1. Selection of T Cell Epitopes Using an Integrative Approach Mette Voldby Larsen cand. scient. in biology ph.d. student

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

6. Predicting proteasomalcleavage NetChop (Ke ş mir et al, 2002, Nielsen et al, 2005) Artificial Neural Networks (ANN) trained on different kinds of data. - NetChop 20S: Trained on in vitro data - NetChop C-term: Trained on 1110 MHC I ligands SLYNTVATL Output: All aa in a protein are assigned a value between 0 and 1. Low values correspond to low probability of cleavage, high values to high probability of cleavage.

7. N1N2N3C A1,560,250,1-0,55 C-0,050,010,020 D-1,37-1,42-1,83 E-1,65-0,02-1,51-1,58 F-1,030,451,052,52 G-0,28-1,14-1,70-1,41 H-0,21-0,330,23-0,55 I0,110,490,620,52 K1,030,41-0,090,45 L0,50-0,090,110,94 M0,380,460,580,29 N1,43-0,69-1,01-1,33 P-1,43-3,00-0,220,09 Q-0,470,97-0,39-0,12 R1,341,470,421,47 S0,560,34-0,11-2,26 T0,120,04-0,43-0,72 V0,490,500,710,30 W-0,540,641,650,87 Y-0,500,671,802,91 Predicting TAP transport efficiency...… Peters et al, 2003 SLYNTVATL RSLYNTVATL 0.56-0.09+1.80+0.94 = 3.21 2.73 SLYNTVATL 2.97 The score for a given peptide is an average over the 9mer, 10mer

8. HLA-AHLA-B A1B7 A2B8 A3B27 A24B39 A26B44 B58 B62 Predicting MHC class I binding Different ANN predict binding affinity to different MHC class I supertypes Output: Each peptide is assigned a value between 0 and 1. Low values correspond to low binding affinity, high values to high binding affinity.

9. In theory, integrating all three steps should lead to improved identification of peptides capable of eliciting CTL responses Integration? How should we do it?

10. Dataset –148 9meric epitopes collected from the SYFPEITHI Database –69 9meric epitopes collected from the Los Alamos HIV Database -The epitopes were grouped according to which MHC class I they bind - The complete aa sequence of each sourceprotein was found in Swiss-Prot - All other 9mers in the proteins were considered to be nonepitopes

11. Collecting and combining the parameters Hypothetical protein: MTSSAKRKMSPDNPDEGPSSKV INT ? ? ? ? ? ? ? ? ? ? ? ? ? ? Proteasomal cleavage Pos1Pos2Pos3Pos4Pos5Pos6Pos7Pos8Pos9TAPMHC-IEpi/nonepi MTSSAKRKM0,870,000,170,060,590,890,960,760,972,140,760 TSSAKRKMS0,000,170,060,590,890,960,760,970,021,010,320 SSAKRKMSP0,170,060,590,890,960,760,970,02 3,050,440 SAKRKMSPD0,060,590,890,960,760,970,02 -0,020,210 AKRKMSPDN0,590,890,960,760,970,02 0,002,220,540 KRKMSPDNP0,890,960,760,970,02 0,000,01-1,090,330 RKMSPDNPD0,960,760,970,02 0,000,010,561,040,050 KMSPDNPDE0,760,970,02 0,000,010,560,040,030,120 MSPDNPDEG0,970,02 0,000,010,560,040,250,720,430 SPDNPDEGP0,02 0,000,010,560,040,250,140,830,110 PDNPDEGPS0,02 0,000,010,560,040,250,140,082,010,110 DNPDEGPSS0,020,000,010,560,040,250,140,080,061,700,660 NPDEGPSSK0,000,010,560,040,990,140,080,060,980,710,431 PDEGPSSKV0,010,560,040,250,140,080,061,000,981,010,020

12. Best performing combination: 1*MHC-I + 0.05*TAP + 0.15*C-term cleavage

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

14. 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

15. Results

17. NetChop 20s NetChop C-term 2.0 TAPMHC-IIntegrated method 0.6430.7890.7860.9330.948 AUC-values

19. 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 Instituto Malbran, Argentina)