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Genome – and HLA-wide scanning and validation for cytotoxic CD8 T cell responses against

Genome – and HLA-wide scanning and validation for cytotoxic CD8 T cell responses against Mycobacterium tuberculosis. EU6-FRAME PROJECT Sheila Tuyet Tang, Ph.D stud. Biologist. Outline. 2. Vaccine4TB I) peptide prediction

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Genome – and HLA-wide scanning and validation for cytotoxic CD8 T cell responses against

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  1. Genome – and HLA-wide scanning and validation for cytotoxic CD8 T cell responses against Mycobacterium tuberculosis EU6-FRAME PROJECT Sheila Tuyet Tang, Ph.D stud. Biologist

  2. Outline • 2. Vaccine4TB • I) peptide prediction • II) Human cytotoxic CD8 T cell responses against novel TB epitopes 1. Introduction to Tuberculosis (TB)

  3. Introduction

  4. Introduction - Pandemic l http://www.stoptb.org/countries/

  5. What is tuberculosis? • Tuberculosis disease (TB) is caused by a bacterium Mycobacterium tuberculosis (Mtb, also known as tubercle bacillus) • Eradication of tuberculosis is difficult since Mtb is able to remain in the host for a long time as latent or chronic state • Bacille Calmette Guérin (BCG), the current vaccine prevent the spread of TB but do not give full protection against pulmonary TB in adult( the initial infection) • BCG is given to infant in endemic areas • Thorough immunological knowledge of BCG and Mtb are lacking and necessary for the development of a more efficacious TB vaccine www.sudantribune.com

  6. TB TB TB Tuberculosis: Transmission Primary infection Death ~2 mill 10% Infection (2 bill, ~ 9 mill/yr) 30% 90% Latent TB Exposure/Infection Reactivation 5-15% First 2yrs highest chance of developing TB disease Treatment with several drugs for 6 months or more can cure more than 95% of patients If not treated 60 % dies Clearance 70%

  7. IFN-g CD8 T cells Tubercle bacilli TB peptide TCR Lysosome +TB ER MACROPHAGE Cellular immune response Tubercle bacilli enter aveoli Infect* macrophages Within few weeks Th1 immune response CD4+/CD8+ T cells Recruit to lung Cytokines: IL-2, TNFa and IFN-y

  8. Cellular immune response • Granulomas prevent spread of infection by confining bacteria within a compact collection of several types of immune cells and activated macrophages • Role of these cells: specific ways to isolate inhibit the replication of, and destroy the bacteria http://www.granuloma.homestead.com/tb_microscopic.html • Bacilli engulfed by macrophages • Replicate within the macrophages 2-3 weeks before spreading throughout the body • 95% contain the bacteria in macrophages • But due to Mtb. complex waxy cel wall the bacteria are protected inside the macrophages

  9. Outline • 2. Vaccine4TB • I) peptide prediction • II) Human cytotoxic CD8 T cell responses against novel TB epitopes 1. Introduction to Tuberculosis (TB)

  10. Vaccine4TB: Peptide prediction

  11. Bioinformatics strategy • Read a GenBank file. • Extract the proteins from the file • Predict binding to HLA supertypes • A2 (A0201), A3 (A0301), B7 (B0702) • (coverage approx. 80% of the worlds population) • 3. Predict proteasome cleavage and TAP binding • 4. Calculate combined score using the method of Larsen et al., 2005 - have been used to predict and select potential epitopes based on a number of different criteria

  12. Selection criteria for potential vaccine candidates….(1) Best predicted epitopes in the entire proteom(67) Proteins with CD8 epitopes(25) TBVAC epitopes(14) Proteins from vaccine trials (Michel Klein WP3) Selected in proteins -previously described by other groups to have CTL epitopes (Michel Klein WP3) Selected as the best predicted epitopes In the entire Mtb proteome 3 epitopes/protein used in vaccine trials (21) Additional epitopes from proteins with CD8 epitopes (43)

  13. Selection criteria for potential vaccine candidates….(2) Conserved epitopes (69) T cell epitopes in proteins with B cell epitopes (60) LAGS/Dos regulon sequences (71) Conservation defined from multiple alignment that have the 9mer epitope: Selected as best predicted epitopes in the LAGS/Dos regulon (Michel Klein Fatima Kazi - WP3) These proteins found by Ugur Sahims group WP4 Selected as the most conserved epitopes with ANN prediction >0.42(<500nM) binding affinity

