problems  could  be  responsible  for  some  nondef initive 
but  suggestive  findings,  and  the  subject  requires  further  study 
that  is  under  way.   Adjunct  studies  did  not  reveal  production  of 
ETs  A,  B,  or  C  by  streptococci  of  Groups  C  (7  strains)  or  G  (8 
strains) ,  despite  reported  clinical  studies  associating  such 
streptococci  with  infection  and  an  erythematous  rash  in  humans. 
(Colon-Whitt,  Cole) 

Interactions  of  Genetically  Competent  Bacteria  with  DNA 

Competent  Haemophilus  influenzae  cells  interact  very  specifically 
with  DNA  (Scocca,  Poland,  and  Zoon;  J.  Bacteriol.  118:  369-373, 
1974).   It  has  recently  been  found  that  competent  H.  influenzae 
cells  recognize  specific  base  sequences  in  DNAs  (Sisco  and  Smith; 
PNAS  76:  972-976,  1979) .   One  study  (Ceglowski,  Fuchs,  and 
Soltyk;  J.  Bacteriol.  124:  1621-1623,  1975)  showed  that  naturally 
occurring  glucosylated  DNAs  (derived  from  T-even  bacteriophages 
of  Escherichia  coli)  competed  poorly,  if  at  all,  with  homologous 
transforming  DNA  in  a  system  using  competent  Streptococcus 
sanguis  (strain  Challis)  cells  as  recipients.   This  result 
resembled  the  one  obtained  with  H.  influenzae  cells  and  was  at 
variance  with  previous  data  from  this  laboratory.   Therefore,  the 
reactions  were  investigated  in  more  detail,  with  the  following 
results.   T-even  phage  DNAs  reacted  except  for  expression,  with 
competent  S.  sanguis  (strain  Wicky)  cells  in  the  same  fashion  as 
homologous  DNA.   The  reactions  examined  were  (1)   DNAse-sensitive 
DNA  binding,  (2)   DNAse-resistant  DNA  binding,  and  (3)  degradation 
of  DNA  by  competent  cells.   From  an  analysis  of  the  initial  rates 
for  the  above  3  reactions,  it  was  concluded  that  T-even  glucosylated 
DNAs  reacted  at  much  slower  rates,  which  is  why  they  did  not 
compete  with  homologous  DNA  for  transformation.   The  rates  were 
inversely  related  to  the  amount  of  diglucosylation;  for  example, 
the  most  extensively  diglucosylated  DNA  (T6  DNA;  70%  glucosylated) 
reacted  the  slowest.   T2  DNA,  the  next  most  extensively  digluco- 
sylated (5%)  reacted  next  slowest,  and  T4  DNA  (0%  diglucosylated) 
reacted  the  fastest  of  the  three.   However,  its  rate  was  still 
slower  than  that  of  Wicky  DNA.   Although  these  data  do  not 
suggest  that  specific  base  sequences  are  also  recognized  by 
competent  S.  sanguis  cells,  other  data  to  be  developed,   may. 

26-4 


Reactions  of  competent  S_.  sanguis  cells  with  DNAs  derived  from 
HeLa  cells,  T5  bacteriophages,  and  Bacillus  subtilis,  were  also 
poor.    Study  of  such  reactions  is  continuing  in  our  efforts  to 
define  the  mechanisms  of  DNA  binding,  uptake  and  dispositon 
during  genetic  transformation  of  bacteria.   (Ranhand) 

Plasmids  of  Spiroplasmas 

Spiroplasmas  (motile  helical  mycoplasmas  infecting  plants, 
insects,  and  ticks,  and  capable  in  some  instances  of  producing 
disease  in  embryonated  eggs  and  rodents)  are  poorly  understood 
metabolically  and  pathogenetically.   All  carry  one  or  more  of  3 
viruses,  but  other  possible  extrachromosomal  nucleic  acid 
elements  were  previously  unknown.   We  examined  12  spiroplasma 
strains  from  diverse  natural  sources  by  dye-buoyant  density 
gradient  centrifugation  followed  by  electron  microscopy  and 
agarose  gel  electrophoresis.   Ten  strains  contained  covalently 
closed  circular  (CCC)  DNAs  ranging  in  size  from  1.0  to  greater 
than  12  megadaltons  molecular  weight,  and  each  strain  carried 
from  2  to  at  least  6  size  classes  of  these  circular  molecules. 
The  presence,  sizes,  or  number  of  size  classes  per  strain  do  not 
correlate  with  either  the  known  carriage  or  state  of  active 
production  of  the  viruses,  and  the  CCC  DNAs  are  therefore  not 
considered  to  be  viral  genomes.   Since  no  host  phenotypic  traits 
have  yet  been  associated  with  the  presence  of  any  CCC  DNAs,  these 
latter  are  currently  believed  to  be  crytpic  plasmids  such  as 
those  often  found  in  bacteria.   No  systems  of  genetic  transfer 
among  spiroplasmas  have  been  developed;  future  work  will  be 
directed  to  this  aspect,  as  well  as  to  definition  of  phenotypic 
markers,  effects  of  plasmid  curing  techniques,  development  of 
mutants,  rapid  methods  of  detecting  and  sizing  plasmids  in 
spiroplasma  surveys,  and  more  precise  physical  characterization 
of  the  plasmids  already  discovered.   (Ranhand,  Mitchell,  Cole, 
Popkin) 

Viruses  of  Spiroplasmas 

The  survey  of  old  and  newly-acquired  strains  of  spiroplasmas 
(helical  motile  mycoplasmas)  for  carriage  of  one  or  more  of  the 
3  morphologically  distinct  viruses  previously  described,  was 
continued  by  electron  microscopy  (EM)  and  plaquing  methods. 
Cumulative  data  indicate  that  all  strains,  regardless  of  source, 
carry  at  least  one  virus,  and  there  are  some  differences  in  their 
distributions.   Spiroplasmas  from  bees  and  flowers  carry  only  the 
rod-shaped  virus,  SpVl;  those  from  corn  stunt  disease,  ticks,  or 
Drosophila  carry  SpVl  or  SpV3  (polyhedral  virus,  short  tail)  or 
both;  and  isolates  from  citrus  stubborn  disease  or  otherwise 
identified  as  Spiroplasma  citri,  may  carry  1,  2,  or  all  3  viruses- 
including  SpV2  (polyhedral  virus,  long  tail)  which  is  not  found 
in  the  other  groups  of  spiroplasmas.   SpV2  has  not  been  isolated, 
propagation  and  characterization  of  SpV3  was  previously  described, 
and  emphasis  this  year  was  on  study  of  SpVl  that  was  first 


26-5 


propagated  only  late  last  year.   EM  showed  that  more  than  50%  of 
spiroplasmas  carry  SpVl  representatives,  and  8  new  isolates 
showing  host  range  differences  but  no  host  modification  or 
restriction,  were  made  from  spiroplasmas  of  bees,  corn,  and 
citrus.   One  bee  isolate  studied  in  detail  has  particles  of 
characteristic  size  (230-280  by  10-15  nm)  with  buoyant  densities 
of  1.39  g/cm   in  CsCl  and  1.21  g/cm   in  Metrizamide  gradients. 
Its  nucleic  acid  is  DNA  and  appears  by  EM  to  be  double-stranded 
and  linear,  but  confirmation  is  needed.   Antiserum  to  this 
isolate  neutralized  other  isolates  equally  well.   Virus  infectivity 
is  also  sensitive  to  chloroform,  ether  (somewhat),  some  detergents, 
temperatures  above  60  C,  or  4  C  or  lower  (if  prolonged) ,  and  pH 
below  6  or  above  9.   It  is  not  affected  by  nonionic  detergents, 
Genetron,  DNAase,  RNAase,  or  drying.   Growth  studies  showed  a 
single-hit  infection,  an  adsorption  constant  of  2.55  x  10 
cm  /min,  a  60-90  min  latent  period,  release  of  10  progeny  viruses 
per  cell  by  6  hrs,  and  some  delayed  cell  death  not  attributable, 
as  in  many  other  phage-host  systems,  to  viral-induced  lysis. 
Host  cultures  can  be  selected  for  resistance  to  one  SpVl  isolate, 
and  are  also  resistant  to  all  other  SpVl  isolates.   This  result, 
the  inhibition  of  different  isolates  by  a  single  antiserum,  the 
obtention  of  isolates  with  these  common  properties  from  different 
host  strains,  and  the  apparent  absence  of  host  modification, 
suggest  that  SpVl  common  in  many  diverse  spiroplasmas  may  be 
identical.   Further  work  will  examine  possible  SpVl  group  charac- 
teristics, characterize  the  DNA,  classify  the  viral  proteins  (of 
which  there  appear  to  be  7  to  9) ,  and  investigate  the  intra- 
cellular state  of  this  virus  and  its  genome.   (Liss,  Cole,  Popkin) 

Streptococcal  Hemolysin  (Streptolysin  S) 

Streptolysin  S,  an  extracellular  protein  that  is  the  most  powerful 
membranolytic  agent  known,  is  non-antigenic  and  of  uncharacterized 
importance  in  the  pathogenesis  of  streptococcal  infections.   We 
found  it  to  exist  as  an  inactive  intracellular  precursor  in 
streptococci  of  Group  A,  C,  and  G,  but  not  in  B.   In  Group  A,  it 
is  membrane-associated;  highest  titers  were  found  in  nephritogenic 
strains,  where  the  greatest  amounts  occurred  in  membrane  fractions 
and  the  highest  specific  activity  in  mesosomes.   The  precursor 
was  previously  shown  to  be  activated  by  vortexing  with  glass 
beads  in  the  presence  of  RNA-core,  and  recent  work  showed 
improvements  in  results  by  use  of  small  beads,  and  beads  washed 
with  60%  acetic  acid,  and  phosphate  buffer.   Although  detergents 
alone  fail  to  activate  precursor,  it  can  be  activated  (to  a  very 
labile  form)  by  vortexing  with  glass  beads  in  the  presence  of  a 
nonionic  detergent  and  then  stabilized  by  addition  of  RNA-core. 
The  precursor  is  neither  activated  nor  degraded  by  streptococcal 
proteinase,  but  this  enzyme  inactivates  active  hemolysin — thus 
suggesting  a  possible  role  in  regulation  of  hemolysin  production. 
Current  emphasis  is  on  purfication  and  characterization  of  both    : 
precursor  and  active  hemolysin  by  use  of  a  lipid  carrier, 
chromatography  in  organic  solvents,  transfer  to  a  synthetic 
nucleotide  carrier,  and  affinity  column  chromatography.   (Calandra, 
Theodore) 

26-6 


Administrative,  Organizational,  and  Other  Changes 

The  number  of  research  projects  increased  by  one  to  a  total  of  7, 
with  the  return  to  the  Laboratory  of  Dr.  Gary  Calandra  from 
detail  to  LCI.   Dr.  David  Williamson's  appointment  as  Visiting 
Investigator  through  the  Intergovernmental  Personnel  Act  expired 
in  February  1979,  and  he  returned  to  the  State  University  of  New 
York  at  Stoneybrook  to  resume  academic  duties.   The  appointment 
of  Dr.  Daniel  Moynet  as  Visiting  Fellow  from  France  was  renewed 
to  February  1980.   During  the  summer,  Ms.  Jodi  Abramowitz  (COSTEP; 
New  Jersey)  and  Ms.  Maria  Velazquez  (Minority  Biomedical  Science 
Student  Program;  Ponce,  Puerto  Rico)  received  training  and 
experience  in  microbiological  techniques  from  Dr.  Alan  Liss 
(Senior  Staff  Fellow)  while  participating  in  our  project  on 
spiroplasmas  and  their  viruses.   It  is  anticipated  that  Dr.  Liss, 
having  completed  two  years  here  in  his  present  appointment,  will 
move  to  the  Rocky  Mountain  Laboratory  in  another  capacity  in 
October  1979.   No  other  changes  occurred  or  are  forecast.   The 
two  positions  lost  last  year  by  reassignment  and  retirement  have 
not  been  restored,  and  it  is  expected  that  a  third  position  (Dr. 
Liss)  will  also  be  lost  because  of  increasingly  severe  last- 
minute  and  unanticipated  cuts  in  personnel  ceilings  imposed  by 
OMB.   The  total  loss  to  LSD  brought  about  by  Institute  distribution 
of  such  cuts  is  disproportionate  (18%)  and  will  result  in  an 
authorized  strength  of  only  14  positions,  of  which  only  5  are 
occupied  by  doctoral  level  investigators.   The  future  of  the 
Laboratory  is  in  some  doubt,  since  the  Laboratory  Chief  will 
retire  mandatorily  in  September  1981  and  the  Institute  leadership 
is  meanwhile  strengthening  bacteriological  research  only  at  RML 
while  maintaining  only  routine  support  of  LSD  in  the  interim.   As 
a  result,  new  directions  of  reasearch,  except  as  may  be  engendered 
to  a  minor  extent  by  new  findings  in  current  projects,  cannot  be 
expected  and  the  prospects  for  bringing  in  new  blood  seem  dismal. 
Nevertheless,  we  shall  attempt  to  continue,  and  complete  as 
possible,  the  basic  microbiological  research  projects  already  in 
progress. 


26-7 


26-8 


Honors  and  Awards 


R.  M.  Cole 


Member,  Editorial  Board,  Infection  and  Immunity. 

Member,  Study  Group  on  Viruses  of  Mycoplasmas  and  Spiroplasmas : 
Subcommittee  on  Bacteriophages,  International  Committee  on 
Taxonomy  of  Viruses. 

J.  M.  Ranhand 

Invited  participant,  23rd  Wind  River  Genetics  Conference  (Estes 
Park,  Colorado) ,  June  12-15,  1979. 

D.  Moynet 

Invited  participant,  23rd  Wind  River  Genetics  Conference  (Estes 
Park,  Colorado),  June  12-15,  1979. 

A.  Liss 

Invited  seminar,  University  of  Montana,  Missoula,  June,  1979. 


26-9 


26-10 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00001-18  LSD 


PERIOD  COVERED 

October  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Biochemical  Studies  on  Staphylococcal  and  Streptococcal  Membranes, 
Cell  Walls,  and  Mesosomal  Vesicles. 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 

Other : 


Huff 


Scientist  Director 


T.  S.  Theodore  Res.  Microbiologist 
T.J.  Popkin    Chemist 


LSD,  NIAID 

LSD,  NIAID 
LSD,  NIAID 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Streptococcal  Diseases 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Md , 


20205 


TOTAL  MANYEARS: 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 
C  (a)   HUMAN  SUBJECTS 


□  (b)  HUMAN  TISSUES 


3  (c)  NEITHER 


□  (al)  MINORS 


(a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  long  range  purpose  of  this  project  is  to  study  the  biochemical 
processes  involved  in  the  growth  of  bacterial  cell  wall  and 
membrane  and  the  coupling  of  these  processes  to  DNA  replication 
during  cell  division.   The  present  goal  is  to  determine  the 
functional  role  of  lipoteichoic  acid  in  Staphylococcus  aureus  and 
other  Gram-positive  bacteria.   Lipoteichoic  acids  are  polymers  of 
glycerol  phosphate  found  intracellularly  closely  associated  with 
plasma  membranes  and  with  membranous  bodies  known  as  mesosomes.   A 
minor  component  of  the  lipoteichoic  acid  fraction,  lipoteichoic 
carrier,  has  been  implicated  by  others  in  the  synthesis  of  cell 
wall. teichoic  acid.   The  function  of  the  remainder  of  the  lipo- 
teichoic acid  (LTA)  is  not  known  and  this  is  the  area  of  current 
interest.   Present  studies  are  directed  at:  1)   determining  total 
intracellular  LTA  content  during  growth;  2)  intracellular  location 
of  LTA  in  bacteria  other  than  S_.  aureus;  3)  determining  specific 
mesosomal  protein-LTA  interactions. 


PHS-6040 
(Rev.    10-76) 


26-11 


Project  No.  Z01  AI  00001-18  LSD 
Project  Description: 

Lipoteichoic  acids  are  antigenic  glycerol  phosphate  polymers 
found  intracellularly  closely  associated  with  membranous  or- 
ganelles known  as  mesosomes  in  Staphylococcus  aureus.   The  purpose 
of  this  study  has  been  to  determine  the  function  of  intracellular 
lipoteichoic  acid  (LTA) .  Lipoteichoic  acid  was  isolated  from 
chloroform-methanol  extracted  cells  by  treatment  with  hot  aqueous 
phenol  and  separated  on  Sepharose  6B  into  peak  I  (micellar  LTA) 
and  peak  II  (non-micellar  LTA)  after  treatment  with  DNase  and 
RNase.    We  have  previously  reported  large  changes  in  the 
observed  LTA  level  in  S.  aureus  during  growth.   Since  LTA  is 
always  closely  associated  with  isolated  mesosomes  from  this 
organism,  we  tested  whether  large  changes  in  the  number  of 
mesosomes  could  also  be  observed  during  growth.   Cells  of  S_. 
aureus  (Lafferty  strain)  were  harvested  at  different  stages  of 
growth,  negatively  stained  with  phosphotungstic  acid  and  examined 
in  the  electron  microscope.   No  changes  were  observed  in  the 
number  or  size  of  mesosomes  during  growth,  while  10-fold  changes 
in  LTA  content  were  observed.   This  result  prompted  us  to  examine 
our  extraction  procedure.   We  found  that  undisrupted  exponentially 
growing  cells  gave  full  yields   of  LTA  as  compared  to  disrupted 
cells.   However,  undisrupted  stationary  cells  yielded  only  10%  of 
the  LTA  obtained  in  cells  disrupted  by  shaking  with  glass  beads. 
No  differences  were  observed  in  the  total  intracellular  LTA  yield 
whether  cells  were  disrupted  in  the  Braun  homogenizer  with  glass 
beads  or  by  protoplasting  with  lysostaphin. 

Previous  studies  had  also  shown  large  variations  in  the  amount  of 
LTA  extracted  from  Streptococcus  faecium  and  Bacillus  licheniformis 
during  growth.   Experiments  were  run  to  determine  if  disruption 
was  necessary  to  completely  extract  LTA  from  these  organisms.   In 
S.  faecium  (ATCC  9790)  the  LTA  yield  (measured  as  peak  I  material 
from  Sepharose  6B  columns)  for  stationary  cells  went  from  0.6% 
(of  the  dry  weight  of  the  cells)  in  undisrupted  to  1%  in  disrupted 
cells.   Throughout  the  growth  cycle  the  LTA  level,  as  determined 
in  Braun-disrupted  cells,  remained  constant  at  1.0  to  1.4%  of  the 
dry  weight  of  the  cells.   The  yield  of  LTA  from  early  logarithmic 
undisrupted  cells  was  higher  (2.2%)  than  disrupted  cells  (1.4%) 
probably  as  a  result  of  enzymatic  destruction  of  LTA  during  Braun 
disruption.   In  B.  licheniformis  (ATCC  9945)  the  LTA  yield  from 
18  hour  cells  went  from  0.3%  in  undisrupted  to  1.8%  in  disrupted 
cells.   During  growth  the  LTA  level,  as  determined  in  Braun 
disrupted  cells,  remained  at  1.8  to  2.8%  of  the  dry  weight  of  the 
cells.   The  highest  level  of  LTA  from  undisrupted  cells  was  1.8%. 

These  results  show  large  changes  in  the  extractability  of  LTA 
from  Gram-positive  organisms  dependent  on  the  growth  state  of  the 
organism.   In  all  the  species  studied,  the  level  of  LTA  obtained 
from  undisrupted  stationary  cells  was  very  low.   After  disruption, 
the  level  of  LTA  was  very  high.   The  high  levels  obtained  after 
disruption,  the  constancy  of  these  high  levels  during  growth,  and 

26-12 


Project  No.  Z01  AI  00001-18  LSD 

the  independence  of  the  level  on  the  method  of  disruption  (mech- 
anical versus  enzymatic  lysis)  argue  that  these  figures  represent 
total  LTA. 

These  results  also  point  to  the  need  for  complete  disruption  of 
an  organism  as  a  necessary  first  step  in  establishment  of  the 
intracellular  LTA  content.   Once  whole  cells  have  been  treated 
with  chloroform-methanol  followed  by  hot  aqueous  phenol,  the  LTA 
appears  to  be  fixed  in  the  residue  and  not  extractable. 

It  is  not  known  what  changes  occur  at  the  onset  of  exponential 
growth  to  make  the  intracellular  LTA  more  extractable  from 
undisrupted  cells.   This  could  be  due  to:   1)  an  increase  in  the 
permeability  of  the  cell  walls  to  LTA  as  the  cells  start  to 
divide   or,  2)   the  dissociation  of  LTA  from  an  unextractable 
protein-LTA  complex  at  the  onset  of  cell  division.   It  seems 
unlikely  that  mechanical  disruption  would  break  such  a  complex. 
If  this  complex  was  cleaved  enzymatically ,  it  would  have  to  occur 
within  the  mesosome  during  protoplasting  since  LTA  added  during 
protoplasting  does  not  exchange  with  mesosomal  LTA.   Although 
peak  I  LTA  occurs  predominantely  in  the  mesosome  and  peak  II  LTA 
occurs  predominantly  in  the  periplasm,  the  extractability  of 
these  two  substances  from  S.  aureus  (Lafferty)  increases  and 
decreases  coordinately  during  growth.   It  seems  unlikely  that 
mechanical  disruption  could  cause  a  protein-peak  I  LTA  dissociation 
in  mesosomes  and  also  a  similar  protein-peak  II  LTA  dissociation 
in  the  periplasm.   A  much  simpler  explanation  is  that  the  cell 
wall  of  an  exponentially  growing  cell  is  more  permeable  to  both 
peak  I  and  II  LTA.   Another  possibility  is  that  cell  wall  disruption 
can  occur  during  extraction  of  exponential  cells  with  chloroform- 
methanol  and  hot  phenol  but  this  does  not  happen  with  stationary 
cells . 

Determination  of  the  intracellular  location  of  LTA  has  been 
difficult  and  is  controversial.   In  S.  aureus  (Lafferty  strain) 
the  LTA  is  predominantly  in  the  mesosomes  in  cells  protoplasted 
in  the  presence  of  magnesium,  20%  NaCl  and  lysostaphin.   If  cells 
are  frozen  before  protoplasting  there  is  a  shift  of  peak  I  LTA  to 
the  periplasm.   This  shift  is  20%  in  stationary  cells  and  60%  in 
exponential  cells.   Braun  disintegration  of  either  frozen  or 
fresh  cells  also  causes  the  LTA  to  shift  into  the  100,000  x  g 
supernatant  fraction.   Omission  of  magnesium  results  in  an  even 
greater  shift  of  LTA  into  the  soluble  fraction.   This  is  parti- 
cularly true  in  exponential  cells  where  Braun  disintegration  in 
the  absence  of  magnesium  yields  9  0%  of  the  LTA  in  the  soluble 
fraction.   Similarly,  when  B.  licheniformis  (ATCC  9945)  cells 
were  frozen  and  Braun  disintegrated  in  the  absence  of  added 
magnesium  ion,  none  of  the  LTA  could  be  recovered  in  the  membrane 
or  mesosome  fraction  at  any  stage  of  growth.   When  S_.  faecium 
frozen  cells  were  Braun  disintegrated  in  the  absence  of  added 

26-13 


Project  No.  Z01  AI  00001-18  LSD 

magnesium  ion  about  50%  of  the  LTA  was  recovered  in  the  mesosomal 
and  2  0%  in  the  supernatant  fraction.   These  ratios  were  constant 
at  all  growth  stages.   The  peak  II  LTA  varied  from  0.1%  of  the 
dry  weight  of  the  stationary  cells  to  2%  in  late  exponential 
cells  and  was  primarily  in  the  100,000  x  g  supernatant  fraction 
of  Braun  disintegrated  frozen  cells  without  magnesium.   Planned 
are  studies  on  LTA  in  B.  lichenif ormis  and  S_.    faecium  using  fresh 
cells,  magnesium,  and  Braun  disintegration. 

An  attempt  was  made  to  determine  if  the  protein  found  at  much 
higher  levels  in  mesosomes  than  in  membranes  was  associated  with 
LTA  during  SDS-gel-electrophoresis .   P   -labeled  mesosomes  and 
membranes  were  treated  with  SDS-buffer  and  run  on  slab  gels. 
Duplicate  portions  of  the  gels  were  stained  for  protein  in  one 
sample  and  exposed  for  radioautography  in  the  other.   LTA  was 
found  to  run  as  a  diffuse  band  in  a  region  free  of  protein 
bands.   Thus  if  LTA  is  attached  to  a  protein  in  the  mesosome  this 
complex  is  dissociated  by  SDS. 

The  ubiquitous  presence  of  lipoteichoic  acids  in  Gram-positive 
organisms,  the  location  of  micellar  (peak  I)  LTA  external  to  the 
bacterial  membrane  (in  mesosomes  and  periplasm)  and  the  presence 
of  non-micellar  (peak  II)  LTA  in  the  periplasm  and  extracellularly , 
point  up  the  importance  of  these  LTA's  as  bacterial  antigens.   We 
have  previously  shown  the  presence  of  LTA  antibodies  in  the  sera 
of  staphylococcal  endocarditis  patients.    Recently,  the  lipo- 
teichoic acids  of  Streptococcus  mutans  have  been  implicated  in 
the  formation  of  dental  plaque.    The  present  study  demonstrates 
large  changes  in  extractability  of  LTA  from  Gram-positive  bacteria 
during  growth.   The  need  for  complete  disruption  of  these  organisms 
for  extraction  of  LTA  has  been  established.   Using  these  new 
methods,  high  levels  of  LTA  (1-3%  of  the  bacterial  dry  weight  as 
peak  I  LTA  and  1-3%  as  peak  II  LTA)  have  been  measured  at  all 
stages  of  growth.   Lipoteichoic  acids  are  thus  major  end  products 
of  bacterial  growth  in  Gram-positive  organisms. 

Planned  and  ongoing  work  includes:   1)   determining  if  LTA  is  a 
cellular  end  product  or  if  there  is  LTA  turnover;  2)   testing 
mesosomal  protein  and  lipid  to  see  if  it  arises  from  old  or  newly 
synthesized  membrane  in  pulse-labeled  experiments;  3)   characterizing 
enzymes  involved  in  LTA  degradation;  4)   characterizing  the 
nature  of  the  interaction  between  LTA  and  mesosomal  protein  and 
lipid;  5)  continued  study  on  the  intracellular  location  of  LTA  in 
S.  faecium  and  B.  lichenif ormis . 

Publications : 

None  : 


26-14 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZQ1  AI  00002-14  LSD 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Studies  on  the  Nutrition,  Physiology  and  Ultrastructure  of 
Staphylococcus  aureus 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


Theodore 


Res.  Microbiologist 


LSD,  NIAID 


COOPERATING  UNITS  (if  any) 

Bureau  of  Biologies,  FDA  (B.  Fraser) 

Laboratory  of  Infectious  Diseases,  NIAID  (H.  Greenberg,  A.  Kalica) 


tory  of  Streptococcal  Diseases 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Md. 


20205 


TOTAL  MANYEARS: 

2.9 


PROFESSIONAL: 

0.9 


2.0 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   fj  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


CRc 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Distinct  features  of  most  Gram  positive  bacteria  are  the  presence  c 
lipoteichoic  acid  and  a  membrane  system  consisting  of  a  plasma 
membrane  and  me so somes.   The  purpose  of  this  project  is  to  relate 
membrane  structure  with  function  using  Staphylococcus  aureus  as 
the  model  test  system.   The  major  emphasis  is  to  define  the  functiqn 
(or  functions)  of  mesosomes  which  contain  all  of  the  lipoteichoic 
acid  present  in  the  cell.   To  corroborate  the  chemical  localization 
of  lipoteichoic  acid  in  mesosomes  by  immunochemical  methods,  an 
antisera  specific  for  the  polyglycerol  phosphate  protion  of  the 
molecule  was  prepared.   This  antisera  failed  to  react  with  ribitol 
teichoic  acid  and  gave  one  precipitin  band  with  deacylated  lipo- 
teichoic acid  and  cardiolipin.   Passive  hemagglutination  inhibition 


tests  showed  the  exclusive  localization  of  lipoteichoic  acid  in 
mesosomes;  verifying  our  previous  chemical  findings.   Also,  pre- 
liminary data  on  a  radioimmunoassay  procedure  for  the  quantitation 
of  lipoteichoic  acid  is  presented. 


PHS-6040 
(Rev.    10-76) 


26-15 


Project  No.  Z01  AI  000Q2-14  LSD 


Project  Description: 


Morphologically,  the  most  distinct  feature  of  Gram  positive 
bacteria  are  the  mesosomes  which  arise  from  the  membranous 
spetum  and  precede  crosswall  formation.   The  most  distinct 
chemical  feature  of  isolated  mesosomes  is  the  presence  therein  of 
almost  all  of  the  cell's  lipoteichoic  acid  (LTA:   a  glycerol 
phosphate  polymer  containing  a  phosphatidyl  glycolipid) .   LTA 
interacts  strongly  with  divalent  cations,  participates  as  a 
carrier  molecule  in  cell  wall  synthesis,  and  acts  as  a  regulator 
of  autolytic  activity.   Also,  it  is  immunologically  active  and 
binds  to  mammalian  cells.   Using  chemical,  enzymatic  and 
serological  methods,  as  well  as  subcellular  fraction  techniques, 
the  overall  objective  of  this  study  was  to  ascertain  whether 
mesosomes  are  functional  organelles. 

1)    Immunological  studies  with  staphylococcal  LTA 

Subcellular  fractionation  and  chemical  analysis  have  shown  that 
most  of  the  LTA  of  Staphylococcus  aureus  in  the  cell  resides  in 
the  mesosomal  portion  of  the  membrane.   However,  the  location 
within  the  cell  is  not  precise,  since  it  is  possible  that  during 
fractionation  procedures,  some  movement,  reattachment  or 
degradation  of  the  LTA  can  occur.   Therefore,  a  more  direct 
approach  to  this  problem  would  be  by  immunochemical  localization 
of  the  LTA  in  intact  cells.   Initial  attempts  to  prepare  antisera 
for  this  purpose,  using  whole  cells,  mesosomes,  membranes,  or  hot 
water  and  cold  phenol  extracts  of  LTA,  unsuccessful.   Multiple 
precipitin  bands  were  detected  by  Ouchterlony  technique,  but,  the 
specificity  of  these  antisera  appeared  to  be  directed  toward  the 
protein  portion  of  the  antigens  rather  than  the  polyglycerol- 
phosphate  backbone.   These  antisera  failed  to  react  with  hot 
phenol  extracted  LTA,  deacylated  LTA,  and  deacylated  cardiolipin. 
Recently,  using  LTA  extracted  with  hot  phenol  and  coupled  to 
methylated  bovine  serum  albumin,  we  succeeded  in  preparing  an 
antisera  specific  for  the  backbone  portion  of  LTA.   This  antigen 
contained  no  protein,  nucleic  acids  or  hexosamines.   Glycerol  and 
phosphate  were  present  in  a  1:1  ratio  and  the  only  amino  acid 
detected  was  d-alanine.   The  preparation  was  devoid  of  any  cell 
wall  contaminants  adn  the  only  carbohydrate  present  was  glucose. 
Of  the  five  fatty  acids  detected,  C,-  was  the  predominant 
species.   (In  collaboration  with  B.  Fraser,  BOB,  FDA)  This 
antiserum,  when  tested  by  immunodiffusion  against  LTA  extracted 
by  hot  water,  or  by  hot  and  cold  phenol,  gave  single  precipitin 
bands  that  showed  lines  of  identity  among  the  several  extracts. 
It  also  reacted  with  deacylated  LTA  and  cardiolipin.   No 
precipitin  bands  were  observed  with  ribitol  teichoic  acid. 
Immunoelectrophoretic  analysis  showed  that  the  antigen  migrated 
to  the  anode  and  reacted  strongly,  forming  only  one  precipitin 
band.   Crossed  and  rocket  Immunoelectrophoresis  were  also  used  to 


26-16 


Project  No.  Z01  AI  00002-14  LSD 

further  verify  the  specificity  of  this  antiserum:  over  a  wide 
range  of  antigen  concentrations,  only  one  precipitin  band  was 
detected.   Quantitative  precipitin  analysis  showed  that  1  ml  of 
antisera  contained  2 . 5mg  of  antibody  that  could  be  precipitated 
with  100  ug  of  antigen.   Passive  hemagglutination  inhibition 
tests  on  the  various  subcellular  fractions  verified  our  chemical 
analyses  that  most  of  the  LTA  was  present  in  the  mesosomes. 
Mesosomal  titer  (reciprocal  of  dilution)  was  1024,  periplasm,  16; 
membrane,  2;  and  cytoplasm,  0.   No  LTA  was  detected  in  any  of  the 
culture  fluids.   Deacylated  LTA  and  cardiolipin  titers  were  256 
while  ribitol  teichoic  acid  failed  to  react.   At  present,  we  have 
prepared  the  IgG  fractions  of  normal  and  immune  sera  and  will  now 
attempt  to  localize  LTA  in  thin  sections  by  immuno electron 
microscopy,  using  the  horseradish  peroxidase  enzyme  system. 

Although  the  evidence  is  still  indirect  and  pending  on  the 
outcome  of  the  immunoelectron  microscopy,  chemical  and  sero- 
logical data  support  the  localization  of  LTA  in  mesosomes.   Since 
these  organelles  are  most  preminent  at  the  time  of  cell  division, 
arise  from  the  membranous  septum,  precede  crosswall  formation, 
and  contain  LTA,  it  is  quite  probable  that  they  play  an  active 
role  in  the  biosynthesis  of  LTA,  cell  wall  and  membrane. 

The  planned  course  of  this  project  will  include  immunoelectron 
microscopic  localization  of  LTA  in  intact  cells;  characterization 
of  the  LTA  structure;  and  biosynthesis  of  LTA  and  its  function  as 
it  relates  to  the  mesosome. 

2)  Radioimmunoassay  for  the  Determination  of  LTA 

Serological  methods  used  so  far  are  extremely  sensitive  for  the 
detection  of  small  amounts  of  LTA;  however,  they  are  only  semi- 
quantitative in  comparison  to  chemical  analysis.   The  drawbacks 
of  chemical  analysis  are  that  they  are  difficult,  time  consuming, 
and  require  much  larger  samples.   For  this  reason,  we  attempted 
to  develop  a  radioimmunoassay  (RIA)  method  for  the  quantitative 
determination  of  LTA.   Microti ter  plates  were  coated  with  varying 
dilutions  of  antigen  and  then  reacted  directly  with  radiolabeled 
IgG    .   By  this  method  it  was  possible  to  detect  quantitatively 
between  10  and  100  nanograms  of  LTA.   Present  studies  are  ' 
directed  toward  refining  this  procedure  and  increasing  the 
sensitivity  of  the  assay.   (in  collaboration  with  H.  Greenberg 
and  A.  Kalica,  LID,  NIAID) 

3)  Characterization  of  Rotaviral  Proteins 

Simian  rotavirus  (SA-11)  was  selected  for  the  study  of  rotavirus 
polypeptides  since  it  grows  well  in  cell  culture  and  possesses  a 
hemaglutinin  associated  with  its  outer  capsid.   CsCl  density 


26-17 


Project  No.  Z01  AI  QQ0Q2-14  LSD 

gradient  centrifugation  yielded  two  peaks  of  rotavirus.   The 
first  peak  contained  mostly  single  capsid  particles  and  yielded  5 
polypeptides  on  phosphate  buffered  SDS  polyacrylamide  gels.   The 
second  peak  contained  almost  exclusively  double  capsid  particles 
and  exhibited  8  polypeptides  on  polyacrylamide  gels.   The 
molecular  weights  ranged  in  size  from  52,000  to  118,000  daltons. 
The  3  polypeptides  present  in  the  second  peak  are  assumed  to  be 
associated  with  the  outer  capsid  of  the  simian  rotavirus.   The 
phosphate  buffered  continuous  gel  system  used  in  this  study 
appears  to  be  comparable  to  the  more  commonly  employed  discontinuous 
tris-glycine  gel  system  and  the  reported  resolution  of  fewer 
bands  in  the  former  gel  system  was  probably  due  concentration 
differences  of  double  capsid  virus  rather  than  the  gel  system 
employed.   (In  collaboration  with  A.  Kalica;  LID,  NIAID) 

Publications : 

Kwon-Chung,  K.  J.,  Bennett,  J.  E.,  and  Theodore,  T.  S.: 
Cryptococcus  bacillisporus  sp.  nov.   Int.  J.  Syst.  Bact.  2_8_,  616- 
620,  1978. 

Kalica,  A.  R. ,  and  Theodore,  T.  S.:   Polypeptides  of  simian 
rotavirus  (SA-11)  determined  by  a  continuous  polyacrylamide  gel 
electrophoresis  method.   J.  Gen.  Virol.  43:  463-466,  1979. 


26-18 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00004-19  LSD 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Electron  Microscopy  of  Bacteria  in  Relating  Structure  and  Function 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 
Other: 


R.  M.  Cole 
T.  J.  Popkin 


Chief 
Chemist 


LSD,  NIAID 
LSD,  NIAID 


COOPERATING  UNITS  (if  any) 

See  following  page  for  cooperating  units . 


LAB/ BRANCH 

Laboratory  of  Streptococcal  Diseases 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda, 


Md.  20205 


TOTAL  MANYEARS: 


PROFESSIONAL: 


1.0 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 


b)  HUMAN  TISSUES 


3  (c)  NEITHER 


□  (a1  )  MINORS 


(a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

This  project  provides  support,  through  preparative  techniques 
(negative  staining,  thin  sectioning)  and  subsequent  examination, 
for  Laboratory  and  Collaborative  microbiological  studies 
requiring  transmission  electron  microscopy.   Objects  of 
ultrastructural  study  include  mycoplasmas  and  their  viruses; 
streptococci  and  their  bacteriophages ;  staphylococci;  bacilli; 
gram-negative  bacteria;  fungi;  and  various  cell  fractions 
thereof,  examined  by  means  of  cytochemistry  and  immunoelectron 
microscopy. 


PHS-6040 
(Rev.    10-76) 


26-19 


Project  No.  Z01  AI  00004-19  LSD 
Cooperating  Units: 

Dept.  of  Biochemical  Sciences,  University  of  Tampere,  Finland  (E. 
Jansson) 

Laboratory  of  Clinical  Investigations,  NIAID  (Clinical  Mycology 
Section;  K.  J.  Kwon-Chung) 

Laboratory  of  Infectious  Diseases,  NIAID  (Mycoplasma  Section;  J. 
G.  Tully) 

Office  of  the  Scientific  Director,  NIAID  (R.  Repaske) 

Leprosy  and  Rickettsia  Branch,  Virology  Division,  Bureau  of 
Laboratories,  CDC,  Atlanta  (C.  C.  Shepard) 

Division  of  Infectious  Diseases,  Medical  College  of  Virginia, 
Richmond  (G.  L.  Archer) 

Laboratory  of  Microbiology  and  Immunity,  NIDR  (B.  M.  Chassy) 

Pathology  Division  Bureau  of  Laboratories,  CDC,  Atlanta  (F.  W. 
Chandler) 

Project  Description: 

Objectives  are  to  provide,  as  required,  electron  microscopy 
facilities  in  support  of  other  projects  in  the  Laboratory;  and  to 
conduct  such  original  and  in  depth  studies  of  the  ultrastructure 
of  microorganisms  as  may  be  engendered  by  collaborative  beginnings 
or  by  independent  approach  to  problems. 

1.    New  strains  of  spiroplasmas  examined  for  viruses  (SpV) 
included  1  S.  citri  (Iran:  SpV3+) ,  2  corn  stunt  (Rio  Grande, 
Chen,  and  Jamaica,  Eden-Green:  both  SpVl+) ,  1  Drosophila  hydei 
(Oishi:  SpV3+) ,  1  bee  (AS576C  Davis:   SpVl+) ,  1  flower  (Gl  Davis: 
SpVl+) .   Aggregate  evidence  from  repeated  EM  examinations  of  over 
5  0  strains  indicates  that  bee  and  flower  spiroplasmas  carry  only 
virus  SpVl;  tick,  corn  stunt,  and  Drosophila  spiroplasmas  carry 
either  or  both  SpVl  and  SpV3 ;  and  citrus  stubborn  disease  strains 
(S_.  citri)  carry  any  of  the  3  viruses  alone  or  in  double  or 
triple  combination,  and  is  the  only  spiroplasma  group  in  which 
SpV2  is  found.   Continued  monitoring  of  all  strains  carried  in 
our  collection  reaffirmed  the  erratic  occurrence  from  passage  to 
passage,  of  massive  virus  production;  and  revealed  several 
instances  of  production  of  viruses  never  before  observed  in  large 
numbers  in  that  particular  strain.   The  stimulus  for  sudden  virus 
production  is  still  unknown. 


26-20 


Project  No.  Z01  Al  00004-19  LSD 

New  emphasis  on  propagation  and  characterization  of  SpVl  (Z01  AI 
00007-5  LSD)  required  extensive  EM  monitoring  of  spiroplasmas  as 
possible  donors  or  indicator  strains;  frequent  examination  of 
plaques,  washes,  inocula,  and  infected  broth  cultures  to  confirm 
the  nature  of  the  virus  produced  in  8  new  SpVl  isolates;  and 
constant  EM  checks  on  stages  of  SpVl  purification.   In  addition, 
many  Kleinschidt-type  spreads  of  SpVl  DNA  were  examined  and 
photographed  in  efforts  to  ascertain  molecular  size  and  conformation. 

2.  Stages  of  attempts  to  isolate  and  purify  the  internal 
filaments  of  spiroplasmas  were  followed  by  EM.   Clean  preparations 
of  filaments  have  not  yet  been  obtained,  although  several  agents 
(Non-Idet  P40,  Triton  X-100,  Zwitterionic  detergents)  disrupted 
cells  and  gave  good  yields  of  crude  filaments. 

3.  Other  mycoplasmas  examined,  both  by  negative  stains  and 
sections,  included  4  human  isolates  from  Finland  (Dr.  E.  Jansson) 
and  3  rod-like  viruses  derived  therefrom,  a  new  species  of 
Acholeplasma  (72-043:  Dr.  Tully) ,  a  new  mouse  mycoplasma  (RIII-4: 
Dr.  Tully),  and  flower  spiroplasma  BNRl  growing  in  chick  amniotic 
fluid  (Dr.  Tully) . 

4.  Bacteriophages  examined  after  induction  of  parent  strains, 

or  during  stages  of  purification,  included  several  from  streptococcal 
Groups  A  and  P  (Ms.  Colon-Whitt) ,  B  (Dr.  Calandra) ,  and  H  (Dr. 
Moynet)  .   Others  were  coliphage  lambda  from  an  iii  vitro  system  of 
DNA  packaging  (Dr.  Repaske:  Z01  AI  00132-12  OSD)  and  several  new 
phages  isolated  from  Lactobacillus  casei  (Dr.  Chassy,  NIDR) . 

5.  Preparations  of  the  Legionnaires1  Disease  Organism  were 
reexamined  and  photographed,  and  reports  prepared.   (See 
Publications) 

6.  Facilities  for  spreading  films  of  purified  nucleic  acids 
(Kleinschmidt  technique)  were  developed  and  applied  by  Mr. 
Popkin,  and  used  extensively  to  examine  SpVl  spiroplasma  virus 
nucleic  acid  (Dr.  Liss:  Z01  AI  00007-5  LSD) ,  DNA  and  restriction 
endonuclease  fragments  thereof,  from  a  Group  H  streptococcal 
phage  (042)  and  some  group  A  streptococcal  phages  (Dr.  Moynet: 

Z01  AI  00006-8  LSD) ,  and  newly-discovered  plasmids  of  spiroplasmas 
(Dr.  Ranhand:  Z01  AI  00006-8  LSD). 

7.  Cocci  examined  included  Group  B  streptococci  in  stages  of 
protoplasting  by  a  cell  wall-active  enzyme,  mutanolysin  (Dr. 
Calandra:  Z01  AI  00181-01  LSD) ;  staphylococci  at  various  stages 
of  growth  to  ascertain  age-related  changes  in  number  of  mesosomes 
per  cell  (not  found)  (Dr.  Huff:  Z01  AI  00001-18  LSD);  staphylococci 
after  treatment  with  various  lipid  solvents  to  determine  effects 

on  cell  wall  structure  (not  found)  and  purified  staphylococcal 
lipoteichoic  acid  (Dr.  Theodore:  Z01  AI  00002-14  LSD) ;  and 


26-21 


Project  No.  Z01  Al  00004-19  LSD 

staphylococci  stained  by  immunolabelling  after  sectioning  in 
unsuccessful  attempts  to  find  the  intracellular  location  of 
lipoteichoic  acid  (Dr.  Theodore,  Mr.  Hochberg:  Z01  AI  00002-14 
LSD)  . 

8.  Preparations  of  Mycobacterium  leprae,  in  various  stages  of 
purification  from  armadillo  livers  and  after  different  treatments 
(e.g.,  trypsin)  were  examined  in  thin  sections  for  differences  in 
structure  of  cell  walls  and  surfaces.   The  results  were  incon- 
clusive, and  more  highly  purified  and  concentrated  samples  have 
not  yet  been  received  (Dr.  Shepard,  CDC) . 

9.  Samples  of  blood  showing  by  Giemsa  and  positive  Gram  stains, 
bacteria-like  bodies  adherent  to  erhthrocytes,  were  received  from 
Dr.  G.  Archer  (Medical  College  of  Virginia).   The  blood  came  from 

a  middle-aged  white  male  with  a  history  of  splenectomy,  arthralgias, 
anorexia,  weight  loss,  chills,  sweats,  and  fever,  and  purpuric 
lesions  on  the  feet.   The  presumptive  bacteria  were  also  found  in 
the  skin  lesions  and  in  bone  marrow.   Electron  microscopy,  by 
both  negative  staining  and  sections,  showed  short  rods  adherent 
to  but  not  within,  erythrocytes;  the  rods  had,  with  the  exception 
of  a  unique  outer  membrane,  the  ultrastructural  features  of  a 
Gram-positive  bacillus.   They  could  not  be  cultured,  either  here 
or  in  Richmond;  the  non-cultivability ,  the  Gram-positive  character- 
istics, and  negative  serological  tests,  ruled  out  known  red-cell 
affecting  bacteria  such  as  Bartonella,  Haemobartonella , 
Eperythrozoon ,  Anaplasma,  and  Grahamella.   The  patient  responded, 
in  several  episodes,  to  vancomycin  and  cephalosporins,  but 
relapsed  and  was  finally  cured  by  chloramphenicol.   The  causative 
organism  appears  to  be  a  new  bacterium,  and  its  description  and 
the  case  report  will  be  presented  in  the  literature.   (See 
Publications) 

10.  A  wild-type  melanin-producing  strain  of  Cryptococcus 
neoformans  and  a  pigment-less  mutant — both  grown  on  standard  malt 
agar  and  on  an  agar  stimulating  melanin  production — were  supplied 
by  Dr.  Kwon-Chung  (LI,  NIAID)  for  comparisons  of  the  presence  and 
cellular  location  of  the  pigment.   Thin  sections,  showed  the 
wild-type  cells  to  have  a  much  thicker  and  layered  cell  wall  and 
a  sub-membranous  location  of  granules  (absent  in  the  mutant) 
assumed  to  be  melanin.   However,  the  study  is  incomplete,  and  may 
require  use  of  cytochemical  methods  to  confirm  the  nature  of  the 
granules. 

Publications : 

Archer,  G.  L. ,  Coleman,  P.  H.,  Cole.  R.  M.  ,  Duma,  R.  J.,  and 
Johnston,  C.  L. ,  Jr. :   Human  infection  with  an  unknown  erythrocyte- 
associated  bacterium.   N.  E.  J.  Med.  (in  press) 


26-22 


Project  No.  Z01  AI  00004-19  LSD 

Chandler,  F.  W. ,  Blackmon,  J.  A.,  Hicklin,  M.  D. ,  Cole,  R.  M. , 
and  Callaway,  C.  S.:   Ultrastructure  of  the  agent  of  Legionnaires' 
disease  in  the  human  lung.   Amer.  J.  Clin.  Path.  71:  43-50,  1979. 

Chandler,  R.  W. ,  Cole,  R.  M. ,  Hicklin,  M.  D.,  Blackmon,  J.  A., 
and  Callaway,  C.  S.:   Ultrastructure  of  the  Legionnaires'  disease 
bacterium.   A  study  using  transmission  electron  microscopy.   Ann. 
Int.  Med.  90:  642-647,  1979. 


26-23 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION.  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00005-14  LSD 


PERIOD  COVERED 

October  1,  197  8  to  September  30,  197  9 


TITLE  OF  PROJECT  (80  characters  or  less) 

Studies  on  Bacteriophages  and  Genetics  of  Streptococci 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:        A.  Colon-Whitt 
R.  M.  Cole 

Other:     G.  B.  Calandra 


Microbiol . 
Chief 

Medical  Officer 


LSD,  NIAID 
LSD,  NIAID 

LSD,  NIAID 


COOPERATING  UNITS  (if  any) 

Laboratory  of  Parasitic  Diseases,  NIAID  (T.  Mercadol 


lab/branch 

Laboratory  of  Streptococcal  Diseases 


INSTITUTE  AND  LOCATION 

NTATD,  NIHr  Bethesda. 


Md.  20205 


TOTAL  MANYEARS: 

1.5 


PROFESSIONAL: 

0.1 


1.4 


check  appropriate  box(es) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


b   HUMAN  TISSUES 


*-■   (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  long  range  goal  of  this  project  is  to  study  bacteriophages 
and  genetic  exchange  (mainly  lysogenic  conversion,  transduction 
and  transformation)  in  streptococci,  and  to  investigate  how  such 
mechanisms  may  affect  the  pathogencity  and  immunology  of  these 
organisms.   Currently  we  are  searching  for  the  nature  and  site  of 
the  genetic  coding  for  a  rare,  Group  A,  non-lysogenic ,  erythrogenic 
toxin   (ET)  producing  strain.   We  have  attempted  1)  to  isolate  a 
defective  prophage  by  ghage-genetic  recombination,  2)  to  induce 
ETB   production  in  ETB   strains  by  lysogenization  with  phages 
from  ETB_r  strains,  and  3)  to  find  toxin  production  in  a  selected 
number  of  Group  C  and  G  streptococci.  Other  studies  concern 
extracellular  product  of  Pseudomonas  f luorescens  which  causes 
morphological  changes  and  lysis  in  strains  of  Trypanosoma  cruzi . 
We  are  trying  to  identify  the  factor  and  assess  its  significance. 


?H 3-60 40 
(Rev.  10-76) 


26-24 


Project  No.  Z01  AI  QQ0Q5-14  LSD 

Project  Description: 

1)    Erythrogenic  toxin  of  a  non-lysogenic  Group  A  streptococci 

Erythrogenic  toxin  (ET)  or  streptococcal  pyrogenic  exotoxins 
(SPE)  are  extracellular  proteins  produced  by  a  number  of  Group  A 
streptococci.   Four  distinctive  antigenic  types  (A,  B,  C,  and  D) 
have  been  described  and  lysogeny  has  been  considered  essential 
for  their  production.   We  have  been  studying  a  rare,  non- 
lysogenic  strain  (64  x  4  02)  which  produces  ET-B  toxin  and,  upon 
lysogenization  or  vegetative  infection  with  a  phage  (0)  isolated 
from  strain  C203U  (producing  ETs  A  and  C) ,  synthesizes  ETC  in 
addition  to  ETB,  but  not  ETA.   These  results  suggested  that 
lysogeny  may  not  be  essential  for  the  production  of  all  toxins, 
and  in  strain  64  x  4  02  the  genetic  coding  for  ETB  could  be  (1) 
an  integrated  portion  of  an  incomplete  prophage  genome  or  (2)  an 
integral  segment  of  the  streptococcal  chromosome  or  (3)  a 
transduced  fragment  or  (4)  an  integrated  plasmid  or  transposon  or 
(5)  a  free  plasmid  or  transposon.   These  hypothesis  were  tested 
and  some  of  the  results  were  presented  in  the  last  annual  report. 
In  summary,  we  were  unable  to  induce  production  of  ET  (by  al 
transformation  [using  strains  64  X  4  02  Sm   (ETB)  and  NY5  Sm   (ETA 
and  B)  as  DNA  donors  and  Group  H  strain  Wicky  as  DNA  recipient] 
or  by  (b)  vegetative  infection  of  Group  A  and  C  non-toxinogenic 
strains  with  phages  isolated  from  strains  producing  ETs  A,  B,  or 
C.    No  plasmids  or  transposons  were  found  and  treatment  of  64  x 
402  cells  with  acridine  orange  did  not  inhibit  synthesis  of  the 
ETB  toxin. 

In  recent  studies  we  have  attempted  (1)  to  induce  synthesis  of 
ETB  in  a  number  of  ETB~   strains  by  lysogenization  with  phages 
isolated  from  ETB   strains  and  (2)  to  isolate  a  defective  phage 
from  strain  64  x  4  02  by  infection  and  recombination  with  a 
virulent  phage  (0V2O3U) .   In  (1) ,  52  Group  A  strains  were  tested 
for  sensitivity  to  4  phages  isolated  from  ETB   strains.   Plaques 
or  lysis  were  observed  on  50%  of  the  tested  strains.   Colonies 
were  picked  from  the  center  of  the  lytic  area  streaked  on  P-agar 
plates  through  5  consecutive  passages;  and  then  tested  for 
lysogenicity  by  induction  with  Mitomycin  C  and  by  sensitivity  to 
the  homologous  phage.   Lysogenic  colonies  were  obtained  from  only 
eight  strains.   Numerous  attempts  to  isolate  true  lysogenic 
colonies  from  the  other  strains  were  unsuccessful.   The  eight 
lysogenic  strains  were  grown  for  24  hours  in  10  ml  dialysate 
media,  centrifuged,  and  the  supernatants  were  filtered,  con- 
centrated lOOx  in  Amicon  cells  and  tested  for  ETB  production  by 
the  Ouchterlony  test  against  monospecific  ETB  antitoxin.  None 
synthesized  detectable  ETB. 

For  isolating  a  defective  phage,  both  non-lysogenic  parent  64  x 
402  and  lysogenized  strain  64  x  402  (0AT2O3U)  were  used.   Eight 

26-25 


Project  No.  Z01  AI  00005-14  LSD 

strains  (including  C203U,  64  x  4Q2  and  64  x  402  (0AT2Q3U) 
were  used  as  phage  indicators.   The  test  strains  [64  x  402 
and  64  x  402  (0AT2O3U) ]  were  grown  in  P-broth  to  log  phase. 
The  cultures  were  divided  into  two  aliquots.   One  was  non- 
induced,  the  other  was  induced  by  adding  Mitomycin  C  (for  15 
min)  or  by  U.  V.  irradiation  (for  60  sec) .   The  cells  were 
centrifuged  and  resuspended  in  fresh  P-broth.   One  ml  of 
0VAT2O3U  (virulent  mutant)  was  added  at  a  MOI  of  10,  the 
tubes  were  incubated  for  3  hrs,  centrifuged  and  0.1  ml  of 
0AT2O3U  antisera  or  normal  sera  was  added  to  0.9  ml  of  the 
supernatant.   After  3  0  min  incubation,  0.01  ml  of  the  supernatant 
was  dropped  on  P-agar  plates  previously  seeded  with  log  phase 
cultures  of  the  indicator  strains.   Controls  were  supernatants  of 
4  hr  cultures  of  both  strains  with  and  without  phage  antisera. 
After  overnight  incubation  the  plates  were  examined  for  plaques. 
There  were  no  plaques  on  strains  C2  03U  or  64  x  4  02  (0AT2O3U)  in 
samples  with  or  without  phage  antisera.   Samples  without  phage 
antisera  plaqued  in  four  of  the  indicator  strains,  following  the 
same  lytic  pattern  as  0AT2O3U.   Samples  from  Mitomycin  C  induced, 
with  and  without  phage  antisera,  plaqued  in  strain  K56.   These   g 
plaques  were  picked,  the  phage  was  propagated  to  high  titers  (10 
pfu/ml)  in  K56  and  compared  for  lytic  and  immunological  patterns 
with  0AT2O3U.   Both  were  identical.   Unsuccessful  attempts  were 
made  to  propagate  this  phage  in  K56  in  the  presence  of  different 
concentrations  of  AT203U  phage  antisera.   These  results  indicate 
that  the  phage  obtained  was  0AT2O3U.   This  phage  is  very  virulent 
for  strain  K5  6,  and  needs  higher  concentrations  or  longer 
exposure  to  specific  phage  antisera  to  inactivate  it  and  prevent 
it  from  plaquing  in  this  strain. 

Recently,  there  has  been  increased  interest  in  the  production  of 
ET  by  Group  C  and  G  streptococci.   Several  clinical  cases  have 
been  reported  in  which  only  streptococci  of  these  groups  have 
been  isolated.   We  studied  a  selected  number  of  strains  (7  Group 
C  and  8  Group  G)  from  our  collection  for  production  of  ETA,  B, 
and  C  by  the  method  previously  described.   There  was  no  evidence 
of  ET  synthesis  in  any  of  the  strains. 

Our  failure  to  detect  ETB  production  in  cells  lysogenized  with 
phages  isolated  from  ETB   strains,  or  in  Group  C  and  G  strains, 
indicates  that  either  these  strains  do  not  produce  the  toxins  or 
they  are  produced  at  such  low  levels  that  we  need  a  more  sensitive 
method  of  detection.   Recently  the  Elisa  test  has  been  successfully 
used  by  other  investigators.   In  future  work  we  will  focus  on  the 
use  of  more  sensitive  methods  for  toxin  detection  in  studying  the 
phage-cell  relationships  in  ETB   producing  strains. 

2)    Screening  for  a  Group  B  muralysin 

There  is  no  satisfactory  method  for  lysing  Group  B  streptococci. 

26-26 


Project  No.  Z01  AI  00QQ5-14  LSD 

Such  a  method  would  be  highly  desirable.   In  collaboration  with 
Dr.  G.  Calandra  (LSD)  we  investigated  the  possibility  of  a  phage 
muralysin  in  Group  B  streptococci  comparable  to  the  phage 
associated  lysin  found  in  Group  C  streptococci.   To  that  purpose 
we  propagated  8  Group  B  phages  to  high  titers,  (1Q   -10   pfu/ml) . 
The  lysates  were  tested  for  lytic  activity  against  Group  B  type 
la,  lb,  Ic,  II,  and  III  strains,  using  different  buffers,  at 
different  pH,  with  and  without  a  reducing  agent  added.   No 
muralysin  was  found.   This  project  was  terminated  when  Dr.  Calandra 
found  another   source  of  an  effective   lysin. 

3)    Lysis  of  Trypanosoma  cruzi  by  Pseudomonas  f luorescens 

In  collaboration  with  Dr.  T.  Mercado  (LPD)  we  are  currently 
studying  the  effect  produced  by  P.  f luorescens  on  T.  cruzi .   It 
was  observed  that  this  gram  negative,  motile  rod  appears  to  be 
attracted  to  the  kinetoplast-f lagella  site,  where  it  attaches, 
eventually  causing  morphological  changes  of  the  parasite. 
Filtered  and  concentrated  (lOx)  supernatants  of  nutrient  broth 
cultures  of  the  bacteria  caused  the  same  effect,  with  complete 
lysis  of  the  parasite  within  10  min  after  exposure.   Supernatants 
of  sonicated  cells  have  some  effect  but  not  to  the  extent  of 
culture  supernatants,  which  indicates  that  the  factor  is  mainly 
extracellular.   Its  peak  of  activity  is  found  in  stationary  phase 
cultures  (48  hrs) .   Its  activity  is  not  inhibited  by  heating  (56 
C  for  30  min) ,  freezing  and  thawing,  or  trypsin  digestion,  but  is 
inhibited  by  boiling  for  5  min.   Concentrated  dialy sates  of  the 
supernatants  retained  their  lytic  activity,  which  indicates  that 
its  molecular  weight  is  less  than  12,000.   Future  work  will  be 
directed  to  identifying  the  extracellular  factor  and  further 
investigation  of  its  activity  in  vivo. 

We  consider  these  observations  to  be  significant  not  only  in 
terms  of  the  general  biology  and  physiology  of  the  parasite  but 
because  of  their  possible  impact  on  parasite  chemotherapy  and/or 
immunology. 

Publications: 

Colon-Whitt,  A.,  Whitt,  R.  S.,  and  Cole,  R.  M. :   Production  of  an 
erythrogenic  toxin  (streptococcal  pyrogenic  exotoxin)  by  a  non- 
lysogenized  Group  A  streptococcus.   In  Parker,  M.  T.  (ed.): 
Pathogenic  Streptococci.   Chertsey,  Surrey;   Reedbooks  Ltd., 
1979, pp.  64-65. 


26-27 


'SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00006-08  LSD 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Competence  Development  and  Genetic  Exchange  Mechanisms  in 
Streptococci. 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:        Jon  M.  Ranhand 

Other:     Daniel  Moynet 
W.  0.  Mitchell 
T.  J.  Popkin 
R.  M.  Cole 


Sr.  Scientist 

Visiting  Fellow 

Biologist 

Chemist 

Chief 


LSD,  NIAID 

LSD,  NIAID 

LSD,  NIAID 

LSD,  NIAID 

LSD,  NIAID 


COOPERATING  UNITS  (if  any) 

Macromolecular  Biology  Section,  LBV  (F.  DeFilippes) 
ZOl  AI  00126-06  LBV 


LAB/BRANCH 

Laboratory  of  Streptococcal  Diseases 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  Md, 


20205 


TOTAL  MANYEARS: 

2.05 


PROFESSIONAL: 

1.2 


0.85 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

G  (a!)  MINORS   Q  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


XJ  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  project  as  originally  defined,  "to  study  the  mechanism  by  which 
competent  group  H  streptococci  take  up  deoxyribonucleic 
acid  (DNA)  molecules  from  their  surroundings,"  is  continued.   I 
examined  further  the  interaction  between  competent  Wicky  cells 
(Streptococcus  sanguis)  and  homologous  or  heterologous,  native  or 
denatured,  DNAs.   DNAs  that  contain  chemical  modifications, 
such  as  alpha  or  beta  glucosylation,  or  both,  as  seen  in  some 
bacterial  viruses,  interact  with  competent  cells  at  a  slower 
rate.   This  observation  explains,  in  part,  the  reasons  why 
certain  DNAs  do  not  compete  with  homologous  DNA  in  transformation 
(Ceglowski,  Fuchs,  and  Soltyk,  J.  Bacteriol.  124:  1621-1623,  1975) 
In  addition  to  the  above,  I  showed  the  effect  of  temperature  on  the 
competent-cell  DNA  reaction.   I  also  showed  that  the  amount  of 
DNA  bound  to  competent  cells  depends  upon  (1)  the  amount  applied, 
(2)  the  source,  and  (3)  the  time  for  interaction.   These  results 
further  our  understanding  of  the  competent-cell  DNA  reactions. 


PHS-6040 
(Rev.    10-76) 


26-28 


Project  No.  Z01  AI  00006-08  LSD 

Project  Description: 

Part  I 

The  object  of  this  work  is  to  study  the  competent  Streptococcal 
cell-DNA  reaction.   Only  competent  cells  are  physiologically 
capable  of  interacting  with  DNA  molecules  and  four  intereactions 
are  defined.   (1)   DNA  binding  to  competent  cells  in  a  DNAse- 
sensitive  fashion;  this  binding  is  thought  to  be  on  the  cells' 
surface,   (2)   DNAse-resistant  DNA  binding;   this  binding  is 
thought  to  be  inside  the  cells'  cytoplasm,  (3)   the  production  of 
acid-soluble  products  (ASPs)  from  the  DNA  that  has  reacted  with 
competent  cells;  this  reaction  is  thought  to  be  due  to  specific 
competent  cell  associated  nucleases,  and  (4)  genetic  recombination 
and  expression  of  homologous  DNA  (transformation) . 

