Analysis of the adaptive immune response to West Nile virus

Transcription

1 Analysis of the adaptive immune response to West Nile virus Jonathan Bramson (PI) Robin Parsons Alina Lelic Galina Denisova Lesley Latham Carole Evelegh Dmitri Denisov

2 Strategy for characterizing T cell function in patients following West Nile virus infection 1. Identify specific West Nile epitopes by IFN-γ ELISPOT using peptide pools covering the entire polyprotein 2. Quantify West Nile-specific response by ELISPOT 3. Use cytokine flow cytometry y and tetramer e staining to phenotype the peptide-specific T cells Are there quantitative or qualitative differences among the patient populations?

3 847 peptides were synthesized : residues in length overlapping by 11 amino acids WNV polyprotein C M E NS1 NS2B NS3 NS4A NS aa NS2A NS4B Peptide library Etc couplin ng efficiency xxxxxxxxxxxxxx xxxxxxxxxxxxx xxxxxxxxxxxx xxxxxxxxxxx xxxxxxxxxx xxxxxxxxx xxxxxxxx Etc ~70% of product ~30% of product Optimal CD8+ T cell epitopes are 8 10 residues Optimal CD4+ T cell epitopes are residues

4 847 peptides were synthesized : residues in length overlapping by 11 amino acids WNV polyprotein C M E NS1 NS2B NS3 NS4A NS aa NS2A NS4B Peptide library Etc Other methods for T cell epitope discovery: 1) Mass Spec analysis of peptides p bound to MHC class I (collaboration with W. Hildebrand, U. Oklahoma) 2) In silico prediction combined with in vitro affinity analysis (collaboration with O. Lund, Technical University of Denmark).

5 Achievements: Defined numerous CD8+ T cell epitopes for common HLA No relationship between disease severity and magnitude or breadth of memory CD8+ T cell response - Functional studies are pending. Developed multi-fluorochrome (polychromatic) phenotypic cocktails and tetramer reagents for 3 epitopes (more coming) Established methods for highly-sensitive detection of CD4+ T cell responses using CFSE dilution.

6 Publications McMurtrey, C., Lelic, A., Piazza, P., Chakrabarti, A.K., Yablonski, E.J., Wahl, A., Bardet, W., Eckerd, A., Cook, R.I., Buchli, R., Loeb, M., Rinaldo, C.R., Bramson, J., and Hildebrand, W.H. Epitope discovery in West Nile virus infection: Identification and immune recognition of viral epitopes. PNAS 105: Parsons, R., Lelic, A., Hayes, L., Carter, A., Marshall, L., Evelegh, C., McMurtrey, C., Hildebrand, W., Loeb, M. and Bramson, J.L. The Memory T cell Response to West Nile Virus in Symptomatic Humans Following Natural Infection is not Influenced by Age and is Dominated by a Restricted Set of CD8+ T Cell Epitopes. J. Immunol., in press. Lund, Bramson, Lelic, Parsons et al. Selection and validation of West Nile virus CD8+ T cell epitopes predicted by NetCTL. In preparation

7 Strategy for characterizing antibody responses in patients following West Nile virus infection 1. Identify epitope mimetics using phage display 2. Characterize breadth of antibody recognition by phage microarrays 3. Quantify antibody response to specific mimotopes by ELISA Are there quantitative or qualitative differences among the patient populations?

8 Estimated that 90% of antibody epitopes are conformational Defies the application of short linear peptides for epitope mapping Random phage display libraries presenting epitope mimetics (mimotopes) may offer a solution Recombinant phage fd Complexity different inserts Recombinant fd protein VIII, with 12-mer looped random peptide Can mimic unmodified and glycosylated peptides C C pviii

9 Plating onto selective medium Infection E.coli F+ Washing, Elution Incubation with phage library Incubation with corresponding serum Coating with anti mouse (or anti-human) IgG Fc specific Plastic plate

10 Remove anti-phage antibodies Remove phage with specificity to environmental antigen and vaccine antigens Pool serum from multiple patients to enrich for Abs with common specificities 1 pool for non-neuro patients 1 pool for neuro patients Pan for specific phage with each pool Verify that phage are not bound by Abs from non-wnv patients Model epitopes using computer algorithms New epitopes identified No new epitopes identified Prepare epitope microarrays Screen with individual patient sera to identify over- or under-represented epitopes Deplete serum of Abs with specificity to identified phage

11 Growing and purification of the phages Transfer to membrane Sequencing of inserts SS12.1 CQKAEERFHEVRAC SS12.2 CYWNIFQSLSSPGC SS12.3 CRRVAATAAREESC SS12.4 CETPEPLEEAASKC SS12.5 CADHVFKRPQPSNC SS12.6 CVETCVEKNEADQC SS12.7 WRRDGC SS12.8 CAYMDPHTQREALC S12.11 EWYTPQG S12.12 CSEPVRDNCAPSGC S12.13 CRVERDIATRPWPCC S12.14 CDERPEIEDVCQAC S12.15 CPQKTLNTSNANNC S12.19 CAWGHCSQGMIEYC S12.20 CEPETCRYGQRNVC Picking the plaques Analysis

12 Achievements: Project advancement has been limited by restricted funds due to change in USD exchange rate. Observed no relationship between disease status and magnitude or specificity of antibody response against WNV E protein (collaboration with M. Diamond, Washington U.) Developed novel algorithms for epitope prediction using monoclonal antibodies and polyserum.

13 Publications Oliphant, T., Nybakken, G.E., Austin, K., Xu, Q., Bramson, J., Loeb, M., Throsby, M., Fremont, D.H., Pierson, T.C., and Diamond, M.S. The Induction of Epitope-Specific Neutralizing Antibodies against West Nile virus. J. Virol., 81: , Denisova, G.F., Denisov, D.A., Yeung, J., Loeb, M., Diamond, M.S., and Bramson, J.L. A novel computer algorithm improves antibody epitope prediction using affinity-selected mimotopes: A case study using monoclonal antibodies against the West Nile virus E protein. Submitted to Molecular Immunology. Denisov, D.A., Lelic, A., Diamond, M., Denisova, G.F, and Bramson, J.L. Pattern recognition method for epitope analysis of polyclonal serum antibodies. In preparation for Bioinformatics.