  14. New set of peptides selected (after meeting in Leiden) Exp verified secreted epitopes (67) Predicted secreted epitopes (68) Selected as best predicted from TubercuList secreted epitopes (67) proteins containing a predicted signal peptide or predicted to be secreted by other pathways

  15. TBVAC epitopes (14)

  16. 3 epitopes /protein used in vaccine trials (21)

  17. LAGS/Dos regulon sequences (71) from 13 proteins Continue....

  18. Summary • 505 peptides of the 800 to be selected in this project have been chosen – binding affinity have been measured and received in Leiden • What next ? • new selection of the remaining 295 peptides have recently been made in Mainz • Diagnostic peptides • New antigens dicovered by Ganymed • More LAGS

  19. predictions Verification of prediction - Wet lab experiments

  20. Outline • 2. Vaccine4TB • I) peptide prediction • II) Human cytotoxic CD8 T cell responses against novel TB epitopes 1. Introduction to Tuberculosis (TB)

  21. Vaccines4TB Genome- and HLA-wide scanning and validation of cytotoxc CD8 T cell responses against Mycobacterium tuberculosis WP3 Human cytotoxic CD8 T cell responses against novel TB epitopes Fatima Kazi, Pascale van Weeren, Corine Prins, Tom Ottenhoff, Michèl Klein Department of Immunohematology and Blood Transfusion Leiden University Medical Center Leiden, The Netherlands

  22. Identification of novel CD8 T cell epitopes • Establishment of blood panel for screening peptides • Design of assay for testing immunogenicity of peptides • Analysis of CD8 response to peptides

  23. Blood panel • 41 Buffy coats – more coming up • Approx 50% PPD+ (purified protein derivative cell culture extract used as the classical Tubeculin/Mantoux test) • non-BCG vaccinated • A2+, A3+, B7+ (full HLA-typing details) • IFN-g ELISA testing for PPD response • 6 day assay • > 100 pg/ml = PPD+

  24. Assay for peptide screening 7 days FACs (cfse /CD56/CD3/CD8) PBMC (Peripheral blood mononuclear cells) CFSE labelled 1.5x105 cells/well + 10ug/ml peptide R1=gate on lymphocytes R2=gate on CD8 T cells Acquire 10,000 events in live CD8 gate + SSC SSC FSC CD8

  25. 1.33% 9.96% 74.7% 9.73% Analysing peptide screening R1=gate on lymphocytes (PBMCs) medium SSC PPD FSC PHA CD8 CD8 peptide CD3 CFSE

  26. Blood panel • 21 donors tested (cfse) • PPD+ = 9 donors (43%) • PPD- = 12 donors • 7/21 (33%) donors responded • to Mycobacteria specific Ags

  27. Epitope Prediction Proteins with CD8 epitopes(25) TBVAC epitopes(14) Proteins from vaccine trials (Michel Klein WP3) Selected in proteins -previously described by other groups to have CTL epitopes (Michel Klein WP3) 3 epitopes/protein used in vaccine trials (21) Additional epitopes from proteins with CD8 epitopes (43)

  28. Peptides TBVAC peptides: Ag85A/B, ESAT6, PPE, HBHA TB-CD8 peptides: Mycobacteria tuberculosis H37Rv strain

  29. CD8 T cell proliferation to A2 motif bearing peptides

  30. CD8 T cell proliferation to A3 motif bearing peptides

  31. A3-peptidesBinding versus peptide immunogenicity 3/5 donor recognition

  32. CD8 T cell proliferation to B7 motif bearing peptides

  33. B7-peptidesBinding versus peptide immunogenicity

  34. PPD-ve individuals do not respond to peptides A2 donors A3 donors B7 donors • PPD responses < 1% cfse+ve • No responses to peptides

  35. Peptides Recognised by CD8 T cells B7 peptides A3 peptides A2 peptides 4/11 (36%) 9/13 (70%) 6/14 (43%)

  36. SUMMARY • 19/38 predicted peptides induced a CD8 proliferative response • The frequency of proliferating CD8 T cell response to peptides varied between individuals • Heterogenous response to peptides • For A3-peptide responses, 3/5 donors recognised the same peptide: QINELHHSK (CD8-#108-76), suggesting it may be immunodominant peptide