Reactions  (1)  and  (2)  above  are  essentially  completed  by  10  min, 
at  37  C;  reaction  (3)  continues  to  increase  beyond  60  min  with 
some  DNAs  and  with  other  DNAs  it  is  completed  by  60  min.   These 
reactions  are  measured  by  using  radiolabeled  DNA. 

The  first  3  reactions  above  have  now  been  shown  to  have  specific 
temperature  requirements.   Reaction  (1)  occurs  optimally  between 
3  0  and  42  C;  reaction  (2)  has  a  temperature  optimum  of  3  0  C; 
reaction  (3)  has  no  optimum  between  25  and  42  C.   However, 
between  37  and  42  C,  there  was  a  change  in  slope  in  the  Arrhenius 
plot.   This  indicates  that  2  or  more  nucleases  are  responsible 
for  ASP  formation.   From  Arrhenius  plots  of  the  data  the 
respective  activation  energies  (AE)  were  calculated. 

Reactions  (1)  and  (2)  above  occurred  with  AEs  of  23  KCal/mole; 
reaction  (3)  had  an  AE  of  4  6  KCal/mole,  reflecting  a  summation  of 
2  or  more  nucleases.   AEs  of  23  KCal/mole  suggest  enzyme  mediated 
reactions.   Therefore,  DNA  binding  to  competent  cells,  which  was 
thought  previously  to  be  a  physical  process  (electrostatic) ,  does 
not  occur  at  o  C  and  is  enzymatic. 

It  was  also  observed  that  when  DNA  was  added  to  competent  cells 
at  concentrations  below  saturation  (less  than  2  ug/ml)  the  amount 
of  DNA  that  was  (a)  cell-bound,  (b)  DNAse-resistant,  and  (c) 
degraded  to  ASPs  was  independent  of  the  concentraiton.   For 
example,  when  DNA  was  applied  at  concentrations  between  0.12  to 
1.8  ug/ml,  about  24%  was  cell-bound,  9%  was  DNAse-resistant,  and 
70%  was  degraded  (results  for  T  7  phage  DNA) .   It  was  shown  that 
DNA  preparations  do  not  contain  a  fraction  of  reactive  molecules. 
It  appears  that  competent  WE4  cells  can  control,  in  some  unknown 
way,  the  amount  of  DNA  that  they  will  react  with. 


26-29 


Project  No.  Z01  AI  00006-08  LSD 

In  reciprocal  experiments,  where  a  nonsaturating  concentration  of 
DNA  was  added  to  various  concentrations  of  competent  cells, 
again,  essentially  a  constant  fraction  of  the  DNAs  was:   cell- 
bound,  DNAse  resistant,  degraded. 

The  initial  rates  for  the  above  reactions  varied  and  depended 
upon  the  DNA  source.   In  the  present  study,  the  rates  (molecules 
of  DNA/cell/min/  ug  DNA  added)  in  descending  order  were:   Wicky 
DNA  >  T7  DNA  >  E.  coli  B  DNA  >  T7  DNA  >  T2  DNA  >  T6  DNA. 

T  even  phage  DNA  is  glucosylated.   The  glucose  residues  are 
covalently  attached  to  hydroxymethyl  cytosines.   Seventy  percent 
of  T6  phage  DNA  is  diglucosylated;  5%  of  T2  phage  DNA  is 
dislucosylated;  and  0%  of  T4  phage  DNA  is  diglucosylated.   It 
appears,  from  their  order  of  reactivity,  that  the  more  digluco- 
sylation  of  the  DNA,  the  slower  it  reacts.   In  general,  gluco- 
sylation  may  cause  the  DNA  to  interact  at  a  slower  rate.   This 
observation  explains  why  T-even  phage  DNAs  compete  poorly  with 
homologous  DNAs  for  transformation. 

The  rate  limiting  reaction  for  transformation  is  the  DNAse- 
resistant  DNA  binding  step.   This  slow  rate  was  also  observed 
with  labelled  DNA.   Reactions  (1)  and  (3)  above  occur  2  to  7 
times  faster  than  reaction  (2) ,  depending  on  the  DNA  used. 

This  study  also  showed  that  the  amount  of  DNA  that  was  bound  per 
cell  depended  upon  the  amount  of  DNA  applied.   The  same  was  true 
for  the  other  reactions.   At  DNA  concentrations  below  saturation, 
the  amount  of  DNA  that  was  cell  bound  varied  from  0.5  to  4 
molecules/cell;  the  amount  of  DNA  that  became  DNAse  resistant 
varied  from  0.1  to  1  molecule/cell;  and  the  amount  of  DNA  that 
was  degraded  varied  from  0.6  to  7  molecules/cell.   At  DNA 
concentrations  above  saturation,  the  amount  of  DNA  that  was  bound 
to  cells  varied  from  3-33  moelcules,  the  amount  of  DNA  that 
became  DNAse  resistant  varied  from  0.7  to  5  molecules,  and  the 
amount  of  DNA  that  was  degraded  varied  from  3  to  7  5  molecules. 
At  low  DNA  concentrations  as  well  as  high  DNA  concentrations   the 
amount  of  DNA  that  was  bound  and  taken  up  at  10  min  equaled  the 
amount  that  was  bound  and  taken  up  at  3  0  min.   In  other  words, 
these  reactions  were  completed  by  10  min,  at  37  C. 

From  the  above  data,  it  has  been  calculated  that  competent  cells 
can  take  up  in  a  DNAse  resistant  form  almost  10%  of  their  total 
DNA  content,  and  can  bind  up  to  50%  of  their  total  DNA  content. 

(These  calculations  assume  that  S.  sanguis  Wicky  cells  contain  1 
x  10   daltons  of  DNA.)   Therefore,  if  a  competent  Wicky  cell  is 
0.5  um  to  1  urn  in  diameter,  then  in  1  min,  at  37  C,  a  single  cell 
can  take  up  a  molecule  of  a  length  8  to  17  times  its  own  diameter. 
A  remarkable  feat! 


26-30 


Project  No.  Z01  AI  00006-08  LSD 

I  also  showed,  for  the  first  time,  that  heat  denatured  (single 
stranded)  DNA  is  bound  to  competent  cells  in  a  reaction  analogous 
to  native  (double  stranded)  DNA.   Calcium  ion  is  essential.   In 
addition,  it  was  also  shown  by  competition  experiments  that 
competent  cells  contain  specific  binding  sites  for  denatured  DNA. 

It  is  concluded  from  the  work  above  that  competent  cells  react  to 
a  given  concentration  of  DNA  in  such  a  way  that  they  only  take  up 
a  fraction  of  it.   How  this  is  accomplished  is  yet  unknown. 

I  will  continue  to  examine  the  competent-cell  DNA  reactions  as 
well  as  continue  studies  on  the  physiology  and  enzymology  of 
competence. 

Part  II 

Mr.  W.  O.  Mitchell  and  I  isolated  satellite  DNAs  by  dye-buoyant 
density  gradient  centrifugation  of  cell  lysates  from  10  of  12 
strains  of  the  helical  motile  mycoplasmas  known  as  spiroplasmas. 
(See  also  Projects  Z01  AI  00007-5  and  Z01  AI  00004-19  LSD) . 
Electron  microscopy  (T.  J.  Popkin  and  R.  M.  Cole,  LSD)  and 
agarose  gel  electrophoresis  revealed  that  the  satellite  materials 
were  composed  of  2  to  6  classes,  per  strain,  of  covalently 
closed  circular  (CCC)  DNA  molecules  ranging  in  molecular  weights 
from  1.0  to  at  least  12  x  10  daltons.   No  functions  are  yet 
known  for  these  extrachromosomal  elements.   Accumulated  indirect 
evidence  suggests  that  they  are  not  viral  genomes,  and  they  are 
considered  to  be  cryptic  plasmids.   Presumptive  plasmids,  though 
of  larger  sizes  and  also  without  demonstrated  functions,  have 
been  reported  in  a  few  other  mycoplasmas,  but  were  previously 
unknown  in  spiroplasmas.   Future  efforts  will  be  directed  to 
clarification  of  size  classes  and  their  distributions  among  these 
and  other  spiroplasmas,  to  possibilities  for  transfer  among 
strains,  and  to  studies  of  antibiotic  resistances  and  other 
phenotypic  traits  that  may  aid  in  elucidating  the  functions  of 
these  plasmids. 

Dr.  Moynet  continued  studies  of  streptococcal  phages.   042,  a 
temperate  phage  of  lambdoid  morphology,  was  propagated  in  a  Group 
H  streptococcus  (Streptococcus  sanguis:  strain  Wicky) .   By 
contour  lengths  from  electron  microscopy   (EM)  and  by  summation 
of  the  sizes  of  fragments  generated  with  Hind  III  restriction 
endonuclease   separated  by  agarose  gel  electrophoresis,  042 
nucleic  acid  was  conf irmed,to  be  a  linear  double-stranded  DNA  of 
molecular  weight  24.1  x  10   daltons.   Limited  treatment  with  E. 
coli  Exonuclease  III  produced  molecules  with  51  single-stranded 
ends;  the  molecules  subsequently  circularized,  providing  the 
classic  evidence  of  terminal  repetition  of  nucleotide  sequences. 
EM  of  DNA  after  denaturation  and  reassociation  showed  double- 


26-31 


Project  No.  Z01  AI  00006-08  LSD 

stranded  artificial  circles,  thus  giving  evidence  of  circular 
permutation  of  the  genome.   Comparisons  of  contour  lengths  of 
such  circles  with  the  length  of  the  intact  linear  molecule 
indicated  the  extent  of  the  terminal  repetition  to  be  between  15% 
and  2  0%  of  the  genome.   Treatment  of  the  shortened  duplex  DNA 
resulting  from  exposure  to  Exonuclease  III,  by  restriction 
enzymes  Hind  III  (8  cuts  in  the  intact  molecule)  and  Pvu  II  (20 
cuts) ,  produced  fragments  which  were  of  equal  intensities  and  all 
present  after  gel  electrophoresis.   In  controls  of  adenovirus  2 
DNA,  which  is  neither  terminally  redundant  nor  circularly 
permuted,  a  number  of  terminal  bands  disappeared  after  similar 
treatment.   The  evidence  not  only  indicates  the  presence  of 
permutation  in  042  DNA,  but  also  suggests  that  its  location  in 
the  molecule  is  random. 

The  04  2  DNA  was  also  treated  with  several  other  restriction 
enzymes  (Xba  I,  Sal  I,  Sac  I,  Pst  I,  Msp  I)  and  the  fragments 
ordered  by  gel  electrophoresis  to  initiate  physical  mapping  of 
the  genome.   In  conjunction  with  A.  Colon-Whitt,  DNAs  of  several 
lambdoid  phages  of  Group  A  streptococci  were  examined; g these  were 
of  approximately  similar  molecular  weights  (24-26  x  10   daltons) 
and,  after  treatment  with  several  of  the  above  restriction 
enzymes,  showed  gel  patterns  of  fragments  that  were  similar  to 
one  another  but  different  from  those  of  similarly  treated  phage 
DNAs  of  Group  H  phages.   Comparisons  of  streptococcal  phages  and 
their  DNAs,  on  which  little  information  exists,  will  be  continued 
as  time  permits.   (In  collaboration  with  F.  DeFilippes,  LBV) 

Publications : 

Ranhand,  J.  M. :   DNA  binding  and  uptake  by  competent  Streptococcus 
sanguis  Wicky  cells.   In  Glover,  S.,  and  Butler,  L.  0.  (eds.). 
Transformation  1978.   Cotwsold  Press,  Oxford,  1979  (in  press). 


26-32 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00007  -  nd  |_.SP 


PERIOD  COVERED 

October  1,  197  8  to  September  30,  197  9 


TITLE  OF  PROJECT  (80  characters  or  less) 

Viruses  of  Spiroplasmas  and  Mycoplasmas 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


Other: 


M.  Cole 
Liss 


W.  0.  Mitchell 
T.  J.  Popkin 


Chief 

Sr.  Staff  Fellow 

Biologist 
Chemist 


LSD,  NIAID 

LSD,  NIAID 

LSD,  NIAID 

LSD,  NIAID 


COOPERATING  UNITS 


LAB/BRANCH 

Laboratory  of  Streptococcal  Diseases 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda, 


Md.  20205 


TOTAL  MANYEARS: 

4.3 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   Q  (*2)  INTERVIEWS 


PROFESSIONAL: 

1.8 


n  (b)  HUMAN  TISSUES 


[j^c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

This  project  aims  to  characterize  the  nature,  distribution,  and 
ecologic  importance  of  viruses  carried  by  spiroplasmas.   Recent 
studies  of  the  rod-shaped  virus  SpVl  show9single-hit  infection 
kinetics,  an  adsorption  constant  of  2.55    cm  /min,  a  latent 
period  of  60  to  90  min,  and  delayed  host  cell  death  unrelated  to 
virus-induced  lysis.   Virus  infectivity  survives  non-ionic 
detergents,  Genetron,  nucleases,  heterologous  viral  antiserum, 
and  drying;  but  is  sensitive  to  other  detergents,  chloroform, 
ether  (to  some  extent) ,  homospecific  antiserum,  pH  <6  or  >9,  and 
temperatures  >60  C  or  (if  prolonged)  4  C.   Eight  new  isolates  of 
SpVl  from  spiroplasmas  from  bee,  corn,  and  citrus  diseases  showed 
host  range  differences  but  no  evidence  of  host  modification  or 
restriction.   All  were  equally  sensitive  to  specific  antiserum 
raised  against  the  original  isolate  from  a  bee  spiroplasma. 
Viral  DNA  by  electron  microscopy  appears  to  be  duplex  and  linear, 
but  its  size,  precise  conformation,  and  other  characteristics  are 
under  cent  inning  study. — _ 

PHS-6040 

(Rev.   10-76)  26-33 


Project  No.  Z01  AI  00007  -  04  LSH 

Project  Description: 

The  object  of  these  investigations  continues  to  be  a  description 
of  the  distribution,  host  range,  biological  and  physico-chemical 
characteristics,  and  possible  roles  of  viruses  found  in  the 
helical  mycoplasmas  known  as  spiroplasmas. 

Three  virus  groups,  now  designated  as  SpVl  (rod-shaped) ,  SpV2 
(polyhedral,  long-tailed) ,  and  SpV3  (polyhedral,  short-tailed) 
are  known  from  electron  microscopic  observations.   Propagation 
and  characterization  of  SpV3  isolates  was  previously  reported. 
SpV2  has  not  yet  been  isolated  nor  propagated.   Emphasis  this 
year  was  on  further  characterization  of  SpVl,  which  was  initially 
propagated  only  late  last  year. 

The  original  SpVl  isolated,  designated  SpVl/KC3 (BC3) ,  was  made  by 
plaquing  of  filtered  culture  supernate  from  one  spiroplasma  (KC3) 
that  kills  honeybees,  on  lawns  of  another  honeybee  strain  (BC3) . 
Extension  of  prior  electron  microscopic  studies  showed  that  50% 
of  2  5  spiroplasmas  from  diverse  natural  sources  carry  SpVl 
particles.   Additional  isolates  of  SpVl  were  obtained,  by  use  of 
filtered  washes  of  lawns  grown  on  solid  media,  from  spiroplasmas 
cultured  from  moribund  honeybees  (3  isolates  from  3  strains), 
from  corn  stunt  disease  (2  isolates  from  2  strains) ,  and  citrus 
stubborn  disease   (3  isolates  from  3  strains) .   Host  range 
studies  of  the  8  isolates  revealed  distinct  differences,  but  the 
presence  of  classic  host  modification  and  restriction  systems  has 
not  been  shown.   On  the  other  hand,  spiroplasma  host  strains  or 
derivatives  selected  for  resistance  to  one  SpVl  isolate  were  also 
resistant  to  all  other  SpVl  isolates  tested  to  date,  suggesting 
other  mechanisms  for  resistance  such  as  lack  of  surface  receptors 
or  failure  of    adsorption  or  of  nucleic  acid  injection  for  other 
reasons.   Antiserum  to  purified  SpVl/CK3 (BC3)  was  obtained  by 
immunization  of  rabbits,  and  was  of  the  same  efficiency  in 
inactivation  (determined  by  plaque  assay)  of  other  SpVl  isolates 
as  in  inactivation  of  the  homologous  isolate.   Aggregate  data 
from  plaquing  and  electron  microscopic  studies  are  being  incorp- 
orated into  a  manuscript  on  the  distribution  of  all  3  viruses 
among  the  increasing  numbers  of  spiroplasmas  cultured  from  such 
diverse  sources  as  diseased  plants,  healthy  flowers,  sick  bees, 
ticks,  and  other  arthropods. 

Physico-chemical  studies  of  SpVl/KC3 (BC3)  previously  showed  that 
the  purified, 230-280  by  10-15  nm  particles  have  buoyant  densities 
of  1.39  g/cm   in  cesium  chloride  and  1.21  g/cm   in  Metrizamide 
gradients.   Chemical  and  radiolabelling  studies  indicated  its 
nucleic  acid  to  be  DNA.   Recent  electron  microscopic  studies  of 
both  aqueous  and  formamide  spreads  of  the  extracted  DNA  suggest 
that  it  is  probably  double-stranded  and  linear,  but  consistent 


26-34 


Project  No.  Z01  AI  00007-04  LSn 

peculiarities  in  the  preparations  examined  prevent  a  definitive 
description  at  present  of  its  precise  size  and  molecular  confor- 
mation.  Polyacrylamide  gel  electrophoresis  of  disaggregated 
virions  showed  7  to  9  presumptive  structural  proteins,   and 
calculations  from  virion  buoyant  densities  indicate  that  approxi- 
mately 23%  of  the  particle  should  consist  of  nucleic  acid. 
Infectivity  of  the  virus  particles  was  found  to  be  sensitive  to 
specific  antisera,  chloroform,  some  detergents,  temperatures 
above  60  C  or  prolonged  storage  at  4  C,  and  pH  outside  the  range 
of  6  to  9.   There  was  moderate  sensitivity  to  ether,  and  insensi- 
tivity  to  non-ionic  detergents  (Non-Idet  P40,  Triton  X-100) , 
Genetron,  antisera  to  SpV3,  DNAase,  RNAase,  and  drying. 

Growth  studies  of  SpVl/KC3 (BC3)  on  the  original  propagating  host 
strain  BC3,  are  being  described  in  a  manuscript  in  preparation. 
Infection  followed  a  single-hit  model,  with  a  particle  adsorption 
constant  of  2.55    cm  /min.   At  a  low  multiplicity  of  infection 
(1.0  or  less) ,  a  60  to  90  minute  latent  period  was  followed  by  an 
extended  period  of  gradual  release  of  progeny  virus.   Six  hours 
after  infection,  each  infected  cell  had  released  10  progeny 
viruses,  and  there  was  no  concomitant  cell  death  nor  lysis; 
however,  at  a  much  later  time  in  the  infectious  cycle,  there  is 
death  of  infected  cells  which  appears  to  be  secondary  and 
limited.   Short  range  goals  of  this  portion  of  the  study  include 
study  of  the  intracellular  state  of  this  virus  which  is  not 
detectable  within  infected  cells  by  electron  microscopy. 

Other  aims  include  further  analysis  of  SpVl  DNA  and  proteins,  as 
well  as  identification  of  group  characteristics  of  SpVl  isolates. 
Other  recent  studies  of  this  Laboratory  (see  Project  No.  Z01  AI 
00006-8  LSD)  have  shown  a  range  of  small  to  moderate-sized  (1.0 
to  12  x  10   daltons)  circular  DNAs  in  10  of  12  spiroplasmas — all 
of  which  were  carrying  one  or  more  viruses,  and  some  of  which 
were  actively  producing  a  virus  at  time  of  examination.   However, 
the  sizes  of  these  presumptive  cryptic  plasmids,  the  numbers  of 
size  classes  found,  and  their  distributions  by  strain,  do  not 
correlate  with  virus  carraige  or  production,  and  the  available 
evidence  suggests  that  the  circular  molecules  do  not  represent 
viral  genomes. 

These  studies  furnish  information  essential  to  determining  the 
nature,  and  ultimately  the  importance,  of  new  viruses  that 
occur  commonly  in  a  widespread  group  of  recently  recognized  and 
poorly  understood  mycoplasmas.   Both  viruses  (which  appear 
similar  to  bacteriophages)  and  newly-found  plasmids  may  be 
expected  to  play  roles  in  the  host  ecology  similar  to  those  of 
bacteriophages  and  bacterial  plasmids,  but  the  existence  and 
influence  of  their  effects  in  spiroplasmas  remain  to  be  elucidated, 


26-35 


Project  No.  Z01  AI  00007-  04  LS" 

Publications : 

Cole,  R.  M. :   Mycoplasma  viruses.   In  Laskin,  A.  I.,  and 
LeChevalier,  H.  A.  (eds.):   CRC  Handbook  of  Microbiology,  ed.  2. 
West  Palm  Beach,  CRC  Press  Inc.,  1978,  Vol.  II,  pp.  683-690. 

Cole,  R.  M. :   Mycoplasma  and  spiroplasma  viruses:   Ultrastructure, 
In  Barile,  M.  F.,  and  Razin,  S.  (eds.):   The  Mycoplasmas.   New 
York,  Academic  Press,  1979,  Vol.  1,  pp.  385-410. 


26-36 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  AI  00181-01  LSD 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Isolation,  Purification,  and  Characterization  of  Toxins  and 
Inflammatory  Factors  of  Streptococci. 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:        Gary  B.  Calandra 

Other:  T.  S.  Theodore 
A.  Colon-Whitt 
T.  J.  Popkin 


Medical  Officer 

Res.  Microbiol. 

Microbiol. 

Chemist 


LSD,  NIAID 

LSD,  NIAID 

LSD,  NIAID 

LSD,  NIAID 


COOPERATING  UNITS  (if  any) 

Arthritis  and  Rheumatology  Branch, 


NIAMDD  (Dr.  R.  Wilder) 


lab/branch 
Laboratory   of   streptococcal    Diseases 


INSTITUTE  AND  LOCATION 

NIAID,    NIH,    Rethftsrifl, 


TOTAL  MANYEARS: 

2.3 


Mri.  20205 


PROFESSIONAL: 

1.3 


1.0 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


C?£(c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  purpose  of  this  work  is  to  identify  the  toxic  factors  of 
streptococci,  especially  Group  A,  that  play  a  role  in  streptococcal] 
pathogenesis.   The  major  emphasis  is  to  purify  and  characterize 
the  hemolysin,  streptolysin  S  (SLS) .   Using  the  cellular  precursor 
to  SLS,  the  hemolytic  moiety  is  to  be  purified  and  used  to 
serologically  assess  host  response  to  this  toxin  in  acute  and 
chronic  streptococcal  disease  such  as  glomerulonephritis.   Enzymatic 
methods  to  degrade  Group  B  streptococci  are  being  developed  to 
better  study  its  toxins  [hemolysin (s)  apparently  different  than 
SLS] .   Various  toxins,  such  as  erythrogenic  toxin,  and  cell 
components  such  as  lipoteichoic  acid,  are  being  compared  between 
Groups  A,  B,  C,  and  G  streptococci  to  determine  potential 
relationships  in  disease  production.   The  cell  walls  of  Group  A 
streptococci  are  being  studied  in  the  production  of  arthritis  to 
study  the  genetic  determinants  of  host  responses. 


PHS-6040 
(Rev.    10-76) 


26-37 


Project  No.  Z01  AI  00181-01  LSD 

Project  Description: 

Streptolysin  S  is  the  most  powerful  membranolytic  agent  known. 
Yet  the  role  of  this  hemolysin  of  Group  A  streptococci  in 
pathogenesis  is  unknown  because  no  antibody  response  has  been 
detected  in  either  man  or  test  animal.   The  toxin  is  made  as  an 
inactive  precursor  intracellularly — the  majority  associated  with 
the  membranous  fraction  of  the  cell.   It  has  been  found  in  high 
titer  in  all  nephritogenic  strains  tested.   The  active  hemolysin 
is  likely  a  very  small  peptide  which  is  very  cationic  and  has 
high  affinity  for  lipids.   The  objective  is  to  purify  the  hemo- 
lytic moiety  using  the  different  chemical  characteristics  of  the 
precursor  and  active  hemolysin,  to  characterize  it,  and  to 
determine  its  potential  role  in  streptococcal  disease  such  as 
glomerulonephritis.   This  and  other  toxins  and  inflammatory 
factors  from  streptococci  are  being  characterized  to  determine 
potential  roles  in  disease  production. 

(1)   Streptolysin  S 

SLS  precursor  (measured  in  the  presence  of  a  carrier,  RNA-core) 
was  characterized  in  1974-1975  in  multiple  types  of  Group  A 
streptococci  from  both  nephritogenic  and  non-nephritogenic 
strains  and  found  to  be  in  highest  titer  in  the  membranes  and  of 
highest  specific  activity  in  mesosomes  of  nephritogenic  strains. 
On  resuming  this  work  in  1978,  it  was  initially  not  possible  to 
obtain  these  same  high  titers  and  strain  differences  from  most  of 
the  previously  lyophilized  strains.   The  effect  of  different  lots 
of  media,  additives  (sugars  and  ions),  buffers,  cell  fractionation 
steps,  and  even  water  source  were  studied  to  determine  the  reason 
for  the  difference,  since  fractions  frozen  in  1975  still  showed 
the  same  high  titers  as  when  measured  at  that  time.    It  was 
found  that  some  strains  had  lost  their  high  titer  cellular  SLS 
production  for  reasons  that  are  yet  uncertain.   In  other  strains, 
the  previously  obtained  high  titers  could  be  obtained  by  using 
muralysin  (to  degrade  the  cell  wall  to  protoplast  the  streptococci) 
prepared  in  a  slightly  different  way  than  before.   Using  new 
materials,  the  highest  percentage  of  hemolysin  precursor  was 
again  found  in  the  membranes  and  the  highest  specific  activity  in 
the  mesosomes  of  various  types  of  streptococci.   The  absence  of 
any  hemolysin  precursor  in  walls  has  also  been  confirmed.   All 
precursor  in  the  periplasmic  fraction  is  sedimentable  and 
probably  reflects  that  associated  with  membrane  fragments.   A 
significant  percentage  of  the  precursor  is  also  found  in  the 
cytoplasmic  fraction  and  is  sedimentable  under  appropriate 
conditions.   Part,  at  least,  appears  to  be  ribosomally  associated 
and  may  be  a  ribonucleoprotein  since  7  5%  of  the  activity 
precipitates  from  solution  when  treated  with  ribonuclease.   The 
precise  characterization  of  this  cytoplasmic  precursor  is 
continuing. 

26-38 


Project  No.  Z01  AI  00181-01  LSD 

That  the  precursor  can  be  activated  by  vortexing  with  glass  beads 
in  the  presence  of  RNA-core  was  previously  reported.   Studies  of 
the  chemical  preparation  and  physical  properties  of  the  beads  has 
shown  the  importance  of  small  bead  size  (10-40  microns)  and 
enhancement  of  bead  utility  by  prewashing  with  water,  by 
increasing  %  acetic  acid  to  60%,  and  by  phosphate  buffer  but  not 
by  other  acids  or  base.   In  some  preparations  of  precursor, 
streptococcal  RNA  probably  enhances  the  final  titer.   Numerous 
different  detergents  (nonionic,  anionic,  zwitter ionic)  have  been 
tested  but  none  alone  activates  the  hemolysin  precursor  and  those 
that  solubilize  the  membrane  inactivate  the  precursor.   However, 
when  precursor  is  vortexed  with  glass  beads  in  the  presence  of  a 
nonionic  detergent  such  as  Nonidet  P4  0,  active  hemolysin  is 
obtained  in  the  absence  of  RNA-core.   The  activity  of  this 
hemolysin  is  very  labile  but  can  be  stabilized  by  RNA-core.   Work 
is  in  progress  to  further  characterize  this  hemolysin  in  terms  of 
its  usefulness  in  a  scheme  to  purify  SLS.   How  the  precursor  is 
activated  in  vivo  is  uncertain,  but  studies  are  in  progress  to 
further  investigate  this.   Since  proteinases  activate  certain 
extracellular  enzymes,  the  effect  of  streptococcal  proteinase 
(obtained  from  Dr.  Darrell  Liu,  BOB)  was  studied.   Interestingly, 
although  it  had  no  effect  to  activate  or  degrade  the  precursor, 
it  inactivated  the  active  hemolysin  suggesting  a  possible 
regulatory  role  of  the  proteinase  on  production  of  the  extra- 
cellular hemolysin.   The  effects  of  other  types  of  enzymes  such 
as  phospholipases  on  precursor  activation  remain  to  be  determined. 

The  future  course  of  this  work  will  be  to  purify  and  characterize 
the  hemolytic  moiety,  utilizing  the  differing  characteristics  of 
the  precursor  and  active  hemolysin.   After  purification  of  the 
precursor,  it  will  be  activated  in  the  presence  of  a  lipid 
carrier,  purified  by  various  means  including  chromatography  in 
organic  solvents,  and  then  transferred  to  a  synthetic  nucleotide 
carrier  bound  to  a  thiopropyl  Sepharose  affinity  chromatography 
column.   After  elution  and  further  purfication  by  gel  filtration 
and  ethanol-acid  treatment,  the  peptide  and  amino  acid  content 
will  be  determined. 

(2)   Toxins  of  Groups  B,  C,  and  G  Streptococci 

The  development  of  a  method  to  prepare  protoplasts  of  Group  B 
streptococci  was  needed  to  study  various  cellular  toxins  (such  as 
hemolysins) ,  since  no  suitable  or  efficient  method  had  been 
previously  described.   Examination  of  some  50  to  100  different 
strains  of  streptococci  of  all  groups  and  their  phages  for  a 
phage-associated  muralysin  was  unsuccessful.   However,  a 
Streptomyces  globisporus  enzyme,  mutanolysin,  supplied  by 
Japanese  investigators,  degraded  walls  of  all  5  Group  B  types  as 
well  as  walls  of  Groups  A,  C,  G,  and  H.    Preliminary  studies 
suggest  that  protoplasts  of  Group  B  can  be  prepared  using  this 

26-39 


Project  No.  Z01  AI  00181-01  LSD 

enzyme.   Interestingly,  it  was  found  by  electron  microscopy  that 
when  the  enzyme  was  added  in  excess  to  cells  of  the  different 
groups  in  hypotonic  buffer,  the  gross  cell  form  remained  intact 
and  leakage  of  cytoplasmic  contents  occurred  from  one  area  of  the 
cell.   Why  the  cell  was  not  totally  disrupted  and  whether  only  a 
part  of  the  wall  (instead  of  all)  was  degraded  is  uncertain,  but 
under  further  study. 

The  presence  of  potential  pathogenic  factors  in  Groups  A,  B,  C, 
and  G  is  being  investigated.   Streptolysin  S  precursor  has  been 
found  in  Groups  A,  C,  and  G  streptococci,  but  not  Group  B. 
Whether  or  not  there  are  differences  in  cellular  location  among 
Groups  A,  C,  and  G  has  not  yet  been  evaluated.   Erythrogenic 
toxin  production  has  been  studied  immunologically  in  Groups  A,  C, 
and  G,  but  has  been  found  thus  far  only  in  Group  A.   Lipoteichoic 
acid  (LTA) ,  suggested  as  important  in  streptococcal  adherence, 
was  found  in  all  four  groups  in  approximately  equal  amounts. 
Studies  to  determine  the  cell  surface  distribution  of  LTA  are  in 
progress. 

(3)   Peptidoglycan  Induced  Arthritis 

The  cell  walls,  or  more  specifically,  the  peptidoglycan,  of 
streptococci,  cause  a  severe  arthritis  in  certain  strains  of 
rats.  Although  much  is  known  about  the  effects  of  enzymatically 
modifying  the  peptidoglycan  on  its  pathogenic  potential,  little 
is  known  about  the  determinants  of  the  host  in  terms  of  whether 
arthritis  will  be  produced.   Using  Group  A  streptococcal  cell 
walls  with  various  enzymatic  and  chemical  modification,  arthritis 
production  is  being  evaluated  in  outbred  and  inbred  strains  of 
rats  to  determine  what  genetic  factors  are  responsible,  how  the 
peptidoglycan  is  transported  to  the  joints,  how  the  inflammatory 
response  is  generated  there,  what  specific  bacterial  components 
modulate  this  process,  and  how  the  process  can  be  stopped  by 
treatment  of  the  host.  (In  collaboration  with  Dr.  R.  Wilder) 

Publications : 

None 


26-40 


LABORATORY  OF  VIRAL  DISEASES 

1979  Annual  Report 

Table  of  Contents 

Z01-AI 
Project  Number  Page 

Summary  27-1 

00011-14  Studies  of  Small    DNA  Containing  Viruses   Belonging     27-3 

to  the  Family  Parvoviridae.   —  Hoggan 

00012-17  Studies  of  Mouse  Leukemia  Viruses.—  Hartley  27-7 


PHS-NIH 

SUMMARY  REPORT 

ANNUAL  REPORT  OF  THE  LABORATORY  OF  VIRAL  DISEASES,  NIAID 
October  1,  1978  -  September  30,  1979 

Dr.  Wallace  P.  Rowe 
Chief,  Laboratory  of  Viral  Diseases 

There  was  a  major  administrative  reorganization  of  LVD  during  the  past  year, 
consisting  chiefly  of  removal  of  the  LVD  units  located  in  Building  5.  The 
units  of  Drs.  Mattern,  Takemoto,  Lewis,  and  Jaouni  have  been  reassigned. 
In  effect  Dr.  Levy's  unit  has  also  been  reassigned,  but  the  formal  transfer 
has  not  yet  taken  place.  This  reorganization  has  the  salutary  effect  of  con- 
solidating LVD  into  a  more  coherent  group  both  spatially  and  programrnatically, 
and  of  greatly  simplifying  administrative  responsibilities  for  the  Laboratory 
Chief.  The  laboratory  now  consists  of  the  unit  of  Drs.  Rowe  and  Hartley  plus 
that  of  Dr.  Hoggan. 

In  addition  to  the  change  in  the  lab  structure,  there  is  a  significant  change 
in  program.  Both  Drs.  Rowe  and  Hoggan  are  now  involved  in  generation  of  a 
new  program  involving  molecular  cloning  of  C-type  viral  genomes  by  recombinant 
DNA  techniques.  This  program  is  under  the  general  direction  of  Dr.  Malcolm 
Martin,  and  involves  collaboration  with  workers  in  LVD,  NCI,  Johns  Hopkins, 
and  MIT.  This  work  is  in  part  an  outgrowth  of  the  recombinant  DNA  risk 
assessment  experiments  of  Drs.  Martin  and  Rowe  and  will  involve  both  risk 
assessment  and  basic  scientific  studies. 

The  involvement  of  the  laboratory  chief  in  the  scientific  and  policy  making 
aspects  of  recombinant  DNA  research  again  occupied  a  major  portion  of  his  time 
during  the  past  year,  but  his  term  on  the  NIK  Recombinant  Advisory  Committee 
has  now  expired  and  hopefully  the  time  spent  in  this  activity  will  decrease 
drastically. 

Staff  changes  in  LVD  during  the  past  year  included  the  arrival  of  a  Research 
Associate,  Dr.  Susan  Light,  and  the  return  of  Charles  Buckler  to  work  in  LVD 
following  virtual  completion  of  his  Ph.D.  program  at  Johns  Hopkins. 

Dr.  Rowe  received  a  major  award,  the  Paul  Ehrl ich-Ludwig  Darmstaedter  prize, 
which  was  shared  with  Drs.  Graffi  and  Muhlbock.  The  prize  was  presented  by 
President  Scheel  of  The  Federal  Republic  of  Germany  at  a  ceremony  in  Frankfurt. 

Some  of  the  highlights  of  the  scientific  accomplishments  of  the  past  year 
follow. 

Additional  MuLV  chromosomal  loci  mapped.  Seven  ecotropic  virus-inducing  loci 
have  now  been  precisely  located  on  mouse  chromosomal  DNA.  In  addition  to  the 
five  previously  reported,  Akv-2  has  been  mapped  to  chromosome  16  (as  determined 
by  somatic  cell  hybrid  analysis)  and  one  of  the  C58  loci  has  been  identified  on 
chromosome  8.  A  fifth  xenotropic  virus  inducing  locus,  that  of  C57L,  has  been 
found  to  be  at  the  same  site  on  chromosome  1  as  the  xenotropic  loci  in  other 
strains  previously  studied.  (Kozak,  Rowe) 


27-1 


Virus-inducing  chromosomal  locus  identified  by  restriction  blotting 
techniques.  The  Akv-1  locus  can  be  specifically  detected  in  cellular  DNA  by 
restriction  enzyme  analysis  using  the  Southern  blot  technique.  Using  this 
technique  it  will  eventually  be  possible  to  analyze  viral  chromosome  loci  in 
various  mouse  strains  with  great  precission.  (Steffen,  Weinberg,  Rowe) 

Correlates  of  lymphomagenicity  identified.  Certain  MCF  viruses,  which  repre- 
sent genetic  recombinants  of  ecotropic  and  xenotropic  MuLV,  markedly  accelerate 
the  appearance  of  lymphoma  in  AKR  mice  while  others  are  inactive.  Comparisons 
of  biological  and  biochemical  characteristics  of  leukemogenic  and  non-leukemo- 
genic  isolates  indicate  that  the  former  are  isolated  from  thymic  tissue  or 
neoplasms  of  thymic  origin,  replicate  efficiently  in  AKR  thymus,  are  relatively 
restricted  in  ability  to  infect  NFS  mouse  embryo  cells  as  compared  to  SC-1 
cells,  and  have  in  common  certain  characteristics  of  their  RNA  oligonucleotide 
fingerprints.  Non-1 eukemogenic  isolates  are  non-thymic  in  origin,  unable  to 
replicate  in  thymus,  show  the  reverse  pattern  of  infection  of  NFS  and  SC-1  cells, 
and  have  closely  similar  RNA  fingerprint  patterns  which  are  clearly  distinct 
from  those  of  leukemogenic  isolates.  Recognition  of  these  differences  should 
assist  in  identifying  the  portion  of  viral  genome  controlling  thymotropism. 
(Cloyd,  Hartley,  Rowe;  Hopkins,  MIT) 

Genetic  control  of  sensitivity  to  infection  by  MCF  viruses  under  study. 
Ability  of  the  virus  to  replicate  in  a  given  host  is  a  necessary  event  in  MCF 
virus  induced  lymphomagenesis,  and  evidence  of  genetic  control  of  sensitivity 
to  infection  has  been  obtained  both  in  vivo  and  in  vitro.  In  addition  to  Fv-1 , 
several  other  genes  are  clearly  involved  in  determining  susceptibility. 
(Hartley,  Cloyd) 

Identification  of  xenotropic  viruses  with  MCF-related  sequences.  Although 
the  ecotropic  parentage  of  MCF  viruses  can  be  clearly  identified  as  the  endog- 
enous ecotropic  virus  of  a  given  mouse  strain,  the  putative  xenotropic  parent 
is  unknown.  Certain  xenotropic  viruses  but  not  others  have  been  found  to 
contain  genome  sequences  that  complement  ecotropic  viral  DNA  probes  in  saturat- 
ing the  entire  genome  of  an  AKR  MCF  virus.  The  significance  of  this  finding 
in  precisely  identifying  the  second  partner  in  the  origin  of  an  MCF  recombi- 
nant is  not  yet  clear,  but  these  studies  confirm  involvement  of  xenotropic 
virus  in  the  genesis  of  recombinant  MuLV.   (Chattophadhyay,  Hartley,  Rowe) 

Protein  sequence  homology  among  AAV  proteins.  Tryptic  and  chymotryptic  maps 
of  the  adeno-associated  virus  (AAV)  structural  proteins  show  extensive  areas 
of  sequence  homology  further  suggesting  that  all  three  arise  from  a  common 
precursor.  (Lubeck,  Lee,  Hoggan,  Johnson) 

Appearance  of  extrachromosomal  viral  DNA  in  carrier  culture.  Long  term  passage 
(40-80  cell  transfers)  of  AAV  integrated  human  cell  clones  show  small  amounts 
of  extrachromosomal  viral  DNA  while  earlier  transfers  show  only  integrated 
viral  DNA.  (Chan,  Berns ,  Hoggan,  Houseworth) 

Relatedness  of  human  parvovirus  to  rat  virus.  Three  human  cell  specific  parvo- 
virus  isolates  (Kirk,  HS-3  and  PVZgd)  demonstrate  strong  one  way  immunologic    • 
cross  reactivity  with  the  rodent  rat  virus  (RV)  and  each  possess  the  same  three 
structural  polypeptides.  (Hoggan,  Sears) 


27-2 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  AI  00011-14  LVD 


PERIOD  COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Studies  of  Small  DNA  Containing  Viruses  Belonging  to  the  Family  Parvoviridae. 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:  M.  D.  Hoggan 


Staff  Scientist,  LVD,  NIAID 


COOPERATING  UNITS  (if  any) 

K.  I.  Berns,  Univ.  of  Florida  College  of  Medicine,  Gainesville,  Florida. 
F.  Brent  Johnson,  Dept.  of  Microbiology,  Brigham  Young  Univ.,  Provo,  Utah. 


LAB/BRANCH 

Laboratory  of  Viral  Diseases 


SECTION 

Viral  Oncology  Section 

st i tiitp   aMn  i  nr.LT\ riN 


INSTITUTE    AND    LOCATION 

NIAID,  NIH,  Bethesda,  Maryland  20205 


TOTAL  MANYEARS: 

3 


PROFESSIONAL: 
1 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


Xj(b)  HUMAN  TISSUES 


□  (c)    NEITHER 


SUMMARY    OF    jJORK    (200   words    or   less   -   underline    keywords) 

Ongoing  studies  of  parvovirus  have  shown  that  long  passage  of  integrated  AAV 
carrier  cell  clones  may  show  small  amounts  of  extrachromosomal  viral  DNA.  Studies 
of  the  polypeptides  of  "human"  parvoviruses  demonstrate  that  these  viruses  are 
structurally  and  immunologically  closely  related  to  rodent  (RV)  parvovirus  and 
that  these  viruses  undergo  a  post  assembly  maturation  process  involving  protolytic 
cleavage.  It  has  been  further  shown  that  the  immunogenic  expression  of  a  cross 


reacting  antigen  between  large  plaque  rat  virus  (LPRV)  and  minute  virus  of  mice 
is  linked  to  the  host  cells  in  which  these  viruses  are  grown.  Continued  studies 
of  AAV  proteins  have  shown  that  each  of  the  three  recognized  structural  polypep- 
tides are  closely  related  and  show  a  great  amount  of  protein  sequence  homology. 
Dne  unexpected  finding  is  that  all  of  the  polypeptides  of  canine  AAV  (CAAV)  appear 
10-20%  larger  than  those  of  all  other  AAV's  thus  far  studied.  We  have  some 
evidence  that  suggests  this  is  the  result  of  a  block  in  CAAV  virion  maturation. 


27-3 


PHS-6040 
(Rev.  10-76) 


Z01  AI  00011-14  LVD 

Project  Description 

Objectives:  To  use  molecular  biological  as  well  as  classic  physical -chemical , 
immunological  and  epidemiological  techniques  to  elucidate  the  mechanisms  of 
virus  replication,  persistence  and  genetic  evolution  of  viruses  belonging  to 
the  Family  Parvoviridae  for  a  better  understanding  of  their  natural  history 
and  role  in  disease. 

Major  Findings 

Appearance  of  extrachromosoma!  viral  DNA  after  prolonged  culture  of  inte- 
grated AAV  carrier  clones:  Soon  after  initiation  of  AAV  carrier  clones, 
multiple  copies  of  the  virus  genome  can  be  detected  in  host  cell  DNA  with  no 
detectable  virions  or  extrachromosomal  viral  DNA.  However,  after  prolonged 
cultivation  (40-80  cell  passages)  small  amounts  of  unintegrated  viral  DNA 
appear  in  the  cell  cytoplasm  and  can  be  detected  using  the  Southern  blotting 
technique.  These  results  explain  why  virus  is  much  easier  to  induce  with 
helper  virus  in  some  high  passage  clones. 

Polypeptides  of  "human"  parvovirus  strains  Kirk,  HS-3,  and  PVZgd  indistin- 
guishable from  those  of  rodent  parvovirus  (RV):  Kirk,  HS-3,  and  PVZgd  viruses 
replicate  only  in  human  cells  yet  they  show  strong  one  way  serological  reac- 
tivity with  sera  produced  against  various  strains  of  RV  which  replicate  to 
high  titer  only  in  rodent  cells.  Comparative  studies  of  the  proteins  from 
these  "human"  and  "rodent"  viruses  showed  them  to  be  indistinguishable  from 
each  other  although  they  each  could  be  easily  distinguished  from  other  members 
of  the  genus  parvovirus.  Further  studies  showed  that,  as  with  other  members 
of  this  genus,  the  major  polypeptide  on  the  virus  capsid  at  the  time  of 
assembly  is  a  middle  sized  protein  of  about  70-72,000  MW.  Later  during  the 
course  of  the  infection,  during  maturation,  this  protein  is  proteolytically 
trimmed  to  a  slightly  smaller  protein  about  68-70,000  MW.  Both  the  latter 
are  more  stable  in  CsCl  and  demonstrate  a  higher  efficiency  of  infection. 

Immunogenic  expression  of  a  cross  reacting  antigen  between  large  plaque 
variant  of  rat  virus  (LPRV)  and  minute  virus  of  mice  (MVM)  linked  to  host 
cells:  We  have  carried  out  detailed  studies  of  the  LPRV  variant  which  show 
that  this  variant  exhibits  striking  differences  from  wild  type  RV  in  virulence 
in  the  experimental  animal  as  well  as  in  the  size  of  plaque  produced  in  tissue 
culture.  The  number  of  plaque  forming  units  (PFU)  required  for  killing  new- 
born hamsters  is  50  to  1000  times  greater  for  LPRV  than  it  is  for  wild  type 
RV  even  though  it  produces  plaques  which  are  3-20  times  larger  in  rat  nephroma 
cells.  For  these  studies  we  have  relied  heavily  on  a  cross  reacting  antigen 
between  LPRV  and  MVM  which  is  not  normally  expressed  by  small  plaque  (wild 
type)  rat  virus  (SPRV).  This  cross  reacting  antigen  is  expressed  on  the  sur- 
face of  the  virus  and  can  be  measured  using  hemagglutination  inhibition, 
immune  electron  microscopy  and  serum  neutralization  and  is  coded  for  by  the 
virus.  However  we  find  that  only  hyperimmunized  animals  injected  with  puri- 
fied MVM  prepared  from  virus  produced  in  rat  cells  develop  the  cross  reacting 
antibodies  to  LPRV.  On  the  other  hand,  all  sera  from  animals  hyperimmunized 
with  purified  LPRV  grown  in  rat  nephroma  cells  react  with  all  strains  of  MVM 


27-4 


Z01  AI  00011-14  LVD 

while  no  sera  from  SPRV  immunized  animals  show  cross  reacting  antibody. 
Using  polyacryl amide  gel  analysis  the  virion  associated  polypeptides  from 
LPRV  and  SPRV  cannot  be  distinguished  but  both  are  easily  separable  from  MVM 
polypeptides. 

Adeno-associated  virus  (AAV)  structural  protein  sequence  homology:  AAV 
structural  proteins  have  been  purified  and  examined  for  areas  of  sequence 
homology  between  each  of  the  3  virion  associated  polypeptides.  Tryptic  and 
chymotryptic  maps  demonstrate  extensive  areas  of  homology  and  have  a  similar 
amino  acid  composition.  Although  we  showed  earlier  that  each  polypeptide 
could  be  distingui sherd  immunologically  using  FA  antigen  induction,  there  is  a 
large  amount  of  antigenic  identity  when  using  the  more  sensitive  radio-immuno- 
precipitation  assay.  With  one  exception,  all  peptides  contained  in  the  small- 
est polypeptide  are  found  in  the  middle  and  largest  size  peptides.  These 
data  further  suggest  that  either  a  large  precursor  (120,000  MW)  protein  is  cut 
with  overlapping  segments  or  the  largest  polypeptide  is  itself  modified  by 
some  proteolytic  action  into  each  of  the  two  smaller  units.  Tertiary  structure 
on  the  virion  is  also  important  in  specificity  since  various  AAV  show  a  high 
degree  of  specificity  when  antibody  is  raised  against  whole  virions  while  much 
crossing  occurs  when  antibody  is  raised  against  dissociated  virus. 

Canine  adeno-associated  virus  (CAAV)  exhibits  uniquely  large  constituent 
polypeptides  when  compared  to  other  members  of  the  same  genus:  As  has  already 
been  noted,  various  types  of  primate,  bovine,  avian,  and  canine  AAV's  show 
little  or  no  serological  cross  reactivity  when  tested  using  sera  produced 
against  whole  virions,  even  though  their  genomes  share  between  60-80%  homology. 
On  the  other  hand  studies  utilizing  sera  produced  against  capsid  polypeptides 
all  show  cross  reactivity.  Our  recent  studies  using  l"j  exogenously  labelled 
polypeptides  from  a  number  of  different  AAV  types  showed  very   little  differences 
in  MW  between  the  polypeptides  of  the  various  AAV  with  the  exception  that  CAAV 
polypeptides  were  10-20%  larger.  The  CAAV  polypeptides  can  be  easily  distin- 
guished from  the  others  when  coelectrophoresed  on  the  same  gel.  It  is  unlikely 
that  this  represents  an  increase  in  coding  capacity  since  the  genomes  of  all 
AAV's  are  quite  similar  in  size  rather,  it  probably  represents  a  block  in 
some  maturation  step.  We  have  previously  shown  that  2  populations  of  AAV 
capsids,  each  containing  a  complete  viral  genome,  are  produced  during  an 
adenovirus-helped  lytic  infection;  these  can  be  separated  based  on  their  re- 
lative specific  density  in  CsCl .  We  have  further  shown  that  the  more  dense 
population  can  be  immunologically  distinguished  from  the  mature  population, 
is  less  stable  and  shows  a  reduced  infection  efficiency.  These  results  and 
the  results  on  the  maturation  process  of  members  of  the  genus  parvovirus 
taken  in  concert  that  AAV  maturation  may  indeed  involve  a  similar  post  assembly 
proteolytic  cleavage.  Although  normally  this  process  may  not  be  seen  because 
of  rapid  turnover,  for  some  yet  to  be  determined  reason  it  is  blocked  in  CAAV. 
Other  data  which  supports  this  theory  is  the  fact  that  we  have  never  seen  a 
major  CAAV  band  in  the  density  range  of  1.40  g/cc  and  the  very  small  full 
band  which  is  seen  is  in  the  density  range  of  1.45  g/cc.  This  may  also  account 
for  the  extremely  low  infectivity  titers  we  find  for  this  virus. 


27-5 


Z01  AI  00011-14  LVD 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute 

The  Institute  has  long  supported  a  program  on  slow  viruses,  persistence  and 
their  role  in  disease.  Parvoviruses  not  only  provide  simple  (genomic  size 
less  than  2xl05  MW)  virus  models  for  elucidating  basic  mechanisms  of  virus/ 
cell  interaction  and  persistence,  they  are  wide  spread  in  nature  and  may  hold 
the  key  to  our  understanding  of  gene  expression.  They  may  also  provide  answers 
regarding  the  etiology  of  as  yet  poorly  understood  slow  progressive  diseases 
such  as  amyotropic  lateral  sclerosis.  We  have  in  fact  found  that  1%  of  all 
human  embryonic  tissues  tested  contain  the  parvovirus  AAV-2  in  an  integrated 
occult  state.  What  is  this  virus  genome  doing?  The  recent  finding  that  a 
"new"  parvovirus  has  been  isolated  from  a  number  of  fatal  epidemics  of  canine 
enteritis  which  is  indistinguishable  from  the  parvovirus  known  to  cause  pan- 
leukopenia  of  cats,  is  of  great  interest.  Is  it  possible  that  the  canine  virus 
is  in  fact,  a  recently  evolved  variant  of  the  feline  virus?  With  man  living 
so  close  to  his  pets  is  it  possible  for  a  similar  mutant  to  evolve  which  infects 
him?  Are  the  "human"  parvoviruses  which  are  so  closely  related  to  the  rat 
virus  actually  variants  of  that  virus?  Could  they  become  pathogenic  for  man? 
These  questions  are  not  only  fascinating  they  could  obviously  be  important  to 
public  health. 

Proposed  course 

Because  of  the  rapidly  developing  area  of  recombinant  DNA  research  and  the 
need  to  develop  expertise  in  this  area  for  the  full  exploitation  of  the  pro- 
blems in  parvovirus  research.  Our  section  has  devoted  about  60%  of  our  effort 
to  learning  these  techniques  during  the  last  3-4  months.  We  have  done  this  in 
collaboration  with  Dr.  W.  P.  Rowe  and  Dr.  M.  A.  Martin  using  an  ongoing  problem 
on  retroviruses.  Since  many  of  the  problems  in  retrovirus  research  involve 
integrated  sequences,  host  restrictions,  etc.,  and  are  in  many  ways  analogous 
to  the  problems  of  parvovirus  carriage  it  is  felt  this  temporary  collaborative 
arrangement  will  result  in  great  benefit  to  both  projects. 

Publ i cations 


Bachmann,  P. A.,  Hoggan,  M.D.,  Kurstak,  E.,  Melnick,  J.L.,  Pereira,  H.G., 
Tattersall ,  P.  and  Vago,  C:  Parvoviridae:  Second  Report.  Intervirology 
11_:  248-254,  1979. 

Lubeck,  M.D.,  Lee,  H.M.,  Hoggan,  M.D.  and  Johnson,  F.B.:  Adenovirus-associated 
virus  structural  protein  sequence  homology.  Virology,  in  press. 


27-6 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01    AI   00012-17  LVD 


PER  I  00    COVERED 

October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Studies  of  Mouse  Leukemia  Viruses, 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Dr.  Wallace  P.  Rowe,  LVD,  NIAID 

Dr.  Janet  W.  Hartley,  LVD,  NIAID 

Dr.  Christine  Kozak,  LVD,  NIAID 

Dr.  Miles  W.  Cloyd,  LVD,  NIAID  (Untile  August  1979) 

Dr.  Janet  M.  Ramseur,  LVD,  NIAID 

Dr.  Susan  E.  Light,  LVD,  NIAID  (From  July  1979) 


cooperating  units  (if  any)  S.  Chattopadhyay ,  DCT,  NCI;  M.  Martin,  OSD,  NIAID;  H.  Morse, 
T.  Chused,  LMI,  NIAID;  M.  Potter,  LCBGY,  NCI;  L.  Old,  E.  Stockert,  Memorial 
Sloan- Kettering  Inst.;  M.  Collins,  MBA;  P.  Pitha,  S.  Staal ,  Johns  Hopkins  Univ.; 
N.  Hopkins,  MIT;  R.  Weinberg,  D.  Steffen,  MIT   


LAB/BRANCH 

Laboratory  of  Viral  Diseases 


SECTION 


SECTION 

Viral  Oncology  Section 


INSTITUTE  AND  LOCATION 

National  Institute  of  Allergy  and  Infectious  Diseases,  Bethesda,  Maryland 


TOTAL  MANYEARS: 

11.8 


PROFESSIONAL: 

4.8 


7.0 


CHECK  APPROPRIATE  BOX(ES) 
C  (a)   HUMAN  SUBJECTS 

□  (a1  )  MINORS   Q  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


2  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  natural  biology  of  the  complex  group  of  murine  C-type  RNA  viruses  is  being 
studied  using  virological,  genetic,  and  biochemical  approaches.  Continuing 
studies  of  the  genetic  transmission  of  both  ecotropic  and  xenotropic  virus 
classes  have  led  to  the  chromosomal  mapping  of  several  additional  virus-inducing 
loci.  Studies  of  the  recombinant  MCF  viruses  in  relation  to  murine  leukemo- 
genesis  have  delineated  certain  biological  and  biochemical  correlates  of  the 
ability  of  a  given  MCF  isolate  to  induce  or  accelerate  leukemia,  leading  to  the 
recognition  of  target  tissue  host  range  as  the  major  determinant  of  leukemo- 
genicity.  Genetic  control  of  sensitivity  to  MCF  virus  infection  is  under 
investigation  in  vitro  and  in  vivo. 


27-7 


PHS-6040 
(Rev.  10-76) 


Z01  AI  00012-17  LVD 

The  major  goal  of  this  project  is  the  analysis  of  the  natural  biology  of  C- 
type  viruses  of  mice.  In  particular  the  genetic  transmission  of  these  viruses 
is  investigated  by  the  use  of  inbred,  hybrid,  and  congenic  mouse  strains. 
Construction  of  such  strains,  chromosomal  mapping  by  Mendelian  and  somatic 
cell  genetic  techniques,  and  analysis  of  the  interplay  of  genes  that  regulate 
expression  of  the  virus-inducing  loci  is  a  large  program  area.  In  addition, 
recombinant  DNA  techniques  are  now  being  employed  to  obtain  molecular  clones 
of  viral  genomes  and  of  the  endogenous  genetic  loci.  Related  to  this  project 
on  leukemia  viruses  is  the  collaborative  work  on  risk  assessment  of  recombi- 
nant DNA  research  using  tumor  viruses  cloned  in  IE.  coli  as  models  of  possibly 
hazardous  post-vector  systems. 

Chromosomal  mapping  of  endogenous  MuLV  genomes.  We  have  continued  our  analysis 
of  the  chromosomal  loci  in  various  mouse  strains  that  are  responsible  for  in- 
duction of  ecotropic  and  xenotropic  MuLV.  We  have  previously  mapped  the  Akv-1 
locus,  as  well  as  ecotropic  loci  in  BALB/c,  C57BL,  BIO. BR  and  C3H/Fg,  and 
xenotropic  loci  in  4  inbred  strains.  During  the  past  year  we  were  successful 
in  identifying  the  chromosomal  location  of  two  more  ecotropic  virus-inducing 
loci:  Akv-2  (on  chromosome  16  as  determined  by  somatic  cell  hybrid  analysis) 
and  one  of  the  loci  of  C58  (on  chromosome  8  near  but  probably  not  allelic  with 
the  locus  carried  by  C57BL).  In  addition,  we  have  mapped  a  fifth  xenotropic 
locus,  of  C57L,  and  have  found  that  it  is  at  the  same  chromosome  1  site  as  in 
the  previously  mapped  strains. 

A  major  objective  over  the  next  year  will  be  to  map  similar  loci  occurring  in 
a  wild  mouse  population  that  is  probably  the  progenitor  of  many  laboratory 
mouse  strains;  that  is  the  Kyushu  mouse  (Mus  molossinus)  which  carries  an  eco- 
tropic virus  indistinguishable  from  that  carried  by  AKR  with  respect  to  its 
restriction  map.  Another  major  goal  of  the  next  year  will  be  to  characterize 
the  endogenous  loci  by  restriction  enzyme  analysis  using  the  Southern  blot 
procedure.  In  collaborative  studies  with  Drs.  Weinberg  and  Steffen  at  MIT 
we  have  shown  that  the  Akv-1  locus  can  be  recognized  in  restriction  blots  as 
a  distinct  band  in  the  gel  that  segregates  with  the  inducibility  of  virus. 
Once  radiolabeled  probes  corresponding  to  specific  regions  of  the  AKR  viral 
genome  are  available  from  the  molecular  cloning  studies,  it  will  be  possible 
to  characterize  all  of  the  ecotropic  endogenous  virus-inducing  loci  by  the 
same  technique.  The  question  of  allelism  of  various  ecotropic  genomes  can 
then  be  studied  in  precise  detail. 

A  particularly  strong  feature  of  our  program  in  this  regard  is  the  avail- 
ability, through  Dr.  Kozak,  of  segregating  somatic  cell  hybrids  containing 
only  one  or  two  mouse  chromosomes,  thus  making  it  possible  for  us  to  examine 
particular  genomes  biologically  and  biochemically,  separately  from  loci  on 
other  mouse  chromosomes. 

A  number  of  the  chromosomal  genomes  are  being  bred  into  NFS  Swiss  mice  for 
production  of  congenic  mice  carrying  various  ecotropic  and  xenotropic  virus 
loci.  With  the  increasing  success  of  mapping  these  loci  we  have  found  that  a 
number  of  them  are  closely  linked  to  morphologic  markers  such  as  genes  for 
coat  color  or  skeletal  abnormalities;  we  are  now  making  a  number  of  congenic 

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Z01  AI  00012-17  LVD 

lines  carrying  the  virus  loci  in  coupling  with  the  morphological  markers.  This 
greatly  simplifies    analysis  of  segregants.  Also,  breeding  the  morphological 
markers  unlinked  to  virus-inducing  loci  into  the  mouse  strain  that  carries  such 
a  locus  at  the  closely  linked  site  provides  a  highly  efficient  way  of  producing 
mice  congenic  for  the  absence  of  a  virus-inducing  locus. 

An  outgrowth  of  the  multi disciplinary  genetic  studies  has  been  further  con- 
firmation that  the  virus-inducing  loci  consist  of  integrated  viral  genomes. 
We  have  shown  previously  that  the  Akv-1  locus  contains  at  least  a  portion 
of  the  MuLV  genome,  but  it  was  not  possible  to  say  if  it  contained  the  entire 
set  of  sequences.  The  restriction  blots  from  the  Stef f en-Wei nberg  study  in- 
dicate that  the  viral  sequences  at  Akv-1  generate  the  same  internal  fragments 
when  cleaved  with  a  multiple  cut  enzyme  as  does  the  viral  genome  obtained  from 
cells  exogenously  infected  with  AKR  virus.  This  provides  strong  evidence  that 
the  locus  contains  the  viral  genome  in  unpermuted  orientation  and  without  any 
major  intervening  sequences.  Also,  the  somatic  cell  hybrid  studies  of  Dr. 
Kozak  have  shown  that  a  segregating  hybrid  cell  line  containing  only  chromo- 
some 1  derived  from  BALB/c,  which  carries  a  xenotropic  inducing  locus  on  this 
chromosome,  is  inducible  for  xenotropic  virus;  thus  the  genetic  information 
must  be  on  that  chromosome  and  is  presumably  at  the  locus  shown  previously  to 
determine  inducibility. 

Biology  of  MCF  viruses.  The  MCF  (Mink  cell  focus-forming  viruses),  discovered 
in  LVD  several  years  ago,  constitute  a  major  area  of  study  in  spontaneous  viral 
leukemogenesis.  These  viruses  are  genetic  recombinants  between  ecotropic  and 
xenotropic  viruses  that  arise  in  somatic  cells  during  the  preleukemic  period 
in  a  number  of  mouse  strains. 

The  collaborative  studies  with  Dr.  Chattophadhyay  have  now  identified  several 
xenotropic  viruses  that  contain  genome  sequences  that  complement  the  ecotropic 
viral  probes  in  saturating  the  entire  genome  of  AKR  MCF  viruses.  Other  xeno- 
tropic viruses  do  not  contain  this  set  of  sequences.  These  studies  are  of 
importance  in  confirming  that  MCFs  are  indeed  derived  from  xenotropic  viruses 
and  for  indicating  that  there  is  significant  heterogeneity  between  xenotropic 
virus  strains  with  respect  to  this  important  set  of  sequences.  Whether  this 
means  that  some  xenotropic  viruses  cannot  become  involved  in  generation  of  MCF 
viruses  remains  to  be  determined. 

