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Ociit 2011


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  • Our lab studies the human antibody response to Streptococcus pneumoniae Serotypes are determined by the structure of the pneumococcal capsular polysaccharide
  • The lab’s two RO1 grants are aimed to study the elderly and HIV positive populations
  • While our lab focuses on the specific antibody response to pneumococcal polysaccharides there are also natural antibodies
  • Identified during screening
  • These antibodies did not have a striking trend in gene usage. PPS-specific antibodies classically express only one or two genes. For example, reported PPS23F specific antibodies variable light chain is restricted to L6 and A23.
  • Analysis of these polyreactive clones revealed several unique trends
  • Went on to analyze amino acid groups that would influence binding to pneumococcal polysaccharide Greater number of flexible amino acids in the VH CDR3
  • After observing these trends in polyreactive antibodies we wanted to compare these findings to PPS-specific antibodies Analyzed all reported human PPS-specific antibodies
  • Went on to determine specific avidity to PPS14 and PPS23F using SPR Polyreactive clones were expressed as both IgG1 and IgG2
  • Mention reversed y-axis Smaller number the better A specific monoclonal antibody measured a avidity of 70pM Generally, IgG1 bound both polysaccharides more avidity than IgG2
  • Upon cleavage of the constant region, the differences between antibody avidity was no longer observed
  • Transcript

    • 1. Analysis of Human Polyreactive Pneumococcal Antibodies Rebecca Thompson OCIIT 2011
    • 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. 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. 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. 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. Isolation of Polyreactive Antibodies
    • 7. Variable Chain Sequence Analysis
    • 8. Conservation of Vk CDR3 Length
    • 9. Characteristics of Amino Acids in CDR3
    • 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. 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. Surface Plasmon Resonance
    • 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. F(ab)’2 Fragment Avidity
    • 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. Future Work• Cloning of isolated PPS-specific antibodies• Comparison of PPS binding kinetics – Polyreactive vs PPS-specific
    • 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