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Rotation Talk – Janoff Lab Kristin Shotts November 23, 2011

Streptococcus pneumoniae Evades the Immune System by Subverting Host Defense: Developing a New Vaccination Target. Rotation Talk – Janoff Lab Kristin Shotts November 23, 2011. Infections Caused by Streptococcus pneumoniae. Mucosal. Systemic. Meningitis Endocarditis Arthritis

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Rotation Talk – Janoff Lab Kristin Shotts November 23, 2011

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  1. Streptococcus pneumoniae Evades the Immune System by Subverting Host Defense: Developing a New Vaccination Target Rotation Talk – Janoff Lab Kristin Shotts November 23, 2011

  2. Infections Caused by Streptococcus pneumoniae Mucosal Systemic • Meningitis • Endocarditis • Arthritis • Bacteremia/sepsis • Colonization • Otitis media • Sinusitis Pneumonia

  3. Host response to S. pneumoniae by IgA • IgA1 antibodies are generated in the mucosa that bind specifically to the polysaccharide capsule of S. pneumoniae • S. pneumoniae bacteria have over 90 different serotypes, which are the current targets for vaccinations Ref 1

  4. Mechanism of Killing by IgA1-Mediated Opsonization Ref 2 KILL Ref 3 Macrophage

  5. IgA1 Protease as a Vaccination Target • Highly conserved protease across pneumococcal serotypes Ref 4 Ref 2 IgA1P KILL Ref 3 Macrophage

  6. Enhanced Adherence to and Colonization of Lung Epithelium Ref 4 Ref 2 IgA1P IgA1 protease KILL NO KILL Ref 3 Macrophage Macrophage

  7. Hypothesis: Capsule-specific IgG in patient sera will bind to IgA1 protease to inhibit IgA1 cleavage IgG (from patient sera) Ref 2 4 KILL Ref 3 Macrophage

  8. Specific Aims • Optimize an ELISA assay to detect IgA1 cleavage using IgA1 protease purified from H. influenzae and four strains of S. pneumoniae. • Determine the frequency of IgA1 protease-specific IgG generation in 23 patients’ sera after acute and 2-4 weeks post S. pneumoniae infection vs. 46 control subjects.

  9. Immunoglobulin Structure CH1 kappa CH2 Fc region CH3 Ref 5

  10. ELISA to Detect IgA1 Cleavage kappa AP AP AP + IgA1 Protease CH3 Cleaved IgA1 Kappa specific-AP detector AB Uncleaved IgA1 IgA-CH3 specific Capture Ab

  11. IgA1 Cleavage by H. influenzae Protease Detected using SDS-PAGE Protease Conc. (ug/mL) IgA1 alone Protease alone 42.8 14.3 1.43 0.428 0.143 4.28 250 150 100 75 Heavy chain 50 37 Cleaved Heavy chains 25 20 Light chain 15

  12. Optimized ELISA Detects IgA1 Cleavage • IgA1 concentration decreases 25 fold in the presence of IgA1 • protease, as detected by the ELISA • Addition of IgG does not interfere with the assay

  13. S. Pneumoniae Bacterial Strain Information IgA1 protease gene diagram Made by: Ron Gill • Characterize the phenotype of the S. pneumoniae strains using PCR and gel electrophoresis F2  erm F3  iga  REV

  14. Determination of Strain Phenotype by PCR Lanes 12-16 F3 + rev primer set Lanes 6-10 F2 + rev primer set DNA ladder DNA ladder 1517 1200 1000 800 700 600 500 400 300 200 100 89 (-) 89 (-) 88 (+) 88 (+) 87 (-) 87 (-) 86 (+) 86 (+) D39 (+) D39 (+) • The F2 + rev primer set generated IgA1P- fragments in 2 strains and the F3 + • rev set generated IgA1P+ PCR fragments in 2 strains and the positive control. • There are two mutant and two wild type S. pneumoniae strains.

  15. ELISA Detects IgA1 Cleavage by Wild Type IgA1P S. pneumoniae Strains • Wild type (IgA1P+) strains show a 4- 7 fold reduction in optical density • (OD), indicating IgA1 is being cleaved • The OD for mutant (IgA1P-) strains remains constant

  16. Neutralization using IgG Purified from Patient Sera • Inhibition of IgA1 cleavage is seen by protease-specific IgG purified from acute and 2-4 weeks post infection sera samples drawn from the same patient • The other patient appears to be a non-responder

  17. Conclusions • The optimized, neutralization ELISA can detect inhibition of IgA1 cleavage in the presence of IgA1P-neutralizing IgG purified from patients infected with S. pneumoniae. • The next steps are to analyze the rest of the patient samples using the neutralization assay as well as to express IgA1 protease in E. coli, in order to characterize where the patient IgG binds to the protein to inhibit cleavage of IgA1.

  18. Acknowledgements • Dr. Edward Janoff, M.D. • Dr. Ronald Gill, P.H.D. • Dr. Dan Frank, P.H.D. • Jeremy Rahkola • Claire Gustafson • Leah Feazel • Diana Ir • Jana Palaia

  19. References • http://www.chori.org/Principal_Investigators/Test_Samuel_T/test_research.html • http://www.immunopaedia.org.za/index.php?id=750 • http://en.wikipedia.org/wiki/File:Macrophage.png • http://www.sayers.staff.shef.ac.uk/igap/ • http://www.biology.arizona.edu/immunology/tutorials/antibody/structure.html

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