In  the  continuing  studies  of  the  natural  history  of  the  MCF  viruses  it  is  now 
apparent  that  they  can  occur  in  most  mouse  strains,  that  carry  high  levels  of 
ecotropic  virus,  and  that  they  are  found  in  a  high  proportion  of  spontaneous 
hematopoietic  neoplasms  arising  in  mice  that  carry  ecotropic  MuLV.  We  have 
now  established  that  the  MCFs  isolated  from  various  mouse  strains  fall  into  two 
sharp  categories.  The  strains  obtained  from  thymic  tissue  or  neoplasms  or 
thymic  origin  are  a  distinct  family  as  opposed  to  those  derived  from  nonthymic 
neoplasms  or  from  nonthymic  lymphoid  tissue  of  normal  animals.  The  strains 
recovered  from  thymus  are  able  to  accelerate  thymic  lymphomagenesis  in  AKR 
mice,  they  replicate  in  the  AKR  thymus,  they  show  a  relative  restriction  of 
growth  in  NFS  mouse  embryo  cells  as  compared  to  SC-1  cells,  and  as  shown  by 
our  collaborator  Dr.  Hopkins  of  MIT  they  share  several  features  of  their  RNA 

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Z01  AI  00012-17  LVD 

oligonucleotide  fingerprint.  With  one  exception  the  nonthymic  MCF  strains 
show  the  opposite  pattern  with  respect  to  these  phenotypes,  that  is  they  are 
unable  to  replicate  in  AKR  thymus  or  to  accelerate  AKR  leukemia,  they  infect 
NFS  cells  more  efficiently  than  SC-1  cells  and  they  have  a  different  pattern 
of  RNA  fingerprints.  One  exceptional  strain  is  intermediate  with  respect  to 
all  of  these  properties.  These  findings  are  of  particular  interest  in  that 
they  point  out  the  importance  of  tissue  host  range  as  the  major  determinant 
of  leukemogenicity  and  they  allow  us  to  examine  several  regions  of  the  viral 
genomes  as  the  possible  determinant  of  thymotropism. 

Among  the  nonthymotropic  MCF  strains,  several  are  clearly  able  to  infect  of 
splenic  cells;  this  property  is  not  shared  by  the  thymotropic  strains.  Attempts 
will  be  made  to  identify  the  target  cells  of  this  subclass  of  MCF  viruses. 

The  nonthymotropic  MCF  viruses,  although  recovered  in  many  cases  from  hemato- 
poietic neoplasms,  have  been  completely  devoid  of  ability  to  induce  leukemias. 
These  viruses,  having  arisen  by  recombination  between  endogenous  viruses,  may 
not  have  been  selected  for  ability  to  initiate  infection  from  without  in  their 
target  cells.  We  will  attempt  to  induce  leukemias  with  these  strains  by  using 
phenotypic  mixing  as  a  mechanism  for  introducing  these  viruses  into  potential 
target  cells . 

Genetic  studies  of  susceptibility  to  MCF  virus  infection.  Studies  have  been 
carried  out  both  in  vivo  and  in  vitro  on  the  ability  of  cells  of  different 
mouse  strains  to  be  infected  by  MCF  viruses. 

The  studies  in  vivo  have  been  confined  primarily  to  tests  for  leukemogenicity 
by  various  MCF  isolates  in  a  variety  of  mouse  strains.  The  only  strain  that 
was  consistently  sensitive  is  AKR,  in  which  thymotropic  MCF  strains  consistently 
accelerated  thymic  lymphomagenesis.  C3H/Bi ,  the  strain  that  is  maximally  sensi- 
tive to  Gross  passage  A  virus,  was  partially  susceptible  to  MCF-247,  as  were 
NFS  mice  carrying  AKR  virus-inducing  loci  and  showing  the  high  ecotropic  virus 
expression  phenotype.  NFS  mice  without  the  virus-inducing  loci  were  refractory 
to  MCF-247  leukemogenesis  as  were  C57BR  and,  surprisingly,  the  high  virus  strain 
C58.  In  crosses  between  AKR  and  NFS,  susceptibility  to  MCF-247  was  dominant; 
and  in  backcrosses  to  NFS  half  of  the  mice  were  sensitive.  This  50%  ratio  did 
not  signify  a  simple  one  gene  Mendelian  effect,  however,  since  all  of  the  mice 
developing  lymphoma  were  from  the  segregants  inheriting  AKR  ecotropic  virus. 
This  confirms  the  earlier  observation  with  the  NFS  congenics  that  presence  of 
ecotropic  virus  facilitates  MCF  lymphomagenesis.  We  infer  that  several  other 
genes  segregating  in  this  cross  must  be  involved  in  determining  susceptibility 
to  MCF  leukemogenicity.  H2  type  was  not  associated  with  sensitivity  nor  was 
the  ability  to  mount  a  humoral  immune  response  to  the  247  virus. 

In  crosses  between  AKR  and  the  resistant  C58  strain,  resistance  to  lymphoma- 
genesis was  dominant  and  in  backcrosses  to  AKR  again  a  one  gene  Mendelian  ratio 
was  observed.  In  this  case  all  of  the  segregants  carried  ecotropic  virus. 
Preliminary  data  indicate  that  susceptibility  correlates  with  inheritance  of 
the  Fv-l-Gpd-1  region  from  AKR.  Further  preliminary  studies  suggest  that  the 
subline  of  C58  employed  in  these  studies  may  actually  carry  a  unique  resistance 


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Z01   AI   00012-17  LVD 

allele  at  Fv-1 . 

In  the  in  vitro  studies  of  susceptibility  of  tissue  cultures  to  infection  by 
MCF-247  and  other  N-  or  NB-tropic  MCF  virus  strains,  the  unexpected  observa- 
tion was  made  that  DBA/2  and  DBA/1  mice  are  30-  to  100-fold  less  sensitive  to 
the  infection  than  other  Fv-1 n  mice.  This  resistance  does  not  segregate  with 
Gpd-1  and  hence  must  be  due  to  a  gene  other  than  Fv-1 .  A  possible  association 
with  xenotropic  virus  expression  in  these  mice  will  be  investigated. 

Studies  of  tumori genes  is  in  mice  by  restriction  fragments  of  polyoma  viral  DNA. 
In  the  course  of  the  polyoma  risk  assessment  studies  carried  out  in  collabora- 
tion with  the  Recombinant  DNA  Unit,  OSD,  the  striking  observation  was  made  that 
polyoma  DNA  inactivated  by  restriction  enzyme  cleavage  in  the  distal  portion  of 
the  early  region  showed  enhanced  oncogenicity  for  hamsters.  To  further  in- 
vestigate this  phenomenon,  we  have  carried  out  pilot  studies  in  mice  using 
polyoma  DNA  cleaved  with  enzymes  that  prevent  it  from  being  infectious.  This 
was  considered  a  possibly  interesting  area  of  study  since  polyoma  tumorigenesis 
in  mice  is  accompanied  by  high  levels  of  productively  infected  cells,  and  the 
tumors  consequently  produce  infectious  virus.  The  cleaved  DNA  preparations 
should  permit  study  of  non-infectious  mouse  tumors.  To  date,  non-infectious 
DNA  preparations  have  induced  a  small  number  of  tumors  including  one  osteoma, 
one  sarcoma,  and  two  endotheliomas.  The  sarcoma  has  been  established  in  serial 
transplantation  passage  and  may  be  of  much  use  for  analysing  transplantation 
antigens. 

Future  Course  of  the  Project.  The  major  components  of  the  program  will  con- 
tinue along  the  same  lines  as  in  the  past  few  years.  In  addition  much  effort 
will  be  devoted  to  the  collaborative  program  on  recombinant  DNA  technology  as 
applied  to  the  analyses  of  endogenous  C-type  viral  genomes.  It  is  expected 
that  this  area  of  study  will  develop  rapidly  and  move  into  a  wide  variety  of 
areas  involving  the  genetic  nature  of  endogenous  viral  loci,  the  role  of 
recombination  in  generating  viruses  from  them,  the  control  mechanisms  involved 
in  regulating  expression  of  ecotropic  and  xenotropic  endogenous  viral  loci, 
further  insights  into  the  origin  of  MCF  viruses,  and  the  role  of  various 
genetic  regions  of  the  virus  in  determining  thymotropism  and  lymphomagenicity. 

Publications 

Chan,  H.W.,  Israel,  M.A.,  Garon,  C.F.,  Rowe,  W.P.  and  Martin,  M.A.:  Molecular 
cloning  of  polyoma  virus  DNA  in  Escherichia  coli:  Lambda  phage  vector  system. 
Science,  203:  887-892,  1979. 

Chattopadhyay,  S.K.,  Jay,  G.,  Lander,  M.R.,  and  Levine,  A.S.:  Correlation  of 
the  induction  of  transcription  of  the  AKR  mouse  genome  by  5-iododeoxyuridine 
with  the  activation  of  an  endogenous  murine  leukemia  virus.  Cancer  Res.,  39: 
1539-1546,  1979.  ~ 

Cloyd,  M.W.,  Hartley,  J.W.,  and  Rowe,  W.P.:  Cell-surface  antigens  associated 
with  recombinant  mink  cell  focus-inducing  murine  leukemia  viruses.  J.  Exp. 
Med.,  149:  702-712,  1979. 

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Z01   AI   00012-17  LVD 

Ishimoto,  A.,  Hartley,  J.W.,  and  Rowe,  W.P.:  Fv-1  restriction  of  xenotropic 
and  amphotropic  murine  leukemia  virus  genomes  phenotypically  mixed  with  eco- 
tropic  virus.     Virology,   93:   215-225,   1979. 

Ishimoto,  A.,   Hartley,  J.W.,  and  Rowe,  W.P.:     Phenotypic  mixing  between  murine 
leukemia  viruses:     characteristics  of  ecotropic  virus   infection  of  heterologous 
cells.     Virology,   9J_:   464-471,   1978. 

Israel,  M.A.,  Chan,   H.W.,  Hourihan,  S.L.,  Rowe,  W.P.,  and  Martin,  M.A.: 
Biological   activity  of  polyoma  viral   DNA  in  mice  and  hamsters.     J.  Virology, 
29:    990-995,   1979. 

Israel,  M.A.,  Chan,  H.W.,  Rowe,  W.P.,  and  Martin,  M.A.:  Molecular  cloning  of 
polyoma  virus  DNA  in  Escherichia  coli:  Plasmid  vector  system.  Science,  203: 
883-887,   1979. 

Israel,  M.A.,   Simmons,  D.T.,  Rowe,  W.P.,  and  Martin,  M.A.:      Interrupting  the 
early  region  of  polyoma  virus  DNA  enhances  tumorigenicity.     Proc.   Natl.  Acad. 
Sci  . ,  USA,   in  press. 

Kozak,  C.A.  and  Rowe,  W.P.:     Genetic  mapping  of  the  ecotropic  murine  leukemia 
virus-inducing  locus  of  BALB/c  mouse  to  chromosome  5.     Science,  204_:   69-71, 
1979. 

Moll,  B.,  Hartley,  J.W.,  and  Rowe,  W.P.:  Induction  of  B-tropic  and  N-tropic 
murine  leukemia  virus  from  B10.BR/SgLi  mouse  embryo  cell  lines  by  5-iodo-2'- 
deoxyuridine.     J.   Natl.   Cancer   Inst.,   63:   213-217,   1979. 

Morse,   H.C.,  Chused,  T.M.,   Boehm-Trui tt,  M.,  Mathieson,   B.J.,   Sharrow,   S.O., 
and  Hartley,  J.W.:     XenCSA:     Cell   surface  antigens  related  to  the  major  glyco- 
proteins  (gp70)  of  xenotropic  murine  leukemia  viruses.     J.    Immunol.,  122: 
443-454,   1979. 

Morse,   H.C.,   III,  Chused,  T.M.,   Hartley,  J.W.,  Mathieson,   B.J.,   Sharrow,   S.O., 
and  Taylor,   B.A.:     Expression  of  xenotropic  murine  leukemia  viruses  as  cell- 
surface  gp70  in  genetic  crosses   between  strains  DBA/2  and  C57BL/6.     J.    Exp. 
Med.,  ±49:    1183-1196,   1979. 

Morse,  H.C.   Ill,  Chused,  T.M.,  Sharrow,  S.O.,  and  Hartley,  J.W.:     Variations 
in  expression  of  xenotropic  murine  leukemia  virus  genomes   in  lymphoid  tissues 
of  NZB  mice.     J.    Immunol.,   122_:   2345-2348,   1979. 

Risser,  R.,   Potter,  M.,  and  Rowe,  W.P.:     Abelson  virus-induced  lymphomagenesis 
in  mice.     J.   Exp.  Med.,   148:   714-726,   1978. 

Rowe,  W.P.  and  Hartley,  J.W.:  Chromosomal  location  of  C-type  virus  genomes 
in  the  mouse.  In  Morse,  H.  C.  Ill  (Ed.):  Origins  of  Inbred  Mice,  Academic 
Press,   Inc.,  New  York,  N.Y.,   1978,   pp.   289-295. 


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Z01    AI   00012-17  LVD 

Steffen,  D.,   Bird,  S.,   Rowe,  W.P.,  and  Weinberg,   R.A.:      Identification  of  DNA 
fragments  carrying  the  ecotropic  proviruses  of  AKR  mice.     Proc.   Natl.  Acad. 
Sci . ,   USA,   in  press. 


27-13 


LABORATORY  OF  MICROBIAL  STRUCTURE  AND  FUNCTION 

Rocky  Mountain  Laboratories 

Hamilton,  Montana 

1 979  Annual    Report 

Table  of  Contents 


Z01   A I 
Project  Number 

Summary 

00065-06 

00070-14 

00071-09 

00076-21 

00077-23 

00078-07 

00087-02 

00182-01 

00183-01 


Antigens  and  Classification  of  the  Rickettsiae  — 
Anacker,   Philip 

Biological   Activities  of  Substances  in  Bordetella 
pertussis  —  Bergman 

Purification  and  Activities  of  Pertussigen  -  a 
Substance  from  Bordetella  pertussis  --  Munoz 

Mechanisms  of  Immunopotentiation  by  Components 
of  Microbes  —  McLaughlin 

Structure  and  Biological   Activity  of  Endotoxins  — 
Von  Eschen,  Ribi 

Microbial    Components  in  Cancer  Immunotherapy  ~ 
Ribi 

Immunological   Activity  of  Eukaryotic  and  Prokaryotic 
Cellular  Components  —  Cantrell 

Biochemical    and  Genetical   Mechanisms  of  Obligate 
Intracellular  Parasitism  --  Williams   (new  project) 

Structural   and  Functional    Relationships  of  Bacterial 
Antigens  in  the  Immune  Response  —  Williams 
(new  project) 


Page 
28-1 

28-13 

28-19 

28-22 

28-28 

28-32 

28-38 

28-44 

28-49 

28-54 


Annual  Report 

Laboratory  of  Microbial  Structure  and  Function 

Rocky  Mountain  Laboratories 

Hamilton,  Montana 

National  Institute  of  Allergy  and  Infectious  Diseases 

October  1,  1978  to  September  30,  1979 

RESEARCH  HIGHLIGHTS 

The  following  sections  provide  brief  descriptions  of  significant 
findings  during  the  past  year.  Eighteen  publications  have  appeared  in  print, 
seven  have  been  accepted  for  publication,  and  several  others  are  in  prepara- 
tion. 

PERTUSSIS 

One  of  the  most  promising  findings  has  been  the  development  of  specific 
affinity  chromatography  columns  for  the  purification  of  pertussigen.  With 
the  aid  of  these  columns,  highly  purified  preparations  of  pertussigen  have 
been  obtained.  This  method  should  allow  us  to  fully  characterize  this  sub- 
stance in  the  near  future.  Another  important  contribution  was  the  development 
of  methods  to  purify  and  crystallize  the  fimbria!  hemagglutinin  (FHA)  from 
Bordetella  pertussis.  It  was  shown,  contrary  to  what  has  recently  been  pub- 
lished, that  when  FHA  is  totally  free  of  pertussigen,  it  does  not  protect 
mice  from  experimental  pertussis,  while  highly  purified  pertussigen  does. 
(Munoz,  Arai ,  Sone) 

It  has  been  established  that  pertussigen,  agglutinogen  factor  1,  and 
fimbria!  hemagglutinin  are  three  distinct  substances „  (Munoz,  Arai) 

One  of  the  many  biological  properties  of  pertussigen  is  its  ability 
to  increase  antibody  production  to  antigens  given  with  it.  An  unusual  char- 
acteristic of  this  adjuvant  action  is  that  the  IgE  response  is  almost  selec- 
tively increased.  In  our  studies  on  the  mechanism  by  which  pertussigen 
increases  the  IgE  response  to  hens'  egg  albumin,  we  have  found  that:  1) 
pertussigen  acts  differently  than  endotoxin  or  Concanaval in  A,  two  substances 
that  also  can  stimulate  IgE  production;  2)  pertussigen  given  3  days  before 
antigen  still  acts  as  an  adjuvant  for  IgE  production;  3)  pertussigen  seems 
to  act  directly  on  lymphocytes  probably  by  eliminating  suppressor  T  cells; 
4)  anti -pertussigen  serum,  when  given  simultaneously  with  or  3  days  after 
pertussigen,  can  suppress  the  adjuvant  action  of  pertussigen;  and  5)  in  spleen 
cells  from  mice  that  had  received  radioactivity-labeled  preparations  of 
pertussigen  3  days  prior  to  removal  of  the  spleens,  a  peptide  of  molecular 
weight  18,500  to  19,000  was  demonstrated,  indicating  that  pertussigen  may 
remain  in  spleen  cells  for  some  time.  (Sadowski) 

We  have  had  success  in  culturing  the  s-cells  of  the  pancreas  of  neo- 
natal mice.  These  cultures  will  be  used  to  study  the  mechanisms  by  which 
pertussigen  stimulates  production  of  insulin.  (Bergman) 

28-1 


MICROBIAL  COMPONENTS   IN  CANCER  IMMUNOTHERAPY 

The  goals  of  this  project  are  to   fractionate  microorganisms,   isolate 
those  components  that  possess  immunopotentiating  ability,  and  to  provide 
acceptable  agents  to  clinicians  for  testing. 

We  have  reported  previously  that  a  high  incidence  of  regression  of 
line-10  tumors  was  obtained  with  materials  unrelated  to  tubercle  bacilli 
provided  they  were  combined  with  trehalose  dimycolate   (P3  or  purified  cord 
factor  provided  by  Dr.   R.   Parker,  Hamilton,  MT,  under  NIAID  contract).     Par- 
ticularly rapid  destruction  of  tumors  was  observed  when  they  were  treated 
with  endotoxic  glycol i pids   (ReGl)   from  0  antigen  (polysaccharide)-deficient 
Re  mutant  strains  of  Enterobacteri aceae  combined  with  P3  and  up  to  100%  cures 
were  obtained,,     Furthermore,  ReGl    synergistically  enhanced  the  potency  of 
CWS  or  CWS  +  P3  combinations.     However  surprisingly,  endotoxic  lipopoly- 
saccharides   (LPS)   from  all   wild  type  strains  so  far  tested  failed  to  cause 
tumor  regression.     Other  experimental    evidence  accumulated  indicated  that 
no  correlation  existed  between  endotoxic  potency  per  se  and  tumor  regressive 
potency.     Therefore,  from  a  practical    point  of  view  our  interest  centered 
upon  the  importance  of  the  toxicity  of  ReGl.     We  explored  chemical   modification 
techniques  hoping  to  selectively  reduce  the  toxicity  of  endotoxic  bacterial 
extracts  and  yet  retain  their  ability  to  synergistically  enhance  the  action 
of  CWS.     Whereas  highly  endotoxic  LPS  from  wild  type  strains  failed  to  cause 
tumor  regression,  we  now  report  that  acid  hydrolysis  of  such  LPS  led  to  a 
residual    fraction  (RESI)  that  serologically  cross-reacted  with  ReGl    samples 
and  provided  high  cure  rates   (90%)   in  the  line-10  tumor  model.     RESI  from 
Serratia  marcescens  was  essentially  nonpyrogenic  for  rabbits  and  100  times 
less  toxic  for  chick  embryos  than   potent  endotoxins.     Both  RESI  and  ReGl   were 
found  to  contain  peptidic  substances;  among  the  major  amino  acids  were  muramic 
acid,  alanine,  and  glutamic  acid.     The  amino  acid  content  of  ReGl   was  reduced 
by  raicroparticulate  silica  gel   chromatography,  and  this  procedure  provided 
an  endotoxic  fraction,  B4  (prepared  by  Dr.   R.  Parker  under  NIAID  contract), 
with  reduced  tumor-regressive  activity.     We  considered  that  precursor  or  auto- 
lysis products  of  the  peptidoglycan  moiety  of  CWS  may  have  been  co-extracted 
with  the  endotoxic  glycol i pids.     Indeed,  antitumor  activity  could  be  restored 
to  the  endotoxic  B4  by  the  addition  of  synthetic  N-acetyl-muramyl-L-seryl- 
D-isoglutamine  (MDP),  which  is  considered  the  minimal   structural    unit 
responsible  for  the  adjuvant  action  of  the  microbial   cell   wall.     It  is 
suggested  that  MDP  may  act  as  an  adjuvant  to  an  antigen  which  may  have  cross- 
reactive  determinants  associated  with  the  endotoxin  itself.     Such  antigens 
appear  to  be  cryptic  or  sterically  hindered  from  being  effective  in  poly- 
saccharide-rich  LPS  from  wild  type  bacteria  but  are  exposed  in  ReGl    from 
polysaccharide-deficient  mutant  bacteria  and  RESI  which  was  prepared  from 
LPS  by  acid  hydrolysis.     Dr,   Cantrell  ,   in  collaboration  with  Dr.   Springer, 
found  line-10  tumor  cells,  ReGl,  and  B4  to  have  components  that  cross-react 
with  T  antigen,  the  precursor  of  human   blood  group  MN  antigen.     Studies  by 
these  workers  are  in  progress  to  determine  whether  the  cross-reactive  antigens 
shared  by  line-10  tumor  cells  and  microbial    components   play  a  role  in  tumor 
regression. 


28-2 


Microbial  Components  in  Cancer  Immunotherapy  (cont'd) 

Whereas  synthetic  MDP  appears  to  be  a  very  effective  adjuvant  which 
can  replace  CWS,  it  was  surprising  to  find  that,  in  contrast  to  CWS,  MDP 
greatly  enhanced  the  susceptibility  of  guinea  pigs  to  endotoxic  shock.  Results 
of  our  studies  indicated  that  caution  must  be  exercised  in  the  application 
of  MDP  as  a  tumor  therapeutic  ingredient,  or  as  an  adjuvant  in  general,  in 
combination  with  endotoxic  products,  such  as  Pseudomonas  vaccine  (Pseudogen) 
whose  antitumor  activity  is  presently  being  evaluated  by  others  in  human 
patients.  (Ribi,  McLaughlin,  Cantrell ,  Schwartzman) 

MECHANISMS  OF  IMMUNOPOTENTIATION  BY  MICROBIAL  COMPONENTS 

The  primary  aim  of  this  project  is  to  define,  at  the  cellular,  bio- 
chemical ,  and  molecular  levels,  the  mechanisms  involved  in  immunopotentiation 
by  microbial  components.  It  was  found  that  mycobacterial  cell  wall  skeleton 
when  combined  with  endotoxic  extracts  from  Salmonella  rough  mutants  (Re  gly- 
col ipid)  (ReGl)  produced  synergistic,  not  additive,  tumor-regressive  responses. 
N-acetyl-muramyl-L-alanyl-D-isoglutamine  (adjuvant  di peptide)  replaced  cell 
wall  skeleton  in  this  synergy  with  endotoxic  extracts.  However,  only  upon 
treatment  of  adjuvant  dipeptide  with  P3  was  the  adjuvant  dipeptide  combined 
with  ReGl  active  against  both  the  dermal  and  metastatic  tumors.  Without  P3, 
regression  occurred  in  dermal  tumors  only,  with  the  treated  animals  succumbing 
to  metastatic  tumor  growth.  A  covalently  lipidated  adjuvant  dipeptide  (provided 
by  Drs.  I„  Azuma  and  Y.  Yamamura)  when  combined  with  endotoxic  extracts  was 
effective  against  both  dermal  and  metastatic  tumors.  These  studies  identified 
critical  structural  components  of  the  mycobacterial  cell  wall  responsible 
for  the  synergistic  antitumor  activity  observed  upon  combination  of  cell  wall 
skeleton  with  ReGl.  It  is  thought  that  the  heightened  efficacy  of  either 
covalently  or  noncovalently  lipidated  adjuvant  dipeptide  reflects  a  critical 
mechanism  of  either  a  prolonged  depot  effect  at  the  site  of  injection  or 
a  preferential  processing  of  the  lipidated  adjuvant  dipeptide  by  immune  cells. 
(McLaughlin,  Ribi) 

As  an  extension  of  these  findings,  a  collaborative  study  with  Dr.  G. 
Jones,  Syntex  Corporation,  was  initiated  to  investigate  the  use  of  various 
synthetic  analogs  of  adjuvant  dipeptide  in  treatment  of  tumors.  Certain, 
extremely  potent,  antitumor  preparations  were  discovered;  all  active  ones 
required  combination  with  P3  in  an  oil-in-water  emulsion  for  expression  of 
antitumor  activity.  (McLaughlin)  In  collaboration  with  Dr.  R0  Toubiana, 
France,  synthetic  analogs  of  trehalose  dimycolate,  such  as  trehalose 
desoxypalmitopalmitate,  were  found  to  act  in  concert  either  with  endotoxic 
extracts  of  synthetic  muramyl  dipeptides  to  produce  significant  incidences 
of  tumor  regression.  Thus,  we  have  successfully  simulated  some  of  the  biological 
activity  of  mycobacterial  components  through  the  use  of  synthetic  substitutes. 
The  use  of  radiolabeled  muramyl  dipeptide  will  be  extremely  important  in 
mechanistic  studies  planned.  Some  success  in  synthesis  of  radiolabeled 
material  for  such  studies  has  been  made,  and  additional  work  is  in  progress. 
CSchwartzman)  These  synthetic  substances  will  greatly  simplify  the  task  of 
visualizing  the  biochemical  steps  leading  to  the  phenomenon  now  called  adjuvant 
activity  or  tumor-regressive  activity. 


28-3 


IMMUNOLOGICAL  ACTIVITY  OF  EUKARYOTIC  AND  PROKARYOTIC  CELLULAR  COMPONENTS 

The  administration  of  Corynebacterium  parvum  has  been  shown  to  have 
diverse  effects  on  the  immune  system.  Although  £.  parvum  has  been  shown  to 
act  as  an  immunoadjuvant  increasing  cell-mediated  and  humoral  immunity,  it 
has  also  been  demonstrated  to  suppress  cellular  immune  functions. 

Through  collaborative  efforts  with  investigators  from  several  institutes, 
we  have  gained  valuable  information  on  the  immunological  activity  of  C.  parvum. 

Several  observations  during  the  past  year  have  strongly  suggested  that 
the  component(s)  of  £.  parvum  responsible  for  antitumor  activity  can  be  isolated 
and  differentiated  from  the  component  that  induces  suppressor  cell  activation 
and  inhibition  of  humoral  and  cellular  immunity.  Since  our  supply  of  strain- 
2  guinea  pigs  was  reduced  the  past  year,  the  biological  and  antitumor  properties 
of  C_.  parvum  fractions  were  evaluated  in  mouse-tumor  models.  Fractions  pre- 
pared by  phenol-water  extraction  of  whole  cells  were  tested  for  lymphoreticular 
stimulation  by  measuring  the  degree  of  splenomegaly  and  blastogenesis.  Using 
three  syngeneical  ly  transplanted  murine  tumors,  antitumor  activity  was  evalu- 
ated by  monitoring  the  rate  of  tumor  growth  in  animals  given  the  fractions 
admixed  with  tumor  cells.  Antitumor  activity  and  lymphoreticular  stimulation 
were  associated  only  with  the  phenol-insoluble  residue.  The  kinetics  of  these 
paralleled  that  observed  with  intact  whole  organisms.  Neither  the  aqueous- 
soluble  nor  the  phenol -soluble  extracts  had  activity  with  respect  to  these 
criteria.  Further  inquiries  were  made  into  the  nature  of  the  active  component 
in  the  residue  fraction.  Following  solvent,  protease,  or  nuclease  treatments 
of  the  residue,  high  antitumor  and  splenomegaly-inducing  properties  were 
retained.  However,  after  the  carbohydrate  moieties  of  the  active  residue 
were  inactivated,  these  properties  were  significantly  reduced.  These  results 
suggest  that  the  active  component  is,  in  part,  carbohydrate  in  nature  and 
not  free  lipids,  proteins,  or  nucleic  acids.  Conceivably,  this  substance 
is  associated  with  cell  walls  since  purified  cell  walls  were  active,  but  the 
degree  of  activity  was  less  than  that  observed  with  whole  cells  or  the  residue. 

We  also  investigated  the  immunosuppressive  properties  of  the  C_,  parvum 
fractions  obtained  by  phenol -water  extraction.  Significant  inhibition  of 
primary  and  memory  cytotoxicity  against  alloantigens  was  observed  in  animals 
given  the  residue  fraction.  In  addition,  this  fraction  stimulated  the  gen- 
eration of  suppressor  cell  activity  capable  of  inhibiting  the  expression  of 
memory  cytotoxicity.  On  the  other  hand,  the  protein-rich  phenol -soluble 
fraction  inhibited  humoral  immunity  as  well  as  primary  and  memory  cytotoxicity, 
but  did  not  enhance  suppressor  cell  activity.  The  aqueous-soluble  fraction 
was  inactive  as  measured  by  these  parameters.  Collectively,  these  findings 
clearly  demonstrate  that  materials  isolated  from  £,  parvum  differentially 
effect  various  aspects  of  the  immune  response. 

Attempts  were  also  made  this  year  to  isolate,  in  soluble  form,  the 
antitumor  component  of  C_.  parvum.  We  found  that  the  pyridine-soluble  extract, 
when  combined  with  adjuvant  active  muramyl  di peptide  (MDP)  and  trehalose 
dimycolate  (P3),  completely  regressed  line-10  tumors  in  syngeneic  strain-2 
guinea  pigs.  The  rate  of  regression  was  superior  to  that  observed  with  either 
whole  cells  or  phenol-insoluble  residue.  In  addition,  the  pyridine-soluble 

28-4 


Immunological  Activity  of  Eukaryotic  and  Prokaryotic  Cellular  Components  (.cont'd) 

extract  did  not  induce  splenomegaly  or  hepatomegaly,  whereas  the  pyridine- 
insoluble  residue  significantly  increased  these  parameters.  This  is  the 
first  report  of  a  soluble  component  isolated  from  C_.  parvum  having  increased 
antitumor  activity  when  compared  to  whole  organisms.  Studies  also  demon- 
strated that  inhibition  of  primary  immunity  was  associated  with  the  pyridine- 
insoluble  residue  and  not  with  the  extract  having  antitumor  activity.  Further 
testing  is  needed  to  determine  whether  these  fractions  stimulate  suppressor 
cell  activation.  In  addition,  we  plan  to  fractionate  the  pyridine-soluble 
extract  with  hopes  to  isolate  a  more  homogeneous  agent. 

In  further  studies  on  the  efficacy  of  specific  immunostimulants  in 
tumor  regression,  attempts  were  made  to  isolate  tumor-associated  antigens 
from  both  murine  tumors  and  guinea  pig  ascites  fluid,  Solubilized  tumor 
antigens  prepared  by  KC1  extraction  of  murine  tumor  cells  induced  high  cure 
rates  in  animals  given  combination  chemoimmunotherapy.  Successful  therapy 
was  dependent  on  emulsifying  the  antigen  in  minute  oil  droplets.  In  addition, 
animals  cured  by  specific  immunotherapy  had  detectable  tumor  specific  immunity 
as  measured  by  antibody  cytotoxicity  and  reaction  of  a  challenge  of  tumor 
cells.  Isolated  tumor  antigens  from  the  ascites  fluid  of  tumor-bearing 
guinea  pigs,  when  combined  with  MDP  and  P3  and  emulsified  in  oil  droplets, 
were  also  efficacious  in  regressing  tumors.  (Cantrell) 

STRUCTURE  AND  BIOLOGICAL  ACTIVITY  OF  ENDOTOXINS 

Several  novel  approaches  were  used  to  describe  further  the  intricate 
relationship  between  the  structure  and  function  of  bacterial  endotoxin. 
Also,  special  efforts  were  made  towards  understanding  the  capacity  of  endotoxic 
materials  to  interact  with  and  stimulate  defined  populations  of  lymphoid 
cells. 

One  major  advancement  was  the  extraction  of  endotoxic  materials  from 
cell  walls  rather  than  from  whole  cells  of  Re  mutant  gram-negative  bacteria. 
These  cell  walls  were  treated  with  EDTA  prior  to  the  extraction  of  endotoxin 
and  the  resultant  endotoxic  material  was  soluble  in  both  aqueous  and  organic 
solvents.  Following  fractionation  by   chromatography  on  microparticulate 
silica  gel  columns,  some  characteristics  of  the  starting  material  (i.e., 
pyrogenicity  for  rabbits,  mitogenicity  and  polyclonal  B  cell  activation  for 
endotoxin  responder  mice,  and  antigenicity)  were  retained  by  the  purification 
products  whereas  other  activities  (i.e.,  toxicity  for  chicks  and  lymphocyte 
stimulation  for  endotoxin  nonresponder  mice)  were  diminished  or  lost0  These 
results  suggested  that  the  starting  endotoxic  glycol i pid  may  have  contained 
at  least  two  components  with  different  biological  activity. 

Other  studies  analyzed  the  chemistry  and  biological  activity  of  endo- 
toxins extracted  from  pathogenic  or  nonpathogenic  strains  of  Yersinia 
enterocolitica.  Potent  endotoxins  were  isolated  and  characterized  from  both 
strains  of  this  organism  which  has  been  gaining  importance  in  certain  enteric 
and  systemic  infections. 


28-5 


Structure  and  Biological  Activity  of  Endotoxins  (cont'd) 

To  assess  the  effects  of  endotoxin  on  the  in  vivo  induction  and  mod- 
ulation of  thymus  derived  lymphocytes  (T  cells),  an  antigen-specific 
proliferation  assay  system  was  used.  Important  findings  using  this  system 
included:  1)  the  existence  of  a  population  of  T  cells  which  specifically 
recognized  and  reacted  to  antigens  associated  with  endotoxin,  2)  that  endotoxin 
was  a  potent  adjuvant  in  the  in  vivo  generation  of  T  cells  specific  for  protein 
antigens,  and  3)  endotoxin-reactive  T  cells  and  the  adjuvant  effects  of  endo- 
toxin were  absent  in  genetic  nonresponder  mice.  These  important  studies 
were  relevant  to  the  following  points:  1)  The  discovery  of  endotoxin-specific 
T  cells  provided  the  potential  that  such  T  cells  participate  in  immune 
responses  to  endotoxin.  Therefore,  we  would  like  to  recommend  a  re-evaluation 
of  the  paradigmatic  idea  that  endotoxin  is  purely  a  "T  cell  independent" 
antigen.  2)  The  capacity  of  endotoxin  to  potentiate  T  cell  responses  to 
other  antigens  may  provide,  in  part,  an  explanation  for  the  immunotherapeutic 
activities  of  endotoxin  described  in  this  and  accompanying  progress  reports. 

Finally,  bacterial  endotoxins  were  tested  for  the  capacity  to  inhibit 
tumor  growth  in  endotoxin  responder  and  nonresponder  strains  of  mice. 
Admixture  of  endotoxin  with  viable  tumor  cells  dramatically  reduced  the 
incidence  of  tumors  in  responder  mice  and  had  little  or  no  effect  in  non- 
responder mice.  Results  of  other  experiments  suggested  that  the  antitumor 
effectiveness  of  endotoxin,  in  genetic  responder  mice,  depended  upon  elicita- 
tion  of  a  host-mediated  response,  possibly  involving  T  cells.  (Ribi,  Von 
Eschen,  McLaughlin) 

ANTIGENS  AND  CLASSIFICATION  OF  RICKETTSIAE 

The  latest  results  from  a  study  of  the  variation  in  properties  of 
eight  strains  of  spotted  fever  group  rickettsiae  reinforce  an  earlier  pre- 
liminary conclusion  that  there  is  considerable  heterogeneity  in  properties 
of  these  rickettsiae  in  nature.  These  strains  varied  in  their  ability  to 
cause  fever  in  guinea  pigs,  the  type  of  plaque  produced  in  Vero  cell  mono- 
layers, and  in  their  reaction  patterns  with  rabbit  antibodies  raised  against 
the  rickettsiae.  One  cluster  of  similar  properties  was  observed:  the 
virulent  rickettsiae  produced  large  clear  plaques  and  belonged  to  the  same 
serotype.  (Anacker) 

Investigations  of  two  types  of  serologic  tests  for  diagnosis  of  Rocky 
Mountain  spotted  fever  indicated  some  promise  of  usefulness  for  these  pro- 
cedures. One  test,  a  hemagglutination  test  employing  human  group  "0" 
erythrocytes  sensitized  with  an  alkali  digest  of  gradient-purified  Rickettsia 
rickettsii ,  detected  spotted  fever  antibody  in  patients  as  early  as  3  days 
and  as  late  as  3  years  after  onset  of  symptoms.  The  second  test  (directed 
by  Dr»  Hechemy  of  the  New  York  Department  of  Health),  the  latex  agglutination 
test,  thus  far  has  successfully  demonstrated  spotted  fever  antibodies  in 
most  patients  tested.  The  latter  test  is  extremely  rapid,  simple,  and 
economical.  (Anacker,  Philip) 


28-6 


BIOCHEMICAL  AND  GENETICAL  MECHANISMS  OF  OBLIGATE  INTRACELLULAR  PARASITISM 

The  primary  objective  of  this  project  is  the  elucidation  of  the  meta- 
bolic cooperation  between  parasite  and  host.  Specifically,  we  are  investigating 
the  biochemical  parameters  involved  in  rickettsial  parasitism  of  eukaryotes 
with  the  ultimate  aim  of  determining  the  growth  factors.  The  underlying 
genetic  determinants  involved  in  the  expression  of  bacterial  functions  are 
being  investigated  by  classical  methods.  Biochemical  parameters  mediated 
by  plasmids,  episomes  and  bacteriophage  are  an  active  part  of  this  program. 
We  have  identified  molecular  interactions  at  the  cell  surface  which  can  be 
considered  as  primary  determinants  of  metabolic  cooperation  between  host 
and  parasite.  Obligatory  features  of  rickettsial  functions  have  been  discovered 
in  purine  nucleotide  metabolism.  Rickettsia  typhi  utilize  host  purine  nucleo- 
tides directly  from  host  cytoplasm  via  a  mechanism  which  requires  the  oxidation 
of  glutamate.  These  experiments  have  shown  that  unusual  membrane  functions 
contribute  to  the  success  of  the  parasite  as  a  scavenger  of  critical  host 
components.  R.   typhi  conserves  the  purine  nucleotide  pool  as  adenosine 
5' -monophosphate  (AMP),  whereas  autonomously  growing  bacteria  degrade  AMP 
to  smaller  compounds. 

Metabolic  comparisons  between  epicellular  (autonomously  growing 
bacteria)  and  rickettsiae  have  revealed  exploitable  differences  in  antibiotic 
sensitivities.  The  cell  wall  directed  antibiotic,  fosfomycin,  which  enters 
cells  via  a  specialized  transport  process  inhibits  the  growth  of  JR.  rickettsii 
but  not  Coxiella  burnetii  or  R.   typhi.  More  importantly,  this  antibiotic 
restricts  the  growth  of  Rochal  imaea  quintana  and  Legionella  pneumophila  at 
an  ED5Q  of  5  and  12  yg  per  ml,  respectively.  The  concentration  of  fosfomycin 
in  the  blood  of  patients  has  been  reported  to  be  as  high  as  33  ug/ml  which 
is  clearly  adequate  for  the  inhibition  of  these  to  epicellular  bacteria. 
We  are  currently  investigating  the  feasibility  of  this  antibiotic  as  an 
effective  chemotherapeutic  for  L_.  pneumophila  infections  in  guinea  pigs. 
(Williams,  McCaul ,  Peacock) 

STRUCTURAL  AND  FUNCTIONAL  RELATIONSHIPS  OF  BACTERIAL  ANTIGENS  IN  THE  IMMUNE 
RESPONSE 

The  objectives  of  this  project  are  to  characterize  the  antigens  of 
the  rickettsiae  and  other  bacteria  which  are  pathogenic  for  humans.  We  have 
been  analyzing  the  structure  and  functional  aspects  of  the  immunomodulation 
by  the  cell  wall  and  soluble  components.  Potential  candidates  for  subunit 
vaccines  and  tumor-regressive  components  have  been  identified.  The  present 
whole  cell  vaccine  for  C.   burnetii  induces  severe  local  and  systemic 
reactions.  Our  objective  was  the  removal  of  the  highly  toxic  factor(s)  from 
whole  cells  via  organic  solvent  extraction  methods.  We  have  discovered  a 
particulate  component  which  is  nontoxic,  provides  protection  against  lethal 
challenge  in  mice  and  guinea  pigs  and  regresses  the  line-10  tumors  in  strain- 
2  guinea  pigs.  This  particulate  material  can  be  derived  from  whole  cells 
or  cell  walls  of  £.  burnetii.  Future  studies  on  the  fractionation  of  cell 
walls  should  yield  information  on  the  nature  of  the  toxic  factorCs)  and 


28-7 


Structural  and  Functional  Relationships  of  Bacterial  Antigens  in  the  Immune 
Response  (cont'd) 

eventually  the  preparation  of  a  nontoxic  subunit  vaccine  against  Q  fever. 
Studies  are  currently  being  conducted  to  determine  both  the  type  and  intensity 
of  the  immune  response  to  antigen.  (Williams,  Ribi,  Cantrell ,  Peacock,  McCaul) 

Soluble  components  derived  from  mechanically  disrupted  C.   burnetii 
have  been  identified  as  putative  early  diagnostic  antigens.  At  least  three 
soluble  antigens  have  been  identified.  Antibodies  to  one  of  these  is  expressed 
only  during  infections,  whereas  the  other  two  are  expressed  during  immunization. 
Attempts  are  being  made  to  purify  the  antigens  and  to  characterize  their 
immunostimulatory  mechanisms.  We  should  be  able  to  prepare  a  soluble  component 
that  will  be  useful  as  a  vaccine  and  early  detection  as  well  as  distinguishing 
between  recent  infections  and  chronically  infected  individuals.  (Williams, 
Kindmark,  Peacock) 


28-8 


Annual  Report 

Laboratory  of  Microbial  Structure  and  Function 

Rocky  Mountain  Laboratories 

Hamilton,  Montana 

National  Institute  of  Allergy  and  Infectious  Diseases 

October  1,  1978  to  September  30,  1979 

ADMINISTRATIVE  REPORT 

The  Laboratory  of  Microbial  Structure  and  Function  (LMSF)  was 
established  during  the  reporting  year.  It  is  comprised  of  three  sections: 
Pertussis  Section,  Molecular  Biology  Section,  and  Rickettsial  Diseases 
Section,,  LMSF  utilizes  sophisticated  biophysical,  biochemical,  analytical 
and  synthetic  chemical  techniques  to  isolate  and  identify  components  of 
bacteria  and  rickettsiae  or  synthetic  analogues  thereof  which  then  are 
studied  in  biological  test  systems  for  their  immunogenicity  and  toxicity 
or  other  hazardous  properties. 

Dr.  Steven  M.  Schwartzman  was  appointed  Senior  Staff  Fellow, 
Molecular  Biology  Section,  to  function  in  his  synthetic  organic  chemistry 
specialty.  Dr„  Thomas  F.  McCaul ,  Visiting  Fellow,  was  reassigned  to  assist 
Dr.  Jimmy  C.  Williams,  Rickettsial  Diseases  Section,  in  electron  microscopic 
studies.  Dr.  Claes  Kindmark,  Sweden,  spent  six  months  as  a  guest  worker 
collaborating  with  Dr.  Williams  in  the  study  of  soluble  rickettsial  antigens. 
During  a  one-month  visit,  Dr.  Emilio  Weiss,  Naval  Medical  Research  Institute, 
Bethesda,  collaborated  with  Dr..  Robert  N.  Philip  and  Mr.  Marius  Peacock 
(Epidemiology  Branch)  and  with  Dr.  Williams  in  studies  dealing  with  biochemical 
and  physiological  aspects  of  legionaires  disease.  Under  Services  Contracts, 
Dr.  Robert  W„  Wheat,  Duke  University,  spent  three  months  collaborating 
with  Drs.  John  L.  Cantrell  and  Edgar  Ribi  (Molecular  Biology  Section)  in 
the  fractionation  of  Corynebacterium  parvum  cells  and  bacterial  endotoxins, 
and  Dr.  Raoul  Toubiana,  Paris,  worked  with  Dr.  Charles  A.  McLaughlin  (Molecular 
Biology  Section)  for  three  months  on  synthesis  of  mycolic  acid  ester  analogues. 
Dr.  Werner  Brehmer,  Robert  Koch  Institute,  Berlin,  spent  six  weeks  as  a 
guest  worker  collaborating  with  members  of  the  Molecular  Biology  Section 
in  studies  on  tumor  regression  with  microbial  components.  Dr.  Gary  Calandra, 
LSD,  NIAID,  spent  a  month  in  LMSF  to  make  use  of  tools  and  techniques  available 
here  for  the  isolation  and  structural  identification  of  peptides.  Dr.  Jin- 
ichi  Sasaki,  Visiting  Associate,  completed  electron  microscopic  studies 
on  a  large  portion  of  his  project  and  returned  to  Japan.  He  will  continue 
to  collaborate  with  members  of  the  Molecular  Biology  Section  from  his 
laboratory  at  Hirosaki  University. 


28-9 


28-10 


Annual    Report 

Laboratory  of  Microbial   Structure  and  Function 

Rocky  Mountain  Laboratories 

Hamilton,  Montana 

National    Institute  of  Allergy  and   Infectious   Diseases 

October  1,  1978  to  September  30,  1979 

HONORS  AND  AWARDS 

The  following  activities  reflect  the  recognition  afforded  staff  of 
the  laboratory  by  their  peers  in  the  scientific  community. 

Awards : 


Dr.  J.   J.  Munoz  received  the  NIH  Director's  Award  for  exemplary 
research  on  the  immunobiology  of  components  of  Bordetella  pertussis  and  his 
contributions  toward  the  development  of  an  improved  vaccine  against  whooping 
cough. 

Editorial   Boards  of  Journals: 

Dr.   E.   Ribi   -  Infection  and  Immunity 

Drs.   J.   L.  Cantrell,   C.  A.  McLaughlin,  J.   J.  Munoz,  E.   Ribi  and  J,   C. 
Williams  reviewed  manuscripts  submitted  to  them  by  Cancer   Immunology  and 
Immunotherapy,   Journal   of  Immunology,   Infection  and   Immunity,  Journal   of 
Infectious  Diseases,   Journal   of  the  National    Cancer  Institute,  Molecular 
Immunology,  Ad  hoc  reviewer  NIAID's  committee,  NID,  on  "RMSF  in  an  Animal 
Model"  and  CRC  Press,   Inc.   "Pyrimidine  Metabolism  in  Bacteria." 

Professional    Posts: 

Dr„   C.  McLaughlin  -  Elected  member,  American  Association  for  Cancer  Research. 

Dr.   J„  Munoz  -  Trustee  of  Stella  Duncan  Memorial    Fund  for  Allergy  Research, 
University  of  Montana,  Missoula;  staff  affiliate,  Microbiology 
Department,   University  of  Montana,  Missoula;  and  chairman-elect  of 
Immunology  Section  of  American  Society  for  Microbiology. 

Dr.  E.  Ribi  -  Visiting  Professor,  University  of  Texas  Cancer  System,  Texas 
Medical  Center,  Houston,  and  staff  affiliate,  Microbiology  Department, 
University  of  Montana,  Missoula. 

Invited  Lectures  and  Participation  in  Meetings  and  Symposia: 

Drs.   R„   K.  Bergman,  C.  A0  McLaughlin,   J.   J.  Munoz,  and  E.  Ribi 
participated  in  internationally  sponsored  meetings  and  symposia. 


28-11 


Honors  and  Awards  (cont'd! 


As  in  previous  years,  many  staff  were  invited  to  present  lectures  at 
universities,  most  of  which  were  done  in  connection  with  travel  to  annual 
meetings  of  national  organizations.  Three  staff  members  presented  invited 
lectures  at  five  different  colleges  and  universities. 


28-12 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl      AI     00065-06   LMSF-EB 


PERIOD    COVERED 


October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Antigens  and  Classification  of  the  Rickettsiae 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:    R.  L.  Anacker 

R.  N.  Philip 

OTHER:  W.  Burgdorfer 

E.  A.  Casper 

T.  F.  McCaul 

L.  A.  Thomas 


Res.  Microbiologist  LMSF  NIAID 

Medical  Director  EB  NIAID 

Res.  Entomologist  (Med.)  EB  NIAID 

Nurse  Director  EB  NIAID 

NIH  Visiting  Fellow  LMSF  NIAID 

Res.  Microbiologist  EB  NIAID 


COOPERATING  UNITS  (if  any) 

Dr.   Robert  Lane,   California  Department  of  Health,  Berkeley,   CA 


lab/branch  Laboratory  of  Microbial   Structure  and  Function  and  Epidemiology  Branch. 
Hamilton,  MT     59840 


SECTION 

Rickettsial  Diseases  Section  and  Epidemiology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  MD 


20205 


TOTAL  MANYEARS: 

5.6 


PROFESSIONAL: 

2.7 


2.9 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 


□  (b)  HUMAN  TISSUES 


(c)  NEITHER 


□  (a1 )  MINORS 


(a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  long-range  objectives  of  this  project  are  to  develop  practicable  procedures 
for  classification  of  spotted  fever-  and  typhus-group  rickettsiae  and  to 
determine  the  nature  and  biological    properties  of  rickettsial   antigens  and 
constituents.     Current  specific  interests  are:     1)   serotyping  rickettsiae 
by  microimmunofluorescence,  2)   relationship  of  rickettsiae  by  means  of  toxin 
neutralization  and  polyacryl amide  gel   electrophoresis  assays,  and  3)   nature 
and  properties  of  rickettsial   antigens  determined  by  other  specialized  tech- 
niques . 


28-13 


PHS-6040 
(Rev.    10-76) 


Project  No.   Z01    AI  00065-06   LMSF-EB 


Project  Description: 


A  variety  of  serological   and  physicochemical    approaches  is  being 
utilized  to  classify  rickettsiae  within  the  spotted  fever  (SFG)   and  typhus 
groups  and  to  isolate  and  characterize  the  antigens  of  these  rickettsiae. 
Included  among  these  procedures  are  microimmunofluorescence  (micro-IF), 
polyacryl amide  gel   electrophoresis,  and  crossed  Immunoelectrophoresis   (CIE). 

(Anacker)  With  the  completion  of  four  sets  of  experiments,  one  phase 
of  our  study  of  the  variations  in  properties  of  eight  strains  of  SFG 
rickettsiae,  isolated  from  several    species  of  ticks,  animals,  or  man,  has 
been  terminated.     By  ordinary  light  microscopy  little  morphologic  difference 
was  found  among  the  various  rickettsiae  in  stained  smears  of  infected  yolk 
sacs.     Diameters  consistently  fell   within  the  range  of  0.3  to  0.4  urn  and 
lengths  averaged  0.7  to  1.1   urn. 

On  the  basis  of  the  other  three  tests,  however,  these  rickettsiae 
were  more  easily  differentiated,,     The  rickettsiae  were  divided  into  three 
virulence  groups  according  to  the  fever  responses  of  guinea  pigs  inoculated 
intraperitoneal ly  with  1000  plaque-forming  units  of  rickettsiae.     The  strains 
of  highest  virulence  induced  temperatures  greater  than  39.6°C  for  6  to  8 
days.     Strains  of  intermediate  virulence  caused  only  a  few  days  of  fever, 
whereas  guinea  pigs  inoculated  with  strains  in  the  third  category  were 
afebrile.     Differences  in  areas  under  the  fever  curves  for  strains  in  the 
different  virulence  groups  were  significant  at  the  0„05  level. 

Plaques  produced  by  these  selected  strains  on  Vero  cell   monolayers 
served  to  distinguish  most  strains.     Some  strains  produced  large  clear 
plaques,  some  large  turbid  or  target-like  plaques,  and  one  strain  a  small 
turbid  plaque.     The  ability  of  the  rickettsiae  to   produce  large  clear  plaques 
was  correlated  with  virulence  of  these  strains   for  guinea  pigs. 

With  the  aid  of  mi croaggluti nation  tests,  employing  antiserum  from 
rabbits  inoculated  with  several    small   doses  of  formalin-killed  rickettsiae 
purified  by  sucrose  density  centrifugation  in  a  zonal    rotor,  these  strains 
were  classified  into  five  distinct  serotypes.     The  four  most  virulent  strains 
fell   within  one  serotype;  the  other  four,  essentially  avirulent  strains  were 
all    serologically  different. 

The  above  tests,  as  well    as  those  mentioned   in  the  1977-78  report, 
indicate  considerable  diversity  in  properties  of  SFG  rickettsiae   isolated 
in  the  U.S.     It  is  unclear  whether  these  differences  are  great  enough  to 
warrant  elevation  of  some  of  these  strains  to  species  rank  or  whether  these 
differences  represent  normal   deviations  from  the  standards  established  for 
classical    strains  of  Rickettsia  rickettsii.     Perhaps  results  from  our  recently 
resumed  study  of  the  antigens  of  these  rickettsiae  will    help  to  clarify  this 
problem. 


28-14 


Project  No.    Z01   AI  00065-06  LMSF-EB 

Preliminary  results  have  been  obtained  from  a  comparison  of  extracts 
of  several   of  the  presumed  more  distantly  related  strains  by  CIE.     It  appears 
most  antigens  demonstrated  by  this  procedure,  particularly  those  which  produce 
the  densest  precipitates,  are  shared  by  these  strains.     Probably,  these 
common  antigens  are  the  "group"  antigens  defined  by  their  ability  to  react 
with  complement-fixing  antibody  in  sera  against  all   SFG  agents.     Type-specific 
antigens  have  not  yet  been  unequivocally  demonstrated  for  these  strains. 

(Philip)   Eight  isolates  by  cell   culture  obtained  during  the  1977 
Bitterroot  Valley  survey  of  Dermacentor  andersoni  ticks   (see  1977-78  report), 
two  representing  each  of  the  four  serologic  types  identified  by  micro-IF 
including  R.  rickettsii ,  JR.  montana,  JR.  rhipicephal  i ,  and  the  369-C  serotype 
were  examined  for  other  distinguishing  biologic  characteristics  including 
pathogenicity  for  chick  embryos,  guinea  pigs,  and  cell   culture;   stability 
during  serial    passage  in  Vero  cells;  antibiotic  sensitivity;  cross- 
immunogenicity  in  guinea  pigs;  and  growth  dynamics  in  cell   culture.     Strains 
within  serotypes  were  similar  to  each  other,  but  the  serotypes  could  be 
readily  distinguished  one  from  another  on  basis  of  several    biologic  markers 
in  addition  to  micro-IF  reactivity.     Some  observations  follow. 

Pathogenicity  for  chick  embryos .     Strains  of  R.  rickettsii   gave 
characteristic  death  patterns   (4  to  5  days)  after  the  first  passage  in 
embryonated  eggs«     R.  montana  strains  were  fully  adapted  after  the  third 
passage  in  eggs,  kiTling  embryos  regularly  on  the  fourth  to  fifth  days. 
R.  rhipicephal i  strains  could  be  maintained  by  serial    passage  in  embryonated 
eggs  but  killed  irregularly  even  after  the  eighth  passage.     The  369-C  strains 
could  be  maintained  in  embryonated  eggs  but  were  not  lethal    for  chick  embryos. 

Pathogenicity  for  guinea  pigs.     JR.  rickettsii   strains  gave  character- 
istic febrile  responses  beginning  on  the  fourth  day  and  accompanied  by 
typical    scrotal    reactions  and  occasional   deaths.     JR.  montana  and  JR. 
rhipicephal i   rarely  gave  rise  to  fever  or  scrotal    reaction.     369-C  strains 
were  completely  nonpathogenic  for  guinea  pigs.     The  numbers  of  plaque-forming 
units  per  guinea  pig  infectious  dose  (determined  by  serologic  response)  were 
approximately  102  for  cell   culture-grown  JR.  rickettsii   strains  and  105   for 
JRo  montana  and  JR.  rhipicephali  organisms.     The  serotypes  could  be  distinguished 
by  the  pattern  of  micro-IF  antibody  response. 

Cytopathogenicity  in  cell   cu1ture0     All    serotypes  were  cytopathic 
for  chick  embryo  cells.     In  Vero  cells  the  serotypes  could  be  distinguished 
on  basis  of  plaque  morphology,  time  of  appearance  of  plaques  and  microscopic 
appearance  of  the  cytopathic  effect. 

Stability  after  serial    passage  in  cell   culture.     Each  strain  was 
examined  by  micro-IF  for  serologic  change  after  50  serial    passages  in  Vero 
cells,,     All    but  one  of  the  filial   strains  were  indistinguishable  from  the 
parental   strains.     The  exception  was  an  instance  where  JR.  rickettsii 


28-15 


Project  No.  Z01  AI  00065-06  LMSF-EB 

apparently  "changed"  to  _R.  rhipicephal i  between  the  40th  and  48th  passages. 
This  is  thought  to  have  resulted  from  spurious  contamination  and  overgrowth 
of  R.  rickettsii  by  _R.  rhipicephal i  during  passage. 

Antibiotic  sensitivity.  The  strains  were  compared  in  Vero  cell  plaque- 
reduction  tests  for  sensitivity  to  penicillin,  streptomycin,  neomycin, 
tetracycline,  erythromycin,  rifampin,  and  gentamycin.  No  major  differences 
in  the  spectra  of  strain  sensitivity  were  noted  except  to  penicillin  G. 
Ten  times  the  dose  of  penicillin  necessary  to  inhibit  plaque  formation  by 
369-C  strains  were  required  to  inhibit  plaque  formation  by  other  serotypes. 

Cross-immunogenicity  in  guinea  pigs.  Some  information  on  immunologic 
relationships  among  serotypes  was  obtained  by  the  ability  of  guinea  pigs 
previously  inoculated  with  graded  doses  of  the  eight  representative  strains 
to  withstand  challenge  by  1000  guinea  pig  ID50  of  virulent  _R.  rickettsii . 
The  R.  rickettsii  strains  conferred  complete  protection.  _R.  montana  and 
R..  rhipicephal  i  strains  gave  partial  protection.  The  369-C  strains  did  not 
infect  and  did  not  protect  against  challenge. 

Growth  dynamics  in  cell  culture.  The  growth  dynamics  in  slide-chamber 
cultures  of  irradiated  Vero  cells  were  determined  for  the  369-C  agents. 
Growth  characteristics  were  similar  to  those  of  R_.  rickettsii  except  that 
intranuclear  location  is  infrequent,  an  extremely  large  population  of  organisms 
can  be  supported  by  infected  cells  before  cell  death  occurs  and  obversely, 
these  organisms  do  not  seem  to  have  a  mechanism  for  egress  from  infected 
cells.  Transfer  of  organisms  appeared  to  occur  via  intercellular  bridges. 

In  1977,  isolates  of  rickettsiae  representing  three  different  serotypes 
(R.  rickettsii ,  R.  rhipicephal  i ,  and  R_.  montana)  were  obtained  from  18  ticks 
collected  in  the  vicinity  of  Como  Lake  in  an  area  less  than  one  kilometer 
square.  In  1978,  this  area  was  systematically  flagged  during  spring  and 
summer  and  the  collected  ticks  were  examined  for  rickettsiae.  The  serotypes 
were  determined  both  by  sequential  FA-staining  of  the  hemolymph  and  isolation 
of  organisms  in  Vero  cell  culture.  Three  hundred  seventy-one  ticks  were 
collected  during  the  season.  Eleven  were  positive  by  hemolymph  test  for 
SFG  rickettsiae.  Isolates  were  obtained  from  nine,  eight  of  which  were  R_. 
rhipicephal i  and  one  is  as  yet  unclassified.  Thus,  no  strains  of  _R.  rickettsii 
or  R_.  montana  were  recovered  in  a  locus  which  readily  yielded  isolates  of 
these  rickettsiae  during  the  previous  year.  Two  strains  of  R_.  rhipicephali 
were  established  in  laboratory  tick  lines  and  carried  through  one  complete 
generation.  R_.  rhipicephali  was  also  identified  in  a  third  female  tick. 
Six  normal  nymphs  that  were  fed  on  the  same  animal  with  the  infected  female 
acquired  JR.  rhipicephal i  infection.  However,  F]  larvae  and  nymphs  were  infected 
with  organisms  identified  as  369-C,  and  F]  adults  were  no  longer  infected 
with  rickettsiae.  Thus,  it  appeared  that  either  the  original  female  was 
dually  infected  with  two  serotypes  of  rickettsiae,  or  antigenic  variation 
among  the  infecting  organisms  occurred  during  or  after  transovarial  passage. 


28-16 


Project  No.    Z01    AI  00065-06   LMSF-EB 

The  former  alternative  appears  to  be  the  most  likely  possibility  in  view 
of  the  fact  that  the  guinea  pig  on  which  the  parental    female  fed  responded 
with  both  JR.  rhipicephal  i   and  369-C  antibodies. 

This  year,   in  collaboration  with  the  California  State  Department  of 
Health  (Lane)  we  are  screening  ticks  obtained  from  coastal   areas  of  California 
for  presence  of  SFG  rickettsiae  and  typing  the  isolates  by  immunofluorescence. 
The  preponderance  of  ticks  obtained  by  flagging  were  D.  occidental  is  from 
three  areas:     U.  of  California  Hopland  Field  Station,  north  of  San   Francisco, 
U.  of  California  Hastings  Natural   History  Reservation,  Monterey  peninsula, 
and  Torrey  Pines  State  Park,  San   Diego.     By  hemolymph  test,  165  (19%)  of 
875  ticks  had  rickettsia-1 ike  organisms.     Thus   far,  46  isolates  of  SFG 
rickettsiae  have  been  recovered.     Sequential    staining  by  direct  immunofluores- 
cence indicated  an  antigenic  relationship  to  R.   rhipicephal  i   by  most  strains 
recovered  from  D.  occidental  is.     One  of  these  isolates  was  nonpathogenic 
for  guinea  pigs  and  embryonated  eggs.     One  isolate  from  JJ.  occidental  is  was 
closely  related  to  JR.  rickettsii   in  cross-micro-IF  tests  of  mouse  antisera. 
This  strain  gave  scrotal   reaction  but  no  fever  in  guinea  pigs  and  was  pathogenic 
for  chick  embryos.     An  isolate  from  one  of  two  JD.  variabilis  collected  sero- 
typed  as  the  369-C  agent.     A  fourth  isolate  from  Ixodes  pacificus  appears 
to  be  different  from  all   of  the  other  isolates  but  has  not  yet  been  charac- 
terized.    Thus,  it  appears  that  SFG  agents  are  widely  prevalent  in  ticks 
from  coastal   areas  of  California  and  on  occasion  certain  of  these  organisms 
may  play  an  etiologic  role  in  the  sporadic  RMSF-like  illnesses  that  occur 
in  areas  where  the  usual    tick  vectors  of  RMSF  are  not  present. 

In  the  future,  we  will   continue  our  attempts  to  identify  group-  and 
type-specific  antigens  in  extracts  of  various  SFG  rickettsiae  with  the  aid 
of  immunoelectrophoretic  procedures.     If  the  existence  of  a  type-specific 
antigen  for  the  virulent  strains  of  R.  rickettsii  can  be  established,  an 
effort  will   be  made  to  prepare  a  type-specific  antiserum.     Conceivably,  such 
a  serum  could  be  of  considerable  value  in  determining  whether  the  rickettsial 
agents  found  in  ticks  feeding  on  humans  have  the  potential    to  cause  disease 
and  thus,  whether  the  individuals  should  be  treated  with  antibiotics.     Our 
second  major  effort  will   be  the  continuation  of  our  attempts  to  isolate  and 
identify  the  protective  antigenCs)   in  a  soluble  rickettsial   extract  that 
we  previously  described. 

