1. Selection of T Cell Epitopes Using an Integrative Approach Mette Voldby Larsen cand. scient. in Biology PhD in Immunological Bioinformatics

2. Outline -Summary of biological processes preceding a CTL response -Summary of the methods available for predicting the processes -Case study: -Obtaining data, generating method, evaluating the method (small exercise – how to make Roc curves) - What can you use the method for?

3. MHC-I molecules present peptides on the surface of most cells

4. CTL response Healthy cell Virus- infected cell MHC-I

5. >Haemagglutinin - Influenza A virus (A/chicken/Jilin/9/2004(H5N1)) MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDL DGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKHL LSRINHFEKIQIIPKSSWSNHEASSGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSY NNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSG RMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGA INSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGW QGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLE RRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELG NGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLNREEISGVKLESIGTYQILSIYSTVA SSLALAIMVAGLSLWMCSNGSLQCRICI Epitope-based vaccines: Only the peptides, which are presented by MHC class I molecules are used in the vaccine

6. >Haemagglutinin - Influenza A virus (A/chicken/Jilin/9/2004(H5N1)) MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDL DGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKHL LSRINHFEKIQIIPKSSWSNHEASSGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSY NNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSG RMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGA INSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGW QGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLE RRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELG NGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLNREEISGVKLESIGTYQILSIYSTVA SSLALAIMVAGLSLWMCSNGSLQCRICI Epitope-based vaccines: Only the peptides, which are presented by MHC class I molecules are used in the vaccine

7. Epitope-based vaccine SLYNTVATL VSRLWWERI

8. NetCTL Combining preexisting prediction methods Proteasomal cleavage: NetChop, Artificial Neural Network Kesmir et al (2002). Immunity. 23. 249-62 TAP transport efficiency: Consensus TAP matrix Peters et al (2003). J Immunol. 171. 1741-9 MHC class I affinity: NetMHC, Artificial Neural Network Buus et al (2003). Tissue Antigens. 62. 378-84 Nielsen et at (2003). Protein Sci. 12. 1007-17

9. Dataset 863 known epitopes from the SYFPEITHI database 216 known epitopes from the Los Alamos HIV database The source proteins are found in SwissProt and split into all possible 9mers. 9mers not known to be epitopes are considered non- epitopes

10. Hypothetical protein: MPADNSELVISISAL PeptideProteasomal CleavageTAP TransportMHC-I AffinityCombined NetCTL score MPADNSELV0.43-0.700.040.06 PADNSELVI0.220.930.540.48 ADNSELVIS0.76-0.850.330.32 DNSELVISI0.881.320.220.36 NSELVISIS0.992.910.500.67 SELVISISA0.01-0.630.220.14 ELVISISAL0.95-0.240.180.27 Calculation of the combined NetCTL score: 0.15 * Prot + 0.05 * TAP + 1* MHC-I

11. Performance measure – Roc curve True positive False positive False negative True negative

12. ThresholdTPFNTP/(TP+FN)FPTNFP/(FP+TN) >0.8 4 10 0.29 1 12 0.08 >0.68 6 0.57 3 10 0.23 >0.411 3 0.79 6 7 0.46 >0.2131 0.93 9 4 0.69 >014 0 1 13 0 1 AUC = 0.5 AUC = 1.0

13. Results

14. Results

15. NetChop 20s NetChop C-term 2.0 TAPMHC-IIntegrated method 0.6430.7890.7860.9330.948 AUC-values What does the numbers mean? For an experimentalist aiming at identifying new epitopes he/she has to test 30% fewer peptides to find the same amount of epitopes

17. Practical use of NetCTL -ongoing projects Prediction of epitopes in: HIV (collaboration with Karolinska Institute in Sweden) Influenza A (collaboration with Panum institute) Tuberculosis (collaboration with Leiden University in the Netherlands) West nile virus (collaboration with Panum institute) Yellow fever virus (collaboration with Panum institute) Rickettsia (collaboration with Argentina) Lassa/Junin virus (collaboration with Panum and Argentina)