ST111 Immune selection and vpu variability in a unique narrow source HIV-Positive village cohort

Abstract

Background: HLA-class I associated selection pressure on HIV-1 evolution has been described in several cohorts and can leave characteristic “footprints” on HIV-1 strains (Moore et al, Science 2002). We had the opportunity to study a narrow source clade B’ HIV-1 infected Former Plasma Donors (FPD) village Cohort. We have previously reported that after 10 years of infection, clear HLA class I associated foot-prints are seen in the gag, pol and nef genes of bulk pro-viral DNA populations of untreated patients (Dong et al, Blood 2011). Here we present further studies from this cohort with the aim of defining and examining the selective pressures exerted on the vpu gene (which has important immune evasion functions). Methods: Samples were studied from 95 FDP in the village cohort using convenience sampling, collected an estimated 13-15 years after infection. We examined the sequences of HIV-1 vpu from plasma RNA, and assembled a consensus sequence, for which overlapping peptides were synthesised. The HLA class I and viral sequence data were analysed to determine the extent of HLA class I associations with sequence variation along the vpu sequence. Sites of strong class I associations were tested as possible HLA class I – restricted T-cell epitopes using overlapping peptides representing the cohort consensus vpu sequence in ELISpot assays, with cells from cohort members with the relevant HLA class I type. As antibody-dependent cellular cytotoxicity (ADCC) (Stratov et al, J Virology 2008) has been reported to select vpu variants a flow-cytometric ICS-based- assay was optimised for ADCC responses directed towards vpu. Results: Phylogenetic Analysis revealed the vpu sequences clustered together to reference B’ clade Chinese sequences. HLA association studies identified twenty-three mutations associated with vpu – amongst them, Position 27 and Position 38 of the vpu translated sequence were associated with HLA-B*13. Preliminary studies have demonstrated T-cell recognition of peptides containing these mutations in donors with appropriate HLA. Further studies underway will determine the optimal epitopes recognised, confirm the HLA restriction and demonstrate that the HLA-associated mutations represent T-cell escape mutations. Position A14 has been shown to be critical to vpu tetherin antagonism (Vigan et al, J Virology 2010), and our initial analysis reveals that there are a substantial proportion of A to V mutations in this position position A18 which may impact on tetherin binding. Further data on vpu T-cell and ADCC responses will be presented.