Three  other  questions  will   continue  to  be  addressed  by  this  project: 
1)  What  is  the  etiologic  relationship  to  human  illness  of  SFG  agents  other 
than  j*.  rickettsii?     2)  Are  the  other  SFG  serotypes  unstable  phenotypic 
variants  of  R.   rickettsii?    3)   If  not,  what  is  their  ecologic  role  in 
determining  the  natural   distribution  of  JR.   rickettsii   and  the  focality  of 
RMSF  in  the  United  States? 


28-17 


Project  No.   Z01   AI  00065-06   LMSF-EB 


Publications : 


Anacker,  R.   L.  and  Ormsbee,  R.  A.:     Rickettsiae:     general   descriptions. 
In  Seligson,   D.   (Ed.):     Handbook  Series  in  Clinical    Laboratory  Science. 
Section  H.     Virology  and  Rickettsiology       Cleveland,   CRC  Press,   Inc., 
1978,  vol.  1,   part  2,   pp.   329-360. 

Philip,  R=  N.,  Casper,  E.  A.,  and  Burgdorfer,  W.:     Current  knowledge 
of  the  distribution  of  serotypes  of  spotted  fever-group  rickettsiae 
in  the  United  States  as  determined  by  mi croimmuno fluorescence.     In: 
Proceedings  VII   International    Congress  of  Infectious  and  Parasitic 
Diseases,  Varna,  Bulgaria,  Oct.  2-6,  1978,  1978,   pp.   500-509. 

Philip,  R.  N.,  Casper,  E.  A.,  Burgdorfer,  W.,  Gerloff,   R.   K.,  Hughes, 
Lo   E.,  and  Bell,  E.   J.:     Serologic  typing  of  rickettsiae  of  the  spotted 
fever  group  by  microimmunofluorescence.     J.   Immunol .  121:     1961-1968, 
1978. 


28-13 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl      AI     00070-14  LMSF 


PERIOD    COVERED 


October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Biological   Activities  of  Substances  in  Bordetella  pertussis 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:    R.  K.  Bergman 
OTHER:  J.  J.  Munoz 


Scientist  Director 
Res.  Microbiologist 


LMSF  NIAID 
LMSF  NIAID 


COOPERATING  UNITS  (if  any) 

None 


LAB/BRANCH 

Laboratory  of  Microbial  Structure  and  Function,  Hamilton,  MT  59840 


SECTION 

Pertussis  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 

2.2 


PROFESSIONAL: 
1.1 


1  .1 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


JTJ  (c)    NEITHER 


SUMMARY    OF    WORK    (200   words    or   less   -   underline    keywords) 

It  is  the  purpose  of  this  project  to  study  the  biological  effects  of  substances 
from  Bordetella  pertussis  with  special  emphasis  on  those  substances  which 
induce  physiological  and  immunological  responses  in  experimental  animals. 
Areas  of  current  investigation  are:  1)  ability  of  substances  from  B_„  pertussis 
to  affect  production  of  insul in  by  mouse  pancreatic  s-islet  cells  in  primary 
tissue  culture,  2)  ability  of  pertussigen  to  induce  production  of  unique 
peptides  that  may  enhance  circulatory  shock,,  and  3)  effect  of  substances  from 
B_0  pertussis  on  insulin  binding  sites. 


28-19 


PHS-6040 
(Rev.    10-76) 


Project  No.    Z01   AI  00070-14  LMSF 


Project  Description: 


The  purpose  of  this  project  is  to  investigate  in  experimental   animals 
the  biological   effects  of  substances  from  Bordetella  pertussis  which  produce 
physiological   and  immunological    responses  that  are  related  to  hypersensitivity 
and  autoimmune  phenomena. 

Much  of  our  previous  work  on  the  physiological   and  immunological 
effects  of  substances  from  B_.   pertussis  has  utilized  in  vivo  techniques. 
The  majority  of  the  work  has  been  done  in  mice  and  rats,  and  while  the  work 
has  been  informative,  we  have  not  had  much  insight  into  what  was  happening 
at  the  tissue  or  cellular  level.     During  this  past  year,  we  have  attempted 
to  develop  in  vitro  models  that  would  allow  us  to  investigate  the  effects 
of  biologically  active  substances  from  B_.   pertussis  on  the  pancreas  and  on 
insulin  binding  sites  of  plasma  membranes. 

Our  attempts  to  cultivate  mouse  parotid  salivary  gland  epithelial 
cells  in  tissue  culture  (as  mentioned  in  last  year's  report)  were  unsuccess- 
ful.    Since  substances   from  B.   pertussis  have  a  profound  effect  on  the 
pancreatic  s-islets  of  rats  Tas  reported  by  Ui   et  al_.  at  the  International 
Symposium  on  Pertussis,  NIH,  1978),  we  decided  to  investigate  the  effects 
of  our  pertussigen  preparations  on  mouse  pancreatic  g-islet  cells   in  primary 
tissue  culture.     We  now  have  a  system  which  utilizes  a  trypsin-collagenase 
digest  of  neonate  mouse  pancreas  and  produces  viable  s-cells  that  grow  in 
a  medium  containing  TCM  199,  NCTC  135  and  fetal   calf  serum.     The  glucose  con- 
centration is  adjusted  to  1605  mM.     The  e-cells   seem  to  grow  well    in  this 
system  for  7  to  10  days  and  produce  insulin  as  detected  by  a  radioimmunoassay 
system.     The  a-cells  from  the  islets  do  not  appear  to  proliferate  in  the 
tissue  culture  since  the  production  of  a-amylase  drops  off  rapidly.     After 
10  to  14  days,   fibroblasts  become  predominant  in  the  culture. 

Since  substances   from  B_.   pertussis  are  known  to  affect  the  pancreas, 
and  pertussi gen-treated  mice  and  rats  are  much  more  susceptible  to  circula- 
tory shock,  we  have  made  some  preliminary  tests  for  the  production  of  a 
peptide  in  the  ischemic  pancreas  of  pertussigen-treated  rats  that  might  be 
analogous  to  the  myocardial    depressant   factor  (MDF)  of  Lefer   (Fed_„   Proc.   37: 
2734-2740,  1978).     Results   from  thin  layer  chromatography  (cellulose)   and 
polyacrylamide  gel   electrophoresis   have  given  some  indication  that  there   is 
a  unique  peptide  in  the  plasma  of  pertussigen-treated  rats  following  1    hour 
of  splanchnic  ischemia  that  may  be  similar  to  the  myocardial   depressant 
factor,  however  we  do  not  feel   that  these  results  have  been  definitive.     We 
plan  on   investigating  this   question  further  by  utilizing  high   performance 
liquid  chromatography  to  purify  and  quantitate  the  plasma  peptides. 

We  have  also  initiated  some  work  to  investigate  whether  or  not 
pertussigen  affects  insulin  binding  sites  on  plasma  membranes  of  mouse 
liver  cells.     We  utilized  the  method  of  Ray  (Biochim,,   Biophys.   Acta  196:   1- 
9,  1970)   to  prepare  plasma  membranes  and  the  methods  of  Caron  et  al . 
(Biochim.   Biophys.   Acta  512:  29-40,  1978)   to  measure  insulin  binding  sites. 

28-20 


Project  No.  Z01  AI  00070-14  LMSF 

Preliminary  results  have  not  shown  any  difference  in  insulin  binding 
between  plasma  membranes  from  pertussi gen-treated  mice  and  those  from  saline- 
treated  mice. 

Future  work  will  investigate  via  in  vitro  methods  the  insulin  pro- 
duction of  mouse  pancreatic  3-islet  cells  following  treatment  with  different 
substances  from  B_.  pertussis  and  also  interactions  with  adrenergic  agonists 
and  antagonists.  We  will  investigate  whether  or  not  pertussi gen-treated  rats 
release  a  unique  peptide  into  the  circulation  during  circulatory  shock  which 
may  further  complicate  the  shock  syndrome.  This  will  be  investigated  by  the 
use  of  high  performance  liquid  chromatography.  We  will  also  continue  our 
efforts  to  measure  the  possible  effects  of  pertussigen  on  insulin  binding 
sites  in  both  mice  and  rats. 


Publications: 

Bergman,  R.  K„,  Munoz,  J.  J.,  and  Portis,  J.  L.:  Vascular  permeability 
changes  in  the  central  nervous  system  of  rats  with  hyperacute 
experimental  allergic  encephalomyelitis  induced  with  the  aid  of  a 
substance  from  Bordetella  pertussis.  Infect.  Immuno  21:  627-637, 
1978. 

Munoz,  J.  J.,  Bergman,  R.  K.,  and  Robbins,  K.  E.:  Comparison  of  the 
histamine  hypersensitivity  and  the  Limulus  amoebocyte  lysate  tests 
for  endotoxin  activity.  Infect.  Immun.  22:  292-294,  1978. 


28-21 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl      AI     00071-09     LMSF 


PERIOD    COVERED 


October  1  ,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Purification  and  Activities  of  Pertussigen  -  a  Substance  from  Bordetella 
pertussis 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  ANO  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 

J, 

J.  Munoz 

OTHER: 

H. 

Arai 

R. 

K.  Bergman 

P. 

L.  Sadowski 

Y. 

Sone 

Eo 

Eo  Ribi 

ReSo  Microbiologist 
NIH  Visiting  Fellow 
Scientist  Director 
Staff  Fellow 
NIH  Visiting  Fel low 
Res.   Chemist 


LMSF  NIAID 
LMSF  NIAID 
LMSF  NIAID 
LMSF  NIAID 
LMSF  NIAID 
LMSF  NIAID 


COOPERATING  UNITS  (if  any) 

S.  M.  Strain,  Hamilton  Biochemical  Research  Lab,  Hamilton,  MT 


LAB/BRANCH 

Laboratory  of  Microbial    Structure  and   Function,  Hamilton,   MT     59840 


SECTION 

Pertussis  Section 


NSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 

5.9 


PROFESSIONAL: 

4.0 


1  .9 


CHECK  APPROPRIATE  BOX(ES) 
G  (a)   HUMAN  SUBJECTS 


Q  (b)  HUMAN  TISSUES 


3  (=)  NEITHER 


G  (a1 )  MINORS 


(a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  objectives  of  this  project  are  to  fully  characterize  and  to  study  the 
activities  of  pertussigen,  a  substance  from  Bordetella  pertussis,  and  to  find 
the  role  that  this  and  other  substances  play  in  the  actions  of  B_,  pertussis. 
Methods  to  obtain  pertussigen  in  highly  purified  form  have  been  developed, 
and  with  these  preparations  we  are  studying  their  ability  to  protect  mice 
from  experimental  pertussis.  The  effect  that  other  substances  from  B_„ 
pertussis  have  on  the  protective  effect  of  pertussigen  is  presently  under 
investigation.  Particular  attention  has  been  given  to  the  mechanisms  by  which 
pertussigen  enhances  the  production  of  immunoglobul in  E  (IgE)  in  mice.  These 
studies  indicate  that  pertussigen  may  act  directly  on  the  lymphoid  cells 
responsible  for  antibody  production., 


28-22 


PHS-6040 
(Rev.    IO-76) 


Project  No.  Z01  AI  00071-09  LMSF 


Project  Description: 


The  primary  objectives  of  this  project  are  to  characterize  the  chemical 
nature  of  pertussigen  and  other  biologically  active  substances  of  the 
Bordetella  pertussis  cell  and  to  uncover  the  mechanisms  of  their  biological 
activities.  The  ultimate  practical  aim  is  to  develop  a  pertussis  vaccine 
free  of  toxicity. 

1,  Purification  of  pertussigen  (Munoz,  Arai  and  Sone),  In  the  1978 
report  we  described  a  method  of  obtaining  highly  purified  preparations  of 
pertussigen.  This  method  employed  Triton  X-100  which  we  found  difficult  to 
remove  from  the  purified  material „  Furthermore,  the  purified  material  was 
not  stable  when  frozen  and  thawed.  For  these  reasons  we  continued  our  efforts 
to  find  better  methods  of  purification.  Two  methods  have  been  tried  with 
promising  results.  Phenyl  sepharose  absorbs  pertussigen  and  it  can  then  be 
eluted  with  a  high  pH  buffer.  This  simple  step  can  then  be  followed  by 
filtration  in  a  BioGel  A, 5  column  and  by  ultracentrifugation  in  a  glycerol 
gradient  to  produce  a  highly  active  purified  pertussigen.  The  other  method 
involves  the  use  of  affinity  chromatography  columns  made  by  conjugating  either 
haptoglobul in  or  specific  anti-pertussigen  serum  to  Sepharose  beads  and  then 
eluting  pertussigen  by  an  appropriate  buffer.  This  latter  method  is  promising 
and  is  presently  under  extensive  experimentation. 

2,  Studies  on  fimbria!  hemagglutinins  (FHA)  (Arai  and  Munoz).  B_. 
pertussis  produces  two  distinct  hemagglutinins,  one  which  is  identical  to 
pertussigen  and  the  other  called  fimbrial  hemagglutinin.  According  to  Sato 
and  Arai,  FHA  protects  mice  from  experimental  pertussis.  We  have  found  that 
under  the  conditions  normally  used  at  RML  to  grow  B_,  pertussis  (shaking 
cultures),  FHA  is  not  found  in  3-  to  5-day-old  cultures.  However,  under  the 
conditions  used  by  the  Japanese  workers  (stationary  incubation  in  shallow 
layers  of  media),  FHA  is  produced.  Employing  stationary  cultures  of  B_, 
pertussis,  FHA  was  purified  and  crystallized  by  a  simple  procedure  which  in- 
volved passing  5-day-old  culture  supernatant  fluids  through  a  CM-Sepharose 
CL-6B  column  equilibrated  in  20  mM  phosphate  buffer  pH  6.5.  After  washing 
the  column  with  20  mM  phosphate  buffer,  FHA  was  eluted  with  0.5  M  NaCl  in 

20  mM  phosphate  buffer  containing  5%  ethanol ,  The  active  fractions  were 
pooled,  concentrated  by  vacuum  dialysis  and  passed  through  Sepharose  6B 
columns  equilibrated  in  50  mM  Tris-HCl  buffer  containing  1  M  NaCl  and  5% 
ethanol.  The  active  fractions  were  pooled  and  concentrated  by  vacuum 
dialysis.  During  this  concentration,  crystallization  of  FHA  occurred.  These 
crystals  were  made  up  entirely  of  amino  acids  and  had,  when  dissolved,  a 
specific  activity  as  high  as  that  of  uncrystallized  purified  FHA. 

3,  Activities  of  purified  FHA  and  pertussigen  (Munoz  and  Arai). 
Although  FHA  was  highly  purified,  it  still  was  able  to  sensitize  CFW  mice 
to  histamine  at  doses  of  2  to  5  yg  per  mouse.  This  crystalline  preparation 
also  protected  mice  from  experimental  infection  with  B_.  pertussis  at 
similar  doses.  Since  the  ability  to  sensitize  mice  to  histamine  is  a 
property  of  pertussigen,  it  was  thought  that  crystalline  FHA  was  still 

28-23 


Project  No.  Z01  AI  00071-09  LMSF 

contaminated  with  pertussigen.  So  we  proceeded  to  remove  pertussigen  from 
these  preparations.  This  was  accomplished  by  column  chromatography  in  BioGel 
in  the  presence  of  Triton  X-100.  Preparations  of  FHA  free  of  pertussigen 
did  not  protect  mice  from  experimental  infection  or  sensitize  them  to 
histamine  at  a  dose  of  25  yg. 

Pertussigen  purified  by  the  method  described  in  last  year's  report 
or  by  methods  described  here  do  not  contain  FHA,  but  are  highly  active  in 
sensitizing  mice  to  histamine  (SD50  about  .01  yg  of  protein)  and  to  protect 
them  from  infection  (PD50  1  to  2  yg). 

4.  Preparation  of  specific  antisera  to  FHA  and  pertussigen  and 
passive  protection  test  with  these  sera  (Munoz  and  Arai).  Antisera  to 
crystalline  FHA  and  purified  pertussigen  were  made  by  repeatedly  immunizing 
rabbits  with  these  substances  either  mixed  with  complete  Freund's  adjuvant 
(for  FHA)  or  mixed  with  erythrocyte  stromata.  The  antisera  obtained  were 
not  specific  to  FHA,  but  were  made  specific  by  passing  them  through  an 
affinity  column  containing  the  appropriate  antigen,  and  then  eluting  the 
adsorbed  antibody  by  appropriate  buffer.  The  specificity  of  the  eluted 
antibodies  was  determined  by  gel  diffusion  tests  against  the  purified  FHA 
and  pertussigen,,  Passive  protection  tests  were  performed  with  these  antisera. 
Anti -pertussigen  serum  protected  mice  from  intracerebral  infection  while  the 
anti-FHA  did  not» 

50  Relationship  among  pertussigen,  FHA  and  agglutinogens  (Munoz  and 
Arai).  The  relationship  of  the  various  agglutinogen  factors  to  pertussigen 
and  FHA  has  not  been  defined.  We  have  studied  these  relationships  and  have 
arrived  at  the  following  conclusions: 

a)  Pertussigen  and  FHA  are  not  associated  with  agglutinogens  2, 
3,  4,  5,  6,  because  we  can  obtain  these  substances  from  B_.  pertussis 
agglutinogen  types  1 ,  3,  6  or  1 ,  2 ,  4. 

b)  Pertussigen  and  FHA  are  two  distinct  substances  which  are 
produced  by  all  smooth  cultures  of  B_.  pertussis  thus  far  tested.  B_. 
bronchiseptica  and  B_.  parapertussis  produce  smaller  amounts  of  FHA  but  not 
pertussigen. 

c)  Agglutinogen  1  is  not  related  to  FHA  because  B_.  parapertussis 
and  B_.  bronchiseptica  produce  FHA  but  not  agglutinogen  1. 

d)  Pertussigen  is  different  from  agglutinogen  1  because  anti- 
pertussigen  serum  fails  to  agglutinate  cells  that  are  strongly  agglutinated 
by  factor  1  antiserum0 

6.  Adjuvant  action  of  pertussigen  (Sadowski  and  Munoz).  One  of  the 
many  biological  properties  of  pertussigen  is  its  ability  to  increase  antibody 
production  to  antigens  given  with  it.  An  unusual  characteristic  of  this 


28-24 


Project  No.  Z01  AI  00071-09  LMSF 

adjuvant  action  is  that  the  IgE  response  is  almost  selectively  increased. 
Understanding  the  mechanism(s)  by  which  pertussigen  exerts  this  effect  may 
reveal  how  IgE  production  is  regulated  and  thus  may  lead  to  a  better  under- 
standing of  the  allergic  response  in  man. 

The  following  observations  have  been  made: 

a)  Relationship  of  pertussigen  to  endotoxin.  Since  the  preparations 
of  pertussigen  used  were  not  free  of  endotoxin,  it  was  necessary  to  rule 

out  the  participation  of  endotoxin  in  stimulation  of  IgE.  Pertussigen  acts 
as  an  adjuvant  for  stimulation  of  IgE  in  C57BL/10ScN  mice  which  are  unrespon- 
sive to  endotoxin.  Doses  of  Escherichia  coli  endotoxins  up  to  100  yg  (which 
also  contains  endotoxin  protein)  do  not  stimulate  an  IgE  response  in  these 
mice,, 

b)  Time  at  which  pertussigen  acts.  Pertussigen  acts  as  well 

in  stimulating  an  IgE  response  when  administered  3  days  prior  to  the  antigen 
as  when  administered  with  the  antigen, 

c)  Effect  of  Concanavalin  A.  In  contrast  to  the  last  observation, 
Concanavalin  A,  which  also  induces  IgE  stimulation  when  given  with  the 
antigen,  does  not  stimulate  production  of  IgE  when  given  3  days  before  the 
antigen.  Most  adjuvants  will  not  stimulate  Ig  response  to  an  antigen  when 
administered  prior  to  the  antigen,  and  often  they  will  suppress  antibody 
response.  Preliminary  work  indicated  that  pertussigen  given  3  days  prior 

to  antigen  may  not  be  as  effective  in  stimulating  IgM  production  as  when 
it  is  given  simultaneously  with  the  antigen. 

d)  Role  of  suppressor  cells.  The  ability  of  pertussigen  to  act 
as  an  adjuvant  for  IgE  production  when  given  3  days  prior  to  antigen  is 
similar  to  the  effect  of  cyclophosphamide.  The  action  of  cyclophosphamide 
is  thought  to  be  due  to  the  elimination  of  suppressor  T  cells  that  are 
responsible  for  exerting  a  negative  control  over  IgE  synthesis. 

e)  Effect  of  anti-pertussigen  sera.  Antisera  capable  of  inhibiting 
the  histamine  sensitizing  action  of  pertussigen  also  inhibit  its  ability 

to  act  as  an  adjuvant.  When  pertussigen  is  administered  3  days  prior  to 
antigen  and  antiserum  is  given  with  the  antigen,  the  adjuvant  effect  of 
pertussigen  is  still  suppressed.  However,  if  antiserum  to  pertussigen  is 
administered  3  days  after  antigen  and  pertussigen,  the  IgE  stimulation  is 
not  inhibited. 

f)  Irradiation  studies.  The  ability  of  pertussigen  to  stimulate 
IgE  response  can  be  transferred  into  irradiated  recipients  by  spleen  cells 
from  mice  which  had  received  pertussigen  3  days  prior  to  the  removal  of  the 
spleens.  The  irradiated  recipient  mice,  when  given  antigen,  produce  IgE 
with  specificity  to  the  antigen.  This  may  mean  that  pertussigen  was  trans- 
ferred with  the  spleen  cells  although  other  explanations  may  be  possible. 


28-25 


Project  No.  Z01  AI  00071-09  LMSF 

g)  SDS  acrylamide  gel  electrophoresis  studies.  In  spleen  cells 
of  mice  receiving  radioiodinated  pertussigen  3  days  prior  to  removing  the 
spleens,  a  component  of  the  pertussigen  preparation  was  demonstrated  by 
extracting  the  cells  with  detergent  and  then  treating  the  extract  with  anti- 
pertussigen  serum.  The  material  precipitated  by  the  antiserum  showed,  by 
analysis  on  SDS-PAGE  gels,  one  radioactive  polypeptide  of  about  18,500-19,000 
mol ecular  weight. 

7.  Miscellaneous  experiments  (Arai  and  Munoz).  Pertussigen  increases 
the  leukocyte  count  in  mice,  but  in  our  hands  the  increase  in  the  number 
of  leukocytes  has  been  generally  of  a  lower  order  of  magnitude  than  that 
reported  by  other  workers.  It  was  possible  that  these  differences  were  due 
to  the  source  of  blood  to  perform  the  leukocyte  counts.  Indeed,  we  found 
that  counts  performed  with  blood  obtained  by  snipping  the  end  of  the  tail 
were  significantly  higher  than  those  counts  performed  with  blood  obtained 
from  the  infraorbital  sinus.  This  difference  was  probably  due  to  hemocon- 
centration  at  the  tip  of  the  tail.  When  blood  was  obtained  from  the  tail, 
after  allowing  blood  to  flow  freely,  the  leukocyte  counts  were  similar  to 
those  obtained  from  the  infraorbital  sinus. 

During  the  following  fiscal  year  we  will  continue  our  efforts  to  fully 
characterize  pertussigen  and  to  find  its  mechanisms  of  action,  especially 
with  respect  to  its  ability  to  stimulate  IgE  and  to  protect  mice  from  exper- 
imental pertussis.  We  will  study  the  role  that  various  purified  B_.  pertussis 
antigens  play  in  protection,  and  will  study  ways  of  detoxifying  the  protective 
antigen  to  see  if  we  can  develop  a  vaccine  with  fewer  side  effects  than  the 
presently  available  pertussis  vaccines.  Much  time  will  be  devoted  in  obtain- 
ing sufficient  quantities  of  pertussigen  for  chemical  identification  purposes. 

Publ ications : 

Munoz,  J,  J.  and  Bergman,  R.  K. :  Mechanism  of  action  of  pertussigen, 
a  substance  from  Bordetella  pertussis.  Microbiology  1 979  193-197, 
1979. 

Munoz,  J.  J.  and  Cole,  R.  L. :  Extraction  of  pertussigen  from 
Bordetella  pertussis  with  aid  of  Triton  X-100.  IRCS  Med.  Sci,  7: 
218,  1979. 

Arai,  H.  and  Munoz,  J.  J.:  Leukocyte  counts  in  blood  obtained  from 
tail  and  infraorbital  sinus  bleedings  in  normal  and  pertussigen-treated 
mice,  IRCS  Med.  Sci.  7:  306,  1979. 

Wardlaw,  A,  C,  Parton,  R. ,  Bergman,  R.  K.,  and  Munoz,  J.  J.:  Loss 
of  adjuvanticity  for  hyperacute  EAE  and  for  reaginic  antibody  pro- 
duction in  a  phenotypic  variant  of  Bordetella  pertussis.  Immunology 
37:  539-545,  1979. 


28-26 


Project  No.    Z01   AI  00071-09  LMS.F 

Publications:     (cont'd) 

In  press: 

Munoz,  J.  J.  and  Bergman,  R.  K.:  Biological  activities  of  Bordetella 
pertussis.  Proceedings  of  International  Pertussis  Conference, 
Bethesda,  MD,  Nov.  1978. 

Arai,  H.  and  Munoz,  J.  J.:  Purification  and  crystallization  of 
fimbria!  hemagglutinin  from  Bordetella  pertussis.  Infect.  Immun. 


28-27 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl      AI   00076-21    LMSF 


PERIOD    COVERED 


October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Mechanisms  of  Immunopotentiation  by  Components  of  Microbes 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 

Co 

A. 

McLaughlin 

OTHER: 

Eo 

E. 

Ribi 

So 

M. 

Schwartzman 

J. 

L. 

Cantrell 

K0 

B. 

Von  Eschen 

Senior  Staff  Fellow 
Res.  Chemist  (Phys. 
Senior  Staff  Fellow 
Senior  Staff  Fellow 
Staff  Fellow 


Chem. 


LMSF  NIAID 

LMSF  NIAID 

LMSF  NIAID 

LMSF  NIAID 

LMSF  NIAID 


cooperating  units  (if  any)  Dr.  E.   P.  Goldberg,   Dept.   Engineering,   Univ.   of  Florida, 
Gainesville;   Dr.   Co  H.   Granatek,  M.D.  Anderson  Tumor  Institute,  Houston,  TX; 
Dr„   R0  Toubiana,  Gif-sur-Yvette,  France;   Dr0   G.  Jones,  Syntex  Corp.,   Palo  Alto, 
CA. 


lab/branch 
Laboratory  of  Microbial    Structure  and   Function,  Hamilton,  MT     59840 


section 
Molecular  Biology  Section 


institute  and  location 
NIAID,  NIH,  Bethesda,  MD     20205 


TOTAL  MANYEARS: 

2.85 


PROFESSIONAL: 

1.25 


1  .6 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


Q   (b)  HUMAN  TISSUES 


(c)    NEITHER 


SUMMARY    OF    WORK    (200   words   or   less   -   underline    keywords) 

It  is  the  purpose  of  this  project  to  delineate,  at  the  cellular  and  molecular 
levels,  the  mechanism  of  action  of  microbial  components  in  immunopotentiation. 
In  the  past  year,  two  major  efforts  have  been  made:  TJ  elucidating  the  nature 
of  biological  responses  to  treatment  of  tumors  with  synthetic  analogs  of  myco- 
bacterial cell  wall  components  combined  with  endotoxic  extracts  obtained  from 
Salmonella  typhimurium  Re  mutants,  and  2)  establishing  the  structural  features 
of  analogs  of  adjuvant  di peptides  required  for  antitumor  activity  when  combined 
with  trehalose  dimycolate. 


28-28 


PHS-6040 
(Rev.    10-76) 


Project  No.   Z01   AI  00076-21   LMSF 


Project  Description: 


In  general,  our  major  objective  is  elucidating  the  structural   require- 
ments and  mechanism  of  action  of  immunopotentiating  components  isolated  from 
microbes. 

Previously  we  had  shown  that  mycobacterial   cell  wall   skeleton,  when 
combined  with  endotoxic  extracts  from  Salmonella  typhimurium  rough  mutants 
(Re  glycol ipid),  produced  synergistic,  not  additive,  tumor-regressive  responses. 
The  mechanism  of  this  synergy  was  found  not  to  be  a  consequence  of  interaction 
of  the  cell  wall   skeleton  with  endotoxin  molecules.     This  was  unlike  the 
synergistic  response  seen  when  trehalose  dimycolate  was  combined  with  Re 
glycol ipids  wherein  we  showed  that  the  trehalose  dimycolate  enhanced  the 
interaction  of  endotoxin  molecules  with  oil  droplets  of  the  oil-in-water 
emulsions  used  as  a  therapeutic  vehicle.     We  had  not  established  the  minimal 
structural   requirement  of  cell  wall  skeleton  to  produce  the  synergistic  anti- 
tumor activity  observed  upon  combination  with  Re  glycol ipid.     Last  year  we 
proposed  that  the  adjuvant  di peptide,  N-acetyl-muramyl-L-alanyl-D-isoglutamine 
might  represent  at  least  one,  if  not  the  only,  minimal   structural   unit  in  cell 
wall   skeleton  for  the  synergy  observed  with  endotoxin.     Indeed  the  adjuvant 
dipeptide  did  replace  the  cell  wall   skeleton  in  producing  synergistic  antitumor 
activity  in  combination  with  endotoxic  extracts.     The  synthetic  molecule  and 
Re  glycol ipid  produced  high  incidences  of  tumor  regression  of  transplanted 
dermal   tumors  in  guinea  pigs.     However,  only  upon  addition  of  trehalose 
dimycolate  (provided  by  Dr.  R„  Parker,  Hamilton,  MT,  under  contract  with  NIAID) 
were  the  treated  animals  cured  of  both  the  dermal   and  metastatic  tumors. 
Another  synthetic  preparation  (.provided  by  Drs.   I.  Azuma  and  Y.  Yamamura), 
6-0-mycoloyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine  in  combination  with  Re 
glycol ipid,  produced  synergistic  tumor-regressive  activity  against  both  the 
dermal   and  metastatic  tumors.     All   of  these  preparations  were  active  only  when 
presented  to  the  tumor-bearing  animal   in  an  oil-in-water  emulsion  constructed 
to  maximize  the  interaction  of  the  synthetic  preparations  with  the  oil   phase 
of  the  emulsions.     The  Re  glycol ipid  was  effective  in  the  aqueous  phase  of 
the  emulsion.     Thus,  we  have  now  defined  the  structural   features  of  the  BCG 
cell  wall  or  cell  wall   skeleton  responsible  for  the  potent  antitumor  activity 
of  these  preparations  when  combined  with  endotoxic  extracts.     The  requirement 
for  a  covalent  or  noncovalent  lipidation  of  the  adjuvant  dipeptide  for  elicit- 
ing antitumor  activity  against  metastatic  tumors  prompts  one  to  speculate 
about  the  mechanism  of  this  phenomenon.     Conceivably  the  complexing  of 
biologically  active  molecules  (adjuvant  dipeptide)  with  oil   droplets  via  a 
lipid  rich  molecule  such  as  mycolic  acid  or  mycolic  acid  esters  would  produce 
a  kind  of  depot  effect  in  tissues  into  which  the  oil-in-water  emulsion  was 
injected.     Another  reasonable  hypothesis  is  that  the  muramyl  dipeptide-oil- 
droplet  complex  was  preferentially  processed  by  a  specific  population  of  cells 
in  the  afferent  or  efferent  arms  of  the  immune  system.     Experiments  conducted 
with  radiolabeled  cell  wall   skeleton  suggest  that  the  later  mechanism  may  be 
more  important  than  a  slow-release  (depot)  effect  at  the  site  of  injection. 
We  found  that  radiolabeled  cell  wall   skeleton  suspended  in  saline  and  injected 
into  dermal   tumors  remained  in  the  tumors  and  in  other  tissues  for  a  period 

28-29 


Project  No.  Z01  AI  00076-21  LMSF 

of  time  equal  to  cell  wall  skeleton  combined  with  trehalose  dimycolate  and 
suspended  in  an  oil-in-water  emulsion0  Cell  wall  skeleton  in  saline  has  no 
tumor  regressive  activity  whereas  cell  wall  skeleton  combined  with  trehalose 
dimycolate  in  an  oil-in-water  emulsion  is  a  very   effective  tumor-regressive 
preparation.  Additional  experiments  are  underway  whereby  we  will  utilize 
radiolabeled  muramyl  dipeptide  in  similar  studies  to  determine  the  distribu- 
tion of  muramyl  dipeptide  in  vivo  following  intratumor  injections,  It  may 
be  that  a  depot  effect  is  not  critical  to  expression  of  the  biological 
activity  of  these  naturally  occurring  and  synthetic  molecules,  but  what  is 
important  is  the  nature  of  their  presentation  to  the  cells  involved  in  that 
expression. 

As  an  extension  of  the  finding  that  N-acetyl -muramyl -L-al any! -D- 
isoglutamine  possessed  some  of  the  biological  activity  of  cell  wall  skeleton, 
we  initiated  a  collaborative  study  with  Dr.  G.  Jones,  Syntex  Corporation, 
Palo  Alto,  CA.  We  have  found  analogs  of  the  original  adjuvant  dipeptide 
which  are  approximately  100  times  as  effective  in  producing  tumor  regression 
as  the  parent  compound.  All  of  the  newly  described  analogs  are  active  only 
when  combined  with  trehalose  dimycolate  in  the  construction  of  oil-in-water 
emulsions  utilized  as  therapeutic  vehicles  for  intratumor  injections.  In 
collaboration  with  Dr„  Raoul  Toubiana,  we  have  found  that  synthetic  analogs 
of  trehalose  dimycolate,  such  as  trehalose  desoxypalmitopalmitate  and  glycerol 
monomycolate,  act  in  concert  either  with  endotoxic  extracts  or  the  synthetic 
muramyl  dipeptides  to  produce  significant  incidences  of  tumor  regression. 
Thus,  we  have  successfully  simulated  some  of  the  biological  activities  of 
mycobacterial  components  through  the  use  of  synthetic  substitutes.  These 
synthetic  preparations  will  hopefully  facilitate  the  elucidation  of  the 
mechanism  of  action  of  microbes  in  modulating  immune  responses  and  in  patho- 
genesis of  disease  processes. 

An  important  step  in  studying  the  mechanism  of  action  of  muramyl 
dipeptides  at  the  molecular  level  is  the  synthesis  of  radiolabeled  muramyl 
dipeptide.  Dr.  Schwartzman  successfully  synthesized  -^-labeled  adjuvant 
dipeptide,  but  the  procedure  utilized  resulted  in  relatively  low  yields  of 
material.  Another  labeling  procedure  is  in  progress  which  will  use  H-acetic 
anhydride  and  require  fewer  steps.  '-^C-labeled  muramyl  dipeptide  to  be 
synthesized  by  Dr.  Schwartzman  will  be  another  useful  tool  in  studying  the 
mechanism  of  action  of  this  important  compound. 

The  future  course  of  this  project  will  be  concentrated  in  two  major 
directions:  1)  the  elucidation  of  the  biochemical  mechanism  of  action  of 
muramyl  dipeptides,  and  2)  construction  of  the  most  active  adjuvant-antigen- 
carrier  complexes  for  treatment  and  prevention  of  infectious  and  neoplastic 
diseases.  Dr.  Schwartzman  has  proposed  that  muramyl  dipeptide  be  conjugated 
to  suitable  carriers,  such  as  Ficoll  or  polyglutaraldehyde,  as  promising 
candidates  in  construction  of  active  adjuvant-carrier  complexes.  Covalent 
coupling  of  trehalose  monoesters  to  antigens  of  interest  will  be  a  project 


28-30 


Project  No.   Z01   AI  00076-21    LMSF 

undertaken  by  Dr.   R.  Toubiana.     Covalent  linkage  of  muramyl   dipeptides  to 
trehalose  dimycolate  or  to  antibodies  directed  against  tumor  cells  are  other 
avenues  to  be  pursued  by  Dr,  Schwartzman0 

We  now  have  synthesized  minimal   structural   components  of  mycobacteria 
required  for  elicitation  of  certain  biological    responses  conceivably  important 
in  disease  processes  and  potentially  useful   in  disease  control.     These 
synthetic  substances  will   greatly  simplify  the  task  of  visualizing  the  bio- 
chemical   steps  leading  to  the  phenomenon  now  called  adjuvant  activity  or 
tumor-regressive  activity. 

Publications: 

McLaughlin,  C,  A.,  Ribi,  E.  E.,  Goren,  M.  B.,  and  Toubiana,  R.:     Tumor 
regression  induced  by  defined  microbial    components  in  an  oil-in-water 
emulsion  is  mediated  through  their  binding  to  oil   droplets.     Cancer 
Immunol .   Immunother.     4:  109-113,  1978. 

McLaughlin,  C.  A.,  Bickel ,  Wo   D.,   Kyle,  J.  S„,  and  Ribi,   E.: 
Synergistic  tumor  regressive  activity  observed  following  treatment 
of  line-10  hepatocellular  carcinomas  with  deproteinized  BCG  cell   walls 
and  mutant  Salmonella  typhimurium  glycol ipid.     Cancer  Immunol , 
Immunother .     5:  45-52,  1978. 

McLaughlin,  C.  A.,  Hargrave,  S.   L.,  Bickel,  W.   D.,  and  Ribi,  E„: 
Synergistic  activity  of  components  of  mycobacteria  and  mutant  Salmonella 
in  causing  regression  of  line-10  tumors  in  guinea  pigs.     Cancer  Res. 
39:   1766-1771,  1979. 


28-31 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  soace) 


U.S.  DEPARTMENT  OF         PROJECT  NUMBER 
HEALTH,  EDUCATION,  AND  WELFARE 

PUBLIC  HEALTH  SERVICE        ,m    nT   „„„,,  -„ 

notice  of  ZOl      AI     00077-23 

INTRAMURAL  RESEARCH  PROJECT 


LMSF 


>ERIOD  COVERED 


October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Structure  and  Biological  Activity  of  Endotoxins 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:     K.  B.  Von  Eschen 

E.  E.  Ribi 

OTHER:  E.  C„  B.  Milner 

C,  A,  McLaughlin 

Jo  U  Cantrell 

S.  Mo  Schwartzman 

J.  C.  Williams 

J.  Sasaki 


Staff  Fellow  LMSF  NIAID 

Res.  Chemist  (Phys.  Chem„)   LMSF  NIAID 

Student  Scientist  LMSF  NIAID 

Senior  Staff  Fellow  LMSF  NIAID 

Senior  Staff  Fellow  LMSF  NIAID 

Senior  Staff  Fellow  LMSF  NIAID 

Senior  Asst.  Scientist  LMSF  NIAID 

Visiting  Associate  LMSF  NIAID 


cooperating  units  (if  ar.y)^  R>  Ao  par|<er  ancj  s.  m.  Strain,  Hamilton  Biochemical 
ReSo  Labo,  Hamilton,  MT;  Dr0  J.  A0  Rudbach,  Dr.  M0  Jo  Nakamura,  and  S.  Wells, 
University  of  Montana,  Missoula;  and  Dr0  R,  Wheat,  Duke  University,  Durham, 
NC. 


lab/branch 
Laboratory  of  Microbial  Structure  and  Function,  Hamilton,  MT  59840 


SECTION 

Molecular  Biology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 

3.7 


PROFESSIONAL: 
1.1 


2.6 


CHECK  APPROPRIATE  80X(ES) 
C  (a)   HUMAN  SUBJECTS 

Q  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


j(  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  main  objective  of  this  project  is  to  determine  the  minimal  chemical  and 
structural  properties  of  bacterial  endotoxins  necessary  to  elicit  several 
biologic  and  immunologic  responses»  The  purpose  is  to  identify,  by  fractiona- 
tion of  different  endotoxins  and  chemical ,  physical ,  and  biological  analyses 
of  the  individual  fractions,  the  components )  of  the  macromolecular  complex 
responsible  for  the  antitumor  effects  of  some  endotoxins.  Techniques  are  being 
developed  to  assess  the  cellular  nature  of  the  immunomodulatory  activities  of 
different  endotoxic  materials.  Also,  this  project  continues  to  perform  standard 
bioassays  of  endotoxic  materials  contributed  by  several  members  of  the  LMSF. 


28-32 


PHS-6040 

(Rev.    IO-76) 


Project  No.  Z01  AI  00077-23  LMSF 


Project  Description; 


Bacterial  endotoxins,  complex  macroraolecules  extracted  from  gram- 
negative  bacteria,  possess  a  great  variety  of  biological  and  immunological 
activities.  Studies  conducted  in  the  Laboratory  of  Microbial  Structure  and 
Function  continue  to  stress  the  potential  use  of  endotoxins  as  potent  immuno- 
therapeutic  reagents  for  the  treatment  of  certain  neoplastic  diseases.  To 
be  effective  in  the  syngeneic  guinea  pig  line-10  tumor  model,  endotoxins  have 
to  be  combined  with  certain  components  of  mycobacteria,  such  as  trehalose 
dimycolate,  cell  wall  skeleton  or  adjuvant  dipeptide  (see  annual  reports 
Z01  AI  00078-07  LMSF,  Ribi;  Z01  AI  00076-21  LMSF,  McLaughlin;  and  Z01  AI 
00087-02  LMSF,  Cantrell).  Whereas  the  mycobacterial  components  responsible 
for  tumor  immune  responses  are  now  defined  and  have  been  chemically  synthesized, 
the  endotoxic  extracts  we  are  utilizing  remain  complex,  poorly  defined  enigmas. 
Some  progress  in  the  fractionation  of  these  extracts  was  made  in  the  last 
year.  Dr.  McLaughlin  successfully  used  equilibrium  cesium  chloride  density 
gradient  centrifugation  to  separate  contaminating  substances,  such  as  nucleic 
acids,  from  endotoxin-rich  fractions.  We  found  that  this  technique  demon- 
strated the  presence  of  phospholipid-endotoxin  complexes  in  a  variety  of 
endotoxic  extracts.  Interaction  of  endotoxin  with  phospholipids  obtained 
from  a  variety  of  bacteria  caused  a  marked  alteration  in  the  density  of  the 
endotoxin.  The  means  of  extraction  produced  varying  degrees  of  complexation 
as  a  function  of  the  amount  of  phospholipid  contaminating  the  endotoxin-rich 
extracts-  These  findings  suggest  that  a  re-evaluation  of  the  proposed 
chemical  structure  of  endotoxin  is  required.  Considerable  effort  will  be 
needed  to  establish  the  purity  of  endotoxic  extracts  before  assigning 
structural  details  to  a  general  formula  for  endotoxin  (McLaughlin), 

The  use  of  purified  cell  walls  from  Re  mutant  strains  of  gram-negative 
bacteria  rather  than  whole  cells  for  the  extraction  of  endotoxic  material 
appeared  to  be  a  step  in  the  right  direction,  Endotoxic  extracts  prepared 
from  cell  walls,  particularly  EDTA-treated  cell  walls,  in  contrast  to  those 
obtained  from  whole  cells,  were  soluble  in  both  water  and  organic  solvents, 
apparently  as  a  result  of  dissociation  of  glycol ipid-phosphol ipid  complexes 
which  had  been  held  together  by  polyvalent  cations.  A  long  standing  problem 
of  endotoxin  solubility  has,  therefore,  been  solved  that  will  facilitate_ 
in  vitro  and  in  vivo  experimentation  as  well  as  fractionation  of  endotoxic 
material . 

When  endotoxic  chloroform-methanol  extracts  of  cell  walls  were 
chromatographed  through  columns  of  microparticulate  silica  gel,  the  pro- 
cedure used  to  prepare  B4  (see  annual  report  Z01  AI  00078-07  LMSF,  Ribi), 
the  major  component,  after  being  rechromatographed,  was  rendered  essentially 
nontoxic  while  pyrogenicity  was  retained.  Studies  on  the  immunostimulating 
activities  of  the  extract  before  and  after  detoxification  also  showed 
discrete  differences.  Lymphocytes  from  genetic  endotoxin  responder 
(C3HeB/FeJ)  and  nonresponder  (C3H/HeJ)  strains  of  mice  were  used.  In 
general,  the  starting  material  and  nontoxic  fractions  were  potent  mitogens 


28-33 


Project  No.  Z01  AI  00077-23  LMSF 

and  polyclonal  B  cell  activators  for  C3HeB/FeJ  mice.  For  C3H/HeJ  mice,  the 
starting  extract  was  again  mitogenic  and  a  polyclonal  B  cell  activator,  but 
the  nontoxic  fraction  was  essentially  inactive  in  these  respects.  Finally, 
gel  diffusion  analysis  revealed  a  line  of  complete  identity  between  these 
samples.  The  results  showed  that  certain  typical  endotoxic  properties  can 
be  selectively  altered  by  purification  of  endotoxic  glycol ipid  on  silica  gel 
columns. 

Other  studies,  done  in  collaboration  with  Mr.  Steven  Wells  and  Dr. 
Mo  J,  Nakamura,  examined  the  chemical  and  biological  characteristics  of 
endotoxins  extracted  from  Yersinia  enterocol itica.  Although  this  gram- 
negative  organism  has  been  gaining  importance  as  a  causative  agent  in  certain 
enteric  and  systemic  infections,  little  is  known  about  the  possible  role 
that  endotoxin  plays  in  its  pathogenicity.  Biologically  active  endotoxins 
were  extracted  from  both  a  pathogenic  strain  (serotype  0:8,  isolated  at 
necropsy  from  a  human  spleen)  and  a  nonpathogenic  strain  (serotype  0:17, 
isolated  from  water)  of  Y.   enterocol  itica ,  The  chemistry  and  biological 
potency  of  these  endotoxins  were  similar  to  those  of  a  standard  endotoxin 
extracted  from  Escherichia  col i  „  In  addition,  the  relative  yield  of 
endotoxin  was  considerably  higher  from  Yersinia  organisms  cultivated  at 
25°C  than  at  37°C.  This  latter  finding  may  be  important  in  that  organisms 
grown  at  25°C  were  reported  to  be  more  pathogenic  than  cells  grown  at  37°C. 
Also,  based  on  similarities  in  endotoxins,  a  taxonomical  relationship  between 
Yersinia  and  at  least  one  other  Enterobacteriaceae  was  established.  Mr. 
Wells  wrote  his  M.S.  thesis  in  microbiology  from  this  work  and  is  currently 
writing  a  manuscript  for  publication. 

Another  project  of  primary  interest  has  been  the  interaction  and 
functional  stimulation  of  lymphocytes  with  bacterial  endotoxin.  Historically, 
endotoxins  were  reported  to  be  potent  stimulators  of  primarily  bursa 
equivalent  lymphocytes  (B  cells).  Experiments  performed  by  Mr„  Eric  Milner, 
a  Ph.D.  student  working  in  my  laboratory,  provided  new  and  exciting  infor- 
mation concerning  the  interaction  of  endotoxins  with  thymus-derived 
lymphocytes  (T  cells),  A  system  of  in  vitro  proliferation  was  used  to  assess 
the  effects  of  endotoxin  on  the  in  vivo  induction  and  modulation  of  antigen- 
specific  T  cells.  It  was  found  that  nylon  wool  nonadherent  lymph  node  cells 
from  C57BL/6J  mice  immunized  with  endotoxin  gave  strong  proliferative  responses 
upon  in  vitro  stimulation  with  endotoxin  whereas  the  responses  of  T  cells 
from  unimmunized  mice  were  weak  or  absent.  Since  endotoxin  is  known  to  be 
a  nonspecific  mitogen  for  B  cells,  it  was  of  prime  interest  to  characterize 
the  cellular  nature  of  the  endotoxin-specific  response.  Results  from  several 
experiments  suggested  that  the  cells  responding  to  endotoxin  were  T  cells, 
including:  1)  endotoxin-specific  responses  were  generated  in  nylon  wool 
nonadherent  cells  which  were  95%  positive  for  theta  antigen,  2)  the  responses 
were  abolished  by  treatment  of  the  cells  with  anti-Thy  1.2  serum  plus 
complement,  3)  the  responses  were  not  diminished  by  treatment  of  the  cells    > 
with  anti-Ig  serum  plus  complement,  4)  virtually  all  responding  cells  stained 


28-34 


Project  No.  Z01  AI  00077-23  LMSF 

positively  with  fluorescent  anti-Thy  1 „2  serum,  and  5)  endotoxin-specific 
responses  were  not  obtained  in  nylon  wool -purified  lymph  node  cells  from 
athymic  nude  mice. 

Using  this  system,  reciprocal  cross-reactivity  was  observed  between 
proliferative  responses  of  T  cells  primed  to  endotoxin  from  E.   col  i  or 
Salmonella  minnesota.  These  data  indicated  that  the  T  cell  response  was 
directed  to  antigens  shared  by  endotoxins  from  different  species  of  gram- 
negative  bacteria. 

Antigen-specific  T  cell  responses  to  endotoxin  were  not  detected  in 
C57BL/10ScN  mice  which  are  genetic  nonresponders  to  endotoxin.  This  discovery 
suggested  that  the  unresponsiveness  of  cells  from  endotoxin  nonresponder 
strains  of  mice,  reported  to  be  restricted  to  B  cells  and  macrophages,  was 
also  extended  to  T  cells. 

The  antigen  specific  proliferative  system  was  also  used  to  study  the 
adjuvant  activity  of  bacterial  endotoxins.  Lymph  node  T  cells  from  mice 
immunized  with  a  mixture  of  ovalbumin  plus  endotoxin  gave  greatly  enhanced 
responses  upon  in  vitro  stimulation  with  ovalbumin,  as  compared  to  T  cells 
from  mice  immunized  only  with  ovalbumin.  Endotoxin  did  not  act  as  an  adjuvant 
in  genetic  nonresponder  strains  of  mice.  These  are  the  first  observations 
on  the  capacity  of  endotoxin  to  potentiate  the  generation  of  antigen-reactive 
T  cells  vn  vivo. 

We  would  like  to  re-emphasize  the  importance  of  the  studies  on  the 
specific  and  nonspecific  interaction  of  endotoxin  with  T  cells  with  respect 
to  the  following  points.  1)  The  discovery  of  endotoxin  specific  T  cells 
provides  the  potential  that  such  T  cells  participate  in  immune  responses  to 
endotoxins.  Based  on  this  finding,  we  would  like  to  recommend  a  re-evaluation 
of  the  paradigmatic  idea  that  endotoxin  is  purely  a  "T  cell  independent 
antigen."  2)  The  capacity  of  endotoxin  to  potentiate  T  cell  responses  to 
other  antigens  may  provide,  in  part,  an  explanation  for  the  immunotherapeutic 
activities  of  endotoxin  described  in  this  and  accompanying  progress  reports. 

This  work,  which  constitutes  a  major  portion  of  Mr.  Milner's  Ph.D. 
thesis,  was  presented  at  the  1979  Montana  Academy  of  Sciences  Meeting  (."best 
paper  by  a  graduate  student  award")  and  also  at  the  annual  meeting  of  the 
American  Society  for  Microbiology.  Mr.  Milner  is  currently  writing  2  manu- 
scripts on  these  findings. 

The  capacity  of  bacterial  endotoxin  (.ET)  to  inhibit  the  growth  of  tumors 
in  mice  genetically  responsive  or  unresponsive  to  the  diverse  biological 
effects  of  ET  was  also  studied.  Admixtures  of  10  to  250  pg  of  ET  extracted 
from  E.   coli  0113  and  viable  MC-93  tumor  cells  resulted  in  a  dose  dependent 
reduction  in  the  incidence  of  tumors  when  the  mixtures  were  injected  into 
C3HeB/FeJ  mice  (ET  responders).  This  suppression  of  tumor  growth  was  not 
the  result  of  a  direct  cytotoxic  effect  of  the  ET  for  the  MC-93  tumor  cells 
and  was  reduced  markedly  by  base  hydrolysis  of  the  ET.  Similar  doses  of  ET 

28-35 


Project  No.  Z01  AI  00077-23  LMSF 

failed  to  reduce  significantly  the  incidence  of  tumors  in  C3H/HeJ  mice  (ET 
nonresponders) .  In  addition,  ET  suppressed  the  growth  of  EL-4  tumors  in 
C57BL/10Sn  mice  (ET  responders)  but  had  no  effect  on  the  incidence  of  tumors 
in  the  ET  nonresponder  C57BL/10ScN  mice.  Results  of  experiments  with  3 
relatively  nonendotoxic  extraction  products  from  gram-negative  bacteria, 
acetone-chloroform  precipitate  (ACP),  free  lipoprotein  (BLP),  and  endotoxic 
protein  (EP)  showed  that  antitumor  activity  did  not  correlate  exclusively  with 
mitogenicity  or  toxicity.  ACP  and  BLP  were  mitogenic  for  both  C3HeB/FeJ  and 
C3H/HeJ  mice  but  did  not  suppress  significantly  the  growth  of  MC-93  tumors 
in  these  animals.  Also,  EP  was  175  times  less  toxic  than  the  standard  ET 
used  for  tumor  suppression  and  still  suppressed  75  to  90%  of  the  tumors  in 
the  C3H  mice.  Subsequent  experiments  showed  that  1)  64%  of  the  mice  which 
suppressed  primary  tumors  rejected  a  second  injection  of  homologous  tumor 
cells,  2)  tumor  immune  lymphocytes  were  detected  by  a  modified  Winn  assay  in 
the  spleens  of  mice  which  rejected  primary  tumors,  and  3)  endotoxin  did  not 
suppress  the  growth  of  tumors  in  congenitally  athymic  nude  mice.  In  general, 
these  data  indicated  that,  in  genetic  responder  mice,  the  antitumor  effective- 
ness of  endotoxin  depended  upon  elicitation  of  a  host-mediated  response, 
possibly  involving  T  cells. 

Future  plans  for  this  project  include  several  experiments  which  will 
hopefully  describe  further  the  relationship  between  the  structure  and  function 
of  bacterial  endotoxins.  Toxic  and  nontoxic  purification  products  from 
endotoxic  glycol ipid  will  be  analyzed  for  additional  biological  activities 
including  the  capacity  of  these  materials  to  suppress  murine  tumors,  act  as 
adjuvants  in  conventional  antibody  systems  and  also  in  the  generation  of 
antigen  specific  T  cells.  Additionally,  attempts  will  be  made  to  restore  the 
activity  of  B4-rechrom  by  combining  this  material  with  other  bacterial  and 
synthetic  products. 

The  antigen  specific  T  cell  proliferative  assay,  in  conjunction  with 
the  judicious  selection  of  endotoxins  extracted  from  several  species  of  gram- 
negative  bacteria  (including  those  from  defined  mutants  of  S.  minnesota) , 
will  be  used  to  study  the  fine  specificity  of  T  cell  responses  to  endotoxin 
antigens.  More  extensive  experiments  on  the  adjuvant  effect  of  endotoxin  on 
T  cell  responses  to  other  antigens  will  be  done  including  the  effect  of 
stimulating  T  cells  with  endotoxin  before  or  after  exposure  to  antigen.  Also, 
this  assay  system  will  be  used  to  evaluate  the  level  of  antitumor  immune  T 
cells  in  mice  which  have  undergone  endotoxin-induced  suppression  of  primary 
tumors. 

With  respect  to  the  suppression  of  tumors,  we  hope  to  elucidate  more 
clearly  the  cellular  requirements  for  the  antitumor  effects  of  endotoxins. 
For  these  studies,  we  plan  to  adoptively  transfer  select  subpopulations  of 
lymphoid  cells  from  endotoxin  responder  mice  to  X-irradiated  endotoxin  non- 
responder recipient  mice.  These  reconstituted  mice  will  then  be  injected 
with  tumor  cells  and  endotoxin.  Also,  we  plan  to  characterize  the  cellular 
nature  of  the  antitumor  lymphocytes  found  in  tumor  immune  mice. 


28-36 


Project  No.  Z01  AI  00077-23  LMSF 


Finally,  experiments  have  been  started  on  the  capacity  of  endotoxin 
to  facilitate,  in  vitro,  the  generation  of  tumor  immune  lymphocytes.  If 
successful,  these  experiments  may  provide  us  with  a  means  of  capitalizing 
on  the  immunopotentiating  activity  of  endotoxin  without  exposing  the  host 
animal  to  the  deleterious  side  effects  endotoxin  has  in  vivo. 


Publications: 

In  press: 

Winters,  W.  D.,  David,  E„,  and  Ribi,  E„:  Dichotomy  of  endotoxin 
action  during  adenovirus  replication  in  human  cells.  J_.  Gen.  Virol . 

Winters,  W.  D.,  David,  E.,  and  Ribi,  E.:  Effects  of  bacterial 
endotoxins  on  human  adenovirus „  J..  Gen.  Virol . 

Appendix  1.  Contract  N01  AI  72525.  Production  of  P3  and  Isolation, 
Fractionation,  and  Purification  of  Lipopolysaccharides 
(Endotoxins),  Hamilton  Biochemical  Research  Laboratory, 
Hamilton,  Montana. 

Total  Man  Years:  3        Professional:  0o7         Other:  2.3 

Annual  Funding:  $55,995.93 

This  project  was  designed,  in  part,  to  study  and  develop  methods  for 
the  isolation,  fractionation,  and  purification  of  bacterial  endotoxins. 
During  the  reporting  year,  the  contractor  has  supplied  the  Laboratory  of 
Microbial  Structure  and  Function  with  about  500  mg  of  P3  and  about  300  mg  of 
endotoxin  which  was  freed  of  peptide  contaminants  (B4)  by  pressure  elution 
chromatography  through  columns  of  microparticulate  gel.  These  refined 
compounds  have  served  as  invaluable  baseline  materials  for  numerous  tumor 
immunotherapeutic  experiments  referred  to  in  four  individual  annual  reports 
of  Laboratory  of  Microbial  Structure  and  Function. 


28-37 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF        I  PROJECT  NUMBER 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


ZOl      AI     00078-07     LMSF 


PERIOD    COVERED 


October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Microbial  Components  in  Cancer  Immunotherapy 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI: 
OTHER: 


E.  Eo  Ribi 

C.  A.  McLaughlin 

J.  L.  Cantrell 

K„  B.  Von  Eschen 

S.  M.  Schwartzman 


Acting  Chief 
Senior  Staff  Fellow 
Senior  Staff  Fel low 
Staff  Fellow 
Senior  Staff  Fellow 


LMSF  NIAID 
LMSF  NIAID 
LMSF  NIAID 
LMSF  NIAID 
LMSF  NIAID 


cooperating  units  (if  any)Dr.  B.  Zbar,  NCI;   Dr.  W»  Brehmer,  Robert  Koch   Inst.,  Berlin; 
Dr„  H.  Huckauf,  Med.  Clinic,  Berlin;  Drs.  S.   Richman  and  C.  R.  Granatek,  M.D„ 
Anderson  Hosp.,  Houston,   Dr.  Go  Vosika,  U.  of  Minn.,  Minneapolis;   Dr.   P.  Minden 
Natl.  Jewish  Hosp.,  Denver;   Dr.   R.   Parker  and  S.  M.  Strain,  Hamilton  Biochem.Lajb 


Laboratory  of  Microbial    Structure  and   Function,  Hamilton,  MT     59840 


ECTION 

Molecular  Biology  Section 


¥l'AmAM,CAB'e°£hesda,  MD     20205 


TOTAL  MANYEARS: 

4.15 


PROFESSIONAL: 

1.05 


3.1 


CHECK  APPROPRIATE  BOX(ES) 
LK(a)   HUMAN  SUBJECTS 

3(al)  MINORS   □  (a2)  INTERVIEWS 


[%   (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


OUMMARY  OF  WORK  (200  *ords  or  less  -  underline  keywords) 

The  major  objectives  of  this  project  continue  to  be  the  identification  of 
nonviable,  chemically  defined  microbial  components  that  are  efficacious 
antitumor  agents  and  to  develop  and  supply  such  preparations  in  stable, 
standardized  form  for  both  experimental  and  clinically  applied  immunotherapy. 
The  intent  of  the  research  is  to  circumvent  the  use  of  infectious  agents  and, 
by  preparing  synthetic  analogues,  provide  alternative  substances  that  are  not 
antagonistic  or  that  do  not  produce  undesirable  biological  responses. 


28-38 


PHS-6040 
(Rev.    10-76) 


Project  No.   Z01   AI  00078-07  LMSF 


Project  Description: 


The  major  objective  of  this  project  is  to  develop  clinically  useful 
immunostimulants  for  tumor  regression.     Delineation  of  the  mechanisms  of 
immunomodulation  by  microbial   components  has  been  the  goal   of  members  of  the 
Molecular  Biology  Section  (see  annual    reports  submitted  individually  by  Drs . 
McLaughlin,  Cantrell   and  Von  Eschen)» 

The  transplantable  line-10  hepatocellular  carcinoma  of  guinea  pigs 
developed  at  NCI  has  been  used  as  a  model    for  the  study  of  weakly  antigenic 
malignant  tumors.     When  6-day-old  (10  mm  in  diameter)   tumors  were  inoculated 
intralesionally  with  viable  BCG,  a  significant  proportion  of  such  tumors 
regressed  permanently  and,  as  subsequently  reported,  these  results  could  be 
equaled  by  treatment  with  nonviable  mycobacterial   cell   wall   components 
associated  with  oil   droplets  in  saline  emulsion.     They  included  purified  cell 
walls  and  deproteinized,  delipidated  cell   walls,  designated  cell   wall    skeleton 
(CWS).     Either  of  these  products,  when  combined  with  trehalose  dimycolate   (P3), 
a  lipoid  component  removed  from  the  cell   walls  which  is  also  known  as  purified 
cord  factor,  was  highly  effective.     ( P3  was  supplied  by  Dr.   R.   Parker, 
Hamilton  Biochemical    Research  Laboratory  under  contract  N01   AI  72525).  We  also 
reported  that  a  high  cure  rate  was  obtained  with  materials  unrelated  to  tubercle 
bacilli,  provided  they  were  combined  with  P3.     Particularly  impressive  in  this 
respect  were  chloroform-methanol   extracted  endotoxic  glycol ipids   (ReGl)   from 
0  antigen-deficient  (Re)  mutant  strains  of  Enterobacteriaceae  which  provided 
up  to  100%  cures. 

Whereas  highly  endotoxic  1 ipopolysaccharide  (LPS)  extracts  from  all 
wild  type  strains  so  far  tested  failed  to  cause  tumor  regression,  acid 
hydrolysis  of  Serratia  marcescens  led  to  residual    fractions  (RESI)  which 
serologically  cross-reacted  with  ReGl    samples  and  provided  a  cure  rate  of  90%. 
This  RESI  was  essentially  nonpyrogenic  and  100  times  less  lethal    for  laboratory 
animals  than  potent  endotoxins,  indicating  that  there  was  no  correlation 
between  the  antitumor  property  and  endotoxicity.     Endotoxic  glycol  ipids  ex- 
tracted from  Re  mutant  bacteria  with  a  monophasic  mixture  of  phenol,  chloroform 
and  petroleum  ether  (Galanos  et_al_.)  were  highly  endotoxic  but  relatively 
ineffective  in  regression  line-10  tumors.     This  also  indicated  that  the  most 
important  ingredient  was  not  endotoxin  itself  but  rather  some  associated 
componentCs)  that  was  extracted  with  the  glycol ipid.     In  addition,  we  noted 
that  the  lower  efficacy  of  such  extracts  was  paralleled  by  a  reduction  of  the 
content  of  muramic  acid,  alanine,  and  glutamic  acid,  which  are  characteristic 
components  of  bacterial   cell   wall    peptidoglycan.     Reduction  of  amino  acid 
content  of  ReGl    by  microparticulate  gel   chromatography  or  by  treatment  with 
Triton  X-100  significantly  lowered  its  ability  to  bring  about  tumor  regression 
without  affecting  endotoxicity.     The  chromatographically  refined  ReGl  was 
designated  B4.     We  considered  that  precursor  or  autolysis  products  of  the 
peptidoglycan  moiety  of  CWS  may  have  been  co-extracted  with  the  endotoxic 
glycolipids.     Indeed,  antitumor  activity  to  B4  could  be  restored  by  the 
addition  of  synthetic  N-acetyl-muramyl-L-alanyl-(L-seryl)-D-isoglutamine  (MDP), 
which  is  considered  the  minimal    structural    unit  responsible  for  the  adjuvant 

28-39 


Project  No.  Z01  AI  00078-07  LMSF 

action  of  microbial  cell  walls  and  CWS.  The  tumor-regressive  activity  of 
B4  could  be  also  restored  by  the  addition  of  a  nontoxic  lipoid  side  fraction 
(ACP)  recovered  during  the  isolation  of  ReGl  which  contained  a  small  amount 
of  peptidic  substances  or,  as  described  in  Dr.  Cantrell's  progress  report 
(Z01  AI  00087-02),  by  the  addition  of  Braun's  lipoprotein  known  to  contain 
covalently  bound  MDP  moieties. 

