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Ociit conference copy Presentation Transcript

  • 1. Immune Response to PneumococcalPolysaccharides in Elderly and Young Rebecca Thompson OCIIT Forum
  • 2. Streptococcus Pneumoniae– Gram-positive bacteria– Colonizes the nasopharynx– Over 90 identified serotypes • Serotypes are determined by capsular polysaccharide– Vaccines induce antibodies against capsular polysaccharide– These antibodies against capsular pneumococcal polysaccharide (PPS) are protective
  • 3. Pneumococcal Disease– Pathogenesis • Pneumonia • Otitis media • Meningitis • Bacteremia– High risk groups include the very young (<5 years old), the elderly (>65 years), the immunocompromised and the asplenic– Responsible for >40,000 deaths annually in the United States
  • 4. Vaccines– 7-Valent Pneumococcal Conjugate Vaccine • Prevnar® – Contains 7 purified capsular polysaccharide covalently conjugated to CRM197, a diptheria toxoid • Recommended for young children– 23-Valent Pneumococcal Polysaccharide Vaccine • Pneumovax® – Contains 23 purified capsular polysaccharides • Serotypes responsible for >85% of invasive disease in the U.S. • Recommended for adults >65 • 80% protective efficacy in healthy young adults • However despite normal antibody levels efficacy in elderly is drastically reduced. This drop in efficacy is thought to be linked to antibody structure.
  • 5. Previous Research– Several research groups have studied this structure-function relationship of anti-pneumococcal antibodies.– Hybridomas and combinatorial libraries have been used to express recombinant anti-pneumococcal antibodies.– The bulk of this information has come from combinatorial libraries. • Reverse transcription PCR of expressed immunoglobulin genes • Cloning of random VH and VL chains into an expression system. • Assumption that these pairings are representative of a natural repertoire.
  • 6. Zhou et al. Recurrent variable region gene usage and somatic mutation in thehuman antibody response to the capsular polysaccharide of Streptococcuspneumoniae type 23F. Infect Immun (2002) vol. 70 (8) pp. 4083-91
  • 7. • Zhou’s combinatorial library possesses a clear over-representation of certain VL genes – Of 30 PPS23F specific clones • 15 clones L6 VL gene • 12 clones A23 VL gene• Is this a result of random pairing producing the most avid clones? Or is this the true representation of natural pairings of VH and VL in response to S. pneumoniae?• We hypothesized that PPS-specific VH could recombine with various VL chains (i.e. not restricted).
  • 8. Methodology• Vaccinate healthy young volunteers with Pneumovax® – Ages 18-30 – Age group with highest vaccine efficacy – Allows us to establish a natural antibody repertoire in response to PPS• Draw blood at day 7 and isolate B cells• Single cell sort B cells into a 96 well plate• Expand B cells in culture• Test culture supernatants by ELISA – Identify Ig secreting B cells and PPS specific B cells• Harvest and lyse cells• Make cDNA and PCR variable heavy (VH) and variable light (VL) immunoglobulin genes• Ligate paired VH and VL into rhuIg expression plasmids and transfect human cells
