SCREENING FOR HIV RESPONSES USING OPTIMAL EPITOPES PREDICTED BY HLA-VIRAL SEQUENCE POLYMORPHISM ASSOCIATIONS.

1. Coat with INF-g capture Ab Add peptide & cells Add biotinylated detection Ab Add streptavidin enzyme Add TMB SCREENING FOR HIV RESPONSES USING OPTIMAL EPITOPES PREDICTED BY HLA-VIRAL SEQUENCE POLYMORPHISM ASSOCIATIONS. ACTG (n=555) + WA cohort (n=245) HLA-alleles (n=800) HIV sequences (n=800) Statistical analysis 874 HLA associations Consensus sequence scanned with ‘Epi-pred’ with non-adapted and adapted amino acids substituted at sites of HLA association Roberts SG1, Almeida CM1, Bronke C1, Ahmad I1, Al Damuk A1,Cooper D1, Corkery M1, Keane N1, Heckerman D3, Chopra A1, Mallal S1,2, John M1,2 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 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 plates1 (see figure 2). 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). Novel epitopes Known epitopes/escape variants: Low priority in screening assays Novel epitopes/escape variants: High priority in screening assays 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 Figure 2. Overview of the ELISpot method. Each of the steps illustrated can be set up as a separate method using the Biomek FX. ELISpot assay Table 1. Seven novel epitopes were seen in the 29 patients tested. 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. 3A 3B No difference in responses 3C Classicalescape Non-classical escape Pol 901-909 HLA-B*2705 Nef 178-185 HLA-B*1801 Tat 42-50 HLA-A*0301 5’-----HNF KRKGGIGGY SAG-----3’ Non-adapted epitope 690 SFU/106 PBMCs 5’-----DPE KEVLVWKF DSR-----3’ 1900 SFU/106 PBMCs Non-adapted epitope 10 SFU/106 PBMCs Adapted epitope 5’-----ITK GLGISYGRK KRR-----3’ 840 SFU/106 PBMCs Non-adapted epitope KRKGGIGEY Adapted epitope* 2800 SFU/106 PBMCs KEVLMWKF Env 209-217 HLA-A*0101 Figure 1. Method used to select patients peptides to perform ELISpots on. Gag 20-29 HLA-B*1501 20 SFU/106 PBMCs Non-adapted epitope 5’----- EKI RLRPGGKKKY KLK -----3’ 5’----- PKV SFEPIPSHY CAP -----3’ 560 SFU/106 PBMCs 900 SFU/106 PBMCs Adapted epitope Non-adapted epitope GLGISYGRR 0 SFU/106 PBMCs SFEPIPSIY 740 SFU/106 PBMCs Adapted epitope Adapted epitope RLRPGGRKKY 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). • 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 Nef 90-97 and 83-91 and HLA-B*0801 Nef 71-81 and HLA-B*3501 Pollyallelic peptide panel 5’-----YKGALDLSHFLKEKGGLEGL-----3’ 3540 SFU/106 PBMCs Non-adapted epitope FLKEEGGL 900 SFU/106 PBMCs Adapted epitope RPQVPLRPMTY 570 SFU/106 PBMCs Non-adapted epitope “imputed” FLKEMGGL 2500 SFU/106 PBMCs Adapted epitope “imputed” 200 SFU/106 PBMCs Adapted epitope RPQVPLRPMTF FLKENGGL 420 SFU/106 PBMCs Adapted epitope “imputed” FLKEQGGL 1540 SFU/106 PBMCs Adapted epitope “imputed” 720 SFU/106 PBMCs Neo-epitope 5’-----YKGALDLSHFLKE(E/M/N/Q)GGL EGL-----3’ Figure 5. Mutations affecting TCR recognition and intracellular processing. Figure 4. Amino acid change in anchor position 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. 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 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 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