1. Effects of HIV-1 Immune Selection on Susceptability to Integrase Inhibitor Resistance Introduction Integrase inhibitors have emerged as an important new antiretroviral agent. The integrase inhibitors Raltegravir and Elvitegravir block strand transfer, preventing the integration of the viral pre-integration complex. Raltegravir (MK-0518) is currently mainly used against multi-drug resistant HIV-1 strains. We examined polymorphisms in integrase sequences from 342 antiretroviral-naïve individuals from the Western Australian and Swiss HIV Cohort Studies, to examine site-specific interactions between HIV-1 subtype, human leukocyte antigen (HLA)-associated immune selection and integrase inhibitor resistance. Study group and Methods Study populations were drawn from the Western Australian HIV Cohort Study (WAHCS), a state-wide observational cohort study established in 1983 and the Swiss HIV Cohort Study (SHCS), a multi-centre national research project initiated in 1988. Standard bulk sequencing and sequence based typing were used to generate integrase sequences and 4-digit HLA genotypes. Viral residues were examined with respect to published drug resistance mutations and a reference dataset of CD8 T-cell escape mutations. ELISpot assays were performed for functional analysis of predicted epitopes. Results Phylogenetic analysis was performed for all integrase sequences of the combined cohorts (coverage median 100%) and indicated the predominance of subtype B in both cohorts (82.8% in SHCS and 74.9% in WAHCS). The subtype distribution among non-subtype B sequences in the SHCS was 3.7% A, 0.6% C, 0.6% CRF01, 1.8% CRF02, 0.6% CRF06, 0.6% CRF14 and 9.2% inter-subtype recombinants and in the WAHCS 4.5% C, 3.9% CRF01, 0.6% CRF02, 0.6% CRF06, and 15.6% inter-subtype recombinants. Monika Tschochner 1, Ian James 1, Coral-Ann Almeida 1, Niamh M. Keane 1, Steven Roberts 1, Corine Bronke 1, Abha Chopra 1, Tanya M. Maiden 1, Imran F. Ahmad 1, Silvana Gaudieri 1,5, Hansjakob Furrer 2, Huldrych F. Günthard 3, Simon Mallal 1,4, Andri Rauch 1,2 and Mina John 1,4 The polymorphism rate in the reference dataset within and flanking the codons are shown in the presence (black bars) and absence (open bars) of the associated HLA allele (Fig.3). 1 Centre for Clinical Immunology and Biomedical Statistics, Institute for Immunology and Infectious Diseases, Royal Perth Hospital and Murdoch University, Perth, Australia 2 University Clinic of Infectious Diseases, University Hospital Bern and University of Bern, Switzerland 3 Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Switzerland 4 Department of Clinical Immunology and Immunogenetics, Royal Perth Hospital, Perth, Australia 5 School of Anatomy and Human Biology, University of Western Australia, Perth, Australia In both predominantly subtype-B cohorts, twelve of 38 sites that mediate integrase inhibitor resistance were absolutely conserved. The primary resistance mutations for Raltegravir and Elvitegravir T66I, E92Q, G140S, Y143C/H/R, Q148H/R/K and N155H were absent. There were 18 codons with non-primary drug resistance associated substitutions at rates up to 58.8% (Fig.1A) and 8 sites with alternative polymorphisms (Fig.1B). V72I and V201I, were the most common resistance mutations and isoleucine was associated for both with a significantly higher viral load than valine (p=0.025, mean delta log VL=0.21 at codon 72, p=0.00003, mean delta log VL=0.39 at codon 201). Five viral residues were potentially subject to dual drug and HLA associated immune selection in which both selective pressures either drove the same amino acid substitution (codons 72, 157, 163) or HLA alleles were associated with an alternative polymorphism that would alter the genetic barrier to resistance (125 and 193) (Tab.1) Contact e-mail; m.tschochner@iiid.com.au Fig. 3 Synergy : HLA-A*0206 - V72I, HLA-A*3303 - E157Q; HLA-A*3303 - G163A/E Antagonism: HLA-B*5701 - T125A, HLA-B*2705 - G93E B) alternative polymorphism Fig. 1 A) non-primary drug resistance associated substitutions Tab. 1 HLA allele-specific epitope variation at drug resistance sites within our cohorts. Only one sample was HLA-A*0206 positive (associated with codon 72) - not shown. aa=amino acid 5’-----HNFKRKGGIGGYSAG-----3’ KRKGGIGEY 690 SFU/10 6 PBMCs Non-adapted epitope 10 SFU/10 6 PBMCs Adapted epitope classical escape shown as an example for HLA-B*2705 integrase 193G/E. Fig. 2 The common polymorphism T125A increased the mutational barrier to the resistance mutation T125K and was both characteristic of carriage of HLA-B*57/*5801 and associated with non-subtype-B. Conclusion and future studies In an antiretroviral-naïve population-based cohort, primary integrase inhibitor resistance mutations were not detected in keeping with these being sites of significant functional, catalytic or structural importance. Viral polymorphisms due to immune selection and/or associated with non-subtype-B and particular HLA alleles may alter the genetic barrier to some non-primary resistance associated mutations. Acknowledgements We would like to thank the investigators of the WAHCS and the SHCS, all clinical and laboratory staff of CCIBS, DCII, RPH and all participants of the Cohort Studies. Financial support: Australian National Health & Medical Research Council program grant ID 384702. Swiss National Science Foundation (SNF grant #3345-062041) and SHCS research foundation. Research Travel Grant from Merck Sharp & Dohme. Predicted epitopes could be confirmed in ELISpots. 0% 10% 20% 30% 40% KRKGGIGGY negative: n=212 positive: n=8 (aa 186 – 194) 193E 0% 20% 40% 60% 80% STTVKAACWW negative: n=220 positive: n=23 125A, P,Q,V,S (aa 123 – 132) 0% 10% 20% 30% ELKKIIGQVR negative: n=223 positive: n=7 (aa 157 – 166) 157Q 163A,E B*57, B*5801 A*3303 B*2705 consensus proportion with non-consensus