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  • 1. Promiscuous  Variable  Light  Chain   Recombina6on  with  Pneumococcal   Polysaccharide  Specific  Variable  Heavy   Chain Rebecca  Thompson OCIIT  ForumTuesday, June 5, 12
  • 2. • Streptococcus  pneumoniae   is  a  significant  cause  of   morbidity  and  mortality   worldwide.   • The  main  virulence  factor  of   S.  pneumoniae  is  the   capsular  polysaccharide.   • An;bodies  elicited  against   PPS  provide  protec;on   against  invasive  disease.  Tuesday, June 5, 12
  • 3. • The  adult  23  valent  purified  PPS  vaccine  has  an  80%   protec6ve  efficacy  in  healthy  young  adults.   • However  efficacy  in  the  elderly  is  dras6cally  reduced   despite  normal  an6body  levels.   • This  phenomenon  is  thought  to  be                                                                         due  to  an6body  structure.Tuesday, June 5, 12
  • 4. • Previous  data  characterizing  the  structure-­‐func;on   rela;onship  of  an;-­‐pneumococcal  an;bodies  has  been   gathered  primarily  from  the  use  of  combinatorial  libraries.   • Combinatorial  libraries  pool  mul;ple  B  cells  and  PCR  the   variable  immunoglobulin  regions  not  conserving  the  natural   VH/VL  pairings. • Combinatorial  libraries  are  used  for  an;body  repertoire   analysis  and  assume  that  the  random  heavy  and  light  chain   pairs  formed  represent  the  na;ve  repertoire.   • This  is  supported  by  data  that  demonstrates  the  in  vivo  use  of   similar  variable  heavy  and  light  combina;ons.Tuesday, June 5, 12
  • 5. • The  consistent  pairing  of  various  VH  with  iden6cal  VL,  as   seen  in  response  to  Haemophilius  influenza  type  B  (Hib)   polysaccharide  and  pneumococcal  polysaccharide,   suggests  a  restricted  VH-­‐VL  pairing  in  nature.   • In  response  to  Hib,  the  predominant  variable  light  chain,   A2  is  used  in  the  repertoire  across  different  individuals   and  within  the  same  individual.   • The  phenomenon  responsible  for  this  pairing  limita6on   has  yet  to  be  elucidated.Tuesday, June 5, 12
  • 6. • The  goal  of  our  study  was  to  explore  the  reported  restricted   gene  usage  in  response  to  polysaccharide  an;gens  and  to   determine  if  this  observa;on  is  the  result  of  physiological  or   structural  limita;ons  of  an;bodies  that  recognize   polysaccharide  an;gens. • To  test  these  concepts,  one  variable  heavy  chain  specific  for   PPS23F  was  paired  with  several  alterna;ve  PPS-­‐specific   variable  light  chains. • Recombinant  human  an;bodies  were  tested  for  func;onality   and  influences  on  pneumococcal  polysaccharide  binding.Tuesday, June 5, 12
  • 7. • Variable  light  gene  fragments  were  obtained  from  PPS-­‐ specific  B  cells  isolated  from  immunized  donors.  Fragments   were  PCR  amplified  to  add  expression  plasmid  specific   restric;on  sites.   • CBE2,  a  PPS23F  specific  variable  heavy  chain  was  obtained   from  Baxendale  et  al  and  restric;on  sites  were  inserted.   • The  variable  chains  were  each  ligated  into  the  appropriate   pHC-­‐huC  plasmid  (McLean  et  al).  pHC-­‐huC  containing  variable   heavy  and  light  sequences  were  then  transfected  into  HEK293   cells. • Surface  plasmon  resonance  (SPR)  measured  the  avidity  of  the   an;body  (Reichert  3700DC).  The  sensor  chip  with   immobilized  an;-­‐human  Ig  an;body  bound  to  its  surface  was   used  to  capture  recombinant  human  an;body.  PPS23F  was   injected  over  the  chip  surface  to  determine  binding  avidity.Tuesday, June 5, 12
  • 8. Tuesday, June 5, 12
  • 9. 14f2 21d3 31f7 23d3 CBE2  IgG1  An6bodies 25b4 24e8Tuesday, June 5, 12
  • 10. Tuesday, June 5, 12
  • 11. Tuesday, June 5, 12
  • 12. Tuesday, June 5, 12
  • 13. Tuesday, June 5, 12
  • 14. • Analysis  of  these  recombinant  an;bodies  by  ELISA   demonstrates  that  these  an;bodies  maintain  their  specificity   for  PPS23F.  However,  analysis  with  SPR  demonstrates  that   these  an;bodies  possess  unique  binding  characteris;cs.   These  findings  were  also  confirmed  by  disrup;on  ELISA. • Although  there  are  certain  capsular  polysaccharides  where  a   specific  variable  light  chain  gene  is  required  for  epitope   binding  this  does  not  seem  to  be  the  case  with  PPS23F.  It  is   plausible  that  the  variable  light  chain  does  not  significantly   contribute  to  the  structural  requirements  of  PPS23F  binding.   • In  contrast  for  Hib  PS,  an  arginine  in  posi;on  95a  of  the  light   chain  is  essen;al  for  binding.  To  date,  we  have  not  iden;fied   an  essen;al  amino  acid  sequence  in  the  CDR3  of  VL  for  PPS.Tuesday, June 5, 12
  • 15. Future  Work • Comparison  of  IgG1  to  IgG2  isotypes o Predominate  an;body  response  to  S.  pneumoniae o IgG1  levels  drop  with  age • Analysis  of  an;body  structure  in  response  to  S.  pneumoniae o Fc,  Fab  and  Fab’2  fragments • Defining  the  binding  requirements  for  pneumococcal   polysaccharide  23FTuesday, June 5, 12
  • 16. Acknowledgements • Major  Advisor • Collaborators – Dr.  Julie  Westerink,  MD – Dr.  Baxendale • Lab  Members – Dr.  McLean – Jason  Mosakowski • Commidee  Members – Jieying  Wang – Dr.  Dignam – Dr.  Noor  Khaskhely,  PhD – Dr.  Malhotra • Funding – Dr.  Ruch – NIH  RO1 – Dr.  WootenTuesday, June 5, 12