Hotspot Hunter: a computational system for large-scale screening and selection of candidate immunological hotspots in pathogen proteomes G.L. Zhang, A.M. Khan, K.N. Srinivasan, A.T. Heiny, K.X. Lee, C.K. Kwoh, J.T. August and V. Brusic

2 Outline Background & Motivations System – Hotspot Hunter Discussion

3 HLA Peptide TCR Identification of T-cell epitopes for the study of vaccines and immunotherapies

4 T-cell epitope clusters (hotspots) for the development of epitope-based vaccines Promiscuous T-cell epitopes relevant to large proportion of the human population Presence of clusters of promiscuous T-cell epitopes (hotspots) in antigens H1 H4H3H2 P1 P2 P3 P4 Promiscuous epitopes One supertype

5 Mapping hotspots experimentally is a challenging task Large size of pathogen proteomes (sequence length versus sequence number) Low natural prevalence of T-cell epitopes (~1-5%) for a given HLA molecule High cost of peptide synthesis Limited access to human PBMC Time-consuming experimental assays HLA Peptide TCR

6 Limitations of existing promiscuous epitope prediction systems Single protein sequence per submission Do not predict for hotspots Impractical for large-scale systematic study of hotspots in large proteomes Existing prediction systems are not suitable for large-scale study of hotspots in pathogen proteomes

7 Outline Background & Motivations System – Hotspot Hunter Discussion

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9 Hotspot Hunter Screen and select of hotspots specific to four common HLA supertypes HLA class I A2, A3, B7  cover ~ 88% of human population HLA class II DR  cover ~100% of human population

10 Hotspot Hunter Implementation Predictive Engines  ANN and SVM methods Predictions results integrated using soft computing principles 10-fold cross-validation results showed that the system is of high accuracy

11 FP FN Hotspot Hunter can reliably identify real hotspots Hotspot Hunter predictions Experimental verified HLA-DR supertype specific hotspots for HCV Core protein sequence

12 Hotspot Hunter Functions Single sequence query Multiple sequence query Target selection Selection of common hotspot across more than one HLA supertype

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15 Khan et al. (2006) BMC Bioinformatics

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17 Outline Background & Motivations System – Hotspot Hunter Discussion

18 Allows prediction of immunological hotspots Combines the strengths of the ANN and SVM  robust prediction performance Multiple sequence query  suitable for large-scale study Provides a utility for selecting candidate hotspots and experimental targets Hotspot Hunter is a new generation computational tool aiding in epitope- based vaccine design

19 Our system can be customized and integrated into specialized databases Tumor Antigen Database: star.edu.sg/Templar/DB/cancer_antigen/ CandiVF - Candida albicans Virulence Factor Database Tongchusak et al., (2005) Int J Pep Res Ther. Application of Hotspot Hunter

20 Funding Agency NIH, USA Acknowledgment

21 Human pappilomavirus type 16 proteins E6 (Kast et al., 1994) E6 hot-spot regions HLA-A2 E (7-15, 18-26, and 26-34) HLA-A2 E (single peptide) HLA-A3 E (33-41, 42-50, and 59-67) E (75-83, 89-97, and ) E ( and ) Validation using experimental binders E6 HLA-A2 HLA-A3

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