We  found  that  lowering  the  dose  of  MDP  from  150  yg  to  1.5  yg  still 
provided  a  cure  rate  of  at  least  90%  when  combined  with  15  yg  of  B4„  This 
small  amount  of  peptidic  material  is  of  the  order  of  magnitude  (about  0.1 %) 
of  the  amount  of  amino  acids  removed  from  ReGl  during  the  preparation  of  B4. 
Skin  testing  data  had  indicated  that  delayed  type  hypersensitivity  developed 
against  the  endotoxin-associated  antigens  as  well  as  specific  tumor  antigens. 
MDP  may  act  as  an  adjuvant  to  an  antigen(s)  which  may  have  cross-reactive 
determinants  associated  with  the  endotoxin  itself.  Such  antigens  appear  to 
be  cryptic  or  sterically  hindered  from  being  effective  in  polysaccharide-rich 
LPS  from  wild  type  bacteria  but  are  exposed  in  polysaccharide-deficient  ReGl 
from  Re  mutant  bacteria.  It  remains  to  be  determined  whether  this  cross- 
reactive  antigen  is  identical  with  that  shared  by  CWS,  ReGl,  the  line-10  tumor 
cell  and  certain  human  tumors,  such  as  malignant  melanoma,  as  observed  by 
Dr.  Christine  Granatek  and  Dr.  Percy  Minden.  If  so,  cross-reactive  antigens 
shared  by  microorganisms  and  tumor  cells,  and  which  are  absent  or  cryptic 
in  normal  cells,  may  represent  the  immune  determinants  against  which  MDP, 
in  concert  with  P3,  augments  antitumor  immunity.  Although  there  is  no  direct 
correlation  between  endotoxic  potency  and  tumor  regressive  activity,  our 
findings  indicate  that  a  low  level  of  toxicity  may  be  required  to  obtain  optimal 
levels  of  tumoricidal  action. 

From  a  practical  point  of  view,  the  findings  reported  here  lend  merit 
to  the  continued  search  for  products  that  synergistical ly  enhance  the  action 
of  cell  walls,  CWS,  or  MDP  but  have  low  endotoxicity.  It  was  logical,  therefore, 
to  examine  the  efficacy  of  combinations  of  clinically  tested  agents,  such 
as  Pseudomonas  vaccine  (Pseudogen),  which  is  a  registered  product  prepared 
by  Warner-Lambert/Parke-Davis,  and  is  an  LPS-containing  extract  whose  relatively 
low  endotoxicity  was,  in  fact,  comparable  to  that  of  RESI  from  S_.  marcescens. 
Unexpectedly,  we  found  that  when  a  combination  of  Pseudogen,  ReGl  or  B4 
(.150  ug) ,  and  MDP  (150  yg) ,  even  in  the  absence  of  P3,  was  inoculated  into 
established  dermal  line-10  tumors,  a  significant  number  of  the  animals  died, 
prohably  of  endotoxic  shock.  All  surviving  animals  suffered  severe  but 
temporary  lethargy.  For  yet  unexplained  reasons,  Pseudogen  was  even  more 
active  in  this  respect  than  ReGl.  These  effects  did  not  depend  upon  the 
presence  of  malignant  tissue.  When  administered  alone  intradermally  at  the 
dose  levels  tested,  none  of  the  components  caused  severe  lethargy  or  lethality. 
Guinea  pigs  inoculated  intravenously  were  even  more  susceptible  inasmuch  as 
the  addition  of  as  little  as  6  yg  of  MDP  to  150  yg  of  Pseudogen,  itself  not 
lethal,  caused  the  death  of  80%  of  the  animals.  Neither  CWS  nor  P3,  in  the 
doses  used  for  tumor  treatment,  measurably  enhanced  endotoxic  lethality. 
It  has  yet  to  be  determined  whether  the  effect  described  here  is  related  to 
the  known  phenomenon  of  increased  susceptibility  (hyperreactivity)  to  endotoxin 


28-40 


Project  No.  Z01  AI  00078-07  LMSF 

observed  in  mice  previously  infected  with  BCG,  In  any  case,  our  results 
indicate  that  caution  must  be  exercised  in  the  application  of  MDP  as  a  tumor 
therapeutic  ingredient,  or  as  an  adjuvant  in  general  ,  even  in  combination 
with  relatively  weakly  endotoxic  products,  such  as  Pseudogen,  whose  antitumor 
activity  is  presently  being  evaluated  by  others  in  human  patients. 

Eight  steps  were  needed  for  the  synthesis  of  MDP  as  prepared  by  French 
workers  in  1975.  Dr.  Steven  Schwartzman  shortened  the  procedure  by  combining 
several  of  the  operations  and  using  alanine  as  a  template  for  the  addition 
of  muramic  acid  and  isoglutamine.  The  efficacy  in  the  line-10  tumor  model 
of  several  analogues  of  MDP  prepared  by  this  simplified  method  followed  in 
the  order  of  aminobutyryl-MDP>seryl-MDP > alanyl-MDP,  These  findings 
paralleled  those  obtained  in  tests  for  adjuvanticity  as  measured  by  the 
ability  of  MDP  to  augment  delayed  type  hypersensitivity  to  arsenilic  tyrosine 
as  well  as  antibody  production  to  BSA„ 

Last  year  we  reported  that  the  combination  of  oil-attached  CWS  + 
P3  was  found  in  clinical  Phase  I-II  trials  (Dr.  Stephen  Richman  and  Dr.  Gerald 
Vosika)  to  be  an  effective  agent  when  used  intralesionally  to  treat  recurrent 
melanoma  and  cutaneous  and  subcutaneous  breast  carcinoma.  Those  studies  which 
were  continued  during  this  reporting  year  have  substantiated  these  results, 
and  a  second  manuscript  describing  treatment  of  recurrent  melanoma  has  been 
accepted  for  publication. 

Development  of  effective  methods  to  eliminate  tumors  remaining  after 
cancer  surgery  is  a  major  goal  of  oncologists.  Many  patients  with  resectable 
neoplasmas  eventually  succumb  to  progressive  growth  of  tumors  originating 
from  occult  metastasis  present  at  the  time  of  surgery.  Immunotherapy,  because 
it  produces  specific  and  systemic  antitumor  responses  is,  in  theory,  capable 
of  eliminating  malignant  disease  remaining  after  surgery.  In  collaboration 
with  Dr.  Zbar  and  his  co-workers  (NCI),  we  have  continued  to  use  the 
metastasizing  line-10  tumor  model  to  help  establish  conditions  for  successful 
post  operative  immunotherapy.  Animals  with  established  dermal  tumors  were 
treated  either  by  surgical  excision  of  the  tumor  or  by  surgery  followed  by 
administration  of  emulsified  cell  wall  preparations  alone  or  mixed  with 
hepatoma  cells.  Animals  treated  by  surgery  alone  developed  malignant  disease 
characterized  by  progressive  lethal  growth  of  lymph  node  metastases.  Injection 
of  cell  walls  or  combinations  of  cell  wall  components  intradermally  between 
the  site  of  excision  and  the  draining  lymph  node  (regional  injection)  prevented 
the  development  of  palpable  metastasis  in  some  animals.  Cell  wall  components 
given  intradermally  on  the  side  contralateral  to  the  surgical  site  (remote 
injection)  cured  no  animals.  Similar  treatments  with  preparations  containing 
both  cell  walls  and  line-10  tumor  cells  prevented  the  development  of  malignant 
disease  in  a  significant  number  of  animals  whether  the  injections  were  regional 
or  remote.  Treatment  with  mixtures  consisting  of  living  BCG  and  tumors  cells 
was  not  as  effective  as  treatment  with  cell  walls  mixed  with  tumor  cells. 
Since  injection  of  living  tumor  cells  with  cell  wall  adjuvant  into  human 
patients  with  minimal  residual  disease  might  not  be  acceptable  because  of 
the  possibility  of  tumor  growth  at  the  vaccine  site,  the  use  of  X-radiated 

28-41 


Project  No.  Z01  AI  00078-07  LMSF 

tumor  cells  was  explored.  Preliminary  data  indicate  that  X-radiated  tumor 
cells  admixed  with  cell  walls  could  replace  viable  tumor  cells  in  eradicating 
microscopic  lymph  node  metastases.  Whether  these  findings  are  relevant  to 
human  cancer  immunotherapy  remains  to  be  seen. 

Dr.  Werner  Brehmer  and  Dr.  Hans  Huckauf  initiated  a  Phase  I  study  using 
CWS  +  P3  in  patients  with  inoperable  carcinoma  of  the  lung.  Three  to  four 
intratumor  instillations  of  up  to  750  yg  of  CWS  and  375  yg  of  P3  were  per- 
formed with  the  aid  of  a  broncoscope  in  4  patients  and  by  means  of  radiologically 
directed  thoracic  puncture  in  3  patients.  Granulomatous  reactions  were  detected 
in  the  region  of  the  tumors,  but  no  ill  effects  were  noted.  Febrile  reactions 
were  either  absent  or  mild  with  the  exception  of  one  case.  Since  patients 
tolerated  these  treatments  well,  pilot  studies  were  begun  in  patients  with 
still  operable  lung  carcinoma.  The  tumors  were  injected  3  to  4  weeks  prior 
to  their  surgical  removal  in  the  hope  that  specific  tumor  immune  responses 
would  be  enhanced.  Of  course,  a  5-year  period  is  needed  to  evaluate  results 
of  this  immunotherapeutic  approach. 

The  major  courses  of  action  planned  in  this  project  include:  1) 
expansion  of  studies  with  RESI  fractions  of  low  toxicity  which,  in  combination 
with  P3,  produced  excellent  regression  of  animal  tumors  without  noticeable 
life-threatening  effects  to  determine  their  chemical  and  fine  structural 
features  responsible  for  immunotherapeutic  properties,  2)  attempts  to  alter 
MDP  so  as  to  minimize  its  ability  to  enhance  host  susceptibility  to  endotoxin 
by  covalently  li pi  dating  it  and  still  retain  its  tumor  regressive  properties, 
and  3)  continued  collaboration  with  scientists  of  other  institutes  in  attempts 
to  identify  immunologic  cross-reactive  components  shared  by  microorganisms 
and  tumor  cells  and  to  provide  effective  antineoplastic  agents  (microbial 
components  and  their  synthetic  analogs)  suitable  for  clinical  evaluation. 

Publications : 

Richman,  S.  P.,  Gutterman,  J„  U.,  Hersh,  E.  M. ,  and  Ribi,  E.  E. : 
Phase  I— 1 1  study  of  intratumor  immunotherapy  with  BCG  cell  wall 
skeleton  plus  P3.  Cancer  Immunol .  Immunother.  5:  41-44,  1978. 

Ribi,  E0,  McLaughlin,  C.  A.,  Cantrell,  J.  L.,  Brehmer,  W.,  Azuma,  I., 
Yamamura,  Y.,  Strain,  S„  M. ,  Hwang,  K.  M.,  and  Toubiana,  R. :  Immunother- 
apy for  tumors  with  microbial  constituents  or  their  synthetic  analogues. 
A  review.  In  Immunotherapy  of  Human  Cancer,  22nd  Annual  Clinical 
Conference  on  Cancer,  The  University  of  Texas  System  Cancer  Center, 
M.Do  Anderson  Hospital  and  Tumor  Institute,  Houston,  Texas.  New  York, 
Raven  Press,  1978,  pp.  131-154. 

Zbar,  B„,  Canti ,  G.,  Ashley,  M.  P.,  Rapp,  H.  J„,  Hunter,  J.  Ta,  and 
Ribi,  E„:  Eradication  by  immunization  with  mycobacterial  vaccines 
and  tumor  cells  of  microscopic  metastases  remaining  after  surgery. 
Cancer  Res.  39:1597-1603,1979. 

28-42 


Project  No.   Z01   AI  00078-07  LMSF 


Publications:     (cont'd) 


Wepsic,  H.  T. ,  Tracey,  R.  S.,  Harris,  S.,   Ribi,  E.,  and  Morris,  H„: 
Bacillus  Calmette-Guerin  cell   wall    immunotherapy  of  intramuscular 
and  metastatic  Morris  rat  hepatomas.     Cancer  Res.     38:  1217-1222, 
1978. 

Brehmer,  W.,  Huckauf,  H„,  and  Ribi,  E.:     Immuntherapie  mit  BCG  sowie 
mykobakteriellen  fraktionen  und  versuche  zur  anwendung  beim  bronchial 
karzinom.     Praxis   Pneumonologie     33:  358-365,  1979. 


In  press : 

Vosika,  G.  Jo,  Schmidtke,  J.,  Goldman,  A.,  Ribi,  E. ,  Parker,  R.,  and 
Gray,  G.:  Phase  I- I I  study  of  intra! esional  immunotherapy  with  oil 
attached  Mycobacterium  smegmatis  cell  wall  skeleton  and  trehalose 
dimycolate.  Cancer  Immunol .  Immunother. 

Ribi,  E.  Parker,  R.,  Strain,  S.  M.,  Mizuno,  Y.,  Nowotny,  A.,  Von  Eschen. 
K.  Bo,  Cantrell ,  J.  L.,  McLaughlin,  C.  A.,  Hwang,  K.  M.,  and  Goren, 
Mo  B.:  Peptides  as  requirement  for  immunotherapy  of  the  guinea  pig 
line-10  tumor  with  endotoxins.  Cancer  Immunol .  Immunother. 

Vosika,  G.  Jo,  Schmidtke,  J.  R.,  Goldman,  A.,  Ribi,  E„,  Parker,  R., 
and  Gray,  G.  R. :  Intralesional  immunotherapy  of  mal ignant  melanoma 
with  Mycobacterium  smegmatis  cell  wall  skeleton  combined  with  trehalose 
d i mycolate  CP3).  Cancer , 


28-43 


SMITHSONIAN  SCIENCE  INFORMATION 
PROJECT  NUMBER  (Do  NOT  use  this 


^XCHANGi 
space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  'OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl      AI     00087-02   LMSF 


PERIOD    COVERED 


October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Immunological  Activity  of  Eukaryotic  and  Prokaryotic  Cellular  Components 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Senior  Staff  Fellow  LMSF  NIAID 

Res,   Chemist  (Phys.  Chem.)       LMSF  NIAID 

Senior  Staff  Fellow  LMSF  NIAID 

Senior  Staff  Fellow  LMSF  NIAID 

Visiting  Associate  LMSF  NIAID 


PI: 

J. 

Lo  Cantrell 

OTHER: 

E. 

E.  Ribi 

C. 

A.  McLaughlin 

So 

Mo  Schwartzman 

Jo 

Sasaki 

cooperating  units  (if  any)  Qr .   R.  Wheat,   Duke  University  Medical    Center,   Durham,  NC; 
Dr.   G.   F.   Springer,  Evanston  Hospital,  Evanston,   IL;   Dr,   R.   T\   Tuttle,  Burrough 
Wellcome,   Research  Triangle  Park,  NC;   Drs.   J.   Zighelboim  and  Ro    I.  Murahata, 
LLC.L-.A-,   School   of  Medicine,   Los  Angeles,   CA„ 


LAB/BRANCH 

Laboratory  of  Microbial    Structure  and   Function,  Hamilton,  MT     59840 


ection 

Molecular  Biology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 

3.5 


PROFESSIONAL: 

1  .0 


2.5 


CHECK  APPROPRIATE  BOX(ES) 
\Z   (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   []  (a2)  INTERVIEWS 


(b)  HUMAN  TISSUES 


Xj  (c 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  main  goal   of  this   project  is  the  elucidation  of  the  immunological   response 
induced  by  isolated  microbial    components  and  tumor-associated  antigens  that 
are  efficacious  antitumor  agents.     In  the  past  year,  attempts  were  made  to 
1)  evaluate  fractions  of  Corynebacterium  parvum  prepared  by  phenol-water 
extraction  for  lymphoreticular  stimulation  and  antitumor  activity,  2)   isolate, 
in  soluble  form,  the  component  from  C_.   parvum  responsible  for  tumor  regression, 
3)   evaluate  inhibition  of  immunity  and  induction  of  suppressor  cells  with 
components  isolated  from  microorganisms,  4)   evaluate  tumor-associated  antigens 
isolated  from  murine  tumor  cells  and  guinea  pig  ascites  fluid  for  antitumor 
activity,  and  5)   determine  the  cross-reactivity  between  tumor  cells  and 
microbial   components. 


28-44 


PHS-6040 

(Rev.    10-76) 


Project  No.  Z01  AI  00087 -02  LMSF 


Project  Description: 


Many  studies  have  demonstrated  that  killed  suspensions  of 
Corynebacterium  parvum  possess  both  lymphoreticular  stimulatory  and  anti- 
tumor properties.  However,  other  reports  show  that  cell-mediated  immune 
responses  attributable  to  T  lymphocytes  are  often  inhibited  by  systemic 
administration  of  C_.   parvum.  Therefore,  in  collaboration  with  Dr.  R.  Wheat, 
we  have  continued  our  efforts  to  isolate  and  characterize  the  components ) 
from  Co  parvum  responsible  for  antitumor  activity  and/or  immune  suppression. 

In  last  year's  report,  preliminary  studies  demonstrated  that  both 
lymphoreticular  and  antitumor  activities  were  associated  with  the  residue 
of  phenol-water  extracted  whole  cells.  Additional  studies  were  made  to 
determine  the  nature  (chemical  or  physical)  of  the  active  component.  Frac- 
tions were  tested  in  mice  for  lymphoreticular  stimulation  by  measuring  the 
degree  of  splenomegaly  and  spleen  cell  blastogenesis  produced  by  an  i.p. 
injection „  Antitumor  activity  was  evaluated  by  measuring  the  extent  of  tumor 
growth  after  a  subcutaneous  injection  of  the  fractions  admixed  with  either 
of  3  syngeneically  transplanted  murine  tumors  (B-16  melanoma,  LI 21 0  leukemia, 
or  MC-93  sarcoma).  Significant  splenomegaly  with  concomitant  increase  in 
blastogenic  activity  was  observed  in  mice  treated  with  the  residue.  The 
kinetics  of  development  of  these  parameters  paralleled  that  observed  with 
whole  organisms.  In  addition,  significant  antitumor  activity  was  observed 
in  mice  inoculated  with  the  residue,  which  was  similar  to  the  activity  observed 
with  whole  cells.  The  soluble  extracts  (phenol  phase  and  aqueous  phase)  did 
not  induce  either  splenomegaly,  increased  blastogenesis,  or  inhibition  of 
tumor  growth.  High  antitumor-  and  splenomegaly-inducing  activities  of  the 
residue  were  retained  following  chloroformrmethanol  extraction,  SDS/pronase 
digestion,  or  RNAse/DNAse/trypsin  digestion,  whereas  these  properties  were 
significantly  reduced  after  the  residue  was  treated  with  metaperiodate,  which 
resulted  in  a  reduction  in  the  carbohydrate  concentration.  Peritoneal  cells 
from  mice  treated  with  whole  cells,  residue,  or  metaperiodate-treated  residue 
were  nonspecifically  cytotoxic  to  B-16  tumor  cells  in  vitro,  but  the  tumor- 
icidal  activity  of  peritoneal  cells  did  not  develop  in  mice  treated  with 
either  the  aqueous-  or  phenol -soluble  extracts.  Collectively,  these  results 
demonstrate  that  the  component  in  C.   parvum  responsible  for  antitumor  activity 
and  lymphoreticular  stimulation  is,  in  part,  carbohydrate  in  nature  and  that 
although  nonspecific  macrophage  activation  by  whole  cells  or  the  residue  may 
be  important,  it  is  not  sufficient  for  inducing  tumor  rejection.  These 
findings  were  reported  at  the  17th  Annual  Meeting  of  the  American  Association 
for  Cancer  Research,  May  16-19,  in  New  Orleans,  LA. 

Studies  were  also  made  this  year,  in  collaboration  with  Drs.  J. 
Zighelboim  and  R.  I.  Murahata,  into  the  ability  of  C_.   parvum  fractions 
derived  by  phenol -water  extraction  to  modulate  the  Immune  response  to  tumor 
alloantigens  in  mice.  When  the  residue  was  injected  i.v.  into  mice  9  days 
after  subcutaneous  immunization  with  allogeneic  tumor  cells,  it  mimicked  the 
effects  resulting  from  an  injection  of  whole  cells.  These  effects  included 
1)  inhibition  of  the  generation  of  primary  spleen  cell  cytotoxicity,  2) 

28-45 


Project  No.   Z01   AI  00087-02  LMSF 

inhibition  of  the  generation  and  expression  of  memory  cytotoxicity  by  spleen 
cells;  and  3)   generation  of  suppressor  cell   activity  capable  of  inhibiting 
the  expression  of  memory  cytotoxicity  by  control   alloimmune  spleen  cells. 
In  contrast,  injection  of  the  phenol-soluble  extract,  which  was  rich  in 
protein,  inhibited  the  generation  of  primary  and  memory  cytotoxicity,  but  did 
not  result  in  the  generation  of  suppressor  cell   activity.     The  aqueous  soluble 
extract,  which  was  rich  in  carbohydrates,  was  inactive  by  all    criteria  used 
in  this  study.     These  results  clearly  show  that  materials  extracted  from  £. 
parvum  differentially  effect  various  aspects  of  the  immune  response  to 
alloantigens.     These  findings  were  reported  at  the  Leukocyte  Culture  Conference, 
May,   in  Ottawa,  Canada ,, 

Studies   in  this  laboratory  have  also  been  devoted  to  isolating,   in 
soluble  form,  the  carbohydrate-like  component  from  C.   parvum  whole  cells  that 
retains  the  antitumor  activity.     Heat-killed  organisms,  supplied  by  Burroughs- 
Wellcome,  were  extracted  with  pyridine,  which  is  a  solvent  used  to  extract 
glycol ipids  from  mycobacteria,  and  the  soluble  extract  was  tested  in  vivo 
for  biological   and  antitumor  activities.     Local   lesional    immunotherapy  of 
carcinogen-induced  hepatocellular  carcinoma  (line-10)   implanted  into  the  skin 
of  syngeneic  guinea  pigs  was  used  to  measure  antitumor  activity.     Although 
not  significant,  tumor  regressive  activity  was  detected  with  the  pyridine  soluble 
extract  provided  that  it  was  combined  with  trehalose  dimycolate  (P3)   in  an 
oil-in-water  emulsion.     Based  on  our  previous  results  that  antitumor  activity 
was  associated  with  a  cell   wall   component  and  the  observation  that  the  com- 
bination of  synthetic  muramyl   di peptide   (MDP);  the  smallest  adjuvant-active 
unit  of  bacterial   cell   walls;  with  purified  endotoxin,  itself  devoid  of 
antitumor  activity,  provided  significant  line-10  tumor  regressive  potency 
(see  annual    report  Z01   AI  00078-07,  Ribi),  the  pyridine-soluble  extract  of 
C.   parvum  was  combined  with  MDP  and  P3  in  an  oil   emulsion.     High  cure  rates 
T"75  to  88%)  were  observed  in  animals  given  the  triple  combination,  a  rate 
superior  to  that  observed  with  whole  organisms.     No  activity  was  seen  when 
the  extract  was  combined  with  MDP  only.     In  studies  designed  to  evaluate  the 
lymphoreticular  stimulating  properties  of  the  pyridine  extracted  fractions, 
no  significant  increase  in  spleen  or  liver  weight  was  observed  in  mice  treated 
with  the  pyridine-soluble  extract.     Conversely,  significant  splenomegaly  and 
hepatomegaly  were  detected  in  animals  treated  with  the  cell    residue.     Thus, 
in  contrast  to  our  previous  results,  no  correlation  could  be  made  between  the 
fraction  having  antitumor  activity  and  its  ability  to  induce  splenomegaly. 
Additional    studies   in  this  laboratory  have  demonstrated  that  the  inhibition 
of  primary  immunity  to  alloantigens  was  associated  with  the  splenomegaly- 
inducing  cell    residue  and  not  the  pyridine-soluble  extract.     These  results 
suggest  that  the  components  of  C.   parvum  responsible  for  inhibition  of  immunity 
and  antitumor  activity  are  distinct  and  separable.     Studies  are  in  progress 
to  determine  whether  the  pyridine-soluble  extract  or  residue  is   involved  with 
the  induction  of  suppressor  cell    activity. 

Another  area  of  interest  to  us  is  the  use  of  specific  immunostimulants 
in  the  regression  of  established  tumors.     As  reported  last  year,   solubilized 
tumor  antigens  prepared  by  KC1   extraction  of  tumor  cells  caused  complete 

28-46 


Project  No.    Z01   AI  00087-02   LMSF 

regression  of  EL-4  lymphoma  in  syngeneic  mice.     High  cure  rates   (80%)  were 
observed  in  animals  given  combination  chemotherapy  and  immunotherapy,  rates 
significantly  higher  than  in  chemotherapy  only  control    groups.     Successful 
therapy  was  dependent  on  emulsifying  the  soluble  tumor  antigen  in  minute  oil 
droplets.     Emulsions  containing  tumor  cell   extracts,  when  tested  in  conjunction 
with  chemotherapy,  had  no  therapeutic  value.     Animals  cured  by  combination 
chemoimmunotherapy  had  detectable  tumor  specific  immunity.     In  addition  to 
the  murine  system,  attempts  were  made  to  isolate  tumor-associated  antigens 
from  the  ascites  fluid  of  strain  2  guinea  pigs  with  progressively  growing 
line-10  tumors.     Butanol -water  extracts  of  the  ascites  fluid  resulted  in  the 
acquisition  of  tumor  antigens  in  the  butanol    phase.     To  test  the  antitumor 
activity  of  the  butanol    phase,  intratumor  injections  of  emulsions  containing 
P3  combined  with  the  extract  were  given  to  guinea  pigs.     Antitumor  activity 
(30%  cures)  was  found  only  in  the  antigen-containing  butanol    fraction.     We 
also  explored  whether  the  synthetic  adjuvant-active  MDP  would  enhance  the 
efficacy  of  the  isolated  tumor  antigens  in  regressing  line-10  tumors.     Pre- 
liminary results  indicate  that  high  cure  rates   (88%)  were  obtained  when  the 
isolated  tumor  antigens  were  combined  with  MDP  and  P3  in  an  oil   emulsion  and 
injected  directly  into  the  growing  tumor.     No  activity  was  seen  in  animals 
given  this  vaccine  on  the  contralateral    side. 

In  collaboration  with  Dr.  G.  Springer,  studies  were  undertaken  to 
determine  whether  line-10  tumor  cells  and  endotoxic  fractions  isolated  from 
Re  mutant  of  Salmonella  typhimurium  cross-react  with  human  blood  group  antigens. 
Results  from  these  studies   indicate  that  both  tumor  cells  and  endotoxins   (Re 
glycol ipids  and  purified  B4,  see  annual    report  Z01   AI  00078-07,  Ribi)   have 
components  that  cross-react  with  T  antigen;  the  precursor  to  human  MN  antigens. 
In  addition,  tumor  cells  have  membrane  components  that  cross-react  with  human 
B  antigen.     Strain-2  guinea  pig  cells  contain  only  components  that  cross-react 
wi tli  human  B  antigen.     Similar  findings  were  observed  with  the  isolated  tumor 
antigens  (the  butanol   extract  of  ascites  fluid).     Studies  in  progress  are 
designed  to  determine  whether  purified  T  antigen  is  efficacious  in  regressing 
established  line-10  tumors.     Only  if  this  is  accomplished  will   we  be  able  to 
determine  the  role  of  these  cross-reactive  antigens   in  tumor  regression. 

Attempts  were  also  made  this  year,  in  collaboration  with  Dr.  Wheat, 
to  isolate  the  nontoxic,  covalently  bound  MDP-like  moieties   (Braun's  lipo- 
protein), and  pepidoglycan-free  endotoxic  fractions   C Porin)   from  Re  mutant  of 
S_,  typhimurium,  which  are  outer  membrane  components,  in  order  to  determine 
their  efficacy  in  regressing  line-10  tumors.     Results  from  these  studies 
indicate  that  the  endotoxin-containing  fraction,   Porin,  was   ineffective  in 
regresstng  tumors  when  administered  alone  or  in  combination  with  P3.     Similar 
results  were  observed  with  Braun's  lipoprotein.     However,  significant  antitumor 
activity  was  observed  in  animals  treated  with  Porin  combined  with  Braun's 
lipoprotein.     Successful   therapy  was  achieved  only  upon  the  proper  combination 
of  these  components  with  P3  in  an  oil   emulsion.     Antitumor  activity  was  also 
restored  to  Porin  by  the  addition  of  synthetic  adjuvant-active  MDP.     These 


28-47 


Project  No.  Z01  AI  00087-02  LMSF 

results  indicate  that  endotoxicity  per  se  is  not  sufficient  to  bring  about 
tumor  regression,  but  when  combined  with  a  cell  wall  adjuvant-like  component 
(MDP  or  pepidoglycan)  antitumor  activity  is  restored. 

The  future  course  of  this  project  will  include  1)  attempts  to  isolate 
a  homogeneous  material  from  the  pyridine-soluble  extract  of  C.   parvum  by 
medium  pressure  silica  gel  chromatography  with  the  eventual  goal  of  delineation 
of  the  structure  of  the  antitumor  component,  2)  attempts  to  isolate  the  com- 
ponent of  C.  parvum  responsible  for  inducing  suppressor  cell  activation  and/or 
inhibition  of  allogeneic  immunity,  3)  attempts  to  further  purify  and  character- 
ize tumor-associated  antigens  isolated  from  guinea  pig  and  murine  tumors  and 
to  evaluate  these  antigens  for  immunoprophylactic  and  immunotherapeutic  value, 
and  4)  continued  collaboration  with  Dr.  Springer  in  attempts  to  determine 
whether  the  cross-reactive  antigens  shared  between  line-10  tumor  cells  and 
microbial  components  play  a  role  in  tumor  regression.  Hopefully,  results  from 
these  studies  will  enable  us  to  better  understand  the  mechanism  of  tumor 
regression  and  immunosuppression,  which  will  enable  us  to  design  immunotherapy 
protocols  that  are  the  most  potent  and  least  harmful. 

Publications: 

Cantrell ,  J.  L„,  McLaughlin,  C.  A.,  and  Ribi,  E.:  Efficacy  of  tumor 
cell  extracts  in  immunotherapy  of  murine  EL-4  leukemia.  Cancer  Res. 
39:  1159-1167,  1979. 

In  press: 

Cantrell,   J.   L„  and  Wheat,   R.  W.:     Antitumor  activity  and  lympho- 
reticular  stimulation  properties  of  fractions  isolated  from 
Corynebacterium  parvum.     Cancer  Res. 


28-43 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl     AI     00182-01      LMSF 


PERIOD    COVERED 


October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Biochemical   and  Genetical   Mechanisms  of  Obligate  Intracellular  Parasitism 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:  J.   C.  Williams 

OTHER:     T.    F.  McCaul 
Mo   Peacock 


Senior  Asst„  Scientist       LMSF  NIAID 
NIH  Visiting   Fellow  LMSF  NIAID 

Microbiologist  EB  NIAID 


cooperating  units  (if  any)  Drs  .    Eo  Weiss  an(j  Go   naSch,  Naval   Medical    Research   Institute 
Bethesda,  MD;  and  Dr.   J.  Wild,   Dept„  of  Plant  Sciences,  Genetics  Section,  Texas 
A&M  University,  College  Station,, 


LAB/BRANCH 

Laboratory  of  Microbial   Structure  and  Function,  Hamilton,  MT    59840 


SECTION 

Rickettsial  Diseases  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 

1  .75 


PROFESSIONAL: 

0.55 


1  .1 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 


□  (b)  HUMAN  TISSUES 


jg  (c)  NEITHER 


□  (a1 )  MINORS 


(a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  objectives  of  this  project  are  to  characterize  the  biochemical  and  genetical 
mechanisms  of  obligate  intracellular  parasitism  between  the  eukaryotic  host 
and  prokaryotic  parasite.  Molecular  interactions  at  the  cell  surface  and  with 
soluble  components  are  being  Investigated  as  primary  determinants  of  metabolic 
cooperation  between  host  and  parasite.  Rickettsia  typhi  utilize  host  purine 
nucleotides  and  in  the  absence  of  glutamate,  adenosine  5'-triphosphate  (ATP) 
is  catabolized  to  AMP,  the  end  product  of  ATP  catabolism.  Therefore,  unusual 
membrane  functions  contribute  to  the  success  of  the  parasite  as  a  scavenger 
of  critical  host  components,,  Metabolic  comparisons  between  autonomously  growing 
bacteria  and  rickettsiae  have  revealed  exploitable  differences  in  antibiotic 
sensitivities.  A  cell  wall  directed  antibiotic  has  been  found  which  inhibits 
the  growth  of  R.  rickettsii  but  not  Coxiella  burnetii  or  R_„  typhi  .  Furthermore, 
this  antibiotic  restricts  the  growth  of  Rochalimaea  quintana  and  Legionella 
pneumophila  at  an  ED™  of  5  and  12  yg  per  ml,  respectively0 


28-49 


PhS-6040 
(Rev.    10-76) 


Project  No.  Z01  AI  00182-01  LMSF 


Project  Description: 


Obligate  intracellular  parasitism  is  an  enigma  which  has  eluded 
scientists  for  several  decades.  In  this  laboratory,  the  nature  of  the 
cellular  interactions  between  host  and  parasite  are  being  investigated  at 
the  biochemical  and  genetical  level.  Conceptually,  metabolic  cooperation  (MC) 
between  host  and  parasite  is  a  mechanism  of  cellular  interaction  in  which  the 
phenotype  of  the  enzyme  deficient  parasite  is  alleviated  by  specified  hosts. 
Studies  on  MC  are  designed  to  elucidate  the  nutritional  requirements  of  the 
parasite  by  applying  the  rule  that  these  organisms  must  express  certain 
bacterial  functions,  whereas  key  host  functions  must  be  preserved.  The 
alleviation  of  nutritional  requirements  may  be  mediated  via  receptors,  infor- 
mational macromolecules,  metabolic  intermediates  and/or  membrane  functions. 

Investigations  into  the  nature  of  pyrimidine  and  purine  nucleotide 
requirements  have  centered  around  enzymatic  and  transport  capabilities  of 
purified  Rickettsia  typhi „  Evidence  has  been  presented  that  these  cells  require 
host  nucleotides  because  they  do  not  possess  the  enzymic  requirements  for  the 
utilization  of  bases  and  nucleosides.  Pyrimidine  metabolism  is  greatly 
restricted  by  the  absence  of  cytidine  5'-triphosphate  (CTP)  synthetase  which 
is  the  only  enzyme  capable  of  forming  CTP  from  uridine  5'-triphosphate. 

Obligatory  features  of  bacterial  functions  have  been  discovered  in 
purine  nucleotide  metabolism.  The  metabolism  of  purine  nucleotides  by  R. 
typhi  is  controlled  by  substrate  Ci.e.,  glutamate)  availability.  ATP  is 
catabolized  by  whole  cells  and  cell -free  extracts  of  R.  typhi  to  ADP  and  then 
to  AMP,  the  end  product  of  ATP  catabolism  under  the  experimental  conditions 
which  were  used.  The  only  intermediate  of  the  pathway  from  ATP  to  AMP  which 
was  identified  by  thin  layer  chromatography  and  quantitated  by  the  '^C-content 
was  ADP.  Products  such  as  adenine,  adenosine,  hypoxanthine,  inosine,  IMP, 
ribose  and  ribose-5-P  were  not  detected.  The  enzymes  which  could  be  responsi- 
ble theoretically  for  the  catabolism  or  anabolism  of  AMP  were  not  detected 
by  standard  assay  procedures.  Most  importantly,  5 '-nucl  eotidase  and  AMP 
nucleosidase  activities  were  undetectable  under  a  variety  of  experimental 
conditions.  Although  these  two  enzymes  in  other  cells  remove  AMP  from  the 
adenylate  pool  ,  they  are  apparently  nonfunctional  with  R_.  typhi  since  the 
adenylate  energy  charge  was  drastically  lowered  by  the  high  proportion  of  AMP. 
The  biosynthesis  of  ATP  was  initiated  by  adenylate  kinase  because  no  adenine 
phosphoribosyl transferase  or  adenosine  kinase  could  be  detected.  Hence,  the 
salvage  of  purine  bases  and  nucleosides  appears  to  be  nonfunctional,  at  least, 
in  R,   typhi .  Adenylate  kinase  was  readily  separated  from  GMP  kinase  by  column 
chromatography  and  sedimentation  in  sucrose  gradients,  whereas  no  IMP  kinase 
activity  was  detected.  Although  no  IMP  formation  was  detected  with  14C-AMP 
as  substrate,  previous  analyses  of  endogenous  nucleotide  pools  by  high  pressure 
liquid  chromatography  indicated  that  IMP  exists  in  R„   typhi .  The  phosphoryla- 
tion of  nucleoside  diphosphates  is  carried  out  by  a  single  enzyme  with  a  pi 
of  5.0.  These  observations  suggest  that  the  in  vivo  activity  of  adenine 
nucleotide  interconversion  is  limited  to  the  nucleotides  with  AMP  being  the 
end  product  of  ATP  catabolism  and  that  the  salvage  of  purine  bases  and 

28-50 


Project  No.  Z01  AI  00182-01  LMSF 

nucleosides  is  not  an  essential  feature  of  purine  metabolism.  Future  studies 
are  designed  to  determine  the  in  vivo  molecular  interactions  between  rickett- 
sial membrane  function  and  host-derived  components. 

Metabolic  comparisons  between  gram-negative  rickettsiae  and  autonomous 
bacteria  have,  until  recently,  provided  little  evidence  that  primary  differ- 
ences could  be  exploited  in  terms  of  antibiotic  sensitivity.  Use  has  been 
made  of  the  cell  wall  directed  antibiotic  fosfomycin  (FFM)  which  enters  cells 
via  the  hexose  phosphate  (uhp)  and  the  L-a-glycerophosphate  (git)  transport 
system.  Three  strains  of  the  genus  Rickettsia  were  tested  for  susceptibility 
to  FFM  by  their  growth  in  tissue  culture,  embryonated  hens'  eggs  and/or  in 
mice.  The  drug  was  not  cytotoxic  to  embryonated  eggs  at  10  mg  per  egg,  mice 
at  0.6  mg  per  mouse  or  in  tissue  culture  LM3  cells  at  0.1  mg  per  ml.  Observa- 
tions with  R.   typhi  grown  in  irradiated  or  unirradiated  LM3  cells  indicated 
that  FFM  may  have  a  growth  inhibitory  action  at  concentrations  that  were 
cytotoxic  for  the  host  cells.  The  biological  activity  (ability  to  hemolyze 
sheep  red  blood  cells)  of  R.   typhi  was  not  impaired  at  concentrations  of 
33  pg  per  ml „  In  embryonated  eggs  10  mg  per  egg  did  not  alter  the  average 
day  of  death  (ADD)  of  the  embryos.  In  mice,  FFM  at  0.6  mg  per  mouse  did  not 
alter  the  ADD  of  mice  injected  with  a  sufficient  dose  of  R.   typhi  to  kill 
100%  of  the  animals  within  1  week0  Coxiella  burnetii  was  resistant  to  FFM 
up  to  10  mg  per  egg  (250  pg/ml ) ,  whereas  R.   rickettsii  was  sensitive0 
Epicellular  organisms  such  as  Rochalimaea  quintana  and  Legionella  pneumophila 
were  sensitive  to  the  effects  of  FFM  at  an  ED50  of  5  yg  per  ml  and  12  pg  per 
ml,  respectively.  Embryonated  eggs  were  easily  eradicated  of  JL.  pneumophila 
at  5  mg  per  egg.  FFM  can  be  employed  during  the  infection  cycle  and  growth 
phase  in  the  presence  of  the  drug  since  _R0  typhi  and  £.  burnetii  are  not 
susceptible.  Therefore,  there  appears  to  be  no  direct  antibiotic  effect  on 
extracellular  rickettsiae  which  have  not  penetrated  cells  and  started  to 
multiply.  More  importantly,  it  is  not  necessary  to  remove  the  antibiotic  for 
the  infection  to  proceed  from  cell-to-cell.  Since  FFM  is  bacteriocidal,  it 
allows  the  complete  elimination  of  epicellular  organisms  and  autonomously 
growing  contaminants.  The  mechanism  of  this  apparent  resistance  of  rickett- 
siae to  FFM  is  currently  under  investigation.  These  studies  will  provide 
information  on  cell  wall  biosynthesis  and  the  molecular  interactions  between 
rickettsial  and  host  determinants „ 

Investigations  carried  out  on  the  rickettsiae  require  that  an  auto- 
nomously growing  gram-negative  bacterium  be  used  whenever  possible  as  a 
positive  and  negative  control.  We  have  routinely  used  Salmonella  typhimurium 
LT2  and  various  strains  to  demonstrate  enzymatic  activity  and  transport 
functions.  If  an  enzyme  or  transport  process  is  undetectable  in  the  rickett- 
siae, then  appropriate  mixing  of  rickettsial  extracts  to  fractions  of  S_. 
typhimurium  must  be  carried  out  so  that  a  determination  can  be  made  about 
specific  or  nonspecific  enzyme  or  transport  inhibition  of  rickettsiae. 
Previous  studies  with  JR.  typhi  indicated  that  the  transport  of  uridine  did 
not  occur;  however,  S_<,  typhimurium  transport  was  clearly  demonstrable. 


23-51 


Project  No.  Z01  AI  00182-01  LMSF 

S_.  typhimurium  LT2  possesses  complex  transport  systems  for  uracil  and 
uridine  which  have  been  analyzed  in  mutations  affecting  pyrimidine  salvage 
of  bases  and  nucleosides.  The  transport  of  intact  uridine  is  characterized 
by  an  apparent  Km  of  4,28  +  0.37  yM  with  an  apparent  Vmax  of  5.35  +  0.18  pmole 
min-1  yg  protein"^.  Uridine  transport  requires  a  binding  protein,  an  enzymatic 
component  (uridine  kinase,  URKase  encoded  by  udk) ,  and  is  subject  to  induction 
by  uridine.  The  transport  of  uracil  depends  upon  a  binding  protein  and  an 
enzymatic  component  (uracil  phosphoribosyltransferase,  UPRTase  encoded  by 
upp).  Four  mutants  which  are  defective  in  components  for  base  and  nucleoside 
salvage  were  employed  to  characterize  the  transport  systems  according  to  1) 
their  transport  of  uracil  and  uridine,  2)  the  presence  or  absence  of  specific 
enzymes,  and  3)  the  localization  of  the  enzymes  and  binding  proteins.  Strains 
S177  and  HD1043  are  UPRTase"  and  do  not  transport  uracil,  whereas  strain  JL411 
(URKase")  transports  uracil  at  89%  and  uridine  at  5%  of  the  rate  of  strain 
LT2„  Periplasmic  binding  proteins  were  identified  as  components  of  both 
transport  systems,  and  the  uridine  binding  protein  was  identified  as  a 
separate  component  from  URKase.  A  schematic  model  for  the  transport  of  uracil, 
uridine  and  uridine  from  uridine  5'-monophosphate  is  presented  in  which  the 
first  step  involves  the  participation  of  a  periplasmic  binding  protein  for 
uracil  or  uridine.  Uridine  may  be  degraded  to  uracil  and  ribose-1-P,  whereas 
uridine  5' -monophosphate  is  degraded  to  uridine  and  inorganic  phosphate  with 
subsequent  binding  of  the  pyrimidine  moiety.  The  transport  system  is  highly 
discriminatory  reflecting  the  components  of  the  model  from  which  a  new  class 
of  mutants  may  be  defined  as  those  cells  lacking  uracil  or  uridine  binding 
activity. 

Since  R_.  typhi  did  not  transport  uracil  or  uridine,  we  tested  the 
transport  of  pyrimidine  nucleotides.  Preliminary  results  indicate  that  the 
nucleotides  are  transported  intact  by  R_.  typhi .  This  is  clearly  a  property 
of  obligate  intracellular  parasitism,  and  more  studies  are  required  to 
determine  the  complex  nature  of  their  transport  system. 

The  future  course  of  this  project  will  center  around  4  areas  as 
specified  below:  1)  The  in  vivo  molecular  interactions  between  rickettsial 
membrane  function  and  host-derived  components  will  be  investigated  by  ultra- 
structural  cytochemistry  and  autoradiography.  The  objective  is  to  show 
metabolic  cooperation  between  host  and  parasite  during  the  infection  cycle 
by  observing  the  expression  of  bacterial  functions.  Cell  organelle  and 
membrane(s)  will  be  examined  for  phosphatase  activities  which  are  absent  in 
the  parasite  but  present  in  the  host.  2)  Utilization  of  host  nucleotide 
pools  by  R.  typhi  in  selected  tissue  culture  strains  will  be  examined.  An 
attempt  will  be  made  to  correlate  rickettsial  growth  with  nucleotide  avail- 
ability. The  bacterial  enzymes  participating  in  nucleotide  pool  interconver- 
sions  will  be  analyzed.  3)  Cell  wall  biosynthesis  by  C.   burnetii ,  R.   typhi 
and  R.  rickettsii  will  be  studied.  The  differential  sensitivity  to  FFM 
already  demonstrated  will  be  used  as  a  probe.  4)  Rickettsial  and  legionaires 
strains  will  be  screened  for  plasmids.  If  plasmids  are  found,  functional 


28-52 


Project  No.  Z01  AI  00182-01  LMSF 


aspects  of  parasitism  may  be  correlated  with  protein  patterns,  metabolic 
differences,  and  such  characteristics  as  enhanced  or  depreciated  virulence, 
invasiveness,  etc. 


Publications ; 


Williams,  J.  C.  and  Weiss,  E„:  Energy  metabolism  of  Rickettsia  typhi 
pools  of  adenine  nucleotides  and  energy  charge  in  the  presence  and 
absence  of  glutamate.  J.  Bacteriol .  134:  884-892,  1978. 

Williams,  J.  C,  Kizaki,  H.,  Weiss,  E.,  and  Weber,  G. :  Improved 
radioisotopic  assay  for  cytidine  5 '-triphosphate  synthetase  (EC 
6.3.4,2)  Anal.  Biochem.  91:  46-59,  1978. 


In  press: 

Williams,  J.   C,  Lee,  Co   E.,  and  Wild,  J„   R. :  Genetic  and  biochemical 

characterization  of  distinct  transport  systems  for  uracil,  uridine  and 

cytidine  in  Salmonella  typhimurium.     Mol .  Gen.  Genet. 


28-53 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF        j  PROJECT  NUMBER 
HEALTH,  EDUCATION,  AND  WELFARE  j 

PUBLIC  HEALTH  SERVICE        „,    .,   „„„„..  „, 

notice  of  ZOl      AI     00183-01 

INTRAMURAL  RESEARCH  PROJECT 


LMSF 


PERIOO    COVERED 


October  1,  1978  to  September  30,   1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Structural  and  Functional  Relationships  of  Bacterial  Antigens  in  the  Immune 
Response 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Senior  Asst„  Scientist  LMSF  NIAID 

Res.   Chemist  (Phys.   Chem.)  LMSF  NIAID 

Senior  Staff  Fellow  LMSF  NIAID 

NIH  Visiting   Fellow  LMSF  NIAID 

Microbiologist  EB  NIAID 


PI: 

J. 

C.  Williams 

OTHER: 

E. 

E.  Ribi 

J. 

L.  Cantrell 

T. 

F.  McCaul 

M, 

Peacock 

cooperating  units  (if  any)    Drs o   £o  Weiss  and  G.   Dasch,  Naval   Medical    Research   Institute;, 
Bethesda,  MD  and  Dr.  C.   0.   Kindmark,  Asstc  Head  of  the  Dept.  of  Infectious 
Diseases,   University  Hospital,  S-75014  Uppsala,  Sweden 


lab/branch 
Laboratory  of  Microbial   Structure  and  Function,  Hamilton,  MT     59840 


SECTION 

Rickettsial  Diseases  Section 


INSJJIUJE   <JND    LOCATION 

NTAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 

3.20 


PROFESSIONAL: 

1.95 


1  .25 


CHECK  APPROPRIATE  BOX(ES) 
G  (a)   HUMAN  SUBJECTS 


□  (b)  HUMAN  TISSUES 


|X|  (c)  NEITHER 


□  (a1  )  MINORS 


(a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  objectives  of  this  project  are  to  characterize  the  antigens  of  the 
rickettsiae  and  other  bacteria  which  are  pathogenic  for  humans.  Structure 
and  functional  aspects  of  the  immunomodulation  by  the  cell  wall  and  soluble 
components  are  currently  being  analyzed  as  potential  candidates  for  subunit 
vaccines  and  as  tumor-regressive  components.  Studies  employing  Coxiella 
burnetii  have  revealed  a  particulate  component  which  is  nontoxic,  provides 
protection  against  a  lethal  challenge  in  mice  and  guinea  pigs  and  regresses 
the  line-10  tumors  in  strain-2  guinea  pigs.  This  particulate  material  is 
potentially  a  nontoxic  vaccine.  Soluble  components  have  been  identified  as 
putative  early  diagnostic  antigens  which  induce  specific  antibody  during 
infection  but  not  after  immunization. 


28-54 


P^S-6040 
(Rev.  IO-76) 


Project  No.  Z01  AI  00183-01  LMSF 


Project  Description: 


The  objectives  of  this  project  are  to  elucidate  the  chemical  structure 
required  for  the  activation  of  the  immune  responses  against  bacterial  infections, 
Specifically,  our  goal  is  the  establishment  of  standard  preparative  procedures 
for  fractionating  whole  cells  into  constituent  components  and  the  analysis 
of  each  component  in  animal  models  and  in  vitro  studies.  Fractionation  pro- 
cedures involve  direct  whole  cell  extractions  with  organic  solvents,  separation 
of  the  cell  wall  components  and  analysis  of  aqueous-soluble  fractions.  The 
constituent  components  of  each  fraction  will  be  evaluated  for  immunological 
efficacy  in  the  mouse,  guinea  pig  and  rabbit.  The  ultimate  aim  is  the  develop- 
ment of  standard  procedures  for  rapid  analysis  of  various  components  of 
microorganisms  pathogenic  for  humans.  Subsequently  the  establishment  of  such 
procedures  will  lead  to  basic  studies  on  the  development  of  limited  use 
vaccines. 

A  new  procedure  has  been  devised  for  the  purification  of  live  and 
infectious  Coxiella  burnetii  in  gram  quantities  from  the  yolk  sacs  of  embryo- 
nated  eggs.  This  technique  allows  purification  to  homogeneity  and  complete 
separation  from  host  components.  Organisms  purified  by  this  procedure  are 
being  employed  for  biochemical,  electron  microscopic  and  immunological  studies. 

Current  investigations  in  collaboration  with  Drs.  Cantrell  and  Ribi 
and  Mr.  Peacock  are  being  conducted  with  C_0  burnetii  o  Studies  designed  to 
evaluate  the  biological  activities  and  immunoprophylactic  capabilities  of 
whole  cells  and/or  cellular  components  have  revealed  the  following  results: 

1)  Killed  whole  cells  of  £,  burnetii  induce  severe  splenomegaly,  liver 
necrosis,  and  blastogenic  activity  in  mice.  The  severity  of  these  phenomena 
was  dose  dependent.  In  fact,  high  doses  (300  yg  per  mouse)  of  killed  whole 
cells  were  lethal  for  C57BL/10  and  not  for  BDFi  (C57BL/6  X  DBA2).  The  live 
organism  was  lethal  for  C57BL/10  with  an  LD50  of  2.6  X  1 0a  organisms  per  mouse. 

2)  The  residual  particulate  material  from  chloroform :methanol  CC:M)- 
or  hot  phenol :water-extracted  whole  cells  did  not  induce  severe  splenomegaly, 
however  lymphocyte  stimulating  activity  was  retained.  More  importantly,  no 
liver  necrosis  was  detected  in  mice  given  1  mg  of  the  C:M  residue. 

3)  Humoral  immunity,  as  measured  by  microagglutinin  and  fluorescent 
antibody  techniques,  was  induced  in  mice  immunized  i.p.  with  either  whole 
cells  or  C:M  residue.  The  antibody  titer  in  sera  from  mice  given  C:M  residue 
was  decreased  following  absorption  with  either  whole  cells  or  C:M  residue. 

A  similar  reduction  in  antibody  titer  was  observed  in  the  sera  of  mice 
immunized  with  whole  cells  after  absorption  with  C:M  residue.  No  decrease 
in  antibody  titer  was  seen  after  these  sera  were  absorbed  with  the  aqueous 
phenol :water  extract. 


28-55 


Project  No.  Z01  AI  00183-01  U1SF 

4)  Immunodiffusion  studies  detected  at  least  2  different  antigens  in 
the  proxoplasm  of  C.   burnetii  which  were  not  identical  to  those  observed  in 
the  aqueous  phenol :water  and  C:M  soluble  extracts.  At  least  one  additional 
antigen  has  been  detected  as  being  related  to  infection,  whereas  immunization 
with  killed  whole  cells  or  with  fractions  does  not  induce  an  antibody  response 
to  this  antigen.  Soluble  antigens  from  live  preparations  have  been  identified 
which  were  not  detected  when  formalin  was  used  to  kill  the  organisms  before 
purification. 

5)  Studies  using  the  guinea  pig  have  revealed  that  £.  burnetii  are 
lethal  at  high  concentrations,  whereas  killed  whole  cells  are  not  lethal. 
The  C:M  residue  induces  humoral  antibody  and  protects  against  a  lethal  chal- 
lenge (10^  organisms  per  guinea  pig). 

6)  Anti-tumor  activity  in  suppression  tests  was  observed  in  mice  given 
tumor  cells  admixed  with  either  whole  cells,  cell  walls,  or  purified  whole 
cells  of  _C.  burnetii.  Conversely,  no  anti-tumor  activity  was  seen  in  tumor 
cells  treated  with  aqueous  phenol :water  extract  or  C:M  residue.  Regression 
of  the  line-10  tumor  in  the  strain-2  guinea  pig  was  observed  with  whole  cells, 
cell  walls  and  the  C:M  residue.  The  C:M  residue  from  extracted  whole  cells 
and  cell  walls  appear  to  be  a  good  candidate  for  a  nontoxic  vaccine  since  the 
present  whole  cell  vaccine  induces  all  of  the  severe  side  effects. 

In  collaboration  with  Dr.  C.  0.  Kindmark,  soluble  protoplasmic  com- 
ponents are  being  investigated  for  the  rapid  diagnosis  of  rickettsial  diseases. 
Currently  we  are  studying  a  soluble  antigen  from  C_.   burnetii  which  is  obvi- 
ously recognized  by  the  immune  system  during  infection  but  not  during 
immunization.  The  detection  of  circulating  antibodies  and/or  antigen  in 
patients  will  be  useful  in  the  clinical  management  of  Q  fever. 

The  future  course  of  this  project  will  center  around  the  3  areas  as 
specified  below:  1)  £.  burnetii  will  be  fractionated  into  cell  wall  and 
protoplasm.  The  protective  antigens  will  be  localized  and  separated  from 
toxic  factors  and  the  efficacy  of  each  component  will  be  compared  to  the 
present  whole  cell  vaccine.  2)  Membrane  proteins,  carbohydrates  and  other 
components  will  be  compared  in  various  strains  of,rickettsiae.  Extracellular 
proteins  and  glycoproteins  will  be  identified  by    I  labeling  patterns  and 
two-dimensional  chromatography.  Carbohydrates  will  be  identified  by  cyto- 
chemical  staining.  3)  Early  detection  of  antigens  and  antibodies  will  be 
attempted  by  employing  homogeneous  antigens  and  monospecific  antisera.  Antibody 
classes  and  subclasses  induced  by  various  homogeneous  antigens  will  be  analyzed 
during  immunization  as  compared  to  infection  carried  out  in  mice  and  guinea 
pigs. 

Publications:  None 


28-56 


Laboratory  of  Persistent  Viral    Diseases 

Rocky  Mountain  Laboratories 

Hamilton,  Montana 

1979  Annual    Report 

Table  of  Contents 


Z01   AI 
Project  Number 

Summary 

00072-08 

00073-14 

00074-07 

00085-02 
00086-02 


Host  Defense  Mechanisms  in  Viral    Diseases 

Mechanisms  of  Immunity  and  Immunopathology  in  Virus 
Related  Diseases 

Host  Defense  Mechanisms   in  Chronic  Viral   and 
Neoplastic  Diseases 

Biology  of  Aleutian   Disease  Virus 

Mechanisms  of  Immune   Recognition  of  Viral   Antigens 
in   Persistent  Viral    Diseases 


Page 
29-1 
29-8 

29-12 

29-17 
29-21 

29-24 


Annual    Report 

Laboratory  of  Persistent  Viral    Diseases 

Rocky  Mountain   Laboratories 

Hamilton,   Montana 

National    Institute  of  Allergy  and  Infectious   Diseases 

October  1,   1978  to  September  30,   1979 

RESEARCH   HIGHLIGHTS 

The  influence  of  the  Rfv-1   and  Rfv-3  genes  on   Friend  virus   leukemia. 
The  Rfv-1    gene,  associated  with  the  H-2D  region  of  the  major  histo- 
compatibility complex,  has  a  strong  influence  on   recovery  from  Friend  virus- 
induced  leukemia.     Recent  studies   using  irradiation  chimeras   indicated  that 
the  Rfv-1   effect  could  be  transferred  by  spleen  and  bone  marrow  cells   to 
irradiated  recipients.     Furthermore,   transfer  of  spleen  cells   from 
neonatally  tolerized  mice  to  nonirradiated  H-2  hemi-syngeneic  mice  suggested 
that  the  Rfv-1    (H-2D)   genotype  of  the  leukemia  cells   did  not  determine  the 
Rfv-1    effect  on  recovery  from  leukemia.     On  the  contrary,   the  Rfv-1    genotype 
of  the  immune  system  appeared  to  control    the  incidence  of  recovery   from 
leukemia.     Our  current  interpretation  is  that  the  Rfv-1    gene   influences   the 
kinetics  of  the  virus-specific  cytotoxic  T  lymphocyte  response  to   FV  leukemia 
cells,  and  thus  appears  similar  to  other  H-2D-associated  immune   response 
genes  which  influence  cytotoxic  T  lymphocyte   responses. 

The  Rfv-3  gene  acts   in  a  complementary  manner  with  Rfv-1    to  influence 
recovery  from  leukemia.     The  exact  linkage  of  Rfv-3  is   not  known,   but  it  is 
not  linked  to  the  H-2,   Fv-2  or  Ig-1    loci.       Rfv-3  may  be  an   "immune   response 
gene",  as   the  Rfv-3r/s   genotype  is  associated  with  a  positive  cytotoxic 
anti-FV  leukemia  cell   antibody  response.     This  occurs   also  in  the   face  of 
persistent  leukemia  in  association  with  the  low  recovery  Rfv-1    genotype. 
These  anti-FV  antibodies   cause  modulation  of  virus-induced  leukemia  cell 
surface  antigens.     Leukemic  spleen  cells   become  insensitive  to  lysis   by 
antibody  and  complement  and  show  a   dramatic  reduction  in  virus   release   in 
spite  of  persistent  infection.     As  a   result,   cell-free  spleen  virus   titers 
are  decreased  and  viremia  is  eliminated.      It  is   currently  unclear  whether 
the  modulation  of  FV  cell   surface  antigens   interferes  with  elimination  of 
leukemia  cells   by  cytotoxic  T  lymphocytes.     However,   the  genetic  com- 
plementation observed  between  the  Rfv-1    and  Rfv-3  genes  which   is   necessary 
for  recovery  from  leukemia  would  suggest  that  these  effects  are  not 
antagonistic  and  may  involve  different  viral   cell   surface  molecules. 
(Britt,   Chesebro,   Doig) 

Variations  in   Friend  virus-expression  in  leukemia  cell    clones. 
FV  leukemia  cell   clones  with  variations   in  expression  of  virus-induced  cell 
surface  proteins  have  been  isolated.     One  clone  is   deficient  in   processing 
of  "gag"   proteins    (plO,  pl2,   p!5,   p30)   but  has   normal    "env"   proteins    (gp70 


29-1 


and  pl5E).     Another  clone  has   normal    "gag"  expression  but  lacks   the   "env" 
proteins.     These  clones  are  being  used  to  characterize  the  specificity  of 
host  humoral   and  cell-mediated  immune   responses   to   FV  leukemia.      In  addition, 
they  are   being  studied  as  models  of  non-virus   producing  persistently  infected 
cells  which  appear  late  in  the  course  of  leukemia  in  mice  with  the  Rfv-3r/s 
genotype.      (Collins,   Chesebro) 

Alteration  of  expression  of  H-2K  and  H-2D  region  molecules   in  a  clone 
of  virus-induced  leukemia  cells.     A  clone  of  leukemia  cells   derived   from 
an  H-2D/b  lymphoma   (FBL-3)  was   found  to  be  devoid  of  expression  of  antigens 
determined  by  the  H-2D  subregion.     On  the  other  hand,   H-2K  determinants 
recognized  by  serological    reagents  were  normally  expressed.     However,   cells 
of  this   clone  were  unable  to  stimulate  or  block  T  cell-mediated  cytolytic 
responses   directed  at  H-2K°  determinants.     The  dissociation  of  antibody- 
mediated  and  T  cell-mediated  recognition  of  H-2KD  determinants   in  a 
homogenous   cell    population   implies  that  unique  determinants  may  be   recog- 
nized by  each  of  these  immune  mechanisms.     The  lack  of  any  H-2DD  determinants 
on   these  cells  suggests  a  possible  role  of  H-2D^  region   in  control   of  some 
aspects  of  H-2KD  recognition  or  expression.     These   findings   are  being 
pursued  by  biochemical   analysis  of  the  H-2KD  molecules   from  this   clone 
compared  to  molecules    from  normal    spleen  cells  and  EL-4  lymphoma  cells. 
Additional    variant  cell    lines   are  also  being  selected  in  the  presence  of 
specific  antibody  or  cytotoxic  T  cells  to  study  the  relationship  between 
H-2K  and  H-2D  subregions.      [Portis,   Kindt   (Laboratory  of  Immunogenetics ) , 
Colligan   (Laboratory  of  Immunogenetics)] 

M.   tuberculosis   vaccine  induces  a   unique  type   II   interferon   in  mice. 
Mice  inoculated  with  an  oil    droplet  emulsion  of  nonviable  M.   tuberculosis 
exhibit  enhanced  resistance  to  encephalomyocardi ti s   virus  TEMCV) .     This 
effect  is  not  virus-specific  since  vaccinated  mice  also  show  increased 
resistance  to  herpes   simplex  virus    (type   II)  and  rabies   virus.     Recent 
experiments   indicate  that  a  soluble  antiviral   mediator  can  be   found  in 
tissue  culture   fluids   from  peritoneal    cells   removed  from  vaccinated  mice. 
This   factor  blocks   the  spread  of  EMCV  and  other  virus   infections   in  cell 
monolayers   in  vitro.     This  mediator  is   neutralized  by  antiserum  to  mouse 
type   II  interferon,  but  differs   from  type   II  interferon   in  that  it  is   acid 
(pH  2)   sensitive  and  heat  labile,  and  is   inactive  in  a  subline  of  mouse   L 
cells   in  which  other  preparations  of  mouse  type   II   interferon  are   fully 
active.      (Lodmell,   Pusateri ,   Cent) 

Characterization  of  Aleutian   disease  virus  of  mink.     The  Gorham 
strain  of  Aleutian  disease  virus    (ADV)   of  mink  has   been   grown   in   Crandall 
feline  kidney  cells,   labeled  with   ^-thymidine  and  purified  in  CsCl 
gradients.     Separate  peaks  of  virus   infectivity  were  observed  at  densities 
of  1.35  and  1.43  gm/ml .     These  densities   and  the  incorporation  of  thymidine 
into  virion  nucleic  acid  suggests   that  these  viruses   are   both  members  of 
the  parvovirus   group.     Although  neither  virus   peak  produced  virulent  in- 
fection in  sapphire  mink,   both  cross-reacted  antigenically  with  a  virulent 
virus   strain   (Utah   I)   purified  from  mink  with  Aleutian   disease.      (Bloom) 


29-2 


Cell -mediated  immune   response  to  Aleutian   disease  virus  of  mink. 
Certai n  strains  of  mink  infected  with  Aleutian   disease   virus    (ADV)   devel op 
very  strong  specific  antiviral   antibody  responses  and  usually  succumb  to 
immune  complex  glomerulonephritis  or  arteritis.     Lack  of  successful    regula- 
tion of  the  immune   response  to  ADV  appears   to  be  a  significant   factor  in 
this  disease  since  serum  immunoglobulin  levels  are  often  elevated  to  levels 
as  high  as   50  mg/ml   and  many  organs  become  massively  infiltrated  with   plasma 
cells.     To  study  cellular  aspects  of  immunoregulation  during  Aleutian 
disease,  an  ADV-specific  T  lymphocyte  proliferative  assay  has  been   developed. 
Mink  with  advanced  disease  have  strong  responses,  and  those  in  early  stages 
of  disease  have  weak  responses.     The  cellular  immune  response  to  ADV  is 
being  compared  to  the  response  to  protein  antigens    ( KLH  and  HGG)   to  search 
for  antigen-specific  suppressor  cells   capable  of  regulating  the  immune 
response  to  these  antigens  and  see  if  such  cells  are  absent  in  ADV-infected 
mink.     (Race,  Coe) 

Structural   and  genetic  analysis  of  hamster  female  protein   (FP). 
A  sex-limited  serum  protein   (FP)  of  female  hamsters   has  been   found  to  exist 
in  electrophoretically  distinct,   but  antigenically  similar,   forms   in   3 
inbred  strains  of  hamsters.     Using  these  electrophoretic  variants,   genetic 
studies  are  now  under  way  to  search   for  possible  linkage  of  FP  to  the  major 
histocompatibility  locus   in  hamsters   because  of  the  similarity  between   FP 
in  hamsters  and  SLP  in  mice.      (Coe) 


29-3 


29-4 


Annual  Report 

Laboratory  of  Persistent  Viral  Diseases 

Rocky  Mountain  Laboratories 

Hamilton,  Montana 

National  Institute  of  Allergy  and  Infectious  Diseases 

October  1,  1978  to  September  30,  1979 

ADMINISTRATIVE  REPORT 

Administrative,  organization  and  other  changes.     The  Laboratory  of 
Persistent  Viral    Diseases  was   created  during  the  past  year  and  has   drawn 
together  in  a  single  group  the  researchers  at  the  Rocky  Mountain  Laboratories 
working  on  various  aspects  of  virus-host  interactions  in  persistent  virus 
diseases. 

