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SCREENING FOR HIV RESPONSES USING OPTIMAL EPITOPES PREDICTED BY HLA-VIRAL SEQUENCE POLYMORPHISM ASSOCIATIONS. Western Australian HIV Cohort Study Roberts SG1, Almeida CM1, Bronke C1, Ahmad I1, Al Damuk A1,Cooper D1, Corkery M1, Keane N1, 3 1 1,2 1,2 Heckerman D , Chopra A , Mallal S , John M 1Centre for Clinical Immunology and Biomedical Statistics, Institute of Immunology and Infectious Diseases, Murdoch University, Perth, Western Australia, 2Department of Clinical Immunology and Immunogenetics, Royal Perth Hospital, Perth, Western Australia, 3Microsoft Research, Microsoft Inc, Redmond, USA 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 HLAassociated 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 HLAcustomised plates1 (see figure 2). Figure 1. Method used to select patients peptides to perform ELISpots on. HIV sequences (n=800) Statistical analysis 3A HLA-B*2705 Nef Protein Start position HLA Epitope Env 209 A*0101 SFEPIPSHY Env 310 Positive responses (n) 1 Gag 406 Nef 9 Nef 178 B*1801 KEVLVWKF 1 Pol 8 Multiple FPQGKAREF 1 Pol 901 B*2705 KRKGGIGGY 1 A*0101/Cw*0401 GPGPGRAFY A*0302 1 RAPRKKGC WK 1 A*0101/A*0302 SVVGWPAVR 1 Table 1. Seven novel epitopes were seen in the 29 patients tested. Non690 SFU/106 adapted PBMCs epitope KRKGGIGGY SAG-----3’ 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 5’----- EKI RLRPGGKKKY KLK -----3’ Pollyallelic peptide panel Known epitopes/novel escape variants: Medium priority in screening assays Test non-adapted and adapted epitopes. 178-185 HLA-B*1801 5’-----DPE No difference in responses 10 Adapted SFU/106 epitope PBMCs 560 SFU/106 PBMCs 0 SFU/106 PBMCs 2800 SFU/106 PBMCs KEVLMWKF Env 209-217 Non-adapted epitope Adapted epitope HLA-A*0301 1900 NonSFU/106 adapted PBMCs epitope KEVLVWKF HLA-B*1501 RLRPGGRKKY Novel epitopes/escape variants: High priority in screening assays Non-classical escape DSR-----3’ KRKGGIGEY Gag 20-29 3C Tat 42-50 5’-----HNF Consensus sequence scanned with ‘Epi-pred’ with non-adapted and adapted amino acids substituted at sites of HLA association 3B Classical escape Pol 901-909 874 HLA associations Known epitopes/escape variants: Low priority in screening assays Novel epitopes 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) Nonclassical escape – the adapted epitope elicits a higher response than the non-adapted epitope. (3C) No difference in responses seen. ACTG (n=555) + WA cohort (n=245) HLA-alleles (n=800) 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). Adapte d epitope* 5’-----ITK GLGISYGRK KRR-----3’ 840 SFU/106 PBMCs Non-adapted epitope 900 SFU/106 PBMCs Adapted epitope HLA-A*0101 5’----- PKV SFEPIPSHY CAP -----3’ SFEPIPSIY 20 SFU/106 PBMCs Nonadapted epitope 740 SFU/106 PBMCs Adapted epitope GLGISYGRR 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). If escape is extra-epitopic, then test non-adapted Nef 71-81 and HLA-B*3501 ELISpot assay Nef 90-97 and 83-91 and HLA-B*0801 5’-----YKGALDLSHFLKEKGGLEGL-----3’ Coat with INF-g capture Ab Figure 2. Overview of the ELISpot method. Each of the steps illustrated can be set up as a separate method using the Biomek FX. RPQVPLRPMTF Add peptide & cells Add biotinylated detection Ab 570 PBMCs 200 SFU/106 PBMCs 900 SFU/106 PBMCs Adapted epitope “imputed” FLKEMGGL 2500 SFU/106 PBMCs Adapted epitope “imputed” FLKENGGL 420 SFU/106 PBMCs Adapted epitope “imputed” FLKEQGGL 1540 SFU/106 PBMCs Adapted epitope “imputed” Non-adapted epitope Adapted epitope Figure 4. Amino acid change in anchor position Add TMB 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 [email protected] [email protected] http://www.ccibs.org Non-adapted epitope FLKEEGGL 5’-----YKGALDLSHFLKE(E/M/N/Q)GGL EGL-----3’ Add streptavidin enzyme 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 RPQVPLRPMTY SFU/106 3540 SFU/106 PBMCs 720 SFU/106 PBMCs Neo-epitope Figure 5. Mutations affecting TCR recognition and intracellular processing. 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+ Tcell responses against HIV and have implications for HIV vaccine design. 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