  • 9. Baxendale et al. Immunogenetic analysis of the immune response to pneumococcal polysaccharide. Eur J Immunol (2000) vol. 30 (4) pp. 1214-23– To this purpose we obtained a well characterized human mono-specific 23F mAb, CbE2 • Reverse transcriptase PCR of CbE2 VH • Ligated CbE2 VH into a human rIg plasmid along with a random VL chain also in a separate human rIg plasmid – Light chains obtained by selecting B cells with a variety of different PPS
  • 10. Variable Light Chains Clone Isolating Antigen VL CDR3 21d3 PPS14 L2 QQYNNRPRT 14F2 PPS4 B3 QQYYSTPVT 23d3 PPS14 B3 QQYYSTPAT 24e8 PPS14 B3 QQYYSTPYT 25b4 PPS14 A27 QQYGSSPPWT 31b5 PPS23 A27 QQYDRSPLT 31F7 PPS23 L6 QQRSNWPPLLT 31E2 PPS23 A20 QKYNGAPFT 32E8 PPS23 O12 QRSSGGPIS• Transient transfection into HEK293 cells• On day 3, culture supernatant was collected from transfected cells• Samples were tested by ELISA for human Ig, PPS6B, PPS14 and PPS23F
  • 11. 14f2 21d331f7 CbE2 Antibodies 23d3 25b4 24e8
  • 12. Ig Binding 2 1.8 1.6 1.4 1.2OD 490 1 0.8 0.6 0.4 0.2 0 21d3 14f2 23d3 24e8 25b4 31b5 31f7 31e2 32e8 Pos. Ctrl Neg. Ctrl 89sf PPS14 L2 PPS4 B3 PPS14 B3 PPS14 B3 PPS14 A27 PPS23F A27 PPS23F L6 PPS23F A20 PPS23F O12 Variable Light Chain
  • 13. PPS6B Binding 2 1.8 1.6 1.4 1.2OD 490 1 0.8 0.6 0.4 0.2 0 21d3 14f2 23d3 24e8 25b4 31b5 31f7 31e2 32e8 Pos. Ctrl Neg. Ctrl 89sf PPS14 L2 PPS4 B3 PPS14 B3 PPS14 B3 PPS14 A27 PPS23F A27 PPS23F L6 PPS23F A20 PPS23F O12 Variable Light Chain
  • 14. PPS14 Binding 2 1.8 1.6 1.4 1.2OD 490 1 0.8 0.6 0.4 0.2 0 21d3 14f2 23d3 24e8 25b4 31b5 31f7 31e2 32e8 Pos. Ctrl Neg. Ctrl 89sf PPS14 L2 PPS4 B3 PPS14 B3 PPS14 B3 PPS14 A27 PPS23F A27 PPS23F L6 PPS23F A20 PPS23F O12 Variable Light Chain
  • 15. PPS23F Binding 2 1.8 1.6 1.4 1.2OD 490 1 0.8 0.6 0.4 0.2 0 21d3 14f2 23d3 24e8 25b4 31b5 31f7 31e2 32e8 Pos. Ctrl Neg. Ctrl 89sf PPS14 L2 PPS4 B3 PPS14 B3 PPS14 B3 PPS14 A27 PPS23F A27 PPS23F L6 PPS23F A20 PPS23F O12 Variable Light Chain
  • 16. 21.81.61.41.2 1 Ig PS23F0.80.60.40.2 0 21d3 14f2 23d3 24e8 25b4 31b5 31f7 31e2 32e8 Pos. Ctrl Neg. Ctrl 89sf PPS14 L2 PPS4 B3 PPS14 B3 PPS14 B3 PPS14 A27 PPS23F A27 PPS23F L6 PPS23F A20 PPS23F O12
  • 17. Summary• As demonstrated by the promiscuity of the CbE2 VH chain to pair with multiple non-specific light chains, combinatorial libraries do not represent specific binding to PPS23F with exclusive VL clones.• Therefore combinatorial libraries do not characterize the natural repertoire of anti- pneumococcal antibodies and natural VH/VL pairings are crucial for the accurate understanding of the immune response to S. pneumoniae.
  • 18. Future Work• Stable transfections – Allow for purification of rhuIg• Subsequent avidity studies – Competition assays – Surface plasmon resonance• Enzymatic digestion of antibodies – Isolate individual antibody regions to determine areas of importance in response to PPS
  • 19. Acknowledgements• Major Advisor • Collaborators – Dr. Julie Westerink, MD – Dr. Baxendale• Lab Members • Committee Members – Jason Mosakowski – Dr. Dignam – Jieying Wang – Dr. Malhotra – Dr. Louise Smithson, PhD – Dr. Ruch• Funding – Dr. Wooten – NIH RO1