During  the  past  year  3  graduate  students   from  the  Department  of 
Microbiology,   University  of  Montana,  Mr.   R.   Cent,  Ms.  A.   Pusateri   and 
Mr.  W.   Tino,  were  appointed  student  workers   to  carry  out  the   research 
portion  of  their  graduate  studies   under  the  preceptorship  of  members   of  the 
laboratory.     Mr.   D.    Doig,   Department  of  Microbiology,  Montana  State 
University,   left  the  laboratory  after  completion  of  research   for  the   Ph.D. 
degree  under  Dr.   Chesebro. 

Dr.    Frank  Waxman  and  Dr.   Miles   Cloyd  joined  the  laboratory  in 
August  to  begin  work  as  Staff  Fellows   under  Drs .   Coe  and  Chesebro, 
respectively. 


29-5 


29-6 


Annual    Report 

Laboratory  of  Persistent  Viral    Diseases 

Rocky  Mountain  Laboratories 

Hamilton,  Montana 

National    Institute  of  Allergy  and  Infectious   Diseases 

October  1,  1978  to  September  30,   1979 

HONORS  AND  AWARDS 

Editorial    Boards  of  Journals: 

Dr.   B.   Chesebro 

Associate  Editor  -   Journal   of  Immunology 

Ad  hoc  Reviewer    -  Journal   of  the  National   Cancer  Institute 

-   Proceedings  of  the  National   Academy  of  Sciences 

Dr.   D.   L.   Lodmell 

Associate   Editor  -   Journal   of  Immunology 

Professional   Posts: 

Dr.   B.   Chesebro  -  Member,   Immunobiology  Study  Section,   Division  of  Research 
Grants,  NIH;  Adjunct  Professor  -   Department  of  Microbiology,   Montana 
State  University,   Bozeman. 

Dr.   D.   L.   Lodmell   -   Faculty  Affiliate,   Department  of  Microbiology, 
University  of  Montana,  Missoula 

Invited  Lectures   and  Participation   in  Meetings   and  Symposia: 

Dr.   B.   Chesebro  -   Invited  Speaker,   Gordon  Research   Conference  on  Viruses 
and  Animal    Cells,   Boston 


29-7 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl      AI     00072-08  LPVD 


PERIOD    COVERED 


October  1,   1978  to  September  30,   1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Host  Defense  Mechanisms   in  Viral    Diseases 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:  D.    L, 

OTHER:  J.    L. 


Lodmel 1 
Port  is 


Senior  Scientist 
Medical    Officer  (SURG) 


LPVD  NIAID 
LPVD  NIAID 


COOPERATING    UNITS    (if    any) 

Anne  M.   Pusateri  ,   Dept.  of  Microbiology,   University  of  Montana,   Missoula,  MT 
Robert  R.   Cent,   Jr.,   Dept.   of  Microbiology,   University  of  Montana,  Missoula,  MT 


LAB/BRANCH 

Laboratory  of  Persistent  Viral    Diseases,   Hamilton,   MT 


INSTITUTE   AND    LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 

2.0 


PROFESSIONAL: 

1  .0 


1  .0 


CHECK  APPROPRIATE  BOX(ES) 
C  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


fe   (c)    NEITHER 


SUMMARY    OF    WORK    (200   words    or   less   -   underline    keywords) 

The  major  objective  of  this   project  is   to   delineate,  at  the  humoral    and 
cellular  level  ,  the  mechanisms  of  host  resistance  to  viral    diseases.     The 
principal   model    for  these  studies   involves   induction  of  nonspecific   resistance 
of  mice  to  encephalomyocarditis   virus   infection  by  an  oil-droplet  emulsion 
of  nonviable  Mycobacterium  tuberculosis.     Additional    studies   have  been 
initiated  to  study  the  pathogenesis  and  neuro immunology  of  rabies   virus- 
infected  mice,  and  to  ascertain  the  mechanism(s)   by  which  mice  abort  central 
nervous  system  infections  and  recover  from  paralytic  disease. 


29-8 


PH 3-6040 
(Rev.    10-76) 


Project  No.    Z01    AI  00072-08  LPVD 

Project  Description: 

Nonspecific  resistance  to  encephalomyocardi  tis   virus   infection. 
Previous  studies   focused  on  an  in  vivo  model    to  investigate  the  humoral    and 
cellular  immune   functions  of  mice  that  exhibit  enhanced  resistance  to  a 
lethal   challenge  of  encephalomyocarditis   virus   (EMCV)   following  administra- 
tion of  an  oil-droplet  emulsion  of  nonviable  Mycobacterium  tuberculosis. 
Similar  concentrations  of  virus   in  plasma  of  normal   and  mycobacteria- 
sensitized  mice   from  1    to  120  minutes  after  EMCV  challenge  indicated  that 
resistance  was   not  a   result  of  rapid  elimination  of  virus   from  the 
circulation,  and  survival   of  viremic  mice  showed  that  protective  mechanisms 
were  operative  after  EMCV  had  replicated.     Additional    studies   determined 
that  neonatal   thymectomy  had  no  effect  on  protection,  but  that  splenectomy 
after  mice  had  been  sensitized  with  mycobacteria,  or  the  intraperitoneal 
injection  of  silica  24  hours  before  virus  challenge  abrogated  resistance 
to  EMCV. 

As  a  continuation  of  this  work,  we  have   focused  our  efforts  on 
determining  the  mechanisms  of  this  nonspecific  resistance  in  an  in  vitro 
assay  system.     In  collaboration  with  Anne   Pusateri  ,   (Master's   Degree 
Candidate,   Department  of  Microbiology,   University  of  Montana,  Missoula),   it 
has  been  confirmed  that  peritoneal   cells   (PC)   from  sensitized  mice,  as 
compared  to   PC  from  normal    animals,   inhibit  EMCV  replication  in  mouse  embryo 
fibroblast  monolayers  by  >2  logio.     PC  from  mice  that  received  mycobacteria 
either  intravenously  or  intraperi toneally   (i.p.)   inhibit  replication 
equally  well   at  an  effector  to  target  ratio  of  20;  at  lesser  concentrations 
PC  from  i.p.   sensitized  animals  were  more  effective.      PC  collected  2  to  6 
weeks  after  sensitization  were  the  most  effective  in  inhibiting  replication 
(>99%  inhibition),   but  marked  inhibition   (>90%)  also  was   detected  with   PC 
harvested  at  7  to  10  weeks.     These  results   correlate  with  the  time   frame  of 
our  previous  in  vivo  protection  studies. 

Kinetic  studies   to   determine  how  rapidly  PC  from  sensitized  mice 
exert  their  inhibitory  effect  showed  that  minimal    inhibition   (50%)   occurred 
8  hours   post-addition  of  PC  to  monolayers  and  that  90%  inhibition  occurred 
if  PC  were  in  contact  with  monolayers  10  hours.     Thereafter,   virus   titers 
in  monolayers  incubated  without  PC  or  normal    PC  continued  to  increase, 
whereas  viral    replication  in  monolayers  incubated  with  PC  from  sensitized 
mice  was  abrogated  (>99%  inhibition).      It  also  was   determined  that   PC  added 
to  monolayers  as  late  as   8  hours   after  infection  inhibited  replication  90%. 

The  release  of  similar  amounts  of  ^'Cr  from  uninfected  monolayers 
incubated  with  media  or  PC  from  sensitized  or  normal   mice  indicated  that  PC 
did  not  kill   target  cells.      Furthermore,   similar  viral   titers   in  supernatant 
fluids   removed  from  macrophage  cultures  prepared  from  PC  of  sensitized  and 
normal   mice,  as  well   as  media  without  cells,  showed  elimination  and/or 
inactivation  of  infectious   EMCV  by  macrophages  were  not  important  in  this 
system.     It  also  was   determined  that  macrophages   from  neither  normal   nor 


29-9 


Project  No.    Z01    AI   00072-08  LPVD 


sensitized  mice  supported  EMCV  replication.     Additional   studies  showed  that 
nonviable   PC   failed  to  inhibit  viral    replication  and  that   factors  such  as   pH 
and  depletion  of  culture  nutrients  were  unimportant. 

At  this  point,  an  antiviral   mediator  was  considered  to  be  responsible 
for  inhibition  of  viral    replication  because  1)  Marked  inhibition   (>90%) 
was   not  detected  unless   PC  were  in  contact  with  infected  monolayers   for 
8  to  10  hours.   2)   Cytotoxicity  was  not  detected  in  monolayers   incubated  with 
PC.   3)    EMCV  was  not  inactivated  and/or  eliminated  by  macrophages.     4)    PC  did 
not  inhibit  viral    replication  if  every  cell    in  the  monolayer  initially 
was  infected.     After  an  extensive  and  exhaustive  search,   an  antiviral 
mediator  was   detected  in  supernatant   fluids  of  PC  cultures   from  sensitized 
but  not  normal   mice.     The  mediator  was   not  present  in  supernatant   fluids 
harvested  2  hours   postincubation,  whereas  media  harvested  at  4  hours   had 
slight  activity  and  media  harvested  "8  hours  markedly  inhibited  replication 
(>90%  to  >99%). 

The  mediator  has  been  characterized  as   a  Type   II  interferon  in  that 
it  is  neutralized  by  anti-Type   II  interferon  but  not  anti-Type   I   interferon. 
However,   it  is  a   unique  Type   II  interferon  in  that  1)    It  is   labile  to  either 
treatment  at  pH  2.0   for  24  hours  at  4°C,  or  heat  at  56°C  for  1/2  hour. 

2)  It  must  be  in  contact  with  monolayers   in  an  interferon  assay  for  a  minimum 
of  18  hours   to  inhibit  VSV  replication,  whereas  Type   I  and  Type   II 
interferons   inhibit  replication  after  2  and  8  hour  incubations,   respectively. 

3)  It  is   inactive  on  the  mouse  L  cell    routinely  used  in  our  laboratory, 
whereas   two  different  mouse  Type   I  and  Type   II   interferons   tested  at  similar 
units/ml   express  activity  on  this   same  cell. 

The  effector  cell    from  the  peritoneal    cavity  of  sensitized  mice 
responsible   for  inhibiting  EMCV  replication  is   presently  being  determined 
(Master's   Degree  project,   Robert  R.   Cent,   Jr.,   Department  of  Microbiology, 
University  of  Montana,  Missoula).     Preliminary  results   indicate   that  the 
cell   adheres   to  plastic,   nylon  wool   and  baby  hamster  kidney  microexudates , 
but  is  not  phagocytic.     The  classical   T  cell    does   not  appear  to  be  the 
effector  cell   because  PC  from  sensitized  athymic  nude  mice  inhibit   EMCV 
replication  more  effectively  [greater  inhibition  at  lower  effector-target 
ratios]  than   PC  from  their  euthymic  littermates  or  the  C57BL/10ScN  mice 
used  i  n  our  assays . 

The   future  course  of  the  project  associated  with  induction  of 
nonspecific  immunity  with  nonviable  mycobacteria  will    focus  on  identification 
of  the  effector  cell(s)   responsible   for  inhibition  of  EMCV  replication.      In 
addition,  a  more  precise  characterization  of  the  unique  Type   II   interferon 
that  is  associated  with  protection  will    be  undertaken  and  a  determination 
made  as   to  whether  the  effector  cell    produces   this   Type   II  interferon  or 
whether  cell    cooperation  is  needed. 


29-10 


Project  No.    Z01    AI  00072-08  LPVD 


Host  defenses  in  rabies   virus  infection.     Studies  initiated  to  study 
the  pathogenesis  and  neuroimmunology  of  rabies  virus-infected  mice,  and  to 
ascertain  the  mechanisms  by  which  mice  abort  central   nervous  system  (CNS) 
infections  and  recover  from  paralytic  disease  are  in  the  developmental   stage. 
The  technique  for  harvesting  cerebral  spinal   fluid  (CSF)   from  mice  has  been 
mastered.     Using  this  technique,  it  was  shown  that  high  titered  interferon 
present  in  blood  is  not  detectable  in  the  CSF.     These  results  indicate  that 
if  antiviral  mediators  are  present  in  the  CNS  and  do  correlate  with  abortive 
infections,  they  probably  are  produced  locally.     It  also  has  been  determined 
that  the  occurrence  of  abortive  infections  in  either  C57BL/10ScN  or  RML 
mice  is  not  increased  if  different  strains  of  street  virus  isolated  from 
bats,  bobcat  or  fox  are  used  for  infection;  abortive  infections  are  still 
most  readily  induced,  as  has  been  previously  established,  by  the  intra- 
peritoneal  inoculation  of  18  to  21   day  old  mice  with  low  passage  street 
viruses.     We  have,  however,  induced  occasional  abortive  infections  in  mice 
following  intramuscular  (i.m.)  inoculation  of  street  and  fixed  viruses. 
This  observation  is  contrary  to  accepted  dogma,  and  indicates  that  an 
experimental  model   for  abortive  infections  following  the  natural   route  of 
infection  (i.m.  inoculation)  may  be  adaptable  for  further  investigation. 

The  rabies  virus  project  will   focus  on  the  induction  of  abortive 
infections  in  various  strains  of  mice  following  i.p.  inoculation  with 
virulent  street  virus,  and  the  use  of  an  attenuated  rabies  virus  vaccine  to 
induce  abortive  infections  after  intracerebral   inoculation.     With  the 
attenuated  rabies  virus  vaccine  model,  >75%  of  the  mice  become  sick,  abort 
infection  and  survive.     Thus,  mechanisms  of  survival   can  be  more   readily 
studied  and  the  results  applied  to  subsequent  investigations  with  virulent 
street  viruses.     Special  emphasis  will   be  placed  on  detection  and  character- 
ization of  antiviral   mediators  and  antibody  present  in  the  CSF  of  mice  that 
have  aborted  infections. 


Publications : 

Lodmell  ,   D.   L.   and  Ewalt,  L.  C:     Induction  of  enhanced  resistance 

against  encephalomyocarditis  virus   infection  of  mice  by  nonviable 

Mycobacterium  tuberculosis:  mechanisms  of  protection.     Infect.   Immun.  22: 
740-745,   1978. 


In  press : 

Lodmell,   D.   L.,   Cent,   R.   R.,   Jr.,   Pusateri ,  A.  M.   and  Ewalt,   L.   C. : 
Nonspecific  inhibition  of  encephalomyocarditis  virus    (EMCV) 
replication  by  a  nonphagocytic  adherent  cell    from  athymic  nude  mice 
sensitized  with  nonviable  Mycobacterium  tuberculosis.     Proceedi  ngs 
Third  International   Workshop  on  Nude  Mice. 


29-11 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANC 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl      AI     00073-14  LPVD 


PERIOD  COVERED 


October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Mechanisms  of  Immunity  and  Immunopathology  in   Virus   Related   Diseases 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:  J.  E.  Coe 

OTHER:  R.  E.  Race 

M.  E.  Bloom 

L.  A.  Thomas 


Medical   Officer 
Veterinary  Officer 
Medical   Officer  (Res 
Res.   Microbiologist 


LPVD  NIAID 
LPVD  NIAID 
LPVD  NIAID 
LPVD  NIAID 


COOPERATING    UNITS    (if    any) 

Dr.   John   Sogn,   Laboratory  of  Immunogenetics ,   NIAID,   Bethesda,   MD 

Dr.   Gregory  Printz,   Laboratory  of  Infectious   Diseases,   NIAID,   Bethesda,  MD 


lab/branch 
Laboratory  of  Persistent  Viral    Diseases,   Hamilton,  MT 


SECTION 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 

3.5 


PROFESSIONAL: 

1  .5 


|OTHER: 

2.0 


CHECK  APPROPRIATE  BOX(ES) 
Q  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   Q  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


&  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Our  studies  have  been  oriented  to  define  the  role  of  Ig  classes   in   the 
immune   response/pathophysiology  in   viral    diseases.     The  immune  complexes  of 
Aleutian  diseases   (AD)  mink  are  being  analyzed  to  explain  the  predominant 
deposition  of  IgA  in   glomerulae  of  terminal   AD  mink.     Also,   the  aberrant 
lymphoprol i feration  in  AD  mink  is  being  studied  by  analyzing  T  cell   mitogen 
responses  to  protein  and  AD  virus   antigens.     SV-40  inoculated  into  Syrian 
hamsters   has   been   found  to  induce  B  cell    lymphomas  which   are   preferentially 
of  an   IgG  class .     Some  of  these  tumors   also  secrete   IgG  which  is  monoclonal 
and  the  product  varies   from  whole   Ig  molecules  with   free   L  chains   to  primarily 
free  H  chains.      In   contrast,    IgM  synthesis  was   preferentially  enhanced  by 
injection  of  Freund's   adjuvant  combinations  into  Syrian  hamsters.     The  essential 
mycobacterial    component  of  Freund's   adjuvant  is   present  in  the   cell   wall   but 
is   not  MDP. 


29-12 


PKS-6040 
(Rev.    IO-76) 


Project  No.    Z01    AI   00073-14  LPVD 


Project   Description: 


Studies  on  the  aberrant  lymphoprol i ferati ve   response  in  Aleutian 
diseased  mink.     Aleutian  disease   (AD)  of  mink  is  a  persistent  virus   infection 
in  which  viremia  is  associated  with  circulating  antibody.      Infected  mink 
usually  die  of  glomerulonephritis  and/or  arteritis  which  is   probably  related 
to  the  deposition  of  immune  complexes. 

Of  particular  interest  is   the  high,  apparently  uncontrolled,   immuno- 
globulin synthesis  that  occurs   in  Aleutian  disease.     Serum  immunoglobulin 
levels  often  reach  50%  or  more  of  the  total    serum  protein  component   (i.e., 
^50  mg/ml ) ,  and  increase  steadily  in  amount  as   the  disease  progresses. 

Our  studies  are  designed  to  elucidate  the  cellular  mechanisms   that 
may  be  important  in   regulating  immunoglobulin  synthesis   and  identifying 
cellular  subtypes   responsible   for  the   lack  of  immune   regulation  in  AD.      In 
addition,  we  intend  to  identify  viral   antigens  or  viral    induced  antigens 
that  may  be  the  initiator  and/or  target  of  the  immunoglobulin  synthesis. 

In  order  to  study  cellular  immunity  in  mink,   a  lymphocyte  histo- 
genesis  assay  has  been   developed  and  adapted  to  mink  lymph  node   populations. 
Optimum  conditions  have  been  defined  and  the  assay  utilized  to  observe 
cellular  responsiveness  of  lymph  node   cells   to  a  variety  of  nonspecific 
mitogens  as  well    as  specific  antigens  with  which  mink  have   been  sensitized 
[keyhole   limpet  hemocyanin   (KLH),   human   gamma   globulin   (HGG)]  and  also 
preparations   containing  Aleutian  disease   virus   (ADV). 

Definitive  proliferative  responses    (3-  to   8- fold  over  background), 
have  been   found  with  specific  antigens.     Comparison  of  various   lymph  nodes 
from  individual   animals  has  shown  some   variation  of  response.      For  example, 
within  individual   mink,  mesenteric  nodes  were   frequently  more  reactive   than 
peripheral   lymph  nodes   to   KLH.     Sequential   observation  of  the   response  in 
individuals  by  lymph  node  biopsy,   reveals   no  decline  in  the  magnitude  of 
response  over  at  least  a  4  month  period  but  some  lymph  node  to   lymph  node 
variation.     Lymphocytes   from  mink  with  advanced  Aleutian   disease  had  a 
strong  blastogenic  response   (mean  increase  over  background  of  11 )   to  ADV 
containing  preparations  but  not  to  appropriate   (non-ADV)   controls.     Smaller 
responses   (2-fold)  were  detected  in  lymphocytes   from  animals   less   severely 
affected  or  shortly  after  infection.     Specific  responses  were  markedly 
enhanced  by  purifying  the   "T"  cell   population;  cells  which  passed  through 
nylon  wool    columns  were  morphologically   (absence  of  membrane  bound   Ig)   and 
physiologically   (response  to  concanavalin  A)   T  cells.     These  cells   had  low 
background  counts  and  were  the  major  responders   to  KLH,   HGG,  and  ADV. 

Immune  complexes   (IC)   are  the  hallmark  of  AD,  and  previous  work  in 
this  laboratory  has  shown  a  preferential    deposition  of  IgA  in  AD  mink 
glomerulae.     Although  serum  IgA  shows   dramatic  increases   in  AD  affected  mink, 
other  classes    (IgG)   almost  certainly  must  contribute  to  the  circulating   IC. 
The  selective  presence  of  IgA  in   glomeruli   suggests   that  circulating   IgA-IC 


29-13 


Project   No.    Z01    AI  00073-14   LPVD 

are  preferentially  deposited  or  selectively  preserved.     One  approach   to  this 
problem  is   to   define  the   Ig  classes   represented  in   the  putative  circulating 
IC  of  AD  and  the  specificity  of  the  antibody   (i.e.,  antigens   responsible   for 
IC).     The  objective  of  current  work  is  to  isolate  and  analyze  the  components 
of  the  immune  complexes.     Qualitative  and  preparative  techniques  we  are 
developing  in  this   regard  include:     1)   The  Raji    cell    radioimmune  assay  which 
quantifies   complexes  binding  to   complement  receptors   on   the   lymphoblastoid 
cells,   2)   Gel    filtration   followed  by  affinity  chromatography  in  which  high 
molecular  weight  serum  components   containing  putative  complexes   are  bound 
by  staphylococcal-protein  A  affinity  chromatography,   and   3)   111  tracentri  fuga- 
tion. 

The  assays   developed  or  being  developed  appear  to  offer  a   reliable 
means   for  studying  and  defining  the  sequential    cellular  and  humoral    events 
that  occur  during  the  pathogenesis  of  Aleutian   disease   in   susceptible  and 
resistant  mink. 

Characteristics  of  SV-40  induced  hamster  lymphomas.      Previous   studies 
in   this   laboratory  have  shown  that  lymphoma   induced  by  SV-40  inoculation  of 
young  hamsters  were  composed  of  B  cells  with  membrane   Ig   (MIg)   detectable  by 
fluorescent  microscopy.     Of  particular  interest  was  the  predominance  of  a   7S 
Ig   ( IgG2 )   in  4  of  5   different  tumors  examined.     Only  1    of  5   tumors   had  surface 
IgM  in  contrast  to  mouse  lymphomas  which  characteristically  have   IgM  or   IgD 
H  chains  expressed  on  the  surface.     These  SV-40  B  cell    lymphomas   have   been 
evaluated  for  their  secretory  products,   i.e.,  whether  the  cell    secretes    Ig 
which  is  identical    to  MIg  and  is  structurally  comparable  to  serum   Ig. 
Analysis  of  35$  methionine  cultured  cells  by  specific  precipitation   followed 
by  SDS-PAGE,   has  shown   3  of  4  tumors  with    IgG2-MIg  are  secretors.      However, 
their  products   differ  as  one  tumor  is   producing  a   large  excess  of  L  chains 
(^40,000  da! tons,  presumably  a   dimer)   in  addition  to  whole   IgG2   (160,000 
daltons)   and  another  tumor  appears   to  produce  only  H  chain   dimers    (found 
predominantly  i ntracel 1 ularly) .      Another  SV-40   tumor   (the  only  non-IgG2  MIg 
which   is    faintly  positive   for  IgA-MIg)   secretes   a   Ig  molecule  with  a  =70   K 
dalton  H  chain  which   reacts  best  with  nonspecific   (anti-L  chain)   antisera 
and  may  represent  hamster  IgD.     Several   other  non-SV-40  hamster  lymphomas 
have  been  defined,   for  example,  a  plasmacytoma  originally  described  by 
Fortner,   has  been  shown  to  secrete   IgA,  although  the  H  chain   is   consistently 
larger  (70  to   100  K  daltons)   than   the   IgA  H  chain  synthesized  by  normal 
hamster  mesenteric  lymph  node   (60  to  65   K  daltons).     This   large   IgA  H  chain 
does  not  appear  to  be  a  carbohydrate  artifact  of  SDS-PAGE  but  may   represent 
a  molecule  with  a  persistently  attached  "secretory"   component  which  should 
be  split  off  by  a   glycosidase.      Further  work  will    identify  the  probably 
monoclonal i ty  of  these  SV-40  induced  tumors  and  attempt  to   clarify  the 
relationship  between  a  SV-40  receptor  and   IgG2-MIg,   as    IgG  B  cell    lymphomas 
are  of  especial    rarity  in  other  model   systems   (mouse)   and  in  man.     Also, 
the  mechanism  and  specificity  of  tumor  immunity,  which  has   been  observed  in 
appropriately  inoculated  hamsters,  will    be  investigated. 


29-14 


Project  No.    Z01    AI   00073-14  LPVD 

Selective  stimulation  of  IgM  synthesis  by  Freund's   adjuvant.      Freund's 
adjuvants   have   a  well    known  effect   to  markedly  amplify  the  immune   response 
to  antigens   contained  in  the  emulsion.     We  have   found  that   Freund's   adjuvants 
also  have  a  profound  effect  on   IgM  synthesis   in  the  Syrian   hamster  under 
certain  circumstances.      Injection  of  incomplete   Freund's   (IF)   adjuvant  intra- 
peritoneal^  (i.p.)   and  complete   Freund's   (CF)   adjuvant  in   footpads    (FP)  will 
induce  a  profound  increase  in  serum  IgM  levels    (values   from  20   to  50  mg/ml 
are   frequently  observed  vis   normal    levels  of  -1.0  mg/ml.     This   particular 
combination   is  necessary  as   the  emulsion  administered  individually,   or  at 
opposite  sites  produce  only  modest    (4  to  8  mg/ml)   increases.     Male  hamsters 
average  higher  levels    (e.g.,  18  mg/ml)   than   females    (e.g.,   4.7  mg/ml)   although 
this   treatment  usually  results   in  a  high  mortality   (50%  by  day  100)   in 
female  hamsters.     This  marked  elevation  of  serum  IgM  is   not  attended  by  a 
similar  increase  in  other  serum   Ig,  although  an  occasional    ascites  observed 
in  these  animals   contains  high  levels    (8  to  10  mg/ml)  of  IgA  and  of  6 
lymphomas   detected  after  this   treatment  4  were  composed  of  IgG2  B  cells 
(one  of  these  has  been  shown  to  secrete  whole   IgG2  and   free   L  chains).     The 
enhanced  IgM  synthesis  is  especially  apparent  in  spleen    (by  FA  and  C'4 
tissue  culture  analysis)   and  limited  attempts   in  passive  transfer  of  enhanced 
IgM  synthesis  into  lethal ly  irradiated  recipients  have  been   unsuccessful. 
The  elevated   IgM  in  some  individual   sera  is  of  restricted  electrophoreti c 
heterogeneity  which  suggests   a  monoclonal   origin,  however  definitive  proof 
by  production  of  idiotype  specific  antisera  and  isoelectric   focusing  of  L 
chains   are   presently  under  investigation.     This   IgM  has   been  shown  to  be  of 
1 9S   size,  although  the  specificity  is   unknown.     The  applicability  of  this    IgM 
stimulation   (?monoclonal    induction)   technique   for  other  rodents   is   currently 
being  evaluated.     The  mycobacterial    factor  necessary  has   been  shown  to   be 
present  in  cell   walls  and  is  not  in  cell   wall    skeleton  and  is   not  MDP. 

Structural    analysis  of  female   protein   (FP).     A  sex  limited  serum 
protein  of  female   Syrian  hamsters    (called  female  protein)   has   previously 
been  defined  in  this   laboratory.     The  generous  quantities    (2  mg/ml)  of  this 
hormonally  controlled   (testosterone  suppression)   serum  protein  suggests   an 
important   function  although  none  has  been  defined  at  present.     Analysis   of 
its  structure,   however,   indicates  a  strong  similarity  to   C-reactive  protein 
(CRP).     That  is,   FP  has  been   found  to  be  composed  of  identical   ^25 ,000 
molecular  weight  subunits  noncovalently  linked  to   form  a  protein  of  ^1 50 ,000 
molecular  weight.     In  addition  to   CRP,   this   unusual    structure  has  also  been 
described  for  two  other  related  proteins   CI t   (a   complement  component)   and 
P-component  of  amyloid.      In  collaboration  with   Dr.   John  Sogn,  we  are  now 
analyzing  FP  for  amino  acid  sequence  similarities   to  the  above  proteins. 
Because  of  the  similarity  of  hormonal    control   of  FP  and  of  SLP  (a  marker  of 
SS  protein  which  is  a   complement  component  with  an  H-2  linked  gene   in  mouse), 
we  have  investigated  FP  as  a   potential   marker  for  the  MHC  locus   of  Syrian 
hamster.     A  search   for  antigenic  or  electrophoreti  c  differences  of  FP 
among  various  outbred  and  inbred  hamster  strains,   commercially  available, 
showed  no   differences   in  these  strains.     However,  among  9  inbred  strains 
derived   from  a   recent  capture  of  wild  hamsters   in   Syria,   3  strains  were 


29-15 


Project  No.    Z01    AI   00073-14  LPVD 

found  with  an  electrophoretically  slower  (but  antigenically  identical)    FP, 
This  electrophoretic  difference  presumably  is   due  to  protein   differences, 
rather  than  sialic  acid  content  as  neuraminidase  treatment  causes  a  similar 
electrophoretic  retardation  in  both  slow  and  fast   FP's.      FP  obtained   from 
female   (or  castrated  male)   hybrids  of  fast/slow  FP  parents,   show  an  inter- 
mediate mobility,  suggesting  fast/slow  hybrid   FP  molecules.      In   collaboration 
with  Dr.  Wayne  Streilein,  we  are  comparing  this  serum   FP  marker  with  histo- 
compatability  data   from  backcross  populations   derived  from  fast  and  slow 
FP  parents   to  determine  if  the   FP  gene  is   linked  to  the  elusive  MHC  of 
hamsters . 

Publications : 

In  press : 

Coe ,   J.    E.:     Serum  proteins:     normal    values   derived  from  normal 
strains.      FASEB  Data  Book  -   Inbred  and  Genetically  Defined  Strains 
of  Laboratory  Animals 


29-16 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  Of 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01      AI     00074-07   LPVD 


PERIOD    COVERED 


October  1,  1978  to  September  30,   1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Host  Defense  Mechanisms   in  Chronic  Viral    and  Neoplastic   Diseases 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

LPVD  NIAID 
LPVD  NIAID 
LPVD  NIAID 


PI: 

B. 

w. 

Chesebro 

Acting  Chief 

OTHER: 

J. 

K. 

Collins 

Staff  Fellow 

W. 

J. 

Britt 

Staff  Fellow 

COOPERATING    UNITS    (if    any) 

Donald  Doig,   Dept.   of  Microbiology,   Montana  State   University,   Bozeman,   MT 
Dr.   Kenneth  Watson,   Dept.   of  Chemistry,   University  of  Montana,   Missoula,  MT 


lab/branch 
Laboratory  of  Persistent  Viral    Diseases,   Hamilton,  MT 


INSTITUTE   AND   LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 
5.0 


PROFESSIONAL: 

3.0 


2.0 


CHECK  APPROPRIATE  BOX(ES) 
Q  (a)   HUMAN  SUBJECTS 

□  (a1  )  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


fi  (c)    NEITHER 


SUMMARY   OF   WORK    (200   words   or   less   -   underline    keywords) 

The  main   goal   of  this   project  is   the  understanding  of  host  defense  mechanisms 
involved  in   recovery   from  vi  rus- induced  leukemia  in  mice.     We  have   used 
selected  breeding  of  mouse  strains   to  isolate  and  characterize  individual 
genetically  controlled  defense  mechanisms   and  to  study  their  actions   and 
interactions   during  recovery  from  virus   infection  and/or  virus-induced  leukemia. 
In  addition,  we  have   analyzed  a   group  of  leukemia  cell    variants  with  alterations 
in   viral   expression.     These   are  being  characterized  biochemically  and  immuno- 
logically to   determine  the   roles  of  expression  of  different  viral    proteins   in 
immune   recognition  and  the   possible   relevance  of  this   variation  in  expression 
to  persistence  of  virus   infected  cells   i  n   vi  vo . 


29-17 


PHS-6040 
(Rev.    10-76) 


Project  No.    Z01    AI  00074-07  LPVD 


Project   Description: 


We  have  previously  identified  3  mouse  genes   (Rfv-1,   Rfv-2,  and  Rfv-3) 
which  are  involved  in  host  defenses  against   Friend  virus   (FV)-induced 
erythroleukemia.     Rfv-1    is   located  in  the  H-2D  region  of  the  major  histo- 
compatibility complex   (MHC),  and  has  a  major  influence  on  the  incidence  of 
recovery  from  leukemia.     The  Rfv-2  gene  is   also  associated  with  the  MHC  in 
the  H-2K,  H-2I  or  Tla   regions,  and  has  a  weak  effect  on   recovery  from 
leukemia.     The  genetic  linkage  of  the  Rfv-3  gene   is  not  known.      It  is   not 
associated  with  H-2,   however,  it  acts   in  a  complementary  fashion  with  the 
H-2-associated  genes,   Rfv-1   and  Rfv-2,   to  influence  recovery   from  leukemia. 
In  the  absence  of  the  appropriate  H-2  genotypes   the  Rfv-3  gene  influences 
recovery  from  viremia,  even  in  the  presence  of  persistent  leukemia.     Our 
current  work  involves   characterization  of  the  mechanisms  of  action  and  roles 
of  these  genes   in  the  host  immune   responses   to  virus  and  leukemia   cells. 

H-2-associated  genes    (Rfv-1    and  Rfv-2).     We  have   recently  identified 
the  organs   in  which  H-2  effects  on   recovery  from  leukemia  occur  by  use  of 
irradiation  chimeras.     When  spleen  and  bone  marrow  cells  were   transferred 
to   lethally  irradiated  semi-al  logeneic  recipients    [H-Z°/a^H-Z^^  and 
H-2b/a-vH_2a/a)   the  H-2   type  of  the  donor  spleen  and  bone  marrow  cells 
determined  the  incidence  of  recovery  from  FV  leukemia.     Thus   H-2-associated 
effects  appeared  to  be  exerted  by  cells   from  these  donor  organs.     Since  both 
the  leukemia  cells  and  the  cells  of  the  immune  defense  system  were   derived 
from  the  donor  stem  cells   in  irradiation  chimeras,  we  have  attempted  to 
distinguish  which  of  these  two  general   cell    categories  was   the  major  site  of 
H-2  influence  on   recovery.     Two   lines  of  preliminary  evidence  suggested  that 
the  main  H-2  effects  operated  via  the  immune  system  rather  than   via  the  H-2 
type  of  the  leukemia  cells.      First,   H-2a/b  mice   recovering   from  FV  leukemia 
were  able  to   reject  transplanted  FV  leukemia  cells  of  H-Z°^,  H-2D/a   and 
H-2a/a   genotypes  with  equal   efficiency.     Second,  nonimmune  H-2b/b  spleen   cells 
from  mice  made  neonatally  tolerant  to  H-2a/°  cells  were   able  to   potentiate 
recovery  of  H-2a/b  mice   from  FV  leukemia.     Thus,   cells  of  the  H-2°/b  immune 
system  were  able  to  eliminate  leukemia  cells  of  the  low  recovery  H-23/'3 
genotype.     Our  present  preliminary  conclusion  is   that  the   H-2  type  of  the 
immune  system  influences   recovery  from  leukemia  by  immune   response   genes 
which  control    the  rate  of  generation  of  FV-specific  cytotoxic  T  lymphocytes 
which  kill    leukemia  cells. 

We  have   also  approached  the   role  of  the  immune   response  in   recovery 
from  leukemia  by  study  of  FV  specific  T  cell    proliferative   responses   in 
vitro .      Preliminary  data  indicate  that   recovery   from  leukemia  is  associated 
with  a  strong  T  cell    proliferative  response,  and  current  experiments   are 
aimed  at  determining  whether  the  lack  of  a  similar  response  in  persistently 
leukemic  mice  is   due  to  suppression  by  virus  or  virus-specific  suppressor 
cells.     In  additional   studies    (see  below),   the  viral    antigens   recognized  by 
both  T  and  B  cells  are  being  studied. 


29-18 


Project   No.    Z01    AI   00074-07  LPVD 

Rfv-3  gene.     30  to  90  days  after  FV  inoculation  of  Rfv-S^13  mice 
[(B10. A  X  A)P|  ],  we  previously  noted  the  association  of  elimination  of 
viremia,  loss  of  FV-induced  cell   surface  antigens   from  splenic  leukemia 
cells,  and  presence  of  cytotoxic  anti-FV  antibody  in  plasma.      Furthermore, 
these  parameters  segregated  together  in  a  backcross  between   Rfv-3r/s   and 
Rfv-3S/S  mice.     Thus,   they  appeared  to  be  linked  by  a  single   genetically 
controlled  mechanism.     This  mechanism  probably  involved  control    of  pro- 
duction of  cytotoxic  anti-FV  antibody,  since  passive  transfer  of  these 
antibodies   could  reproduce  the  loss  of  FV  cell   surface  antigens   i n   vi vo . 
Our  recent  experiments  have  shown  that  antibody-induced  loss  of  viral    cell 
surface  antigens  leads   to  a  marked  reduction  in  virus   release  by  leukemia 
cells.     This  decrease  in  release  is   reflected  as  a  300- fold  decrease  in 
the  percent  of  leukemia  cells   detectable  as  infectious   centers.     At  this 
time  cell-free  virus  in  spleen  homogenates   is   reduced  as   assayed  both  by 
infectivity  and  reverse  transcriptase.     Decreased  virus   release  by 
persistent  leukemia  cells   appears   to   result  in  elimination  of  viremia.     Virus 
neutralizing  antibodies   do  not  seem  to  play  a  significant   role.     Our  present 
evidence  supports   the  idea  that  anti-FV  antibodies   reduce   FV  cell    surface 
antigens  by  the  process  of  antigenic  modulation.     We  now  hypothesize   that 
antibody-induced  rearrangement  of  viral    cell   surface  molecules  may  interfere 
with  important  steric  relationships  among  these  molecules  which  in  turn 
leads  to   reduced  virus   budding  and  release. 

Current  experiments   are  aimed  at  determining  biochemically  which   viral 
cell   surface  molecules  are  decreased  on  these  cells  and  also  whether  these 
cell   surface  events   cause  changes  in  intracellular  viral    protein  synthesis 
and  processing,,     Lastly,  we  are  attempting  to  determine  what  role,   if  any, 
this  phenomenon  might  have  in  interfering  with  or  amplifying  T  cell-mediated 
mechanisms  of  specific  immune  recognition  which  could  be  important  in 
elimination  of  leukemia  cells. 

Leukemia  cell    variants.     We  have  obtained  several   lines   of  cloned   FV 
leukemia  cells  which  have  variations  in   virus  expression.     These   variant 
cells  appear  to  have  existed  in  the  original   leukemic  mouse  spleen   from 
which  they  were  derived  since  no   further  variations  have  been  observed 
following  cell    cloning  in   vitro.     These  clones  have  been  characterized   for 
FV  cell   surface  and  intracellular  antigens,   reinfectibil i ty,   rescuability 
of  spleen   focus-forming  virus,  and  recognition  by  antibodies   and  T  lympho- 
cytes  from  mice  recovering  from  FV  leukemia.     Using  these  clones  we  have 
attempted  to  determine  which   FV-induced  molecules   are  involved  in   different 
components  of  specific  immune   recognition  i  n   vi  vo .     Analysis  of  the  cloned 
variants  obtained  so   far  has   also  enabled  studies  on  mechanisms   involved  in 
normal   and  aberrant  viral    protein  synthesis   and  processing.     These  mechanisms 
may  serve  as   interesting  models   because  some  of  the   "defects"   leading  to  the 
nonproducer  state  may  allow  persistence  of  leukemia  cells   i n   vi  vo . 


29-19 


Project   No.    Z01    AI  00074-07   LPVD 

One  clone  has   been   found  to  be  deficient  in  expression  and/or 
processing  of  "gag"   region  viral    proteins,  whereas   its  expression  of  "env" 
region  protein   (gp70)   is   normal.     Conversely,   another  clone  makes   no   FV 
gp70,  but  appears   to  express   normal    amounts  of  the   "gag"   protein,   p!2. 
Using  these  two  clones  we  have   found  that  the   FV-specific  T  cell   prolifer- 
ative  response  and  cytotoxic  T  cell    response  are  both  mainly  directed  at  the 
"env"  protein,   gp70.     One   "nonproducer"  clone  has  been   found  to  produce  high 
levels  of  virus   completely  lacking  reverse  transcriptase  yet  containing 
normal   levels  of  gp70.     Also  defective  in  this   virus   is  an   uncleaved  "gag" 
polyprotein  precursor.      It  is   currently  under  investigation  whether  this   is 
a  single  mutation  or  defect  giving  rise  to  pleiotropic  effects   on   viral 
synthesis  or  whether  this   cell    clone  has  multiple  defects. 

Other  cells  being  analyzed  by  the  above  techniques   include  clones 
showing  1)   deficient  synthesis  of  viral   core  protein  precursor  synthesis, 
2)  expression  of  endogenous   viral    components  in  addition  to   Friend  specific 
components,   3)  expression  of  intracellular  viral   antigens  which  are  not 
processed  to   cell   surface  antigens,  and  4)   possible  alteration   in  expression 
of  cellular  H-2  antigen   determinants. 


Publications : 

Doig,   D.   and  Chesebro,   B. :     Antibody-induced  loss  of  Friend  virus 
leukemia  cell    surface  antigens  occurs   during  progression  of 
erythro leukemia  in   F]   mice.     J.   Exp.  Med.   148:     1109-1121,   1978. 

Chesebro,   B.   and  Wehrly,   K.:      Identification  of  a  non-H-2  gene 
(Rfv-3)   influencing   recovery  from  viremia  and  leukemia  induced 
by  Friend  virus   complex.      Proc.   Natl.  Acad.   Sci  .   USA  76: 
425-429,   1979.  "  


In   press: 

Chesebro,   B.,  Wehrly,   K.,   Doig,   D.   and  Nishio,   J.:     Antibody-induced 
modulation  of  Friend  virus   cell    surface  antigens   decreases   virus 
production  by  persistent  erythroleukemia  cells:      Influence  of  the 
Rfv-3  gene.      Proc.   Natl  .   Acad.   Sci  .   USA. 

Doig,   D.   and  Chesebro,   B.:     Anti- Friend  virus   antibody  is   associated 
with   recovery  from  viremia  and  loss  of  viral    leukemia  cell    surface 
antigens   in  leukemic  mice.      Identification  of  Rfv-3  as   a   gene  locus 
influencing  antibody  production.     J.   Exp.   Med. 

Chesebro,  B.:     The   influence  of  the  major  histocompatibility  complex 
(H-2)  on  oncorn'avi  rus-induced  neoplasia  in  mice.      In   Kaiser,   H.    E. 
(Ed.):     Comparative   Pathology  of  Abnormal    Growth. 


29-20 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl      AI     00085-02  LPVD 


PERIOD    COVERED 


October  1,   1978  to   September  30,   1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Biology  of  Aleutian   Disease  Virus 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Medical    Officer  (Res.)  LPVD  NIAID 

Medical    Officer  LPVD  NIAID 

Veterinary  Officer  LPVD  NIAID 

Res.   Veterinarian   (Path.)  EB  NIAID 


PI: 

M. 

E. 

Bloom 

OTHER: 

J. 

E. 

Coe 

R. 

E. 

Race 

W. 

J. 

Hadlow 

COOPERATING    UNITS    (if    any) 

Dr.   John  Gorham,  Washington   State   University,   Pullman,  WA 
Dr.    David  Porter,   UCLA  School   of  Medicine,   Los  Angeles,   CA 


LAB/BRANCH 

Laboratory  of  Persistent  Viral    Diseases,  Hamilton,  MT 


INSTITUTE   AND    LOCATION 

NIAID,  NIH,  Bethesda,  MP  20205 


TOTAL  MANYEARS: 

2.2 


PROFESSIONAL: 

1  .2 


OTHER: 

1  .0 


CHECK   APPROPRIATE   BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


(b)  HUMAN  TISSUES 


S  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  goal   of  this  project  is   to  study  Aleutian  disease   (AD),  a  severe,    fatal 
immune  complex  disease  of  mink  associated  with  a  persistent  viral    infection. 
Techniques  of  both   virology  and  immunology  are   focused  on   this   problem. 
The  current  objectives  are  to  characterize  the  Aleutian   disease   virus    (ADV) 
and  to  determine  what  role  viral    components   have  in  the   genesis  of  the 
immune  complexes. 


29-21 


DHS-b040 
(Rev.    1 0-7 


Project   No.    Z01    AI   00085-02    LPVD 


Project   Description: 


Previous  work  at  RML  by  this   PI   and  others   showed  that  the   Utah   I 
strain  of  Aleutian  disease   virus    (ADV)   could  be  successfully  purified   from 
in  vivo   grown  materials   by  equilibrium  ul  tracentri  fugation  in  cesium  chloride 
density  gradients,   if  virus  was    first  released   from  naturally  occurring 
immune  complexes.     These  studies  suggested,  on  the  basis  of  size,  morphology, 
and  density  that  ADV  was   probably  a  member  of  the  parvovirus   group,  small 
icosahedral    viruses   that  encapsidate  single  stranded  DNA  genomes.     Recently, 
Utah   I  ADV  has   been  adapted  to   growth   in  cell    cultures  of  Crandall    feline 
kidney  cells    (CRFK)   at   31  .8°C  and  assayed,   using  immunofluorescence. 

We  have  now  characterized  one  cell   culture  adapted  strain  of  ADV 
originally  derived  from  Utah   I  ADV  by  Dr.   John  Gorham  at  Washington  State 
University.     This  strain  of  ADV   (ADV-Gorham) ,  which   grows   in   Crandall    feline 
kidney  cells    (CRFK)   to  high  titer,   has  been  examined  at  several    in   vitro 
passage  levels.     At  the  5th   CRFK  passage,   the  virus  had  an  in   vitro  titer 
of  10°«04   fluorescence-forming  units   per  ml    (FFU/ml),   an  in   vi  vo  titer 
(based  on  induction  of  antibody  response   in  mink)  of  10^  ."30"  infectious   dose 
50%  per  ml    ( IDso/ml ) ,  and  caused  typical    AD  in  some  mink.     However,   at  the 
10th  passage  in  CRFK,   although   the  in  vitro  titer  was   1 0^.40   FFU/ml,   the 
in  vivo  titer  was   1 0 T - 5   iDsg/ml   and  the  virus  was   nonpathogenic.     Viral 
growth  studies   in  vitro  showed  that  virus   production  was  equivalent  at   31.8° 
or  37°C  for  2   days   after  which  time  virus  yield  at   37°C  decreased.     This 
suggested  that  at  31.8°C,   the  virus  only  proceeds   through  a  single   cycle  of 
infection.     Virus    from  infected  cell    pellets   could  be  purified  on  CsCl 
gradients   revealing  a  major  peak  of  infectivity  at  a   density  of  1 .42  to  1 .44 
gm/ml    and  a  minor  peak  at  1.34  to   1.35   gm/ml  .     Viral    antigen  was   detected 
in  both  peaks  by  counterimmunoelectrophoresis   using  1)   AD  mink  serum, 

2)  rabbit  sera   prepared  against  CsCl    purified  i  n  vi  vo   virulent  ADV  or 

3)  rabbit  sera   prepared  against   CsCl    purified  cell    culture  adapted  ADV,   thus 
suggesting  that  the  cell    culture  virus   is  in   fact   related  antigenically  to 
ADV. 

Experiments   have  also  been  done  with  isotopic  precursors,  and 
-^-thymidine  could  be  incorporated  into  both  1.42   to  1.44  gm/ml    and 
1.34  to  1.35   gm/ml    peaks  of  infectivity.     When   DNA  from  the  major  peak  was 
analyzed  in  alkaline  sucrose   gradients,  a  single  homogeneous   species  of 
approximately  1 5S  was   detected.     The  above   findings  strongly  suggest  that 
this   virus   is  a  parvovirus. 

We  have   also   done   preliminary  characterization  of  another  cell    culture 
adapted  strain  of  Utah   I  ADV  derived  by  Dr.    David  Porter  of  UCLA  School   of 
Medicine.     This   virus   differs  markedly  from  the  ADV-Gorham  isolate   in   that  it 
is  extremely  virulent  for  mink.     Comparative  in  vivo  and  in  vitro   titrations 
showed  an   ID50  of  10^  with  all    mink   developing  clinical    Aleutian   disease, 
but  an  i_n_  vi  tro   titer  of  only  2  X  10^  FFU/ml  .     This   low  i_n_  vitro   titer  has 
seriously  hampered  characterization  so   far. 


29-22 


Project   No.    Z01    AI   00085-02    LPVD 

In  addition,  we  have  also  been  able  to  adapt  the  i  n  vi  vo   Utah   I  ADV 
to  CRFK  cell    culture  passage  in  this   laboratory  and  are  undertaking  studies 
similar  to  those  described  above  with  this  strain  as  well. 

Future  plans   for  this  project  may  be  broken  down  as   follows: 
1)     We  plan  collaborative  studies  with   Dr.   David  Ward  of  Yale  University 
School   of  Medicine  to  define  the  exact  structure  of  the  ADV-Gorham  viral    DNA 
and  its  homology  relationship  to   DNA's  of  other  nondefective  parvoviruses. 
These  experiments  will   involve  nuclease  digestions,   restriction  endonuclease 
analysis,  heteroduplex  mapping,  and  will    provide  definitive  assignment  of 
ADV  to  the  parvovirus   group.      Furthermore,   the   reagents   developed  in  these 
studies  will   allow  us   to  compare  the   DNA  of  the  cell    culture  adapted 
ADV-Gorham  strain,   to   DNA  from  in  vivo  virulent  ADV  as  well   as   that  from 
other  cell   culture  derived  strains  of  ADV.     2)     We  plan  to  compare  the 
polypeptide  composition  of  both  cell    culture  adapted  and  in  vivo  strains  of 
ADV  by  both  extrinsic   (125I)  and  intrinsic  (35S)   labeling  and  subsequent 
analysis  on  SDS  polyacrylami de  gels.     Thus,  we  will    be  able  to  define  the 
viral   protein  structure  and  examine  the  antigenicity  of  each  polypeptide 
constituent.     3)     A  solid  phase   radioimmunoassay  is   currently  under  develop- 
ment in  an  attempt  to  enhance  the  sensitivity  of  detecting  both   viral 
antigens   and  antiviral   antibodies  over  that  afforded  by  CIE.     4)     By 
comparing  the  macromolecular  composition  of  the  various   strains  of  ADV,  we 
may  be  able  to  delineate  differences  between  virulent  and  non virulent 
strains  of  this   virus. 


Publications:     None 


29-23 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOl      AI     00086-02  LPVD 


PERIOD    COVERED 

October  1,   1978  to  September  30,   1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Mechanisms  of  Immune   Recognition  of  Viral    Antigens   in   Persistent  Viral    Diseases 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Medical   Officer  (SR  SURG)  LPVD  NIAID 

Medical    Officer  (SR  SURG)  LPVD  NIAID 

Staff  Fellow  LPVD  NIAID 

Medical    Officer  LPVD  NIAID 

Res.   Veterinarian    (Path.)  EB       NIAID 


PI: 

J. 

L. 

Port  is 

OTHER: 

B. 

W. 

Chesebro 

J. 

K. 

Collins 

J. 

E. 

Coe 

W. 

J. 

Hadlow 

cooperating  units  (if  any)  Dr.   John  Col  1 1  gan  ,   Laboratory  of  Immunogenetics  ,   NIAID, 
Bethesda,  MD;   Dr.   Thomas   Kindt,   Laboratory  of  Immunogenetics,   NIAID,   Bethesda. 
MD;   Dr.   James   T.   Maddox,   St.   Patrick   Hospital,  Missoula,   MT;   Dr.   S.   K.   Wikel, 
Department  of  Microbiology,   University  of  South   Dakota,   Vermillion,  SD 


LAB/ BRANCH 

Laboratory  of  Persistent  Viral    Diseases,   Hamilton,  MT 


INSTITUTE    AND    LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 

2.0 


PROFESSIONAL: 

1  .0 


OTHER: 

1  .0 


CHECK   APPROPRIATE   BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   Q  (a2)  INTERVIEWS 


(b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  major  goal   of  this   project  is   the  definition  of  mechanisms  of  immune 
recognition  of  viral   antigens  on  the  cell    surface  of  virus   infected  cells.     We 
are   continuing  to  study  the  murine  leukemia  line   FBL-3  which   undergoes   a  major 
phenotypic  alteration  in   vivo  converting  from  benign  to  malignant  behavior. 
Cells   grown  in  vitro  or  for  7  days   in  the  ascites    form  produce  transient  tumors 
when  injected  subcutaneously .     The  cells   passed  in  ascites   for  14  days   produce 
lethal   subcutaneous   tumors  which  metastasize.     We  have   found  by  fluctuation 
analysis   that  malignant  tumor  cell    variants  exist  in  the  parent  line  and  that 
as  yet  undefined  selective  pressure  in  the  host  allows   the  preferential    ex- 
pansion of  the  pre-existing  variants.      Detailed  studies  of  the  surface  antigens 
expressed  by  cloned  tumor  cell    variants   have   revealed  important  aberrations 
in   the  major  histocompatibility  antigens  expressed  by  these  cells  which  may 
adversely  affect  their  immune   recognition  by  the  syngeneic  host. 


29-24 


PHS-6040 

(Rev.    10-76) 


Project  No.    Z01    AI  00086-02  LPVD 


Project  Description: 

FBL-3,  a  C57BL/6   (H-2  '    )  murine  leukemia  was  originally  induced  by 
Friend  virus  and  expresses  MuLV   (the  helper  virus)   but  not  the  defective 
SFFV.     The  tumor  cells   grown  in  vitro  or  for  7  days  in  the  ascites   form 
produce  transient  tumors  when  injected  subcutaneously  (s.c.).     These  tumors 
are  uniformly  rejected  by  the  syngeneic  host.     However,  after  14  days  in  the 
ascites   form  the  tumor  cells  were  found  to  have  converted  to  a  highly  malig- 
nant form  which  grew  progressively  after  s.c.   injection,   disseminating  to 
local   and  distant  lymph  nodes   (occasionally  to  the  liver)  and  was   lethal 
within  40  to  50  days. 

In  the  past  year  we  have  examined  two  questions:     1)    Is   the  phenotypic 
alteration  the  result  of  the  expansion  of  a  population  of  malignant  tumor 
cell   variant  pre-existing  in  the  parent  tissue  culture  line  or  did  the 
variants  arise  de  novo?     2)  What  is   the  nature  of  this   phenotypic  change 
specifically  with  respect  to  H-2  and  viral   antigen  expressed  on  the  cell 
membrane? 

Evidence   for  pre-existing  malignant  variants.     To  approach  this 
problem  we  utilized  the  technique  of  fluctuation  analysis  which  has   been 
applied  extensively  in  the  study  of  bacterial   mutants.     The  in  vitro   grown 
parent  line  of  FBL-3  (which  is   uniformly  rejected  by  syngeneic  recipients 
after  s.c.   inoculation)  was   cloned  and  the  individual    clones  were  tested   for 
tumorigenic  potential.     Of  9  clones,  5  showed  malignant  behavior  and  4  were 
benign.     This  strongly  suggests   that  malignant  variants   do  pre-exist  in  the 
parent  population  and  that  as  yet  unknown  in  vivo  selective  pressures   allow 
for  their  expansion  between  the  7th  and  14th   day  in  the  peritoneal    cavity. 
The  relatively  high   frequency  of  malignant/benign  clones  was   unexpected  but 
may  be  explained  by  a  higher  cloning  efficiency  of  the  malignant  variants. 
We  are  currently,  with  the  help  of  a  summer  student,   repeating  the  fluctua- 
tion analysis  with  larger  numbers  of  animals   in  order  to  determine  if  there 
are  any  differences  in  tissue  tropism  of  those  malignant  variants  which 
metastasize.     With  the  phenotype  of  each  clone  defined  we  plan  to  study  their 
cell   surface  expression  of  viral   and  MHC  antigens  which  might  explain 
differences  in  their  immune   recognition  by  the  host. 

Mechanisms  of  immune  recognition  by  the  host  of  tumor  cell    variants. 
To  this  end  we  have  been  studying  the  expression  of  cell    surface  antigens 
by  an  unusual   cloned  variant  (FY7)  of  FBL-3  (H-2b/b).     This   variant  was 
derived  from  cells  that  were  grown   for  7  days  in  the  ascites    form  in 
(B10. A  X  A.BY)F]   mice   (H-2a/°).     This  clone  is  highly  immunogenic  and 
provokes  a  potent  anti-FBL-3  response  when  injected  s.c.     Immune  peritoneal 
exudate  cells   (PEC)   derived  by  secondary  intraperitoneal    challenge  of 
FY7-immune  mice  are  cytotoxic  for  FY7  ascites   cells.     The  effector  cell    in 
the  PEC  population  has  been  identified  as  a  cytotoxic  T  lymphocyte   (CTL). 
However,  these  CTL   fail   to  lyse  FY7  which  has  been  maintained  in  vitro. 
Furthermore,  in  vitro   FY7   fails  to  competitively  inhibit  the  lysis   by  CTL  of 


29-25 


Project   No.    Z01    AI  00086-02   LPVD 

ascites   FY7  indicating  that  in  vitro   FY7   does   not  express   the   relevant 
antigens   recognized  by  CTL.     We  have  concentrated  our  efforts  on   the  in   vitro 
line  since  there  is  no  problem  with  host  cell    contamination,   and  have 
examined  the  cell   membrane  expression  of  FBL-3  neoanti gens   and  MHC  determi- 
nants.    Anti-H-2b   CTL  generated  by  primary  in  vitro   culture  of  (BIO. A  X  A)Fi 
(H-2a/a)   or   (B10.D2   X  BALB/c)Fi    (H-2d/d)    responders  with  mi tomycin-treated 
(subscript  mc)   BIO  spleen  cell   stimulators   fail    to  lyse   FY7  even  at  an 
attacker/target  ratio  of  100/1.     Yet  these  CTL  do  lyse  other  H-2b/b  tumor 
cell    lines  and  B10  splenic  con  A  blasts.      In  addition   FY7  does   not  com- 
petitively inhibit  the  lysis  of  B10  con  A  blasts   by  anti-H-2b   CTL.     Thus, 
FY7  appears   not  to  express  the  relevant  H-2b  determinants    recognized  by 
anti-H-2b   CTL.     We   found  using  serologic  techniques   that  FY7  expresses 
quantitatively  similar  amounts  of  H-2KD  serologic  specificities   but 
completely  lacks   H-2D'3   determinants   detectable  by  antibody.      In  order  to 
rule  out  the  possibility  that  anti-H-2b   CTL  recognizes  only  H-2Db,  we 
generated  CTL  using  B10.A(4R)    (KkDb)   responders   stimulated  by  BlOmc   (KbDb). 
Even  with  CTL  with  specificity  to  H-2Kb   FY7   fails   to  competitively  inhibit. 
This  suggests   that  the  H-2Kb   determinants   detected  by  antibody  are   distinct 
from  those  detected  by  CTL  and  that  FY7  expresses   the   former  but  not  the 
latter.     Although  the  association  between  lack  of  H-2Db  serologic  and 
H-2Kb   CTL  specificities  on   FY7  may  be  coincidental,  it  raises   an   interesting 
question.     Recently,   the  biosynthesis  of  an   I   region  antigen   (IE*)   has   been 
found  to  be  dependent  on  the  expression  of  two  separate   I   region   genes    IAk 
and  IE^.     As  yet  we  have  no  evidence   for  multi-gene  control   of  H-2Kb„ 
However,   FY7  is   unique  in   that  it  is,   to  our  knowledge,  the  only  homozygous 
H-2b   cell    line  that   fails  to  express  an  H-2D  product  and  therefore  provides 
a   useful    tool    to  study  this   possibility. 

The  lack  of  recognition  on   FY7  of  FBL-3  neoantigens  by  anti-FBL-3 
CTL  despite  their  detection  by  anti-FBL-3  antibody  correlates  with   the 
aberrations  in  expression  of  H-2  antigens.     The  phenomenon  of  H-2   restriction 
in  the  recognition  of  viral,   chemical    and  tumor  associated  cell-membrane 
antigens  has  been  extensively  documented.     A  small    number  of  tumor  cell 
variants  have  been  described  which  lack  the  expression  of  any  H-2  antigens 
detected  by  serology  or  CTL.     CTL   fail    to   recognize  antigens   chemically 
coupled  to  these  cells   but  do   recognize  these   determinants  on   the  parent 
populations  which  do  express  H-2.     Since   FY7  is   selectively  missing  an 
H-2Db   gene  product  we  are  examining  other  variants  of  FBL-3  in  order  to 
establish  whether  there  is  a  correlation  between  the  lack  of  H-2Db  and 
absence  of  CTL  recognition  of  neoantigens  on  these  cells. 

