1. ProteinStart position HLAEpitopePositive responses (n) Env209A*0101SFEPIPSHY1 Env310A*0101/Cw*0401GPGPGRAFY1 Gag406A*0302RAPRKKGC WK 1 Nef9A*0101/A*0302SVVGWPAVR1 Nef178B*1801KEVLVWKF1 Pol8MultipleFPQGKAREF1 Pol901B*2705KRKGGIGGY1 SCREENING FOR HIV RESPONSES USING OPTIMAL EPITOPES PREDICTED BY HLA-VIRAL SEQUENCE POLYMORPHISM ASSOCIATIONS. Roberts SG 1, Almeida CM 1, Bronke C 1, Ahmad I 1, Al Damuk A 1,Cooper D 1, Corkery M 1, Keane N 1, Heckerman D 3, Chopra A 1, Mallal S 1,2, John M 1,2 1 Centre for Clinical Immunology and Biomedical Statistics, Institute of Immunology and Infectious Diseases, Murdoch University, Perth, Western Australia, 2 Department of Clinical Immunology and Immunogenetics, Royal Perth Hospital, Perth, Western Australia, 3 Microsoft Research, Microsoft Inc, Redmond, USA Acknowledgements Study population was drawn from ACTG 5142/5128, Beckman Coulter, “Bill & Melinda Gates Foundation”, National Institutes of Health, National Health & Medical Research Council and CCIBS staff S.Roberts@iiid.com.au M.John@iiid.com.au http://www.ccibs.org Pol 901-909 HLA-B*2705 5’-----HNF KRKGGIGGY SAG-----3’ KRKGGIGEY 690 SFU/10 6 PBMCs 10 SFU/10 6 PBMCs Non- adapted epitope Adapted epitope Gag 20-29 HLA-B*1501 5’----- EKI RLRPGGKKKY KLK -----3’ RLRPGGRKKY 560 SFU/10 6 PBMCs 0 SFU/10 6 PBMCs Non-adapted epitope Adapted epitope 5’-----DPE KEVLVWKF DSR-----3’ KEVLMWKF Nef 178-185 HLA-B*1801 2800 SFU/10 6 PBMCs 1900 SFU/10 6 PBMCs Non- adapted epitope Adapte d epitope* 5’----- PKV SFEPIPSHY CAP -----3’ SFEPIPSIY Env 209-217 HLA-A*0101 740 SFU/10 6 PBMCs 20 SFU/10 6 PBMCs Non- adapted epitope Adapted epitope HIV escapes immune recognition by mutating critical amino acid residues in known HLA-restricted epitopes. Such changes may limit the ability of ex-vivo assays to detect and map the epitopes subject to such selection in-vivo. We used HLA- associated polymorphisms in HIV generated from an analysis of a combined cohort of 800 anti-retroviral naïve, HLA-diverse, predominantly subtype B-infected individuals from the US and Western Australia to predict and map HIV-specific T cell responses(see figure 1). A novel epitope prediction program ‘Epi-pred’ was used to predict optimal length epitopes around sites of HLA-allele specific polymorphism and these were tested in IFN-γ ELISpot assays, taking into account the autologous HLA genotype and the autologous viral sequence of 200 US cohort individuals. The ELISpot assay was optimised and automated for high-throughput testing of multiple HLA- customised plates 1 (see figure 2). The screening strategy was based on a genetics directed approach, in which specific sites and epitopes were tested based on their in-vivo polymorphism. The results have enabled us to identify possible novel epitopes that warrant further investigation using confirmatory assays. At a population level, such screening takes into account the most prevalent HLA genotypes and HLA-restricted responses in that population efficiently. These results provide further insights into CD8 + T- cell responses against HIV and have implications for HIV vaccine design. On average, 11 epitopes were tested for each patient and of these 18% elicited an IFN-γ response. In an analysis of the first 29 individuals tested in this system, seven putative novel epitopes were detected (see table 1). In many instances, the HLA-driven change led to loss of reactivity as predicted for classical CD8 + T-cell escape, however more complex patterns of reactivity were seen, particularly in Nef epitopes (see figure 3). Classical escape Non-classical escape Figure 3. Responses to adapted and non-adapted epitopes detected in ELISpot assays. (3A) Classical escape – the adapted epitope elicits a lower response compared to the non-adapted epitope. (3B) Non- classical escape – the adapted epitope elicits a higher response than the non-adapted epitope. (3C) No difference in responses seen. 5’-----YKGALDLSH FLKEKGGL EGL-----3’ FLKEEGGL 3540 SFU/10 6 PBMCs 900 SFU/10 6 PBMCs Non-adapted epitope Adapted epitope Nef 90-97 and 83-91 and HLA-B*0801 FLKEMGGL FLKENGGL FLKEQGGL 2500 SFU/10 6 PBMCs Adapted epitope 420 SFU/10 6 PBMCs Adapted epitope 1540 SFU/10 6 PBMCs Adapted epitope 720 SFU/10 6 PBMCsNeo-epitope 5’-----YK GALDLSHFL KE(E/M/N/Q)GGL EGL-----3’ “imputed” HIV mutates residues that affect HLA binding, TCR recognition or intracellular processing allowing the virus to escape detection by the host immune system (see figure 4 & 5). Figure 2. Overview of the ELISpot method. Each of the steps illustrated can be set up as a separate method using the Biomek FX. Coat with INF-  capture Ab Add peptide & cells Add biotinylated detection Ab Add streptavidin enzyme Add TMB Novel epitopes 3A3B 3C 5’-----ITK GLGISYGRK KRR-----3’ GLGISYGRR Tat 42-50 HLA-A*0301 900 SFU/10 6 PBMCs 840 SFU/10 6 PBMCs Non-adapted epitope Adapted epitope No difference in responses Figure 5. Mutations affecting TCR recognition and intracellular processing. Figure 4. Amino acid change in anchor position Table 1. Seven novel epitopes were seen in the 29 patients tested. Figure 1. Method used to select patients peptides to perform ELISpots on. All associations/epitopes classified as individual cellular “hypotheses” to test with PBMCs. CD8+ T-cell epitopes (8 to 11-mer) predicted by ‘Epi-pred’ Known epitopes from LANL ACTG (n=555) + WA cohort (n=245) Statistical analysis HIV sequences (n=800) HLA-alleles (n=800) Consensus sequence scanned with ‘Epi-pred’ with non-adapted and adapted amino acids substituted at sites of HLA association 874 HLA associations Pollyallelic peptide panel Known epitopes/novel escape variants: Medium priority in screening assays Test non-adapted and adapted epitopes. If escape is extra-epitopic, then test non-adapted Novel epitopes/escape variants: High priority in screening assays Known epitopes/escape variants: Low priority in screening assays ELISpot assay Accredited for compliance with ISO/IEC 17025 interpreted for research using CITAC Guide CG2, for HLA sequence based typing, viral sequencing and ELISpot analysis. Accreditation number 15785 RPQVPLRPMTF RPQVPLRPMTY Adapted epitope Non-adapted epitope 570 SFU/10 6 PBMCs 200 SFU/10 6 PBMCs Nef 71-81 and HLA-B*3501 References 1. Almeida et al. 2009. Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses. J Immunol Methods; 344:1-5