  37. SUMMARY • 19/38 peptides induced a CD8 proliferative response • The frequency of proliferating CD8 T cell response to peptides varied between individuals • Heterogenous response to peptides • For A3-peptide responses, 3/5 donors recognised the same peptide QINELHHSK(CD8-#10876), suggesting it may be immunodominant peptide

  38. Screening of new set of peptides Best predicted epitopes in the entire proteom(67) Conserved epitopes (69) Visiting Ph.D student Conservation defined from multiple alignment that have the 9mer epitope: Selected as the best predicted epitopes In the entire Mtb proteome Selected as the most conserved epitopes with ANN prediction >0.42(<500nM) binding affinity

  39. 0.69% 77.21% 4.47% 0.61% ppd+ in ELISA 67.62% 1.74% 0.57% 3.37% Donor 46(B7) PPD+ CD8 CFSE

  40. o.19% 92.45% 1.01% 6.33% 85.23% 16.50% CD8 Donor 29(HLA-B7) PPD+ CFSE

  41. Preliminary results: 5/23 peptides B7 ~22% proliferative CD8+Tcell response Concerved hypothetical protein SubCell: Cytoplamic ProFun: Structural protein involved in energy metabolism

  42. Preliminary summery For Best predicted epitopes in the entire proteom(67) peptide set: • No proliferative CD8+ T cell response were observed for A3 peptides from the set of peptide from : Best predicted epitopes in the entire proteom(67) • For the A2 responses => un-going experiments • 22% of the B7 peptides were recognized in five of the donors • Two of the B7 peptides were recognized in >1 donors. RPAIVVPAF (#11672) in 3 and RPAPATGAL (#11673) in two of the donors

  43. Preliminary summery For conserved peptide set(69): • Just initiated the screening last week – finished proliferation assay for two donors:# 49 and 53 (HLA-A3/B7) • Feasible to finish before the end of my study-exchange at LUMC, Leiden, Netherlands

  44. FUTURE WORK and MILESTONES • Assays for immunological monitoring of cytotoxic CD8 T cells • (6 months) • CFSE assay • Detected CD8 T cell responses to predicted epitopes • Cytokine production • (on-going) • Increase to 10 donor per supertype group • CD8 T cell epitopes which can elicit HLA class-I restricted cytotoxic T cell responses with potential anti-microbial activity towards M. tuberculosis • (6-8 months) • CD8 T cell lines/clones • Chromium release assay • /blocking Abs • Recognise endogenous peptides • Granzyme, CD69/CD40L

  45. FUTURE WORK and MILESTONES • Understanding the repertoire of CD8 T cell responses induced during (i) natural infection with M. tuberculosis and (ii) BCG vaccination in a cross sectional and retrospective study. • HLA-tetramers - A2 • Frequency • Phenotype • Function • A2 Transgenic mouse vaccine • model • (2/3 months : begin mid 2006) • Test single peptides or multi- • peptides as vaccine • Challenge with BCG/Mtb • Examine efficacy • Tetramers • CD8 expansion • Bacterial burden

  46. Papers planned: • A2/A3/B7 screening peptide responses: • proliferation of CD8 T cells • cytokine • selected peptides-MHC restriction and functional studies • A2 peptide/Transgenic mice: • Tetramer studies (frequency, phenotype) • Vaccine efficacy • CD8 repertoire of BCG vaccinated individuals: • cross-sectional/retrospective study

  47. Acknowledgements Prof. Dr. Tom Ottenhoff Tuberculosis group Immunohematology and Blood Transfusion Leiden University Medical Center Leiden, Netherlands Proliferation assays, FACS analysis and IFN-g-ELISA Leucosep Isolation of PBMC Ole Lund, Ph.D.Associate Prof. Immunological bioinformatic group CBS-BioCentrum, DTU Techinical University of Denmark In silico peptide prediction, NetCTL Michel Klein Tom Søren Buus, MD, Ph.D Prof. IMMI, University of Copenhagen MHC binding Ugur Sahin Ganymed Genetic library Fatima Kazi Pascale van Weeren And the rest of the Ottenhoff’s group

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