An  approach   to  the  difference  between   FY7   grown  in  ascites   vs. 
in  vitro  is  more   difficult.     We  are  interested  in   determining  if  H-2Db  is 
expressed  on  the  ascites   cells.     However,  because  of  significant   contamina- 
tion of  the  ascites   tumor  cells  with  host  cells    (up  to  40%)  we  are  attempting 
to  develop  cell    separation  procedures  which  will    allow  this   comparison.      It 
is   clear,  however,   using  crude  separation  on  nylon  wool    and  limiting  dilution 
techniques,   that  there  is  a   distinct  difference  between   the  expression  of 
tumor  specific  CTL  recognition   determinants   on   ascites    vs.    in   vitro    grown 
cells . 

29-26 


Project  No.    Z01    AI   00086-02   LPVD 

Studies  on  the  H-2K     product  of  FY7.     Much  of  the  biochemical 
analysis  of  H-2   gene  products  has  been  carried  out  on   H-2  mutant  mouse 
strains  and  on  well    defined  tumor  cells  such  as  the  C57BL/6   (H-2b/b) 
leukemia  line  EL-4.     Ultimately  these  studies   are   directed  toward  defining 
the  structural   correlates  of  the  various   biologic   functions  of  H-2  gene 
products.     Since   FY7  presents  a  unique  aberration  in  its  expression  of 
H-2KD  specificities  we  are  comparing  the   KD  product  of  FY7  with  that  of 
the  well   characterized  line  EL-4.     Using  the  technique  of  immunoprecipitation 
and  resolution  by  SDS  polyacrylamide  electrophoresis    (PAGE)   the   Kb  product 
appears   to  have  the  same  molecular  weight   (^44K)   as   that  of  EL-4  and 
BL/10  spleen  cells.     Further  resolution  by  two  dimensional    (2D)    PAGE  will 
allow  comparison  of  the  various   polypeptides  within  this  44K  band.     We  are 
currently  producing  hybridomas  with  anti-H-2K'3  and  H-2Db  specificities   in 
order  to   facilitate  selective  precipitation  of  these  products.     This   is 
particularly  important   for  2D  gels  since  a  wide  array  of  polypeptides   are 
resolved  by  this  technique  and  much  confusion  can  be  eliminated  by  using 
monoclonal   antibodies  of  defined  specificity.     This  work  is   being  done   in 
collaboration  with  Drs.   John  Colligan  and  Thomas   Kindt   (Laboratory  of 
Immunogenetics)  who  are  looking  for  evidence  of  primary  sequence  differences 
between  the   KD  product  of  FY7  and  EL-4. 

Biochemical   studies  of  H-2   variants  will    allow  more   precise  analysis 
of  the  structural   nature  of  CTL  specificities  and  may  also  provide 
information  on  the  H-2  determinants   recognized  by  viral   specific  T  lympho- 
cytes.    We  plan  to  select  for  such   variants   in  vitro  by  repeated  exposure 
of  tumor  cells   to  anti-H-2   CTL.     This  approach  should  allow  the  screening 
of  large  numbers  of  tumor  cells   for  infrequent  aberrations   in  their  ex- 
pression of  H-2  determinants   recognized  by  CTL. 

Publications : 

Portis,   J.   L.:     Changes  in  the  transplantability  of  a   virus-induced 
murine  leukemia  tumor.      J.   Natl  .   Cancer  Inst.   62:     611-617,   1979. 

Portis,   J.   L.,  Wikel,  S.   K.   and  McAtee ,   F.   J.:     A  simplified  rapid 
method  for  purification  of  glomeruli.     J.    Clin.    Pathol  .    32: 
406-409,   1979. 


In  press : 

Portis,   J.   L.   and  Coe,   J.   E.:     Deposition  of  IgA  in   renal    glomeruli 
of  mink  affected  with  Aleutian  disease.     Am.   J.    Pathol. 


29-27 


EPIDEMIOLOGY  BRANCH 

Rocky  Mountain  Laboratories 

Hamilton,  Montana 

1 979  Annual    Report 

Table  of  Contents 


Z01    AI 
Project  Number 

Summary 

00061-17 

00063-09 

00065-06 

00067-14 
00068-18 
00069-48 

0007  9-19 
00080-12 
00081-18 

00082-18 

00083-10 

00084-10 


Natural    History  of  Tick-Borne  Rickettsial   Agents 
and  Their  Public  Health  Significance  -  Burgdorfer 

Immune  Responses  to   Legionnaires'    Bacterium  - 
Ormsbee 

Antigens  and  Classification  of  the  Rickettsiae  - 
Anacker  and   Philip 

Tularemia  -  Bell    (terminated) 

Rabies  -  Bell    (terminated) 

Systematics  and  Vector  Relationships  of  Certain 
Parasitic  Arthropods   -  Clifford 

The  Encephal i tides   (terminated) 

Tickborne  Disease  Agents  -  Yunker 

The  Epidemiology  of  Human   Infections  of  Special 
Interest  -   Phil ip 

Relation  of  Viruses  to  the  Genesis  of  Chronic 
Disease  -  Hadlow 

Host-Ectoparasite  Relationships  -  Wikel 
(terminated) 

Vector-Pathogen   Relationships   -  Yunker 


Page 
30-1 

30-13 

30-18 

30-21 
30-22 
30-23 

30-24 
30-30 
30-31 

30-35 

30-39 
30-44 

30-45 


Annual    Report 

Epidemiology  Branch 

Rocky  Mountain   Laboratories 

Hamilton,  Montana 

National    Institute  of  Allergy  and   Infectious  Diseases 

October  1,   1978  to   September  30,  1979 

RESEARCH  HIGHLIGHTS 

The  following  sections  summarize  highlights  of  our  research  program 
and  discuss  their  significance  in  relationship  to  the  epidemiology  and 
ecology  of  the  diseases  under  investigation.  Some  of  these  findings  and 
others  were  reported  in  20  publications.  An  additional  30  papers  have 
been  accepted  by  scientific  journals. 

RICKETTSIOSES 

Collaboration  with  various  universities  and  health  agencies  in  the 
United  States  and  abroad  has  continued  to  provide  clinical  and  field 
materials  vital  to  development  of  information  on  distribution  and  nature 
of  tick-borne  rickettsiae  and  rickettsial  infections.  Consequently,  we 
have  gained  insight  as  to  the  variety  of  spotted  fever-group  (SFG)  rickett- 
siae encountered  in  ticks  parasitic  for  man  in  the  United  States.  We  can 
now  suggest  preliminary  hypotheses  regarding  distribution  of  these  agents, 
their  ecologic  relationships  to  Rickettsia  rickettsii,  and  their  role  as 
causes  of  human  infection. 

Earlier  tick  surveys  have  provided  information  about  the  prevalence 
and  serotypes  of  SFG  rickettsiae  in  Connecticut,  Massachusetts,  New  York, 
South  Carolina,  Tennessee,  Alabama,  Mississippi,  Ohio,  Oregon,  and  Montana. 
Current  studies  will  give  additional  information  about  the  variety  and 
nature  of  rickettsiae  encountered  in  Dermacentor  occidental  is  and  Ixodes 
pacificus  from  California  and  D.   variabil is  from  North  Carolina.  The 
number  of  SFG  serotypes  in  the  U.S.  is  probably  finite  (10  have  been  identi- 
fied to  date).  Four  are  broadly  disseminated  in  both  eastern  and  western 
states  and  occur  in  more  than  one  species  of  tick-vector  of  Rocky  Mountain 
spotted  fever  (RMSF).  These  include  R_.  rickettsii  ,  R_.  montana,  R.   rhipi- 
cephali  and  the  unclassified  369-C  agent.  Only  R_.  rickettsii  has  thus 
far  been  etiological ly  associated  with  human  illness,  and  indications  are 
that  the  other  three  are  avirulent  for  man  (as  well  as  for  experimental 
animals)  although  tantalizing  evidence  based  on  serologic  responses  in 
residents  from  Long  Island  and  California  suggests  that  inapparent  or 
missed  infection  may  sometimes  occur. 

There  is  also  fragmentary  epidemiologic  evidence  to  suggest  that 
nonpathogenic  members  of  the  SFG  may  have  bearing  on  the  focal ization  of 
R_.  rickettsii .  Usually,  R_.  rickettsii  is  seldom  found  in  areas  where 
prevalence  rates  of  other  members  of  the  SFG  are  high.  We  are  considering 


30-1 


Rickettsioses   (cont'd) 

the  possibility  that  related  nonvirulent  rickettsiae  compete   in  mammalian 
amplifying  hosts  or  otherwise  interfere  with  the  maintenance  cycle  of 
R_.   rickettsi  i ,   perhaps   by  blocking  transovarial    transmission   in  the  arthro- 
pod  host.     These  possibilities  can   be   investigated  in  the  laboratory  and 
will   occupy  much  of  our  attention  during  the  next  several   years. 

An   increasingly  crucial    question  has  to  do  with  the  stability  of 
SFG  rickettsiae.     It   has   been  assumed  on   basis  of  unchanging  characteristics 
of  rickettsiae  under  laboratory  conditions  that  antigenic  differences 
encountered   in  nature  are  slowly  evolving  manifestations  and  are  not  merely 
expressions  of  short  term  phase  variation  or  degradation   induced   by  stresses 
of  uncertain  existence   in  the  arthropod   host  under  varying  environmental 
influences.     However,   it   is   increasingly  evident  that  the  tick   host   is  the 
keystone  to  our  understanding  of  the  ecology  of  R_.   rickettsii .     We  know 
very  little  about  host-parasite  relationships  at  the  cellular  and   sub- 
cellular level.     We  will    need  additional   expertise  to  define  host-parasite 
interactions,  morphologically  by  electron  microscopy,   and   functionally  by 
tick  physiology,    biochemistry,  and  molecular   biology.     These  needs  apply 
to  tickborne  viruses  as  well. 

Specifically,   a   hemolymph-test  survey  in  four  areas  of  North  Carolina 
revealed  SFG  rickettsiae   in  0.6  to  12.4%  of  2,000  ID.  variabilis   examined. 
Isolation  and   identification  of  these  organisms  are  pending.     Rickettsiae 
were  detected   in  as  many  as  35%  of  Amblyomma   americanum  collected   in 
certain  southeastern  areas  of  the  United  States.   One  characterized   strain 
was  antigenical ly  distinct  from  any  hitherto  described  SFG  rickettsia. 
Differences  were  also   detected   by  electron  microscopy,   pathogenicity  for 
experimental   animals,  and  protein  composition.     The  collaborative  study 
with  the  Harvard  School   of  Public  Health  of  D.  variabil is  on   Cape  Cod  was 
concluded.      Forty-three  isolates  of  SFG  rickettsiae  were  recovered   from 
7,000  ticks.     Thirty-nine  were  _R.  montana,  one  was   R_.   rickettsi  i   and  three 
were  unclassified.     Etiologic  association  of  R_.  montana   with  RMSF  could 
not  be  demonstrated.     A  rickettsia  comprising  a  new  SFG   serotype  was 
recovered  from  L   ricinus   in  Switzerland.     The   "Swiss  agent"   is   pathogenic 
for  meadow  voles,  chick  embryos,  and   several    lines  of  tissue  culture  cells, 
but  not  for  guinea  pigs.      It  possesses  antigen(s)  common   to  the  SFG  but 
is   sharply  distinct  from  R_.   rickettsii ,  R_.   sibirica  ,  R_.   slovaca,   and 
R_.  conorii .     £.  andersoni   collected  on  the  east  side  of  the  Bitterroot 
Valley,  Montana,  an  area  free  of  R_.   rickettsii  ,   frequently  contain  rickett- 
siae in  ovarial   tissues  which  are  nonpathogenic  for  various  experimental 
animals  and   indigenous  rodents.      It  is   hypothesized  that  this  organism 
is  maintained   solely  by  transovarial    passage  and  may  render   its  arthropod 
host  refractory  to  established   infection   by  R_.   rickettsii .      (Burgdorfer) 

Eight  isolates  of  SFG  rickettsiae  obtained  by  cell    culture  during 
the  1977  Bitterroot  Valley  survey  of  D_.   andersoni   ticks,   two  representing 
each  of  four  serologic  types   identified   by  microimmunofl  uorescence   (micro- 
IF)   including  _R.   rickettsi  i ,  _R.  montana ,   R_.   rhipicephal  i ,  and  the 


30-2 


Rickettsioses    (cont'd) 

unclassified  369-C  agent  were  examined  for  other  distinguishing   biologic 
characteristics   including  pathogenicity  for  chick  embryos,   guinea  pigs, 
and  cell   culture;   stability  during  serial    passage   in  Vero  cells;  anti- 
biotic sensitivity;  cross-immunogenicity  in  guinea   pigs;  and  growth  dynamics 
in  cell   culture.     Strains  within  serotypes  were  similar,   but  the  serotypes 
could  be  readily  distinguished  one  from  another  on   basis  of  several    biologic 
markers   in  addition  to  serologic  reactivity.     These  findings   give  us  added 
confidence  in  the  micro-IF  test  as  a  practicable  procedure  for  differen- 
tiating rickettsiae.     In  collaboration  with  the  California  State  Department 
of  Health,  we  are  determining  the  kinds  of  rickettsiae  prevalent  in  J). 
occidental  is  and   l_.   pacificus   in  several    coastal   areas   where  sporadic  cases 
of  RMSF  occur  in  absence  of  the  usual    tick  vectors.     Rickettsiae  represent- 
ing four  serotypes   have  been  isolated;  one  similar  to  R_.   rickettsii ,  one 
related  to  the  369-C  agent,  one  indistinguishable  from  an  unclassified 
SFG  rickettsia  found  in  I_.   pacificus  ticks  in  Oregon,  and  numerous   isolates 
similar  to  R_.  rhipicephal  i .      (Philip) 

In  western  Montana,   two  of  three  persons   bitten  by  R_.  rickettsii- 
infected  ticks  acquired  RMSF.     On  the  other  hand,   13  persons   bitten   by 
ticks   infected  with  R_.  rhipicephal  i,   R_.  montana,  or  the  369-C  agent  were 
not  ill   and  did  not  develop  antibodies  against  these  organisms.     However, 
in  a  collaborative  study  in  New  York  state,  14%  of  103  residents  of  Shelter 
Island  at  the  eastern  tip  of  Long  Island  developed  low  but  significant 
levels  of  micro-IF  antibodies  to  R.   rickettsii   and/or  the  369-C  agent 
between  May  and  October  1978„     Since  illness  could  not  be  correlated  with 
antibody  responses,   the  source  of  the  reactivity  is   enigmatic.     Similar 
low  levels  of  antibody  are  occasionally  found  among  coastal    residents  of 
California  with  tick-associated   illnesses  suggesting  that  other  SFG  agents 
may  occasionally  give  rise  to  inapparent   infection  or  abortive  disease. 
We  are  continuing  our   investigations  on  the  serodiagnosis  of  rickettsial 
disease.     A  latex  agglutination  test   (in  collaboration  with  the  New  York 
State  Health  Department),  an  enzyme-linked   immunosorbent  assay   (ELISA), 
and  a  fluoroimmunometric  assay   (FIAX)   were  evaluated   for  the  diagnosis  of 
RMSF.     The  last  two  tests  are  quantitative  and  4  to  8  times  more  sensitive 
than  our  standard  micro-IF  test,   but  otherwise  offer   few  advantages  over 
the  latter.       (Philip,  Casper) 

LEGIONNAIRES'    DISEASE 

Studies  on  Legionnaires'   disease   (LD)   are  continuing,  on  reduced 
scale  after  the  retirement  of  Dr.   Ormsbee.     We  hope  to   increase  activity 
in  this  area  in  the  near  future.     The  parameters  of  infectivity  and  patho- 
genicity of  four  strains    (LAC,   LAP,   LAW,   LAB)   of  Legionella   pneumophila 
representing  three  serologic  types  initially  cultivated  in  chick  embryo 
yolk  sac  were  determined  after  9  and  12  serial    passages  on  Mueller-Hinton 
(MH)  medium.     The  plaquing  ability  on  chick  embryo   cells  and  pathogenicity 
for  guinea   pigs  of  the  LAC,   LAP,  and  LAB  strains  was  greatly  diminished 
after  in  vitro   passage.     The  LAW  strain,  originally  isolated  on  MH  medium, 


30-3 


Legionnaires'    Disease   (cont'd) 

showed  good  plaquing  ability  that  did  not  change  during  passage.     This 
strain  was  only  weakly  pathogenic  for  guinea   pigs   both   before  and  after 
passage.     We  are  now  looking  for   biochemical   and  serologic  markers   to 
explain  the  changes   noted.     Further   passages   in  chick-embryo  yolk  sac 
are  also   being  conducted  to  determine  whether  variations  are  stable  charac- 
teristics.    Work  with  LD  during  the  coming  year  will    include  development 
of  better  laboratory  procedures  for  early  diagnosis  and   definition  of 
growth  dynamics  of  L_.   pneumophila   in  chick-embryo  cell    culture.      (Ormsbee, 
Peacock) 

SYSTEMATICS  OF  TICKS 

Systematic  treatment  of  ticks  of  medical    importance,   including 
taxonomy,   biology,  ecology  and  colonization  continues   to   be  the  major  goal 
of  this   project.     Our   studies  on  the  taxonomy  of  the  genus  Argas   based  on 
the  morphology  of  Haller's  organ   have   been  expanded   to   include  more  species 
in  each  subgenus.     We  hope  that  morphology  of  this  organ  can   be  correlated 
with  host-specificity  and  other  behavioral   characteristics  of  these  ticks. 
Significant  progress  was  made   in  our  continuing  study  of  the  Ornithodoros 
capensis  complex.     Using  the  scanning  electron  microscope   (SEM) ,  we   have 
redescribed  0_.  amblus ,  an  ectoparasite  of  marine  birds   in   Peru,  and  are 
preparing  photos  of  0_.  muesebecki   for  possible  redescription  of  this 
species.     Both  ticks  frequently  bite  man  and  carry  viruses   that  may  cause 
disease   in   humans  as  well   as   birds.     We  are  also  completing  descriptions 
of  0_.  darwini   and  0_.   gal  apagensis ,   ectoparasites  which  are   probable  vectors 
of  i ntraerythrocytic  hemogregarines  of  marine   iguanas  and  lava  lizards  on 
the  Galapagos    Islands.     Practical   guides,  amply  illustrated  with  SEM 
photos  that  will   allow  easier   identification,   have   been  prepared   for  the 
genus   Ixodes   and  are  being  prepared   for   Dermacentor  as  well.     The  magnifi- 
cation of  the  photos   is   well   within   the  range  of  standard   stereoscopic 
microscopes  and  should  enable  health  authorities   to   readily  identify 
vectors  of  RMSF.     We  initiated  a   study  to   clarify  the  status  of  an    Ixodes 
species  of  the  angustus   complex  that  occurs   in  Black  Hills,   South   Dakota, 
where  years  ago,    Dr.   Carl    Eklund   isolated   Powassan  virus   from   Ixodes. 
We  are  also  continuing  our  investigations  of  medically  and  agriculturally 
important  rhipicephal id   species   from  Africa  and   hope  soon  to   initiate  a 
study  of  the  R_.   sanguineus  complex,   some  members  of  which  are  vectors  of 
human   rickettsioses.     Final    editing  of  the  master  list  of  Smithsonian 
computer  data  on  the  RML-NAMRU-3  tick  collection   has   been  completed. 
This   fall,  a  mini   file  will    be  prepared  for  economy  of  data-search,  and 
we  can   begin  to  use  the   information.      In   the  meantime,   new  records  are 
rapidly  accumulating  and  we  have  as  yet  no  means   for  updating  the  computer 
data   bank  on  a  continuing   basis.      (Clifford,    Keirans) 

Results  of  research  on   the  G.H.F.   Nuttall    tick  collection,   the 
world's   third  largest,  deposited   in  the  British  Museum,  are   being  compiled 
for  publication   in   book  form.     This   collection  contains  over  100  type- 


30-4 


Systematics  of  Ticks   (cont'd) 

species  that  are  known  vectors  of  human  and  animal    disease  agents.     Many 
are   in  need  of  taxonomic  clarification.     A  second   important  tick  collection, 
that  of  N.   C.  Rothschild,  was  also  redetermined  and  updated.      (Keirans) 

In  taxonomic  studies  of  mites,  manuscripts  describing  clinical  mange 
in  the  brown  bear  and  laboratory  primates,  as  well   as  the  macronyssid   fauna 
of  Surinam,   have  been  submitted  for  publication.     Parasitic  Acari   of 
Saudi  Arabia  are  being  studied   preliminary  to   preparation  of  a  fauna   list 
and  taxonomic   key  for  public  health  workers.      (Yunker) 

ARTHROPOD-BORNE  VIRUSES 

In  our  continuing  studies  of  distribution,  ecology,   identification, 
characterization  and  classification  of  tick-borne  viruses  and  their  poten- 
tial  relationship  to   human  disease,  102  isolates  were  obtained  from  more 
than  1,100  ticks  collected  in  seven  states.     Agents  identified  thus  far 
included  30  isolates  of  Soldado  virus  from  Ornithodoros  capensis   ticks   in 
Texas,   25  strains  of  Hughes  virus   from  0_.   denmarki    in   Florida,   15   strains 
of  Sapphire  II  virus  from  Argas  cool eyi    in  South  Dakota,  and  16   isolates 
of  Sixgun   City  virus   from  the  Argas  collection.     Both  species  of  Ornitho- 
doros are  parasites  of  migratory  seabirds.     Soldado  and  Hughes  virus  are 
distantly  related  members  of  an  unclassified  taxon  referred  to  as  Hughes 
virus   serogroup.     Some  Hughes  group  agents   have   been   implicated   in   seabird 
die-offs  and  febrile  diseases  of  man.     A_.  cool  eyi    is  a   parasite  of  migra- 
tory passarine  birds,  and  Sapphire   II  virus   is  also  a  member  of  the  Hughes 
serogroup.     Sixgun   City  is  a  reovirus.     Geographical    records  of  tick-borne 
viruses   in  various  vectors  and   in  flyways  of  migratory  birds   enable  us  to 
predict  distribution  and  occurrence  of  these  pathogens.     High  prevalence 
of  several   of  these  viruses   in  ticks    (e.g.,  Soldado  and  Hughes   viruses) 
suggests  that  these  arthropods   serve  both  as   host  and  vector.     Of  particular 
interest  was  the  recent   isolation  of  an   unidentified  virus   from  D_.   occiden- 
tal is  collected   in  two   Oregon  counties.     p_.  occidental  is    is  abundantly 
distributed   in  coastal    areas   from  Washington  to   California  and   frequently 
bites  man.     Fourteen  isolates  were  obtained   in  a  poikilothermic  cell    line 
derived  from  Xenopus     laevis,  South  African  clawed   frog.     None  was  obtained 
in  Vero  cells.     The  virus   is  pathogenic  for  meadow  voles  and  will    propagate 
in  L  cells,   pig  kidney  cells  and  tick  tissue  cultures,   but  it   is  non- 
pathogenic for  suckling  mice  and   hamsters.      Physicochemical    properties  are 
being  characterized.     Xenopus  cells  were  also  superior  to  other  lines  for 
recovery  of  Soldado  virus.      It  may  prove  particularly  useful    for   isolation 
of  temperature-sensitive  variants.     (Yunker,  Clifford,   Thomas) 

Our  studies  of  vector-borne  virus   interaction  with  arthropods, 
particularly  ticks  at  a  cellular  level,  continue.     Last  year,   we   began 
investigation  of  transovarial   transmission  of  Flaviviruses   by  ticks. 
Langat  virus  inoculated   parenterally  into   Dermacentor  females  was   trans- 
mitted  by  bite  by  all    stages  of  the  F]    generation.     However,  virus  could 
be  demonstrated  only  in  a  few  F2   eggs  and  larvae  and  could  not   be  detected 


30-5 


Arthropod-Borne  Viruses   (cont'd) 

at  all    in  subsequent  stages.      It  may  be  that  proviral    DNA  was  not   involved 
in  this   particular  system  in  contrast  to   findings   by  Soviet   investigators 
of  DNA  transcripts  of  Bhanja  virus   RNA  in  eggs  of  Hyalomma  ticks.     Addi- 
tional   experiments  with  Quaranfil   virus   in  Argas  arboreus  and  Colorado 
tick  fever  (CTF)  virus   in  Dermacentor  are  underway.     Carrier  cultures  of 
CTF  virus  were  established   in  Xenopus  cells   grown  at  27°C.      Even  after  50 
passages  apparently  normal   cultures  yielded  low  titers  of  CTF  virus  which, 
unlike  that  grown  in  mosquito  cells,  was  unchanged   in   pathogenicity  for 
suckling  mice.     Thus,   temperature  of  incubation  alone  does  not  seem  to 
determine  CTF  virus  attenuation   in   insect  cells.     Mosquito  cell   cultures 
were  evaluated  for  detection  of  dengue  viruses.     Four  serotypes  of  dengue 
virus  were  inoculated   into  mosquitos   by  Dr.   Rosen   in  Hawaii.     Virus  was 
detected  at  RML  from  all    but  3  of  24  infected  mosquitos  after  they  were 
inoculated  into  Aedes  albopictus  cells.     Two  strains  missed  were  dengue-3 
virus.     Thus,   it  appears  that  mosquito  cells  may  be  useful    for  rapid 
detection  of  3  of  4  dengue  serotypes.     This  year,  we  have  established  a 
serially  propagating  cell    line  from  _D.  variabil  is   embryos.     These  cells 
are  now  in  their  21st  passage,  are  diploid,  and  form  monolayers  within 
one  week.     The  North  American  tick-borne  Flavivirus,   Powassan,  was  shown  to 
replicate   in  these  arthropod  cells.     Transovarial    transmission  of  Flavi- 
viruses   by  ticks,   particularly  St.   Louis  encephalitis   virus,  will    receive 
particular  emphasis  next  year,   based  on  the  recent  discovery  by  French 
investigators  of  natural    infections  of  yellow  fever   in,  and  transovarial 
transmission  of  virus   by,   wild-caught  African  ticks.      (Yunker) 

PATHOGENESIS  OF  SLOW  VIRAL  DISEASES 

This  project,  which  considers  the  unusual    host-virus  relationships 
that  give  rise  to   slowly  evolving  virus  diseases  after  a  long  incubation 
period,   is  being  terminated.     Emphasis   has   been  directed   particularly  to 
scrapie-! ike  diseases  of  sheep,  goats,  mink  and  man.      No  new  experiments 
were  initiated  during  the  year.     However,  a  most  noteworthy  finding  occurred 
in  experiments   still    in   progress.     Four  years  ago,   a   pilot  study  was   begun 
during  which  specimens  of  brain  from  11    patients  dying  of  Creutzfeldt-Jakob 
disease  (CJD)   were  inoculated  into   brains  of  African  pygmy  goats.     One  of 
three  goats   that  received  one  brain  specimen  and  one  of  four  that  received 
another  manifested  a   scrapie-like  disease  43  months  after  inoculation. 
One  goat  was   sacrificed  8  weeks  after  clinical    signs  appeared.     Histologic 
examination  of  the  brain  and  spinal   cord   showed  changes  compatible  with 
those  of  caprine  scrapie.     Astrocytosis  was   prominent   in  diencephalon,  mid 
brain,  and  cerebellar  cortex,   but  spongiform  changes  were  minimal.     The 
other  goat  with  scrapie-like  signs   is   still    being  observed,  and   three  pen- 
mates may  now  be  in   prodromal    stage  of  disease.     Goats   inoculated  with  the 
other  nine  specimens  are  still    normal,   but  most  have  not   been  observed  long 
enough  to  warrant  conclusions  as  to  whether  they  too  may   become  affected. 


30-5 


Pathogenesis  of  Slow  Viral    Diseases   (cont'd) 

Occurrence  of  scrapie-like  disease  in  these  goats   is  a  very  signifi- 
cant finding  in  that  it   indicates  a   possible  relationship  of  CJD  virus  to 
scrapie  virus.     Although  CJD  virus  and  scrapie  virus   induce  similar  neuro- 
logic diseases   in  squirrel   and  rhesus  monkeys,   the  observation  here  that 
CJD  virus  induced  disease  and  pathologic  changes   in  goats   that  are  indis- 
tinguishable from  those  resulting  from  natural    infection  with  scrapie 
virus  may  be  more  significant  than  occurrence  of  similar  disease   in  an 
aberrant  host.     Guinea   pigs  and  mink  inoculated  intracerebral! y  with   brain 
specimens  from  the  same  patients  with  CJ  disease  have  remained   free  of 
recognizable  neurologic  disease  after  2  and  4  years,   respectively.     A 
presumed  neurologic  disease  in  similarly  inoculated  mice  has  recurred  in 
some  of  those  in  second  passage,   but  the  results  are  still    inconclusive. 

Except  for  continuing  observation  of  animals   inoculated  with  CJD 
material,  no  new  experiments  are  currently  contemplated.     However,   it   is 
most  important  that  the  findings  of  scrapie-like  disease  in  CJD  virus- 
inoculated  goats  on  primary  passage  of  brain  material   from  patients  be 
confirmed  by  secondary  passage   in  these  animals.      (Hadlow) 


30-7 


30-8 


Annual    Report 

Epidemiology  Branch 

Rocky  Mountain  Laboratories 

Hamilton,  Montana 

National    Institute  of  Allergy  and  Infectious  Diseases 

October  1,  1978  to  September  30,  1979 

ADMINISTRATIVE  REPORT 

In  March  197  9,  the  Epidemiology  Branch   (EB)  was  officially  estab- 
lished as  one  of  three  laboratories  which  comprise  what  was   formerly  the 
Rocky  Mountain  Laboratory.     Currently,   the  EB  comprises  three  sections, 
viz.:     Arthropod-Borne  Diseases,  Epidemiology,  and  Histopathology.     The  EB 
is  manned   by  8  Ph.D. -level    scientists,   including  4   in  the  Arthropod-Borne 
Diseases  Section,  3  in  the  Epidemiology  Section,  and  one  in  the  Histo- 
pathology Section.     In  addition,  there  are  12  supporting  staff  and  one 
secretary  assigned  to  the  Office  of  the  Chief.     A  permanent  Chief  of  EB 
has  not  yet  been  appointed.     The  total   complement  is  22.     All  ,  at  present, 
are  tenured  personnel.     Prior  to  reorganization,  two  scientists  retired 
(Dr.   Richard  Ormsbee  in  January  1979  and  Dr.  J.   Frederick  Bell    in  February 
1979).     Dr.  Ormsbee's  project  on  Legionnaires'   disease  is  being  continued 
and  hopefully  will    be  expanded   in  the  near  future.     Dr.   Bell's  projects  on 
rabies  and  tularemia  have  been  terminated. 

A  visiting  scientist,   Dr.   Klaes  Kindmark,  Uppsala,   Sweden,  arrived 
in  February  for  a  6-month  period  in  Dr.  Burgdorferi  laboratory.     He  is 
developing  more  satisfactory  techniques  for  early  diagnosis  of  rickettsial 
diseases.     Dr.  William  Todd,  Colorado  State  University,  will    spend  2  years 
in  Dr.  Burgdorfer's  laboratory  doing  tick-transmission  studies  of  SFG 
rickettsiae  under  an  IPA  contract. 

The  EB  has  assumed  the  role  of  WHO  Collaborating  Center  for  Rickett- 
sial  Reference  and  Research  which  was   formerly  the  responsibility  of  RML. 
In  this  regard,  we  provide  reference  rickettsial  antigens  and  antisera 
and  consultative  expertise  to  qualified  foreign  as  well   as  domestic   inves- 
tigators. 

An   International    Symposium  on  Spongiform  Encephalopathies  was   held 
at  RML  in  honor  of  Dr.   Carl    Eklund,  October  1978.     Approximately  57  visitors 
(U.S.  and  foreign)  attended.      In  June  1979,  a  Workshop  on  St.   Louis 
Encephalitis  was   held  at  RML  under  sponsorship  of  the  Viral    Diseases   Panel, 
U.S. -Japan  Cooperative  Medical    Sciences   Program,   NIAID.     Approximately 
54  participants  and  4  staff  members  of  NIH  attended   this  meeting. 


30-9 


30-10 


Annual    Report 

Epidemiology  Branch 

Rocky  Mountain  Laboratories 

Hamilton,  Montana 

National    Institute  of  Allergy  and   Infectious   Diseases 

October  1,  1978  to  September  30,  1979 

HONORS  AND  AWARDS 

The  following  activities  reflect  the  recognition  afforded  staff  of 
EB   by  their  peers   in  the  scientific  community. 

Editorial    Boards  of  Journals: 

Dr.  W.   Burgdorfer  -  Acta  Tropica 

Dr.    C.  M.   Clifford  -  Journal    of  Medical    Entomology 

Dr.    C.   E.   Yunker  -  Journal   of  Parasitology 

Drs.   Burgdorfer,   Yunker,  Clifford,   Hadlow,  and   Philip  reviewed 
manuscripts   submitted  for  publication   in  J.  Med.    Entomol . ,   Am .   J .   Tro p . 
Med.   Hyg. ,   J.   Parasitol. ,  Ann.    Entomol .   Soc.  Am. ,   Vet.    Pathol. ,   New  Eng. 
J.   Med. 

Professional    Posts: 

Dr.   R.   N.    Philip  -  Secretary,   American   Committee  on  Rickettsiae  and 
Rickettsial    Diseases 

Dr.   W.   Burgdorfer   -  Consultant,   University  of  Neuchatel ,   Switzerland 

Dr.  W.   J.   Hadlow  -  Member  of  the  Council,  American  College  of  Veterinary 
Pathologists 

Dr.   C.    E.   Yunker  -  U.S.   representative  to  the  Committee  on  Polar  Viruses 

Invited   Lectures  and   Participation   in  Meetings  and  Symposia: 

Dr.   W.   Burgdorfer  was   invited   by  Rockefeller   Foundation  to  partici- 
pate and  present  a  paper  at  the  Conference  on   Comparative  Aspects  of 
Animal   and   Plant  Pathogen  Vectors,   Bellagio,   Italy;   he  was  also   invited 
by  ASM  Committee  on   Pathogenic  Microorganisms  to  present  a  lecture  on 
"Borreliae  and  Borrel ioses",   Los  Angeles. 

Dr.   R.   N.    Philip  presented  a   paper  and  chaired  a   session  on 
rickettsiae  at  VII   Intl.    Congress  of   Infections  and   Parasitic   Diseases, 
Varna,   Bulgaria. 

Dr.   W.   J.   Hadlow  headed  the  Organizing  Committee  of  Symposium  on 
Spongiform  Encephalopathies,   Hamilton,  Montana,    in   honor  of  Dr.   Carl    Eklund. 

30-11 


30-12 


Ml  HISUNIAN    I..CILNGL    I  Nl  UliMAT  I  UN    LXCIIANUL 
PROJECT   NUMBER    (Do   NOT    use    this    space) 


\K1MLNT    01 
\TI0N,    AND    WELFARE 
C.    'ILALTII    SERVICE 
NOTICE   OF 
INTRAMURAL   RESEARCH   PROJECT 


HEALTH,    EDU 
PUBLI 


PH0JLC1    NUMULH 

Z01    AI   00061-17   EB 


PERIOD  COVERED 


October  1,  1978  to   September  30,   1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Natural    History  of  Tick-Borne  Rickettsial    Agents  and  Their  Public  Health 
Significance 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

Res.    Entomologist   (Med.) 
Medical    Director 
Research  Microbiologist 
Nurse   Director 


PI: 

OTHER: 


W.  Burgdorfer 

R.  N.    Philip 

L.  A.   Thomas 

E.  A.    Casper 

R.  L.   Anacker 


Research  Microbiologist 


EB  NIAID 
EB  NIAID 
EB  NIAID 
EB  NIAID 
LMSF  NIAID 


cooperating  units  (if  any)    Dr _    j_    L>   Benacn>   state  of  New  York   Dept.  of  Health; 
Dr.   J.    N.   MacCormack,   CDC  Branch,   Dept.   Human   Resources,   North  Carolina; 
Dr.  W.    Feng,   Harvard  School   of  Pub.   HI th 


Conn.    Agric.    Res.    Exp.    Station.    Dr. 


Drs.    J.  Anderson   and   L.  Magnarelli, 
C.-O. "Kindmark,   Uppsala,   Sweden. 


LAB/ BRANCH 

Epidemiology  Branch  (RML),  Hamilton,  MT  59840 


SECTION 

Arthropod-Borne   Diseases  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  MD  2C205 


TOTAL  MANYEARS: 


3.6 


PROFESSIONAL: 


1.0 


2.6 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 


(b)  HUMAN  TISSUE 


□  (c)  NEITHER 


□  (a1  )  MINORS 


(a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

This  project   is  concerned  with  studies  of  Rocky  Mountain   spotted   fever  and 
other  tick-borne  rickettsial    diseases   in  the  United   States  and   in  other 
countries  regarding  ecology,   identification,  and  characterization  of  the 
rickettsial   agent(s)   with  their  associated  vectors.     With  collaboration  of 
outside  agencies   (state   health  departments,   public  health  laboratories, 
hospitals,   physicians,  etc.)   source  material    for  comparative  studies  on  the 
various  rickettsial    types  and  information  about  the  distribution  of  vector 
species  associated  with  spotted   fever  in  the  United  States   is  obtained. 
This  project  also  considers  the  cellular  and   subcellular  aspects  of  inter- 
action between  tick-borne  agents,   particularly  rickettsiae,  and  their  vectors, 


30-13 


PHS-6040 
(Rev.    IO-76) 


Project  No.  Z01  AI  00061-17  EB 


Project  Description: 


Rocky  Mountain  spotted  fever  continues  to  be  a  significant  health 
problem  throughout  most  of  the  United  States.  As  a  result,  many  outside 
agencies  called  on  this  laboratory  for  guidance  and  assistance  in  their 
efforts  to  determine  the  epidemiologic,  ecologic,  and  health  impacts  on 
the  public.  Because  of  changes  in  RML's  rickettsial  research  program, 
some  of  our  previous  collaborative  investigations  with  outside  agencies 
had  to  be  terminated  or  were  limited  to  providing  research  guidance  and/or 
diagnostic  reagents  necessary  for  epidemiological  and  ecological  investi- 
gations. With  the  exception  of  a  tick/rickettsial  survey  in  North  Carolina, 
most  of  our  current  projects  are  related  to  the  evaluation  and  characteri- 
zation of  rickettsial  material  collected  from  various  parts  of  the  U.S. 
during  recent  years. 

Research  accomplishments  during  the  past  year  include  the  following 
findings: 

North  Carolina  continues  to  have  the  highest  incidence  of  spotted 
fever  cases  in  the  U.S.  In  1978,  it  reported  204  cases  or  approximately 
20%  of  the  nation-wide  reported  total.  In  cooperation  with  the  Duke  Univer- 
sity Medical  Center  at  Durham,  N.C.,  and  the  Communicable  Disease  Control 
Branch,  Department  of  Human  Resources  at  Raleigh,  N.C.,  a  study  was  initiated 
to  determine  in  selected  areas  of  that  state  the  prevalence  of  spotted 
fever  group  (SFG)  rickettsiae,  particularly  of  Rickettsia  rickettsii  in  the 
American  dog  tick,  Dermacentor  variabilis.  So  far,  2,123  adult  ticks 
from  4  different  areas  were  examined.  The  percentage  of  ticks  infected 
with  typical  pleomorphic  SFG  rickettsiae  in  these  areas  ranged  from  0.6  to 
12.4%.  Isolation  and  serotyping  of  these  rickettsiae  remain  to  be  done. 

Isolation  of  SFG  rickettsiae  from  Ixodes  dammini  from  Shelter  Island, 
New  York,  suggests  that  this  tick,  all  stages  of  which  readily  attack  man, 
may  be  involved  as  a  vector  of  spotted  fever  rickettsiae  in  the  eastern 
parts  of  the  U.S. 

A  SFG  rickettsia  has  been  detected  in  up  to  35%  of  Amblyomma  ameri- 
canum  coll ected  in  several  southern  and  southeastern  states.  The  fact 
that  residents  bitten  by  infected  ticks  did  not  develop  illness  suggests 
that  this  rickettsia  is  nonpathogenic  for  man.  Laboratory  investigations 
(see  1976-77  Project  No.  Z01  AI  00065-04  RML)  with  this  agent  have  now  been 
concluded  and  indicate  that  this  organism  is  related  to,  but  quite  distinct 
from,  any  hitherto  described  SFG  agent.  Differences  include  various  mor- 
phologic (electron  microscopy),  physiologic  (behavior  in  laboratory  animals), 
biochemical  (protein  composition  as  determined  by  SDS-polyacryl amide  gel 
electrophoresis)  as  well  as  serologic  microimmunofl  uorescence  (micro-IF) 
test  characteristics. 


30-14 


Project  No.    Z01    AI   00061-17   EB 

The  final    results  of  the  cooperative  study  with  the  Harvard   School 
of  Public  Health  on   spotted  fever  on   Cape  Cod,  Massachusetts,   indicate  that 
R_.  mo n tana  does  not  play  a  role  in  the  etiology  of  this  disease  on   Cape 
Cod.     In  1977-78,  a  total   of  7,302   D.   variabilis  were  tested.     Seventy- 
nine  (1%)  were  found  to   be  infected  with  SFG  rickettsiae.     Of  these,  43 
isolates  were  made  and  identified.     Thirty-nine  were  R_.  montana ,  one  was 
R_.   rickettsii ,  and  3  were  of  an  unclassified  serotype.     The  _R.   rickettsii 
isolate  came  from  a  tick  removed  from  a  patient  with  spotted   fever.     Of 
117  dogs  with  antibodies  to  SFG  rickettsiae,   65   (56%)   had  at  least  four- 
fold or  higher  titers  to   R_.  montana  than  to  R_.   rickettsii.      Nineteen   (16%) 
had  greater  titers  to   R_.   rickettsii   and  33    (28%)   had  equivalent  titers  to 
both  serotypes. 

A  survey  of  viral,  rickettsial,  and  protozoan  agents  in  ticks   from 
Switzerland  revealed  a   hitherto  undescribed  SFG  rickettsia   ("Swiss  agent") 
in  up  to  11.7%  of  adult   I_.   ricinus.      Infection   in  ticks  was   found  to   be 
generalized  with  intracellular  as  well   as  intranuclear  growth,  and  trans- 
ovarial   passage  to  100%  of  filial    ticks.     The  Swiss  agent  is  nonpathogenic 
for  guinea  pigs  but  in  meadow  voles   (Microtus   pennsyl vanicus)   produces  a 
microscopically  detectable  infection  in  the  tunica  vaginalis.     It  does  not 
grow  in  artificial  media  but  propagates  well    in  tissue  culture  systems 
including  chick  embryo   fibroblast,  Vero,   and  vole-tissue  cells.      It  kills 
100%  of  chicken  embryos  5   to   7  days  after  yolk  sac  inoculation.     Antigenic 
relatedness  of  the  Swiss  agent  to  SFG  rickettsiae   is   indicated   by  direct 
fluorescent  antibody  staining.     Serologic  typing  by  micro-IF  shows  that 
this  agent  is  quite  different  from  R_.   rickettsii ,   R_.    sibirica,   R_.   slovaca, 
and  R_.   conori  i.     So  far,  there  is  only  serologic  evidence  that  the  Swiss 
agent  may  be  pathogenic  for  man. 

Electron  microscopic  investigations  of  the  Swiss  agent  in   its  tick 
vector,  J_.   ricinus,   revealed  rickettsiae   in  early  spermatids.     This  obser- 
vation  induced  us  to   re-evaluate  sexual    transmission  of  rickettsiae  by 
tick  sperms  a   phenomenon  claimed  to   be  an   important  means   for  the  dissemina- 
tion and  maintenance  of  the  spotted  fever  agent,  R_.   rickettsii ,   in  nature. 
The  results  of  this  investigation  are  not  as  yet  available. 

During  a  tick/rickettsial    survey  in  an  area  of  western  Montana 
where  human  cases  of  spotted  fever  have  never  occurred,   up  to  75%  of  adult 
D.   andersoni   were  found  to   be  infected  with  a  SFG  agent  that  was  limited 
in   its  development  to  the  ovarial   tissues  and  occasionally  also  to  certain 
parts  of  the  Malpighian  tubules.     The  organism  has   been  isolated  and  appears 
to  be  related  to  SFG  rickettsiae.     Since   it  is  nonpathogenic   for  laboratory 
animals  and  any  of  several    indigenous  rodents  tested  so   far,   it   is   specu- 
lated that  it  is  maintained   in  nature  by  transovarial    passage  only  and  may 
indeed   be  responsible  for  rendering  its  tick  vector  refractory  to   infection 
with  virulent  _R.    rickettsii .     Experiments  to   clarify  this  question  are   in 
progress. 


30-15 


Project   No.    Z01    AI   00061-17   EB 

Serologic  characterization  of  rickettsial    isolates  from  ticks  taken 
off  residents  in  western  Montana  or  collected  from  vegetation   (see  1977-78 
Project  No.    Z01    AI   00065-05  RML)   revealed  the  presence  of  at  least  4  sero- 
types of  SFG  rickettsiae,  namely  R_.   rickettsii ,   R.   montana,  R_.    rhipicephal  i , 
and  369-C  -  an  organism   indistinguishable  from  a   rickettsia   isolated 
initially  from  JD.   variabil  is   in  Arkansas.      In  cooperation  with  staff  of 
RML's   Laboratory  of  Microbial    Structure  and   Function,   a   long-term  project 
was   initiated  to  determine  the  antigenic  makeup  and   stability  of  these 
serotypes  as  well   as  their  physiologic   behavior   in   tick  and  vertebrate 
hosts.     Additional    investigations  pertain  to  the  susceptibility  of  various 
vertebrate  hosts   indigenous  to  western  Montana  and  to   the   question  of 
interference  among  these  serotypes  of  rickettsiae. 

Male  guinea  pigs  are  being  used  by  Dr.   Kindmark  as  a  model    for  the 
development  of  an  early  diagnostic   procedure   for  rickettsial    diseases  in 
man.     Animals  infected  with  a  virulent  strain  of  R_.   rickettsii   are   bled 
on  day  4  of  fever  and  are  examined  for  rickettsial   antigens   by  direct  and 
indirect  immunofl  uorescent  staining  of  smears   prepared   from  the   buffy  coat 
of  blood  samples,  and  by  immunoadsorbence   in  combination  with  el ectro- 
immuno-osmophoresis  or  electrophoresis   in  agarose  containing  antibodies 
to  R_.   rickettsii.     The  appearance  of  early  specific  antibodies   is   being 
determined   by  the   indirect   immunofluorescence  test.     The  results  obtained 
so   far  are   promising  and  suggest  that  these  methods  may  be  applicable  to 
an  early  diagnosis  of  spotted  fever  and  related  rickettsial    diseases  of  man. 

Publ ications : 

Loving,   S.   M. ,   Smith,  A.    B. ,    DiSalvo,   A.    F.,   and   Burgdorfer,   W. : 
The  distribution  and  prevalence  of  spotted   fever  group  rickettsiae 
in  ticks   from  South  Carolina  with  an  epidemiological    survey  of 
persons   bitten  by  infected  ticks.     Am.   J.   Trop.  Med.   Hyg.   27: 
1255-1260,   1978. 

Burgdorfer,   W. :     The  Rocky  Mountain  wood  tick:     Beware   its   bite! 
Montana  Outdoors  May/ June:     2  9-31,   1979. 

Hayes,   S.    F.   and  Burgdorfer,   W.  :     111  trastructure  of  Rickettsia 
rhipicephal i ,  a  new  member  of  the  spotted   fever  group  rickettsiae 
in  tissues  of  the  host  vector  Rhipicephal us  sanguineus.     J.   Bacteriol . 
137:      605-613,   1979. 


30-16 


Project   No.    Z01    AI   00061-17   EB 


In   press 


Burgdorfer,  W. :  The  spotted  fever-group  diseases.  In  Steele,  J.  H. 
( Ed . ) :  CRC  Handbook  of  Zoonoses 

Burgdorfer,  W. :  Rickettsioses .  Chapter  34.  In  Hubbert,  McCulloch 
and  Schnurrenberger  (Eds.):  Suppl ementum  to  "Diseases  Transmitted 
from  Animals  to  Man" 

Magnarelli,  L.  A.,  Anderson,  J.  F.,  and  Burgdorfer,  W. :  Rocky 
Mountain  spotted  fever  in  Connecticut:  human  cases,  spotted  fever- 
group  rickettsiae  in  ticks,  and  antibodies  in  mammals.  Am.  J. 
Epidemiol . 

Linnemann,  C.  C,  Jr.,  Schaeffer,  A.  E. ,  Burgdorfer,  W.,  Hutchinson, 
L.,  and  Philip,  R.  N. :  Rocky  Mountain  spotted  fever  in  Clermont 
County,  Ohio.  II.  Distribution  of  population  and  infected  ticks  in 
an  endemic  area.  Am.  J.  Epidemiol. 

Aeschl imann,  A.,  Burgdorfer,  W. ,  Matile,  H.,  Peter,  0.,  and  Wyler,  R. 
Aspects  nouveaux  du  role  de  vecteur  joue  par  Ixodes  ricinus  L.  en 
Suisse.  Note  preliminaire.  Acta  Trop. 

Burgdorfer,  W.,  Aeschl imann,  A.,  Peter,  0.,  Hayes,  S.  F. ,  and  Philip. 
R.  N.:  Ixodes  ricinus:  vector  of  a  hitherto  .undescribed  spotted 
fever  group  agent  in  Switzerland.  Preliminary  note.  Acta  Trop. 


30-17 


oMI  IIISUNIAN  3CI  Lt-iCL  INIOUMAIIUN  tXGIIANGL 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  Di.l  ANTMENT  Uf 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01    AI    00063-09   EI 


PERIOD    COVERED 


October  1,   1978  to   September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Immune  Responses  to  Legionnaires'  Bacterium 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI: 


OTHER: 


R.  A.  Ormsbee 

M.  G.  Peacock 

R.  N.  Philip 

E.  A.  Casper 

J.  C.  Williams 


Scientist   Director 
Microbiologist 
Medical    Director 
Nurse  Director 
Biochemi  st 


[retired' 


EB  NIAID 
EB  NIAID 
EB  NIAID 
LMSF  NIAID 


COOPERATING  UNITS  (if  any) 

Dr.    Paul    Fiset,   Dept.  of  Microbiology,   University  of  Maryland  School   of 
Medicine,   Baltimore 


LAB/BRANCH 

Epidemiology  Branch   (RML),   Hamilton,   MT  59840 


SECTION 

Epidemiology  Section 


INSTITUTE  AND  LOCATION 

NIAID,   NIH,    Bethesda,   MD  20205 


TOTAL  MANYEARS: 


1.5 


PROFESSIONAL: 


0.1 


1  .4 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 


(b)  HUMAN  TISSUES 


□  (c)  NEITHER 


□  (al  )  MINORS 


(a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  purpose  of  this  project  is  to  study  immune  responses  in  man  and  experi- 
mental animals  to  natural  and  experimental  infections  of  Legionella 
pneumophila,  the  causative  agent  of  Legionnaires'  disease.  This  past  year, 
we  have  compared  the  changes  in  pathogenicity,  limits  of  infectivity  in 
guinea  pigs,  behavior  in  tissue  culture  systems,  and  other  variations  of 
four  strains  of  L.  pneumophila  when  cultivated  from  embryonated  hens'  eggs 
and  after  numerous  further  passages  on  artificial  growth  media. 


30-13 


FHS-6040 
(Rev.    10- 


Project   No.    Z01    AI   00063-09  EB 


Project  Description: 


Isolates  of  Legionnaires'   disease  bacterium   (LDB)   from  fatal    cases 
of  Legionnaires'   disease  were  furnished   by  Dr.  McDade,   Center  for   Disease 
Control,    (strain  LAC),  Mr.   Sideman,   Pennsylvania   Department  of  Health, 
(strain  LAP),  and  Ms.    Janice  Jernigan,  Washington  State  Department  of 
Health,    (strain  LAW).     A  fourth  strain   (LAB)   was   isolated   from  cord   blood 
of  a   healthy  i  nfant. 

Isolates  of  Legionella   pneumophila  were  cultured   in  yolk  sacs  of 
embryonated   hens'    eggs  and  then  serially  passed  on  Mueller  Hinton   (MH) 
agar  supplemented  with  hemoglobin  and  cysteine  HC1   for   9  and  12   passages. 
Seeds  prepared  from  yolk  sacs  of  infected  chick  embryos  and  from  enriched 
MH  agar  were  titrated   in  guinea  pigs  and  on  primary  chicken  embryo  cell 
cultures.     The  parameters  of  infectivity  and  changes   in  pathogenicity  of 
the  four  strains  of  L_.   pneumophila  on  continued  passage  on  MH  media  were 
compared.     The  LAC,   LAP  and  LAB  strains  of  LDB  lost   their  ability  to   kill 
guinea  pigs  when  passed  numerous  times  on  MH  agar.     The  LAW  strain  of  LDB 
had  been  passed  numerous  times  on  artificial   media  before  being  received 
at  this  laboratory.     This   strain  was  ne^er  lethal    for  guinea   pigs,  even  at 
yery  high  concentrations  of  viable  organisms.     The  plaquing  ability  of 
LAB,  LAC  and   LAP  strains  was  also  greatly  diminished  after  passage  on  MH 
medium.     The  LAW  strain  plaque-forming  titers  remained  unaltered  after 
12  passages  on  the  artificial  media. 

Dr.  Ormsbee  retired   in  January  1979,   but  this   project  will    be  con- 
tinued  by  Mr.   Peacock  under  general    supervision  by  Dr.   Philip.     Current 
investigations  are  underway  to  determine  whether  the  changes   in  pathogenicity 
observed  after  continued   passage  of  L.   pneumophila  on  artificial   media  are 
stable  variations  and  whether  they  are  also  accompanied   by  immunologic 
changes.     Guinea  pigs  and  laboratory  mice  will    be  used   in  challenge  studies 
and  indirect   immunofluorescence  tests  of  convalescent   sera   to  monitor 
immunologic  responses.     We  are  also  looking  for   biochemical   markers  of  the 
MH-passed  strains  to  explain  the  changes  noted.     Future  plans  include 
continued   investigations  on   improvement  of  antigens  used  in  the  micro- 
immunofluorescence  diagnostic  test  for  Legionnaires'   disease.     The  nature 
of  cytopathogenicity  in  cell   cultures  will   also   be  studied. 

Publications: 

Ormsbee,  R.,   Peacock,  M.,   Philip,   R.,  Casper,   E.,   Plorde,   J.,  Gabre- 
Kidan,  T.,  and  Wright,   L.:     Antigenic  relationships   between  the  typhus 
and  spotted  fever  groups  of  rickettsiae.     Am.    J.    Epidemiol .   108: 
53-59,   1978. 


30-19 


Project   No.    Z01    AI   00063-09   EB 


Ormsbee,  R.  A.,   Peacock,  M.   G.,   Lattimer,   G.    L.,   Page,    L.   A.,  and 
Fiset,   P.:     Legionnaires  disease:     antigenic   peculiarities,   strain 
differences,  and  antibiotic  sensitivities  of  the  agent.      J.    Infect. 
Pis.    138:      260-264,   1978. 

Peacock,  M.  G.,   Fiset,    P.,  Ormsbee,  R.  A.,  and  Wisseman,   C.   L.,   Jr.: 
Antibody  response  in  man   following  a   small    intradermal    inoculation 
with  Coxiella   burnetii   phase  I  vaccine.     Acta  Virol .   23,   73-81,   1979. 


In  press: 

Kimbrough,   R.   C,  Ormsbee,   R.,   Peacock,  M.,   Rogers,   W.   R.,   Bennetts, 
R.  W.,   Raff,  J.,    Krause,  A.,  and  Gardner,   C.  :     Q  fever  endocarditis   in 
the  United  States.      J. A.M. A. 

Ormsbee,  R.  A.:     A  review.     Studies   in  Pyroplasma   hominis    ("Spotted 
fever"  or  "Tick  fever")   of  the  Rocky  Mountains  by  Louis   B.  Wilson 
and  William  M.    Chowning.     Rev.    Infect.   Pis. 


30-20 


PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  Uli  m'MMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01    AI   00065- 


LMSF-EB 


PERIOD    COVERED 


October  1,  1978  to  September  30,   1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Antigens  and  Classification  of  the  Rickettsiae 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:     R.  L.  Anacker 

R.  N.  Philip 

OTHER:  W.  Burgdorfer 

E.  A.  Casper 

T.  F.  McCaul 

L.  A.  Thomas 


Research  Microbiologist  LMSF  NIAID 

Medical  Director  EB  NIAID 

Res.  Entomologist  (Med.)  EB  NIAID 

Nurse  Director  EB  NIAID 

NIH  Visiting  Fellow  LMSF  NIAID 

Research  Microbiologist  EB  NIAID 


COOPERATING  UNITS  (if  any) 

Dr.   Robert   Lane,   California   Department  of  Health,   Berkeley,   CA 


lab/branch    Laboratory  of  Microbial    Structure  and   Function  and   Epidemiology  Branch 
RMI ).    Hamilton,    MT   59840 


SECTION 

Rickettsial  Diseases  Section  and  Epidemiology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 

5.6  (1.9  -  EB) 


PROFESSIONAL: 

2.7  (1.0 


eb: 


2.9   (0.9  -  EB) 


CHECK  APPROPRIATE  BOX(ES) 
[j  (a)   HUMAN  SUBJECTS 

□  (at)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


(c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

See  page  28-13  of  Annual    Report  of  Laboratory  of  Microbial    Structure  and 
Function. 


30-21 


PHS-6040 
(Rev.    IO-76) 


MIIIISUNIAN  SCILNCL  1  Nl  ■  UKMA  [I  UN  LXCIIANGL 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  01  I  AIITMLNT  OK 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMULR 

Z01    AI   00067-14 


PERIOD    COVERED 


October  1,  1978  to   September  30,   1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Tularemia 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:  J.  F.  Bell 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Rocky  Mountain   Laboratory 


SECTION 

Medical    Zoology  and   Zoonotic   Diseases  Section 


INSTITUTE  AND  LOCATION 

NIAID,   NIH,   Bethesda,   MD  20205 


TOTAL  MANYEARS: 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 

[]  (a)   HUMAN  SUBJECTS 

Q  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Project  terminated 

Bell,   J.    F.,   Wikel,   S.    K. ,   Hawkins,   W.   W.,  and  Owen,   C.   R.:      Enigmatic 
resistance  of  sheep   (Ovis  aries)   to   infection   by  virulent   Francisel la 
tularensis.      Can.    J.   Comp.    Med.   42:      310-315,   1978. 


30-22 


PHS-6040 
(Rev.    10-76) 


SMITHSONIAN  SCIENCE  INFORMATION  EXCIIANUL 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DU  AHTMLNT  UF 
HEALTH,  EDUCATION,  AND  WLLFARE 
I'UULIC  III  ALTll  SLRVICL 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01    AI   00068-18     EB 


PER  I  00    COVERED 


October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Rabies 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:  J.  F.  Bell 


COOPERATING  UNITS  (if  any) 


LAB/ BRANCH 

Rocky  Mountain  Laboratory 


Medical    Zoology  and   Zoonotic  Diseases  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOx(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Project  terminated 


30-23 


PH 3-6040 
(Rev.    10-76) 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  Of 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01    AI    00069-48   EB 


PERIOD    COVERED 


October  1,   1978   to   September  30,   1979 


TITLE   OF    PROJECT    (80   characters    or   less) 

Systematics  and  Vector  Relationships  of  Certain  Parasitic  Arthropods 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:  CM.    Clifford 

OTHER:     J.    E.    Keirans 
C.    E.    Yunker 


Scientist   Director  EB  NIAID 

Res.    Entomologist   (Med.)         EB  NIAID 
Scientist   Director  EB  NIAID 


cooperating  units  (if  any)  Drs.   H.   Hoogstraal,    NAMRU-3;   D.   Sonenshine  and   P.   Homsher, 
Old   Dominion   Univ.;  A.   J.    Spielman,   Harvard   School   of  Pub.    Hlth;    D.  Stiller, 
USDA:   F.   S.   Lukoschus,   Catholic  Univ.,    Netherlands;  W.   Buttiker,   Ciba-Geigy, 
Riyadh,    Saudi    Arabia;    Miss    Jane  Walker,    Div.   of  Vet.    Services.    Qnderstepoort. 


lab/branch 

Epidemiology  Branch   (RML),   Hamilton,   MT  59840 


section 
Arthropod-Borne  Diseases  Section 


institute  and  location 

NIAID,   NIH,   Bethesda,  MD  20205 


TOTAL  MANYEARS: 


4.4 


PROFESSIONAL: 


2.0 


2.4 


CHECK   APPROPRIATE   BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   []  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


(c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

The  activities  of  this   project  currently  comprise  four  main  functions: 
1)    Identification  of  ticks  received  from  various   individuals  and  government 
agencies  throughout  the  world.      Only  one  other   institution   in  the  world  is 
capable  of  performing  this   service.     2)   Systematic   study  of  certain  groups 
of  parasitic  arthropods.      The  foremost  tool    in   systematic  studies,   the 
scanning  electron  microscope,   has  greatly  aided  in  elucidating  taxonomic 
concepts   in  acarines  actually  or  potentially  involved  in  transmission  of 
disease  agents.      3)   Final    editing  and   storage  of  all    RML  and   NAMRU-3   (Cairo] 
tick  data  in   the  Smithsonian  data   retrieval    system.     4)   Colonization  of 
medically  important  arthropod  vectors,  which  are  furnished  to  outside 
investigators. 


30-24 


PHS-6040 

(Rev.    10-76) 


Project  No.    Z01    AI   00069-48   EB 


Project   Description 


The  primary  objective  of  this  project   is  the  systematic  treatment, 
including  taxonomy  and  classification,   biology,   ecology,  and  colonization, 
of  parasitic  arthropods  of  known  or  potential   medical    importance. 

Arqasid  ticks.     Our  studies  on  the  genus  Argas   have  continued  with 
the  emphasis   being  placed  on  the  morphology  of  Haller's  organ.     During  the 
past  year,   it  was  decided  to  expand  the  investigations  to   include  more 
species  of  each  subgenus.     This  will   necessitate  publishing  three  separate 
manuscripts,   i.e.,  one  on  the  subgenus  Argas,   another  on   Persicargas  and 
Microargas,  and  a   last  paper  on   Carios,   Chiropterargas,   Secretargas,   and 
Ogadenus.     As  we  previously  stated,  we  hope  to  correlate  the  morphology  of 
this  organ  with  the  host-specificity  and  other  behavioral    characteristics 
of  these  ticks. 

Significant  progress  was  made  on  our  continuing   study  of  the 
Ornithodoros  capensis  complex.     We  have  redescribed  0_.  amblus  that  feeds 
mainly  on  marine  birds   in  Peru  and   have  prepared   scanning  electron  micro- 
scope  (SEM)   photos  of  0.  muesebecki   for  possible  redescription  during  the 
next  year.     Clarification  of  the  status  of  these  two  ticks  is  deemed 
important  because  they  frequently  bite  humans  who   invade  their  domain 
causing  severe  reactions.     Also,  viruses  have  been  isolated  from  0.  amblus 
and  0_.  muesebecki   that  may  cause  illness  in  the  humans  and  birds  the  ticks 
feed  on. 

