Iit forum 2008


Published on

Published in: Technology
  • Be the first to comment

  • Be the first to like this

No Downloads
Total views
On SlideShare
From Embeds
Number of Embeds
Embeds 0
No embeds

No notes for slide
  • IgG1 Greater complement activation Higher bactericidal and opsonophagocytic capabilities IgG2 Can form dimers allowing for more avid binding to pneumococcal polysaccharide
  • TH1 T cells activate macrophages while TH2 activates B cells
  • mAb PN31-1 binds to type 4 PPS, Heparin-binding epidermal growth factor (HB-EGF) was immobilized to CM5 chip using amine coupling
  • Iit forum 2008

    1. 1. Role of Constant Regions in Antibody Binding to Pneumococcal Polysaccharide Rebecca Thompson
    2. 2. Streptococcus pneumoniae• Gram-positive bacteria• Colonizes the nasopharynx – Over 90 identified serotypes• Serotypes are determined by capsular polysaccharide• These antibodies against capsular pneumococcal polysaccharide (PPS) are protective• However antibodies are specific to each serotype and are not protective http://contanatura.com against other serotypes
    3. 3. Pneumococcal Disease• Pathogenesis – Pneumonia – Otitis media – Meningitis www.mayoclinicproceedings.com – Bacteremia• High risk groups include the very young (<5 years old), the very old (<65 years), the immunocompromised and the asplenic• Responsible for >40,000 deaths annually in the United States
    4. 4. Vaccines in U.S.• 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• 7-Valent Pneumococcal Conjugate Vaccine – Prevnar® • Contains 7 purified capsular polysaccharide covalently conjugated to CRM197, a diptheria toxoid – Recommended for young children
    5. 5. Antibody Structure• Antibodies have two functional regions• Variable – Capable of specific recognition and binding to epitope• Constant – Classically thought to contribute to effector functions • Complement activation • Mediation of immune phagocytosis • Antibody-dependent cytotoxicity.• Recent studies suggest that the F(ab)2 may influence effector functions such as antibody- dependent cytotoxicity while antibody fine specificity can be a function of isotype/subclass (Fc). Janeway’s Immunobiology, 2001
    6. 6. Characteristics of Subclass IgG1 IgG2 Heavy chain γ1 γ2 Molecular weight 146 146 kDa Mean serum level 9 3 (mean adult mg/mL) Half life (days) 21 20 Classical pathway of ++ + complement activation Binding to + - macrophages and other phagocytes Dimer formation - + Janeway’s Immunobiology, 2001
    7. 7. Subclass Distribution in Reaction to Pneumococcal Polysaccharide• Subclass distribution is age-related – Children <5 years of age show predominately IgG1 antibodies – Adults elicit a predominate IgG2 response• Ratio of IgG1:IgG2 continues to decrease with age – Lottenbach et al showed this ratio is conserved regardless of vaccine (conjugate or PPS)
    8. 8. Previous Studies• McLean et al. expressed the VL and VH of mAb specific for the capsular polysaccharide of Cryptococcus neoformans with human constant regions μ, γ1, γ2, γ3, γ4 and α1. The the mouse human antibodies IgM, IgG3 and IgG4 antibodies clearly showed differences in fine specificity despite identical variable region sequences suggesting that the constant region can affect conformation of the variable region affecting fine specificity and possibly avidity.• Pritsch et al. studied the influence of constant region on the avidity of recombinant human antibodies sharing identical VH and VL to tubulin. Immunoglobulin G and A antibodies and their Fab fragments with identical variable regions bound tubulin with different avidity.• Torres et al. murine IgG3 antibodies noncovalently associate allowing for a concentration dependent increase in antigen binding.
    9. 9. Summary• Isotype distribution of IgG1 and IgG2 in response to PPS is clearly age related• Constant region, which determines antibody isotype, appears to play a role in antibody avidity and fine specificity
    10. 10. Why is isotype so important?• Newly developed adjuvants allow for specific stimulation of TH1 versus TH2 branches of the immune system – For example alum skews the immune system towards a TH2 response • Secretion of IL-4, IL-5 • Generation of IgG1 and IgE isotypes – CpG and MPL are novel adjuvant that promote TH1 response • Secretion of IFN-γ, TNF-α, IL-12 • IgG2• Therefore we can direct either IgG1 or IgG2 production• Thus it is crucial that we first define the most desirable reaction to PPS
    11. 11. Current Project• What role does the constant region have in antibody binding? – Paired VH VL with specificity to PPS regions will be subcloned into expression vector • Isolated from single human PPS specific B cells – Expressed in mammalian cells – Measure binding to PPS using SPR• Thus comparing IgG isotypes with identical variable regions
    12. 12. McLean Vectors• Mammalian expression vectors- Express a recombinant human IgG antibody• The antibody expressed will have either a IgG1 or IgG2 constant region• Variable chain pairs will be cloned into each expression vector and analyzed- Here the variable region is conserved in both recombinant subclasses of Ig allowing for true interpretation of antibody binding to pneumococcal polysaccharide
    13. 13. Surface Plasmon Resonance• Recombinant antibodies will be analyzed by surface plasmon resonance (SPR). - SPR allows for real-time data collection of the interaction between antibody and antigen - Surface of sensor chip is coated with antigen (pneumococcal polysaccharide) - Antibody is passed over the chip’s surface in liquid phase - Interactions between the recombinant antibody and pneumococcal polysaccharide are recorded by the SPR equipment
    14. 14. RU 40 35 30 25 20 15Res 10p.Diff. 5 0 -5 0 50 100 15 0 200 250 3 00 T ime s • Bivalent analyte model • Overlay sensorgrams at different concentrations to determine KD
    15. 15. Analysis of Antibody Affinity• After antibody SPR data collection, IgG1 and IgG2 values will be compared• If there is a statistical difference between IgG1 and IgG2 affinity, Fabs will be created to determine the molecular basis of binding
    16. 16. Clinical significance• If there is an observed difference between IgG1 and IgG2 constant region in fine specificity of antibody binding, then it would be advantageous to design vaccines that allow for the direction of immune response to either IgG1 or IgG2• Adding adjuvants to such vaccines would allow for the stimulation of IgG1 or IgG2 antibodies improving the protective ability of the vaccine
    17. 17. Current Status• Successfully cloned PPS specific naturally paired VH VL chains into rhIgG expression vectors• Transient transfection into human kidney cells• ELISA to test for antibody binding
    18. 18. Binding to PPS23
    19. 19. Future Work• Presently purifying antibodies 113G1 and 113G2 for analysis by SPR• Once this system is optimized several other VH and VL pairs are ready for cloning into the McLean expression vectors
    20. 20. Acknowledgements• Dr. Westerink• Dr. Smithson• Jason Mosakowski• McLean Lab• Dr. Dignam