Ociit 2011


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Ociit 2011

  1. 1. Analysis of Human Polyreactive Pneumococcal Antibodies Rebecca Thompson OCIIT 2011
  2. 2. Streptococcus Pneumoniae– Gram-positive bacteria– Colonizes the nasopharynx– Over 90 identified serotypes • Serotypes are determined by capsular polysaccharide– Antibodies against capsular pneumococcal polysaccharide (PPS) are protective
  3. 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. 4. Natural Antibodies– First line of defense against pneumococcal invasive disease – Also aid in the clearance of pneumococcus if an infection is already present– Weakly polyreactive– In murine studies, polyreactive antibodies are produced by B1 cells– In humans, the origin and characteristics of these antibodies are not well characterized
  5. 5. Methodology• Draw blood from healthy young volunteers 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-binding B cells• Harvest and lyse cells• Make cDNA and PCR variable heavy (VH) and variable light (VL) immunoglobulin genes – Natural VH/VL pairing is maintained – True representation of antibody repertoire• Ligate paired VH and VL into rhuIg expression plasmids and transfect human cells• Harvest recombinant human antibodies, perform avidity measurements on whole and F(ab)’2 fragments
  6. 6. Isolation of Polyreactive Antibodies
  7. 7. Variable Chain Sequence Analysis
  8. 8. Conservation of Vk CDR3 Length
  9. 9. Characteristics of Amino Acids in CDR3
  10. 10. Polyreactive vs. PPS-specific Antibodies Multiple Serotype Vk CDR3 Composition 6.0 5.0 Polyreactive (10) Average number of amino acids 4.0 PPS3 (2) PPS6B (1) 3.0 PPS14 (4) 2.0 PPS23F (35) 1.0 Series6 0.0 Flexible AA - RWY Positive AA - RHK Negative AA - DE
  11. 11. Polyreactive vs. PPS-specific Antibodies Average CDR3 Length 25.0 20.0 Polyreactive PPS3# of amino acids 15.0 PPS6B 10.0 PPS14 PPS23F 5.0 Series6 0.0 VH CDR3 Vk CDR3
  12. 12. Surface Plasmon Resonance
  13. 13. Polyreactive Avidity Avidity to PPS14 0.E+00 2.E-07 4.E-07 6.E-07 8.E-07KD uM 1.E-06 1.E-06 1.E-06 2.E-06 2.E-06 33G8 32E8 31B5 21B2 24F5 IgG1 0.00000102 0.00000057 0.00000067 0.00000076 0.00000028 IgG2 0.00000012 0.00000097 0.00000099 0.0000053 0.0000014
  14. 14. F(ab)’2 Fragment Avidity
  15. 15. Summary• After analysis of our polyreactive antibodies, there are multiple features limited to this group of antibodies. – Conservation of Vk CDR3 length – Increased number of flexible (arginine, tyrosine and tryptophan) amino acids in the VH CDR3 – Longer VH CDR3 length when compared to PPS-specific antibodies• In general, the IgG1 isotype bound more avidity to PPS14 and PPS23F than IgG2• When antibodies were digested into F(ab)’2 fragments the difference in avidity between isotypes was largely diminished
  16. 16. Future Work• Cloning of isolated PPS-specific antibodies• Comparison of PPS binding kinetics – Polyreactive vs PPS-specific
  17. 17. Acknowledgements• Major Advisor • Committee Members – Dr. Julie Westerink, MD – Dr. Dignam• Lab Members – Dr. Malhotra – David Leggat – Dr. Ruch – Dr. Noor Khaskhely – Dr. Wooten• Funding – NIH RO1