We  are  completing  descriptions  of  0_.   darwini   and  0_.   galapagensis , 
ectoparasites  of  land  and  marine  iguanas  and  lava   lizards   in  the  Galapagos 
Islands.      It   is  very  uncommon  to   find  members  of  the  tick  genus  Ornithodoros 
parasitic  upon  reptiles.     Two   species  of  intraerythrocytic   hemogregari nes 
are  found  in  the  blood  of  marine  iguanas  and  lava  lizards  and  ticks  are 
probable  vectors. 

Ixodid  ticks.     A  manual    to  allow  easier  identification  of  ticks  of 
the  genus  Dermacentor  is  still    in  preparation.     This  manual    is  to   be 
similar  to  our  work  on  the  genus   Ixodes  and  will    be  amply  illustrated  with 
SEM  photos. 

A  study  was  initiated  to  clarify  the  status  of  an   Ixodes   species  of 
the  angustus  complex  that  occurs   in  Spearfish  Canyon  in  South  Dakota.     The 
ticks  in  this  canyon  are  particularly  important  because   Powassan  virus, 
a  disease  of  humans,   has   been  isolated  in  this  area   from   Ixodes  ticks. 

Our  continuing  investigations  of  the  medically  and  agriculturally 
important  rhipicephal id  species  of  Africa   is  progressing  nicely.     A  manu- 
script clarifying  the  status  of  Rhipicephalus   kochi    is   being  written  and 
another  on  R_.   tricuspis  and  the  R.   appendiculatus  group   has   been  started. 
Hundreds  of  SEM  photographs   have   been  taken  of  African  ticks  of  this  genus. 


30-25 


Project  No.    Z01    AI   00069-48  EB 

We  also   hope  to   initiate  a   study  of  the  medically  important  R_. 
sanguineus  complex.     Hopefully,   this  will    be  accomplished   by  acquiring  a 
predoctoral   level    student  who   is  familiar  with  this  problem.     We  are 
attempting  to  obtain  funding  for  Mr.   Rupert   Pegram  of  Brunei    University, 
Uxbridge,   England,  who   is  working  on  a  Master's  thesis  on   the  genus 
Rhipicephalus   in   Ethiopia  and  Somali   and  would   have  a  fine  background  for 
our  project. 

Mite  studies.     Manuscripts  describing  clinical   mange  in  the  brown 
bear  and   in  laboratory  primates,  as  well   as  the  macronyssid   fauna  of 
Surinam,   have   been  submitted  for  publication.     The  parasitic  Acari   of  Saudi 
Arabia  are  being  studied  with  the  aim  of  providing  a   fauna  list  and  key  for 
public  health  workers.     Numerous   identifications  of  parasites,   some  involved 
in  human  or  animal    illness,   were  made.      Institutions  requiring   identifica- 
tions were:     Center  for  Disease  Control    (Atlanta),   U.S.   Naval    Medical 
Research   Institute   (Bethesda),   Eppley  Institute  for  Cancer   Research   (Omaha), 
University  of  South  Dakota   (Brookings),   Canada   Department  of  Agriculture 
(Lethbridge) ,  Cambridge  University   (Cambridge),   Panamerican  Health  Organiza- 
tion  (Panama),   Sudanese  Veterinary  Research  Administration   (Khartoum),  and 
University  of  Cairo   (Egypt). 

RML-NAMRU-3  computer  data.      Final    editing  of  the  master  list  of  the 
data  on  the  RML  and  NAMRU-3  tick  collection   has   been  completed  and  all    the 
errors  noted  have   been  corrected.     We  are  currently  awaiting   Dr.   Gautier 
of  the  Smithsonian  to  complete  his  work  on  the  data  file.     This   is   supposed 
to  be  completed   by  September  of  1979.      Following  this  a  mini   file  will    be 
constructed  to   save  money  in  querying  the  file  and  we  will    be  able  to  begin 
using  the   information  for  the  first  time. 

In  the  meantime,  new  records  and  corrections  are  rapidly  accumulating. 
We   have  asked  for  some  technical    help  to  alleviate  this   situation,   but  as 
yet  no  solution  has   been  found.      It  is   imperative  that  the  continuity  of 
entry  of  the  new  data  and  corrections   be  maintained. 

Nuttall    Collection.     Results  of  a  year's   research  on  the  G.H.F. 
Nuttall    tick  collection  are  now  being  compiled.     This   is   the  third  largest 
tick  collection  ever  assembled   (behind  those  of  RML  and   H.   Hoogstraal ) 
and  comprises  roughly  3,900  collections  ranging   from  one  to   several    hundred 
specimens  and  contains  material    from  all    zoogeographic  areas  of  the  world. 
The  collection  contains  over  100  type  species,  many  in  need  of  taxonomic 
clarification,  that  are   known  vectors  of  human  and  animal   disease  agents. 
A  book  cataloging  this  collection   is   being  prepared  under  the  aegis  of  the 
British  Museum   (NH),   the  depository  for  this  collection. 

In  conjunction  with  the  above  project,  a   second   important  tick  collec- 
tion, that  of  Nathanial    C.   Rothschild  of  the  famed   banking  family,   was 
redetermined  and  updated.     He  deposited  197  tick  collections  with   Professor 


30-26 


Project  No.    Z01    AI   00069-48   EB 

Nuttall    and,   between  1911    and  1922,  deposited  an  additional    155  collections 
in  the  British  Museum  (NH).     A  manuscript  on  this  work  has  just   been  com- 
pleted. 

In  1976  we  petitioned  the  International   Commission  of  Zoological 
Nomenclature  to  use  its  plenary  powers  to   conserve  the  name   Dermacentor 
andersoni ,  one  of  the  world's  most  important  ticks  in  terms  of  medical 
importance.     The  Commission  has  just  delivered  a  favorable  verdict.     The 
name   D.   venustus   has   been  suppressed  and  can  no  longer  be  used   for  the 
Rocky  Mountain  wood  tick. 

Service  oriented  activities.     Several    thousand  ticks  were  identified 
for  various  laboratories  throughout  the  world.     Also,  over  222,000  ticks 
were  furnished  to  investigators  at  the  RML  and  elsewhere  for  experimental 
studies  on  ticks  and  tickborne  diseases.     The  ticks  submitted  to  us  for 
identification  continue  to  be  an  excellent  source  of  new  tick  material    for 
our  own  research  interests. 

The  future  course  of  this  project  will   emphasize  the  use  of  the 
SEM  to  clarify  taxonomic  problems  among  various  genera  of  ticks.     Special 
attention  will    be  devoted  to  the  groups  of  ticks  from  which  we  are  isolating 
viruses  and  other  disease  agents.     We  will   also  stress  the  work  on  groups 
of  ticks  like  the  African  rhipicephal ids  that  are  proven  vectors  of  diseases 
of  humans  and  other  animals.     We  plan  to  produce  additional    practical    guides 
to  the  identification  of  ticks  that  are  amply  illustrated  with  SEM  photos. 


Publ  i cations: 

Keirans,   J.    E.   and  Clifford,  CM.:     The  genus   Ixodes   in  the  United 
States:     a   scanning  electron  microscope  study  and   key  to   the  adults. 
J.  Med.   Entomol  .   Supplement  No.   2:     1-149,  1978. 

Clifford,  C.  M.,   Hoogstraal  ,   H.,   Keirans,   J.    E. ,   Rice,   R.C.A.,  and 
Dale,  W.   E. :     Observations  on  the  subgenus  Argas   (Ixodoidea: 
Argasidae:  Argas).     14.      Identity  and   biological   observations  of 
Argas   (A.)  cucumerinus   from   Peruvian  seaside  cliffs  and  a   summary  of 
the  status  of  the  subgenus  in  the  Neotropical    Faunal    Region.     J.   Med. 
Entomol.   15:     57-73,  1978. 

Clifford,   C.  M.,   Keirans,   J.    E. ,   Hoogstraal,   H.,  and  Morel,   P.   C: 
Observations  on  the  subgenus  Argas   (Ixodoidea:  Argasidae:   Argas) . 
15.     Identity  of  Argas    (A.)  ma  gnus   from  Ecuador  and  Colombia. 
J.   Med.   Entomol  .   15:     157-165,  1979. 

Siuda,   K. ,   Hoogstraal,   H. ,   Clifford,   C.  M.,  and  Wassaf,   H.   Y.: 
Observations  on  the  subgenus  Argas   (Ixodoidea:  Argasidae:   Argas) . 
17.     Argas   (A.)   polonicus  sp.   n.   parasitizing  domestic  pigeons   in 
Krakow,   Poland.     J.    Parasitol .    65:     170-181,  1979. 

30-27 


Project  No.    Z01    AI   00069-48  EB 


Keirans,   J.   E. ,   Hoogstraal  ,   H. ,  and  Clifford,   C.   M. :     Observations  on 
the  subgenus  Argas   (Ixodoidea:  Argasidae:  Argas) .     16.     Argas    (A.) 
morel i ,  new  species,   and  keys  to   Neotropical    species  of  the  subgenus. 
J.    Med.    Entomol .   15:     246-252,   1979. 

Spielman,  A.,   Clifford,  C.  M.,   Piesman,    J.,   and  Corwin,   D.:     Human 
babesiosis  on   Nantucket   Island,   USA:     description  of  the  vector, 
Ixodes   (Ixodes)  dammini ,   n.   sp.    (Acarina:    Ixodidae).     J.  Med.   Entomol , 
15:     218-234,   1979. 


In  press: 

Stark,  H.    E. ,   Inlao,   I.,  Clifford,   C.  M.,   Keirans,   J.    E.,   and 
Lakshana,   P.:     A  preliminary  checklist  of  the  ticks  of  Thailand. 
Appl .   Sci.   Res.   Corp  J.   Thailand 

Keirans,   J.    E.,   Clifford,   C.  M.,  and   Yunker,   C.   E. :     Rocky  Mountain 
Laboratory  reference  collection  of  ticks  and  parasitic  mites    (Acari). 
A  Guide  to   the  Parasite  Collections  of  the  World. 

Corwin,   D. ,   Clifford,   C.  M.,  and   Keirans,   J.    E. :     An   improved  method 
for  cleaning  and  preparing  ticks   for  examination  with  the  scanning 
electron  microscope.     J.   Med.   Entomol . 

Hoogstraal,   H.,   Clifford,   C.  M.,   and   Keirans,    J.    E.  :     The  Ornithodoros 
(AI ectorobius)  capensis  group   (Acarina:   Ixodoidea:  Argasidae)  of  the 
Palearctic  and  Oriental    regions.     0_.    (A.)  coniceps:      identity,   bird 
and  mammal    hosts,   virus   infections,  and  distribution   in   Europe, 
Africa,  and  Asia.     J.    Paras i to!  . 

Hoogstraal,   H. ,   Clifford,  C.  M.,   Keirans,   J.    E. ,  and  Wassef,   H.   Y.: 
Recent  developments  in  biomedical    knowledge  of  Argas  ticks    (Ixodoidea: 
Argasidae).      Proc.   5th   Intl.    Congress  of  Acarology 


30-28 


Project  No.   Z01    AI   00069-48  EB 

Appendix  1.     PL-480  Project  03-063N.     Epidemiological    and  Biological 
Aspects  of  Tick-Borne  Infections   in   Egypt  and   Elsewhere, 
NAMRU-3,   Cairo,    Egypt 

Annual    Funding:     $123,000 

The  work  done  in  the  Bibliography  Section  at  NAMRU-3  continues  to 
provide  the  scientific  world  with  excellent  reference  material.     Volume  5, 
Part   II,   has   been  published  and  includes  over  2500  references   for  1974, 
1975  and  1975  and  2400  additions  to  Volumes  1-5,   Part   I.     Attention   is  now 
being  directed  toward  completing  the  Annotated   Bibliography  of  Tickborne 
Viruses.     The  section  on  the  Bunyaviridae  should   be  published   in  1979. 
Numerous   (250)  translations  were  completed  and  several    species  of  ticks  were 
expertly  illustrated. 

The  serological    survey  for  tickborne  viruses  of  potential    public   health 
importance  also  produced  some  excellent  results.     Sera   from  domestic  animals 
were  tested  for  the  presence  of  four  arboviruses,   i.e.,   Dugbe,   Crimean-Congo 
hemorrhagic  fever,  Bhanja  and  Tettnang  virus.     Antibodies  to  all    four 
viruses  were  found  in  Egypt. 

Large  volumes  of  human  sera  were  also  tested   in  this   program  for  the 
presence  of  antibodies  to  Rift  Valley  Fever  (RVF)   -  a  disease  which  infected 
large  numbers  of  people  in   Egypt  in  1977  and  1978.     About  3000  sera  collected 
in  1974,  1975  and  1975  were  negative  for  antibodies,   which  helps  to  establish 
that  RVF  was  not  present  in   Egypt  prior  to  the  recent  outbreak.     Also, 
tests  of  human  sera  collected  in  1977  and  1978  demonstrated  the  presence 
of  RVF  antibodies   in  19%  of  the  samples. 

During  the  next  year,  a  larger  number  of  tickborne  viruses  will    be 
tested  in  human   sera   samples  from  various  parts  of  Egypt.      Further  studies 
on  RVF  fever,   such  as  determining  the  vector  of  this   virus   in   Egypt  and 
where  the  virus  overwinters  will    be  undertaken  during  the  next  year.     As 
mentioned  above,   the  work  being  conducted  at  Cairo   University  is   in  pre- 
liminary stages.     This   is   partly  due  to   the  fact  that  although  funds  were 
received  for  a  full   year,  approval    was  granted  near  the  first  of  the  year. 


30-29 


MITIISUNIAN  SCItNCt  I  HI  ■  UHMA  I  I  UN  tAUIIANGL 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  l:_.  sKTMtfU  01 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJtCI  NUMBER 


Z01    AI   00079-19   EB 


PERIOD    COVERED 


October  1,   1978   to    September  30,   1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

The  Encephal i tides 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:  L.  A.  Thomas 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH  ,         ■-  ■- - 

Epidemiology  Branch  (RML),  Hamilton,  MT  59840 


SECTION 

Epidemiology  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 


0.1 


PROFESSIONAL: 


0.1 


CHECK  APPROPRIATE  BOX(ES) 
Q  (a)   HUMAN  SUBJECTS 

G  (al)  MINORS   □  (a2)  INTERVIEWS 


□  (b)  HUMAN  TISSUES 


□  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

Project  terminated 

In  press: 

Thomas,  L.  A.,  Patzer,  E.  R.,  Cory,  J.  C,  and  Coe,  J.  E.  :  Antibody  develop- 
ment in  garter  snakes  (Thamnophis  spp.)  experimentally  infected  with 
Western  equine  encephalitis  virus.  Am.  J.  Trop.  Med.  Hyg. 


30-30 


PHS-6040 
(Rev.    IO-76) 


SMITHSONIAN  SCIENCE  INFORMATION 
PROJECT  NUMBER  (Do  NOT  use  this 


IXCHANGE 
;pace) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01    AI    00080-12    EB 


PERIOD    COVERED 


October  1,  1978  to  September  30,   1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Tickborne  Disease  Agents 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:  C.  E.  Yunker 

OTHER:     C.  M.  Clifford 

J.  E.  Keirans 

L.  A.  Thomas 


Scientist  Director  EB  NIAID 

Scientist   Director  EB   NIAID 

Res.    Entomologist   (Med.)  EB  NIAID 

Research  Microbiologist  EB  NIAID 


cooperating  units  (if  any)  rjrs.    J.   Casals,   YARU ,   New  Haven;   R.   Gresbrink,   Oregon  State 
Bd.   Hlth.,   Portland;   H.   Hoogstraal  ,   NAMRU-3,   Cairo,   Egypt;   B.    Easterday,   Dept. 
of  Vet.   Sci.,  Univ.   Wisconsin,  Madison;    K.    King,    Patuxent  Wildlife  Res.    Ctr.; 
E.    Easton,   S.   Dakota  State   Univ.:   D.   G.   McKercher,   Univ.    Calif.T    Davis. 


lab/branch 
Epidemiology  Branch   (RML),   Hamilton,  MT  59840 


section 
Arthropod-Borne   Diseases  Section 


institute  and  location 
NIAID,   NIH,   Bethesda,  MD  20205 


TOTAL  MANYEARS: 


2.8 


PROFESSIONAL: 


0.9 


1  .9 


CHECK  APPROPRIATE  BOX(ES) 
G  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   □  (a2)  INTERVIEWS 


H  (b)  HUMAN  TISSUES 


D(c) 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

In   this  project  the  incidence,  distribution  and   ecology  of  tick-borne  disease 
agents,   arboviruses  and  rickettsiae,   are  studied.     These   pathogens  are  isolated 
in  tissue  cultures  or  animal s   from  field-collected  materials  and  are  sero- 
logical 1y  identified.     Viruses,    if  new,  are  physicochemically  and   biologically 
characterized  and  taxonomically  classified.     Of  particular  importance   is 
the  delineation  of  vector-relationships  and   human  disease  potential    of  the 
increasing  number  of  tick-borne  viruses   in  the  United  States. 


30-31 


PHS-6040 
(Rev.    IO-76) 


Project  No.    Z01    AI   00080-12    EB 


Project   Description: 


Over  1100  ticks,  as  well   as  166  blood   samples  from  humans,   rodents 
and   birds,  collected   in   seven  states,  mostly  by  public   health  or  wildlife 
disease   investigators,   were  tested   for  pathogens.     These  tests  resulted  in 
102  isolations  of  viruses  and  22  of  rickettsiae.     All    blood   samples  proved 
negative  for  pathogens. 

Among  the  agents   identified  were  30  isolates  of  Soldado  virus   from 
Ornithodoros  capensis  ticks   in  Texas    (infection  rate   [IR],   10.6%)  and  25 
of  Hughes  virus  from  0_.   denmarki    in  Florida   (IR,  14%).     Both  tick  species 
are  parasites  of  migratory  seabirds  and  their  viruses  are  distantly  related 
members  of  an  unclassified  taxon  of  viruses  currently  termed  the  Hughes 
virus   serogroup.     Some  Hughes  group  agents  have   been   implicated   in   febrile 
disease  of  humans  and  in  seabird  die-offs.      Fifteen  additional    isolations 
of  Sapphire   II  virus   (also  of  the  Hughes  serogroup)   were  made  this  year 
from  South  Dakota  populations  of  Argas  cool eyi ,  a   parasite  of  migratory 
passerine  birds.     These  same  ticks  yielded  16  strains  of  Sixgun   City  virus, 
a  Reovirus  of  the  genus  Orbivirus.     For  each  virus,   the   infection  rate  was 
calculated  to   be  about  3%.     Five  viral    isolates  from  these  ticks  remain 
unidentified. 

A  new  virus  of  the  Uukuniemi   serogroup   (Bunyaviridae) ,  which  we 
discovered  last  year   in  seabird   parasites,    Ixodes  uriae,  on  St.    Paul    I., 
Alaska,   was  physicochemical ly  and   biologically  characterized   in   preparation 
for  publication.      Its  virion   is   between  100  and  220  nm   in  diameter  and 
contains  RNA  as  well   as  a   surrounding  1 ipid-containing  envelope.     While 
fairly  resistant  to  thermal    inactivation,   the  virus   is  readily  denatured 
by  exposure  to  acid   pH.      It   is   highly  pathogenic  for  newborn  mice  by  intra- 
cerebral   and   peripheral    routes  of  inoculation,    but  causes   illness  and  death 
in  weanlings  only  by  intracerebral    route.      It   fails  to   plaque   in  Vero  cells, 
but  does   so   in  Xenopus  cells   regardless  of  exposure  of  the  virus  to  neutra- 
lizing antibody.     No  virus  growth  was  seen   in  mosquito  or  tick  cell   cultures 

This  year,   we  isolated  an  unidentified   virus  from  Dermacentor 
occidental  is  collected   in  two  counties  of  Oregon.     The  tick,  a  man-biting 
species,   has  previously  been  shown  to   harbor  Colorado  tick  fever  virus, 
as  well   as  rickettsiae  of  the  spotted   fever  group,   but  the  existence  of 
this  serologically  unique  virus  was  unsuspected.      Fourteen   isolations  of 
the  new  agent  were  made   in   Xenopus  cells,    but  none  were  obtained   in  Vero 
cells  or  by  injection  of  newborn  mice.     The  virus,   while  not   pathogenic   for 
newborn  white  mice  or  hamsters,  does  cause   illness   in  meadow  voles.      It 
will    propagate   in  L  cells,   pig  kidney  cells,  and  tick  tissue  cultures  and 
is   presently  being  characterized  with  regard  to   its   physicochemical    proper- 
ties.    At  the  request  of  California  researchers,   a  virus   isolated   from 
aborting  cattle  and  their  tick  parasites,   0_.   coriaceus,   was  also   partially 
characterized. 


30-32 


Project  No.  Z01  AI  00080-12  EB 


The  rickettsiae  were  isolated  in  Vero  and  Xenopus  cell  cultures 
from  three  species  of  ticks  known  to  bite  man:  I_.  pacif icus  and  JJ.  occi- 
dental i  s  (from  Oregon)  and  JD.  variabil  is  (from  South  Dakota).  Two  rickett- 
sial entities,  as  determined  by  microimmunofl uorescence  test,  were  involved 
in  16  of  the  isolations.  Both  are  members  of  the  spotted  fever  group  and 
have  previously  been  recorded  by  us  from  the  respective  tick  species. 
Six  additional  isolations  remain  to  be  identified. 


3W 


Last  year,   in  attempts  to  increase  the  sensitivity  of  our  assay 
procedures  for  isolation  and   identification  of  tickborne   pathogens  of  lov 
virulence  and  antigenicity,   we  extensively  tested  an  amphibian  cell    line 
derived  from  Xenopus  laevis.     As  noted  above,  this  cell   line  of  poikilo- 
thermic  origin  was  the  only  system  capable  of  detecting  the  unidentified 
virus  from  D.  occidental  is.     Hence,   it  may  prove  useful    in  the  isolation  of 
temperature  sensitive  variants.      In  addition,    it  alone  accounted  for  all 
but  three  of  the  Soldado  virus  isolations  mentioned  above,  and   is  obviously 
superior  to  Vero  cells  and  newborn  mice  for  isolation  of  this  tickborne 
virus.     Its  advantages   in  the   isolation  of  spotted   fever  group  rickettsiae 
have  previously  been  noted.     Also,   these  cells   have  proven  useful    in  the 
production  of  viral   antigens  for  microimmunofl  uorescence  tests. 

We  plan  to  continue  this  project  as  before  with  the  assistance  in 
the  field  of  collaborating   investigators.     Geographical    records  of  tick- 
borne viruses   in  various  vectors  and   in  flyways  of  migratory  birds  enable 
us  to   predict  the  distribution  and  prevalence  of  these   pathogens.      For 
example,  the  many  isolations  obtained  this  year  of  Hughes  group  viruses 
support  our  earlier  evidence  of  vector-specificity  for  these  viruses. 
From  this  we  may  infer  that  their  distributional    patterns  are  primarily 
those  of  the  vector.     Also,   the   high  infection  rates  for  the  viruses  under 
study  suggests  a   dual    role  of  the  tick  as  vector  as  well   as  maintenance 
host.     Arboviruses  transmitted   by  mosquitoes  and   sandflies  are  relatively 
well    studied   in  comparison  to  those  that  are  tickborne.     Thus,   this   project 
helps  to   fill   an  obvious  void   in  our  understanding  of  the  natural    history 
of  arboviruses. 


30-33 


Project   No.    Z01    AI   00080-12    EB 


Publ  ications : 

In  press: 

Yunker,   C.    E. :     Current  research  on  transmission  of  disease   by  Acari . 
Proc.   Symp.   Vth  Intl.   Congress  of  Acarology,   East   Lansing,  MI, 
Aug.    6-12,   1978 

Yunker,   C.    E. ,  Thomas,   L.  A.,  and  Cory,   J.:      Phenetic  relationships 
of  viruses  of  the  Hughes  serological   group.      Proc.    2nd   Int.   Symp. 
on  Arctic  Arboviruses,   Mt.   Gabriel,   Que.,  Canada,  May  26-28,   1977. 
New  York,  Academic   Press. 

Yunker,   C.    E. ,   Clifford,   C.  M.,    Keirans,   J.   E. ,   Thomas,   L.  A.,   and 
Rice,   R.C.A.:     Isolation  and  characterization  of  a  new  tickborne  virus 
of  the  Upolu   serogroup,  new  serogroup,   from  coastal    Texas.      J.   Med. 
Entomol . 

Clifford,   C.  M.:     Tick-borne  viruses  of  seabirds.      Proc.   2nd   Int.   Symp. 
on  Arctic   Arboviruses,  Mt.   Gabriel,   Que.,   Canada,  May  26-28,   1977. 


30-34 


* 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01    A I   00081-18   EB 


PERIOD    COVERED 


October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

The  Epidemiology  of  Human   Infections  of  Special    Interest 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 

PI:  R.   N.    Philip  Medical    Director  EB   NIAID 

OTHER:      E.  A.   Casper  Nurse  Director  EB  NIAID 

L.   A.   Thomas  Research  Microbiologist  EB  NIAID 

W.   Burgdorfer  Res.    Entomologist   (Med.)  EB  NIAID 

R.   L.   Anacker  Research  Microbiologist  LMSF  NIAID 


cooperating  units  (if  any)    Dr .    Karim  Hechemy,   New  York  State  Dept.   of  Health,   Albany; 
Dr.   Richard   Emmons,   Viral    and  Rickettsial    Disease  Laboratory,   California 
Dept.  of  Health,   Berkeley;   Dr.    Jorge  Benach,    New  York  State  Dept.   of  Health, 
Sfnny   Rrnnk;    Dr.    P.at.haHnp   Uilfprt,    flnkp   Univ.    Mpriical     C.pnt.pr,    Durham,    NC. 


lab/branch 
Epidemiology  Branch   (RML| 


Hamilton,  MT  59840 


SECTION 

Epidemiology  Section 


INSTITUTE   AND   LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 


2.4 


j PROFESSIONAL: 


1  .5 


0.8 


CHECK  APPROPRIATE  BOX(ES) 
3  (a)   HUMAN  SUBJECTS 


(b)  HUMAN  TISSUES 


□  (c)  NEITHER 


i1  )  MINORS 


a2)  INTERVIEWS 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

This  project  emphasizes  epidemiologic  investigations  of   infectious  diseases 
of  particular  interest  to  the  research  program  of  this  laboratory.      Current 
investigations  include  studies  of  the  etiologic  relationship  of  various 
spotted   fever-group  serotypes  to   human   illness,  and   improvement   in  laboratory 
methods  for  diagnosis  of  rickettsial    infections,   particularly  Rocky  Mountain 
spotted  fever. 


30-35 


PHS-6040 
(Rev.    10-76) 


Project   No.    Z01    AI   00081-18   EB 


Project   Description: 


Etiologic  role  of  spotted  fever  group   (SFG)   agents  to   human   infection. 
A  study  to  determine  the  importance  of  SFG   organisms  other  than  Rickettsia 
rickettsii   as  a  cause  of  human   infection  was  continued   in  collaboration 
with  Dr.   Burgdorferi   staff.     Attached   Dermacentor  andersoni,  taken  from 
Bitterroot  Valley  residents  and  shown  to   be   infected  with  SFG  rickettsiae 
by  hemolymph  test,  were  inoculated   into  microtus  and  Vero  cell    cultures. 
Nine  isolates  were  obtained   in  cell   culture  and/or  microtus  from  33   infected 
ticks  that   had   been  stored  for  1    to  2  years  at  65°C.     The   isolates   included 
4  strains  of  R_.   rickettsii ,  2  of  R_.  rhipicephal  i ,  2  of  369-C,  and  one  of 
R_.  montana.      In  addition,  8  ticks  gave  rise  to  antibodies   in  microtus  which 
typed   by  microimmunofl  uorescence   (micro-IF)  as  R^.   rhipicephal  i    (1)  and 
369-C   (7).     Thus,  the  infecting  strain  was   identified   in  17  of  the  33   ticks. 
Aside  from  3  cases  of  Rocky  Mountain  spotted  fever   (RMSF)   from  whom  R_. 
rickettsi  i-infected  ticks  were  obtained,  none  of  the  persons  with  tick  bite 
had  clear-cut  clinical   or  serologic  evidence  of  associated   infection. 

In  collaboration  with   Dr.   Benach,  a   serologic  study  was  conducted 
among  permanent  residents  of  Shelter   Island  located  at  the  eastern   tip  of 
Long   Island.      In  recent  years,  this  locale   has   been  heavily  infested  with 
p_.  variabil  is  and   Ixodes  dammini   due   in   part  to  curtailment   in  clearing  of 
underbrush   by  burning.       Hemolymph  tests   revealed   rickettsia-1  ike  organisms 
in  14%  of  D_.  variabil  is   surveyed  one  year  ago.     A  serosurvey  of  Shelter 
Island  residents  for  micro-IF  antibodies  to  various  members  of  the  SFG  in 
June  1978  revealed  a   significantly  higher  prevalence  of  low  level    reactivity 
than  was   found  among  residents  of  New  York  City  and   Long   Island.     The  same 
population  cohort  of  Shelter   Island  residents  was   retested   in  October  1978. 
An  amazingly  high  14%  of  103  participants   showed   serologic  conversion   by 
micro-IF  against   rickettsiae  of  the  SFG  during  the  4-month   interval,   parti- 
cularly against  R_.   rickettsii   and  the  369-C  agent.     Yet   there  was  no  clini- 
cal   evidence  of  RMSF  in  this  population  group.     The  etiology  of  the  sero- 
logic reactivity  has  not   been  determined,   but  whatever  the  cause,   it  was 
not  associated  with  a  clinical    response. 

Serologic  tests .     The   investigation  of  the  suitability  of  the 
indirect   hemagglutination  test  for  the  detection  of  spotted  fever  antibody 
in  man  and   several    kinds  of  laboratory  animals    (Anacker)   was  completed 
during  the  past  year.     Antibody  in  sera  from  human   patients  was  detected, 
with   human  group  "0"   erythrocytes   sensitized  with  an  alkali   digest  of 
purified  R_.  rickettsi  i  as  antigen,  as  early  as  3  days  and  as   late  as   3 
years  after  onset  of  illness.     Both   human   IgG  and   IgM  antibodies   partici- 
pated  in  the  reaction   but   IgM  antibodies  were  much  more  efficient.     Anti- 
body which  agglutinated   both  sensitized   fresh  and   gl utaral  dehyde-fixed 
sheep  erythrocytes  was   found   in  rabbit  sera  at  all    intervals   tested    (10- 
59  days  postinfection).     Antibody  which  agglutinated  fresh   but  not 


30-36 


Project   No.    Z01    AI   00081-18   EB 

glutaral dehyde-fixed  sheep  erythrocytes  was  detected   in  guinea   pigs   7,   14, 
and  28  days  postinfection.     Antibody  was   found  in  mice  inoculated  with 
a   high  dose  of  R.  rickettsi  i   but  not  with  a  low  dose.     This   hemagglutination 
test  should   be  of  value  for  serologic  diagnosis  of  spotted   fever   infection 
of  humans  and  for  laboratory  studies  of  spotted  fever   infections  of  common 
laboratory  animal  s. 

We  are  assisting  Dr.   Hechemy  in  the  development  of  a   latex  aggluti- 
nation test  for  diagnosis  of  RMSF.     Latex   particles  complexed  with  the 
alkali  digest  of  purified  R_.   rickettsi  i   from  a   stable  suspension  which  can 
be  stored  for  many  months.     This  suspension   is  rapidly  and  specifically 
agglutinated  by  sera  from  most  spotted  fever  patients,  particularly  those 
with   IgM  antibodies.     Presently  the  latex  agglutination  test   is   being 
compared  with  the  micro-IF  and  complement-fixation  tests   for  detection  of 
antibody  in  sera  from  patients  from  different  regions  of  the  United  States. 
The  latex  agglutination  test  may  prove  to  be  the  simplest  and  most  economical 
of  all    tests  for  the  routine  serodiagnosis  of  spotted  fever. 

In  collaboration  with  Dr.   Emmons,   we  are   investigating  the  etiologic 
relationships  of  SFG  agents  with  RMSF-1 i ke   illnesses  associated   particu- 
larly with  D_.  occidental  is   in  the  coastal    areas  of  California  outside  the 
range  of  D.   andersoni .     In  one   instance,  paired  acute  and  convalescent 
serum  samples  obtained  from  a  mild  RMSF-1  ike  illness  associated  with  tick 
bite  acquired   in  coastal   northern  California  were  shown  to  have  a  modest 
micro-IF  antibody  titer  rise  to  various  SFG  agents   including  isolates 
obtained  from  D_.   occidental  is   in  that  area    (see  Project   No.    Z01   AI   00065-06 
LMSF-EB). 

The  attributes  of  an  enzyme-linked   immunosorbent  assay   (ELISA)   test 
and  a   solid-phase  immunofluorescence  procedure  (FIAX)   for  quantitating 
rickettsial   antibody  are  being  defined.     Current  information   indicates 
that  both  test  systems  are  about  4-  to  8-fold  more  sensitive  than  our 
micro-IF  test  for  detection  of  antibodies  against  R_.   rickettsi i    in  con- 
valescent sera  from  patients  with  RMSF.     Comparably  low  concentrations  of 
the  same  soluble  rickettsial  antigen  are  optimal    for   both  test  systems. 
For  this  reason,  we  are  optimistic  that  these  procedures  can   be  used  for 
early  detection  of  antigen   in  body  fluids  of  patients  with   RMSF.     These 
studies  will    begin  shortly  and  will   occupy  much  of  the  activity  in  this 
project  during  the  coming  year.     We  will    also   be   involved   in   providing 
serologic   support  for  an  epidemiologic  study  of  RMSF  in  2  counties   in 
North  Carolina   (Wilfert).     Our  role  will    be  to  obtain  ancillary  information 
on  the  etiologic  relationship  of  various   SFG  agents  to   human   illness. 
Similar   investigations  will    be  continued   in  collaboration  with  the  Cali- 
fornia State  Viral   and  Rickettsial    Disease   Laboratory   (Emmons). 


30-37 


Project  No.    Z01    AI   00081-18   EB 


Pub! ications: 


Hechemy,   K.   E. ,  Stevens,   R.   W.. ,   Sasowski,   S.,  Michaelson,   E.   E., 
Casper,   E.  A.,  and   Philip,   R.   N. :     Discrepancies   in  Weil-Felix  and 
microimmunofluorescence  test  results   for  Rocky  Mountain  spotted 
fever.      J.   Clin.  Microbiol.    9:     292-293,   1979. 

Philip,   R.   N.,  Casper,   E.  A.,   and  Burgdorfer,  W.:     Current   knowledge 
of  the  distribution  of  serotypes  of  spotted   fever-group  rickettsiae 
in  the  United  States  as  determined   by  microimmunofluorescence.      In: 
Proceedings  VII   International    Congress  of  Infectious  and   Parasitic 
Diseases,   Varna,  Bulgaria,   Oct.   2-6,  1978,   1978,   pp.   500-509. 


In  press  : 

Philip,   R.    N.:     Scrub  typhus.      In  Steele,   J.   H.    (Ed.):      CRC  Handbook 
of  Zoonoses 

Philip,   R.   N.:     Typhus   group  rickettsiae.      In  Steele,   J.   H.    (Ed.): 
CRC  Handbook  of  Zoonoses 


50-38 


MITIISONIAN  SCILNCL  I  Nh  OHMAT  I  ON  LXCHANGl 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  1)1  .  MUMLNT  Of 
HEALTH,  EDUCATION,  AND  WLLFARE 
PUBLIC  IILALTfl  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PKOJLCI  NUMBER 

Z01    AI    00082-18    EB 


PERIOD    COVERED 


October  1,  1978  to  September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Relation  of  Viruses  to  the  Genesis  of  Chronic  Disease 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:     W.   J.   Hadlow 


Res.   Veterinarian   (Path.) 


EB   NIAID 


cooperating  units  (if  any)     ^   s>   g>    Prusinerj   Dept>   of  Neurology,   University 
of  California,  San   Francisco 


lab/branch 

Epidemiology  Branch 


'RML),   Hamilton,   MT  59840 


SECTION 

Histopathology  Section 


INSTITUTE  AND  LOCATION 

NIAID,   NIH,   Bethesda,  MD  20205 


TOTAL   MANYEARS: 


2.8 


PROFESSIONAL: 


1.0 


CHECK  APPROPRIATE  BOX(ES) 
□  (a)   HUMAN  SUBJECTS 

D  (al)  MINORS   □  (a2)  INTERVIEWS 


[%  (b)  HUMAN  TISSUES 


(J  (c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

This  project  is  mainly  concerned  with  characterizing  several    naturally 
occurring  slow  viral    diseases  of  domestic  animals  that  resemble  important, 
etiologically  obscure  diseases  of  man.      Two  diseases  receive  most  attention 
scrapie  of  sheep  and  goats  and  Aleutian  disease  of  mink.     Their  clinico- 
pathologic,  virologic,  and   immunologic  features  are  studied  to  provide  an 
understanding  of  their  pathogenesis  and  natural    history.      Information   from 
such  studies  will    help  define  the  unusual    host-virus   relationships  that 
result  in  protracted  disease   in  man  and  animals. 


30-39 


PHS-6040 
(Rev.    10-76) 


Project   No.    Z01    A I   00082-18   EB 


Project  Description: 

This  project  is  concerned  with  learning  more  about  the  unusual    host- 
virus  relationships  that  give  rise  to  slowly  evolving  diseases  after  a 
long  incubation  period.     The  main  diseases   studied  are  scrapie  of  sheep  and 
goats  and  Aleutian  disease  of  mink.     Because  the  primary  objective  of  the 
research  is  the  study  of  the  disease  process   in  each   infection,  most  atten- 
tion  is  directed  to  obtaining  information  on  the  temporal   replication  of 
the  causative  virus  and  on  the  cellular  and   humoral    host  responses   in  rela- 
tion to  the  natural    history  of  the  disease.      Less  attention   is  directed   to 
the  properties  of  the  causative  viruses. 

Because  of  an  administrative  directive  to  do  so,  all   new  work  in 
this  project  was  discontinued  about  mid  April   1978.     Moreover,  the  project 
has  since  been  stripped  of  all    its  supporting  personnel    but  one— a   histo- 
technologist.     Thus,  even  work  in  progress  at  the  time  the  directive  was 
issued  has  suffered  severely— val  uable  observations  on  experimental    animals 
could  not   be  made,   supporting  laboratory  studies  could  not   be  done,  and 
records  could  not   be  kept  up-to-date.     The  main  concern  of  the  principal 
investigator,  therefore,  has  been  to  salvage  what  he  could  from  the  tattered 
remnants,  and  this  effort  has  not   been  too  productive  so   far. 

No  new  observations  on  scrapie  or  Aleutian  disease  were  made  since 
the  last  annual  report.  Only  a  few  experiments,  mostly  virus  titrations, 
remain  active.     And  these  will    be  completed  within  a  year. 

As  mentioned   in  last  year's  report,  a  pilot  study  was   begun  about 
4  years  ago  with  specimens  of  brain  from  11    persons  dying  of  Creutzfeldt- 
Jakob  disease   (CJD).     Guinea   pigs  and  mink  inoculated   intracerebral!  y  with 
these  specimens   have  remained   free  of  recognizable  neurologic  disease  after 
2  and  4  years,   respectively.     A  presumed  neurologic  disease  that  occurred 
in  similarly  inoculated  mice,  mentioned   in  last  year's  report,   has  occurred 
in  some  of  those  in  the  second  passage,   but  the  results  are  still 
inconclusive. 

The  only  certain  positive  results   so   far  were  in  similarly  inoculated 
African  pygmy  goats.     One  of  3  that  received  one   brain  specimen  and  1   of  4 
that  received  another  became  affected  with  a  scrapie-like  disease  about 
43  months  after  intracerebral    inoculation.      In   both,   the  disease  was 
characterized   by  scratching  of  the   skin,  a  depressed  attitude,  and  motor 
difficulties  affecting  gait.     One  goat  became  extremely  depressed,   lost 
weight,  and  had  impaired  vision.      It  was   killed  about  8  weeks  after  onset 
of  clinical    signs,   when   it  spent  much  of  the   time  recumbent.     The  other 
goat,   still   under  observation,   wanders  about   its   pen  and   scratches  continu- 
ally.    Three  other  goats  of  this  group  may  now  be  in  the   prodromal    stage 
of  the  experimental   disease. 


30-40 


Project  No.    Z01    AI   00082-18   EB 

Preliminary  histologic  examination  of  the  brain  and   spinal   cord  of 
the  goat  that  was  killed   indicated  changes  compatible  with  those  of  caprine 
scrapie.     Astrocytosis  was  prominent  in  the  diencephalon,  mid   brain,   and 
cerebellar  cortex,   but  spongiform  change  was  minimal.     A  more  complete 
evaluation  of  these  microscopic  changes  is  in  progress. 

Goats  inoculated  with  the  other  9  specimens  of  brain  are   still 
normal  ,   but  most   have  not  been  under  observation  long  enough  to  warrant  any 
conclusions  about  whether  they  too  may  become  affected. 

The  occurrence  of  scrapie-1 ike  disease  in  these  goats   is  of  consider- 
able interest  vis-a-vis  the  possible  relationship  of  CJD  virus  and   scrapie 
virus.     But  whether  it  provides  any  better  understanding  of  the  origin  and 
identity  of  CJD  virus  is  still   uncertain.     CJD  virus  and  scrapie  virus  also 
cause  indistinguishable  neurologic  diseases  in  the  squirrel   monkey  and  the 
rhesus  monkey.     In  the  absence  of  information  about  the  antigenic  relation 
of  these  viruses,   their  similarity  or  common  identity  no  doubt  will    not   be 
determined   by  a   study  of  their  experimental    host  ranges.     With  both  viruses, 
the  pattern  of  neuropathology  changes  seen  in  experimental    hosts   is   influ- 
enced by  the  passage  history  of  the  viruses  and  by  other  laboratory  manipu- 
lations imposed  upon  them.     Even  so,  the  occurrence  of  an  encephalopathy 
resembling  scrapie  in  a  natural    host  of  that  disease  may  be  more  significant 
than  the  occurrence  of  a  similar  disease  in   some  aberrant  host  like  the 
monkey  or  the  hamster.     At  any  rate,   this  finding  in  goats   inoculated  with 
brain  tissue  directly  from  man  emphasizes  a  long-held  view  of  the  principal 
investigator:     the  susceptibility  of  all    common  farm  animals  to   scrapie 
virus  infection  should   be  examined  not  only  to  clarify  some  epidemiologic 
aspects  of  scrapie,   but  also   to  provide  information  that  may  be  relevant 
to  determining  possible  sources  of  human   infection  with  CJD  virus.      Hosts 
with  inapparent  infections  may  be  more  important  than  those  in  which  overt 
disease  occurs. 

Except  for  continuing  observations  on  animals   inoculated  with  brain 
suspensions  from  persons  dying  of  CJD,   little  more  is  contemplated.     Any 
further  work  at  RML  will    be  largely  contingent  on  what  the  principal    inves- 
tigator may  be  allowed  to  do  with  these  animals.      If  nothing  else,  certainly 
a  second  passage  in  goats  should  be  made.     In  the  coming  year,  then,   time 
will    be  spent  evaluating  the  large  amount  of  experimental   data  already 
collected.     But  this  can  not   be  done  single-handedly.     So,  only  that  which 
is  more  readily  accessible  and   is  more  easily  coped  with  will    be  considered. 
It   is  not  the   intent  of  the  principal    investigator  to  spend  much  of  his 
time  in  largely  clerical   tasks;   he  will   devote  most  of  his  time  to   the 
evaluation  of  the  large  amount  of  histologic  material   that  still    needs  to 
be  studied.     Given  the  present  constraints  imposed  on  the  activities  of 
the  principal    investigator,   this  approach  seems  to   be  the  only  productive 
way  to  deal   with  what  remains  of  this   project. 


30-41 


Project   No.    Z01    AI   00082-18   EB 

Pub!  ications : 

Eklund,   C.  M.  and  Hadlow,  W.   J.:     Animal    viral   diseases  as  models  of 
central    nervous  system  disease   in  man.      In  Vinken,   P.   J.   and   Bruyn, 
G.   W.    (Eds.):     Handbook  of  Clinical    Neurology,   Vol.    34:      Infections 
of  the  Nervous  System,   Part   II.     Amsterdam,   New  York,   North  Holland 
Publishing  Co.,   1978,   pp.   291-305. 

Prusiner,   S.   B.,   Hadlow,   W.   J.,   Eklund,   C.   M.,   Race,   R.   E. ,  and 
Cochran,   S.   P.:     Sedimentation  characteristics  of  the  scrapie  agent 
from  murine  spleen  and   brain.     Biochemistry  17:     4987-4992,   1978. 

Prusiner,   S.   B.,   Hadlow,  W.    J.,   Garfin,   D.    E.,    Cochran,   S.    P., 
Baringer,   J.   R.,  Race,  R.   E.,  and   Eklund,   C.  M.:      Partial    purification 
and  evidence  for  multiple  molecular  forms  of  the  scrapie  agent. 
Biochemistry  17:     4993-4999,1978. 

Prusiner,   S.   B.,  Garfin,   D.   E. ,   Baringer,   J.   R.,   Cockran,   S.    P., 
Hadlow,  W.   J.,  Race,  R.   E.,  and   Eklund,   C.   M. :     Evidence  for  multiple 
molecular  forms  of  the  scrapie  agent.      In   Stevens,   J.    G.,   Todaro, 
G.   J.,  and   Fox,   C.    F.    (Eds.):     Persistent  Viruses.      ICN-UCLA  Symposia 
on  Molecular  and   Cellular  Biology.     New  York,  Academic   Press,    Inc., 
1978,   vol  .   VI,    pp.    591-613. 

In   press: 

Prusiner,   S.   B.,  Garfin,   D.   E.,   Baringer,   J.   R.,   Cochran,   S.    P., 
Hadlow,  W.    J.,   Race,   R.    E. ,  and   Eklund,   C.   M.:     On  the  partial    puri- 
fication and  apparent   hydrophobicity  of  the  scrapie  agent.      In 
Prusiner,   S.   B.   and  Hadlow,   W.   J.    (Eds.):     Slow  Transmissible   Diseases 
of  the  Nervous   System.      New  York,  Academic   Press,    Inc. 

Hadlow,   W.   J.,   Race,   R.    E.  ,   Kennedy,   R.   C,  and   Eklund,   C.  M.: 
Natural    infection  of  sheep  with  scrapie  virus.      In   Prusiner,   S.   B. 
and  Hadlow,   W.   J.    (Eds.):     Slow  Transmissible   Diseases  of  the  Nervous 
System.     New  York,  Academic  Press,    Inc. 

Hadlow,  W.   J.,   Kennedy,   R.   C,   Race,   R.    E.,   and   Eklund,   C.  M. : 
Virologic  and  neurohistology   findings  in  dairy  goats  affected  with 
natural    scrapie.     Vet.   Pathol. 

Hadlow,  W.   J.:     Myopathy  associated  with  congenital    hydrocephalus   in 
Hereford  calves.      In  Andrews,   E.    J.,  Ward,   B.   C,  and  Altman,   N.  A. 
(Eds.):     Spontaneous  Animal    Models  of  Human   Disease.     New  York, 
Academic  Press,    Inc. 


30-42 


Project   No.    Z01    AI   00082-18   EB 


In   press:     (cont'd] 


Eklund,  C.  M.   and  Hadlow,  W.    J.:     Slow  viral   diseases.      In  Steele, 
J .   H .    ( Ed . ) :     Handbook  of  Zoonoses,   Section  B.     Viral    Zoonoses. 
West  Palm  Beach,  CRC  Press,   Inc. 

Hadlow,  W.   J.,   Cheville,   N.    F. ,  and   Jellison,  W.    L. :     Occurrence  of 
pox  in  a  northern  fur  seal   on  the   Pribilof  Islands   in  1951. 
J.   Wild!.   Dis. 


30-43 


SMITHSONIAN  SCIENCE  INFORMATION  EXCHANC 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  D  .,  .illlMtNT  Of 
HEALTH,  EDUCATION,  AND  WELFARE 
PUBLIC  ':EALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01    AI    00083-10 


EB 


PERIOD    COVERED 


October  1,   1978  to   September  30,  1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Host-Ectoparasite  Relationships 


NAMES,  LABORATORY  ANO  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI 


S.  K.  Wikel 


COOFERATING  UNITS   if  ar 


LAB/BRANCH 

Rocky  Mountain   Laboratory 


SECTION 

Medical    Zoology  and  Zoonotic   Diseases  Section 


INSTITUTE  AND  LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


TOTAL  MANYEARS: 


PROFESSIONAL: 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (al)  MINORS   fj  (a2)  INTERVIEWS 


H  (b)  HUMAN  TISSUES 


(c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less 

Project  terminated 


-    underline    keywords) 


30-44 


PHS-6040 
(Rev.    1 0-76) 


'SMITHSONIAN  SCIENCE  INFORMATION  EXCHANGE 
PROJECT  NUMBER  (Do  NOT  use  this  space) 


U.S.  DEPARTMENT  OF 
HEALTH.  EDUCATION,  ANO  WELFARE 
PUBLIC  HEALTH  SERVICE 
NOTICE  OF 
INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01    A I   00084-10   EB 


PER  I  00    COVERED 


October  1,  1978  to   September  30,   1979 


TITLE  OF  PROJECT  (80  characters  or  less) 

Vector-Pathogen   Relationships 


NAMES,  LABORATORY  AND  INSTITUTE  AFFILIATIONS,  AND  TITLES  OF  PRINCIPAL  INVESTIGATORS  AND  ALL  OTHER 
PROFESSIONAL  PERSONNEL  ENGAGED  ON  THE  PROJECT 


PI:     C.  E.  Yunker 
OTHER:  L.  A.  Thomas 


Scientist  Director 
Research  Microbiologist 


EB  NIAID 

EB  NIAID 


cooperating  units  (if  any)    Qr m    Kouka   S .  E.   Abdel  -Wa  ha  b,  Al   Azhar  University,   Cairo, 
Egypt;    Dr.  Mamdouh  Y.    Kamel ,   National    Research  Center,   Cairo,   Egypt; 
Dr.   D.   Knudson,   Yale  University,   New  Haven;   Drs.   L.   Rosen  and  R.   Tesh, 
University  of  Hawaii.   Honolulu. . 

lab/branch 


Epidemiology  Branch 

SECTION 

Arthropod-Borne  Diseases  Section 


jRML),   Hamilton,   MT  59840 


INSTITUTE   ANO   LOCATION 

NIAID,  NIH,  Bethesda,  MD  20205 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)   HUMAN  SUBJECTS 

□  (a1  )  MINORS   □  (a2)  INTERVIEWS 


TOTAL  MANYEARS: 


1.4 


PROFESSIONAL: 
0.4 


1  .0 


□  (b)    HUMAN   TISSUES 


(c)  NEITHER 


SUMMARY  OF  WORK  (200  words  or  less  -  underline  keywords) 

This  project  deals  with  the   interactions  of  vector-borne  microorganisms, 
arboviruses,   rickettsiae  and  protozoans,  and  eel  1 s  of  haematophagus  arthro- 
pods, mainly  ticks.     Model    in  vitro   systems  are  developed  and  applied  to 
studies  of  pathogen   host-range,   growth  dynamics,   persistence  and   interference 
After  prolonged  growth  in  these  systems,   pathogens  are  examined   for  changes 
in  invasiveness,  virulence,  or  antigenic  characteristics.     By  contract  with 
collaborative  laboratories  overseas,   we  are  studying  critical    factors 
influencing  survival   of  ticks  and  of  pathogens  within  ticks  from  a   biochemical, 
histochemical ,   and   physiological    standpoint. 


30-45 


PHS-6040 
(Rev.    10-76) 


Project   No.    Z01    AI    00084-10  EB 


Project   Description 


A  study  of  transovarial    transmission  of  Flaviviruses   by  ticks   begun 
last  year  was  continued.     Soviet  workers   have   identified   DNA  transcripts 
of  Bhanja  virus  RNA  in  eggs  of  Hyalomma  ticks.     A  model    system  of  Langat 
virus  in  Dermacentor  ticks  was  devised   in  which  virus  was  recovered   in  all 
Fi    stages  of  female  ticks  inoculated  parenterally  and  a   small    percentage 
of  ?2  e99S  and  larvae.     All    F]    stages  transmitted  virus   by  bite  to  labora- 
tory hosts.     Virus  could  not   be  detected   in   subsequent   F2  stages,   whether 
fed  or  unfed.     Thus,   proviral    DNA  may  not   be   involved   in  this   particular 
system.     Additional    transovarial    transmission   experiments  with  Quaranfil 
virus  in  Argas  arboreus  and   Colorado  tick  fever   (CTF)   virus  are   in   progress. 

Carrier  cultures  of  CTF  virus  were  established   in  Xenopus  cells 
grown  at  27°C.     After  50  passages   (350  days)  apparently  normal    cell    cultures 
continue  to  yield  low  titers  of  CTF  virus.     Previously  we  observed  a  dimin- 
ished pathogenicity  for  mice  when  the  virus  underwent   prolonged  growth 
in  mosquito  cells  incubated  at  27°C.     However,   in   the  Xenopus  cells  this 
virus  retains  its  pathogenicity.     This   implies  that  growth  at  reduced 
temperature  of  incubation  alone   is  not  a   factor  in  attenuation  of  this 
virus   in  arthropod  cells. 

The  attenuated   strain  of  CTF  virus,   which  we  developed   by  its  pro- 
longed passage   in  arthropod  cells,   as  well   as  a  virulent   strain,   have 
been  sent  to  Yale  University  where  they  will    be  examined   from  a  molecular 
standpoint,  with  a  view  toward  understanding  the  genetic  basis   for  Orbivirus 
virulence. 

The  application  of  mosquito  cell   cultures  to   the  detection  of  dengue 
viruses  was  studied.      Isolation  and   identification  of  these   Flaviviruses 
in  traditional    systems   is  a   slow  and  difficult  process  and   is  often  uncer- 
tain.    Mosquitos  inoculated  in  Hawaii  with  an  unspecified   number  of  4 
dengue  serotypes  were  coded,   frozen  and  shipped   to  RML,  where  they  were 
ground  and  inoculated  into  Aedes  albopictus  cells.     Virus  was  detected 
in  all    but  3  of  24  mosquitos.       Sixteen  of  the   isolates  were  recognized 
by  the  appearance  of  syncitia  4   to  6  days  after   inoculation  and   the  remain- 
der by  microimmunofl  uorescence  to  dengue-2   immune  serum.     Two  of  the 
isolates  that  were  definitely  missed  were  of  the  dengue-3  serotype   (which 
grows  poorly  in  LLC-MK2   cells  also);   the  third  was  lost   because  of  a 
defective  culture  flask.      It   is  apparent  that  the  mosquito  cells  are  a 
satisfactory  system  for  rapid  detection  of  at  least  3  of  4  dengue   serotypes 
and  that  their  use  should   be  further  explored. 

Considerable   progress  in  developmental    tick  tissue  culture  was  made 
this  year,  with  the  apparent  establishment  in  culture  of  a   serially  propa- 
gating line  of  cells   from  D_.   variabilis   embryos.     These  cells,   now  in 
their  21st  passage,  are  diploid  and  capable  of  forming  confluent  monolayers 


30-46 


Project  No.    Z01    AI   00084-10  EB 

within  one  week.     At  this  time,  cultures  may  be  split  on  a   1:2  or  1:3 
ratio.     This  development  of  a  new  arthropod   ("vector")  cell   line  allows 
comparison  of  its  virus  supporting  properties  with  those  of  vertebrate 
("host")  cells.     We  compared  yield  of  a   North  American  tickborne  Flavivirus, 
Powassan,   in  the  two  systems  in  the  presence  of  a  virus   inhibitor,   gluco- 
samine.    This   substance,  which  is  a   basic  component  of  the  glycoprotein  of 
arthropod  cuticle,   is  also   known  to   block  the  replication  of  many  viruses 
in  vertebrate  cells.     Duplicate  sets  of  cell    cultures  of  both  0.   andersoni 
and  Vero,  with  appropriate  controls,  were  infected  with  virus.      One  set 
was  exposed  to  glucosamine  after  virus  adsorption,  while  the  other  did 
not  receive  it.     Virus   increased  exponentially  in  the  untreated  cultures, 
but  not  in  those  given  glucosamine.     Cytotoxicity  of  the  amine  was  not 
a   factor  affecting  virus  growth. 

We  continued  to  train  investigators  from  universities  and  other 
outside  research  organizations,   both  domestic  and   foreign,   and  to   provide, 
upon  request,  arthropod  cell   cultures,  viruses  and  serological    reagents. 

Our  studies  of  arthropod-borne  pathogens  at  the  cellular  level   will 
continue.     These  will    include  viral    propagation  and  comparative  growth 
dynamics  in  vector  and   host  cells,   virus  attenuation   in  arthropod   cells, 
and  vertical   and  horizontal    transmission  of  viruses   by  vectors.     Special 
emphasis  will    be  placed  on  transovarial    transmission,   by  ticks,  of  selected 
viruses.     The  recent  discovery  by  French  workers  of  natural    infections 
of  yellow  fever  in,  and  transovarial    transmission   by,  wild  caught  African 
ticks   has  reawakened   interest  in  the  tick  as  a  reservoir  of  mosquito-borne 
Flaviviruses.     Resulting  epidemiological    implications  impinge  on   the 
possibil ity  of  a   tick-St.   Louis  encephalitis    (SLE)   virus  cycle   in  the 
United  States.     In  collaboration  with  the  University  of  Hawaii,  we  will 
investigate  transovarial    transmission  of  SLE  virus   in  candidate   species  of 
ticks. 

Pub!  ications : 

In  press: 

Anderson,   J.    F.,  Magnarelli,   L.   A.,  and   Kurz,   J.:     Hematozoa   in  rodent 
populations  of  Connecticut:     Babesia  and  Grahamella.      J.    Parasitol. 

Bhat,  U.K.M.   and  Yunker,   C.   E. :     Susceptibil  ity  of  a  tick  cell    line 
(Dermacentor  parumapertus  Neumann)   to   infection  with  arboviruses. 
Proc.   2nd   International   Symposium  on  Arctic  Arboviruses,   Mt.   Gabriel, 
Que.,   Canada,  May  26-28,  1977 

Yunker,   C.   E.   and  Cory,   J.:      Plaque  assay  method  for  arboviruses  in 
mosquito  cells.     Tissue  Culture  Manual 

Yunker,  C.    E.  and  Meibos,   H.:     Culture  of  embryonic  tick  cells    (Acari : 
Ixodidae).     Tissue   Culture  Manual 

30-47 


Project   No.    Z01    AI   00084-10   EB 


Appendix  1.     PL-480  Project  03-030-N.      In  vitro  and   in  vivo   Studies  of 

Certain  Tickborne  Viruses  in  Vector  and  Host  Cells,  Al   Azhar 
University,   Cairo,   Egypt. 

Annual    Funding:      $97,738 

This  project  seeks  to  understand  the  contrasting  behavior  of  a 
tickborne  virus  that  is  pathogenic  for  humans   in  Egypt,   when  grown   in  a 
vertebrate  host  as  opposed  to  the  vector.     During  the  second  year  of  the 
project,  eight  manuscripts  were  completed;   their  subjects  include   immuno- 
suppression of  Quaranfil   virus,  viremia  dynamics  in  cell   culture  and  com- 
parative behavior  of  virus  in  vector  and  non-vector  ticks.     Additional 
lines  of  study  are  in  progress. 


Appendix  2.      PL-480  Project  03-051-N.     Biochemistry  and   Physiology  of  Tick 
Vectors  of  Disease. 

Annual    Funding:      $156,294 

This  project  was  designed  to   provide  basic   biochemical   and  physio- 
logical   information  on  tick  metabolism   in  order  to  define   the  environment 
in  which  tickborne  pathogens  of  man  and  animals  exist  when  not   infecting 
their  definitive  host.     Additionally,   these  data  aid   in  the  refinement  of 
tissue  culture  media  specifically  designed   for  tick  cells.     During  the 
first  year  of  the  project,  embryonic  changes  in  nucleic  acids,   proteins, 
carbohydrates  and  nitrogenous  excretory  products  were  studied.     Results 
revealed  at  what  stage  the  embryo   becomes  maternally  independent.     Manu- 
scripts describing  these  results  are   in   preparation. 


30-48 


Rocky  Mountain  Laboratory 
Adminstrative  and  Operational  Support 
Operations  Branch 

1979  Annual  Report 
Table  of  Contents 


Introduction ■ 

General  Overview  of  the  Responsibilities  of  Operations  Branch. 


30-1 
30-2 


October  1,  1978  -  September  30,  1979 

Administrative  and  Operational  Support  for  Rocky  Mountain  Laboratory 

Operations  Branch 
NIAID 

Introduction 

The  Branch  provides  all  services  necessary  to  the  professional  staff  in 
the  pursuit  of  their  investigations.  This  support  covers  the  following  areas; 
procurement,  personnel,  custodial,  security,  media  preparations,  glassware  re- 
finishing,  photography,  animal  rearing  and  care,  motor  pool,  operation  of 
power  plants,  and  full  maintenance  in  every   area  except  electronics. 


-I 


General  Overview  of  the  Responsibilities  of  Operations  Branch 

The  fiscal  and  procurement  department  manages  a  budget  of  $950,000. 
Payroll  is  not  included  in  this  figure.  It  covers  only  the  purchase  of 
supplies  and  minor  equipment  used  in  the  operation  of  the  laboratories. 
Timekeeping  and  the  correction  of  errors  in  the  payroll  are  also  handled 
in  the  unit. 

Personnel  handles  all  actions  and  advises  on  personnel  matters.  This 
department  also  is  charged  with  the  operation  of  the  Comprehensive  Employment 
Training  Act  (CETA)  in  association  with  the  local  Montana  State  Employment 
Office.  Through  the  year  we  have  averaged  fifteen  (15)  people  on  this 
program  at  all  times.  The  maximum  time  a  person  may  spend  on  the  program 
is  3  months.  Hence,  we  are  constantly  interviewing  and  employing  people 
under  the  program.  Also  handled  by  Personnel  are  persons  under  the  following 
programs:  Stay  in  School,  Work  Study,  Government  Summer  Program,  and  students 
studying  for  advanced  degrees. 

Custodial  services  are  provided  in  the  five  laboratory  buildings  daily. 
Security  is  provided  in  the  form  of  a  guard  on  duty  every  night  of  the 
year. 

Most  of  the  media  used  in  the  laboratories  is  prepared  by  the  branch. 
All  glassware  used  in  the  lab  is  cleaned  and  sterilized  for  reuse  in  the 
laboratories. 

The  photography  department  provides  full  professional  services  necessary 
in  the  laboratories  with  the  exception  of  medical  artistry. 

The  animal  unit  raises  two  strains  of  guinea  pigs,  five  strains  of  mice 
plus  one  strain  of  rats  and  hamsters,  and  one  colony  of  microtus.  They  breed 
and  raise  approximately  145,000  animals  a  year.  An  additional  12,000  animals 
are  purchased  annually  from  outside  sources,  including  mink,  sheep,  rabbits, 
mice,  guinea  pigs,  and  hamsters.  After  rearing  care  is  provided  for  these 
animals  while  they  are  under  experiment. 

The  maintenance  department,  through  the  power  plant,  provides  heat, 
steam,  air  and  vacuum  to  the  laboratories.  Also  provided  is  air  conditioning, 
compressed  air,  vacuum  and  demineralized  and  distilled  water.  A  motor  pool 
consisting  of  10  vehicles  is  maintained  and  grounds  care,  including  snow 
removal,  is  provided. 

With  the  exception  of  the  electronics  work  all  maintenance  is  done  by 
the  staff.  This  includes  plumbing,  electrical,  sheet  metal,  carpentry,  and 
refrigeration,  including  ultra  low  temperature  boxes. 

Labor  management  work  is  handled  by  the  Chief  of  the  Branch. 


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