CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

497 BICTEGRAVIR DISSOCIATION HALF-LIFE FROM HIV-1 G140S+Q148H INTEGRASE/DNA COMPLEXES Kirsten L. White , Anita Niedziela-Majka, Nikolai Novikov, Michael D. Miller, Haolun Jin, Scott E. Lazerwith, Manuel Tsiang Gilead Scis, Inc, Foster City, CA, USA

Background: Long dissociation half-life (t1/2) of HIV integrase strand transfer inhibitors (INSTI) correlates with high antiretroviral activity and barrier to resistance. The dissociation t1/2 of bictegravir (BIC) fromwild-type (WT) integrase (IN)/DNA complexes is longer than that of elvitegravir (EVG), raltegravir (RAL), and dolutegravir (DTG). BIC has improved activity against HIV isolates with INSTI resistance mutations, particularly the 140/148 combination. Here, we evaluate phenotypic resistance and dissociation kinetics using G140S+Q148H mutant IN. Methods: INSTIs were phenotyped with 17 G140S+Q148H clinically-derived isolates with or without other IN mutations. The apparent association and dissociation t1/2 of 3H-labelled RAL, EVG, DTG, and BIC were measured using WT and G140S/Q148H mutant HIV IN/DNA complexes with a scintillation proximity assay. The association and dissociation kinetics were analyzed using both a single exponential decay function and an equilibrium binding model that generates half-lives that may be more representative of the t1/2 in cells. Results: HIV isolates with G140S+Q148H in IN had mean phenotypic fold-change values of 3.6±1.9 for BIC, 8.1±4.7 for DTG (p<0.01 for DTG vs BIC), and >100 for RAL and EVG. The apparent association t1/2 of BIC and DTG to G140S+Q148H mutant HIV IN/DNA complexes were 37±4 min and 34±5 min, respectively, and similar to that for WT. For BIC and DTG, the dissociation t1/2 from G140S+Q148H IN/DNA complexes were both shorter compared to WT (p≤0.01). The BIC apparent t1/2 from the mutant IN/DNA complexes using single exponential fit was longer than DTG (5.5±0.1 h vs 2.0±0.01 h, respectively, p<0.01). Similarly, dissociation t1/2 determined from equilibrium binding models were longer for BIC than DTG (2.5±0.07 h vs 0.65±0.2 h, respectively, p<0.01). In contrast, EVG and RAL had no measurable association or dissociation with the mutant IN/DNA, consistent with high-level resistance. Conclusion: BIC has improved activity compared to DTG, EVG, and RAL against HIV isolates with the 140+148 INSTI resistance mutations. BIC dissociates more slowly than DTG from the G140S+Q148H mutant IN/DNA complex and may explain the improved in vitro activity of BIC compared to other INSTIs against this mutant. 498 PHENOTYPIC SUSCEPTIBILITY OF SIV AND HIV-1 VARIANTS TO BICTEGRAVIR, A NOVEL INSTI Said Hassounah , Thibault Mesplede, Bonnie Spira, Nathan Osman, Mark A. Wainberg McGill Univ, Montréal, Quebec, Canada Background: Animal models are essential to answer key questions pertaining to antiretroviral therapy that include studies on novel drugs, drug resistance-associated mutations (RAMs), and treatment interruption. Given the high cost of animal-based research for the study of drug resistance, there is a need to validate and extend findings in tissue culture that were first obtained with HIV. A novel next generation integrase strand transfer inhibitor (INSTI), Bictegravir (BIC), has shown promising results against HIV-1 infection in vitro and in vivo, and against clinical isolates with INSTI resistance. BIC possesses a higher barrier to resistance than raltegravir (RAL) and elvitegravir (EVG). Our objective was to compare the susceptibilities of SIV and HIV-1 to BIC and to characterize the susceptibility of mutational patterns in HIV-1 IN against BIC. Methods: We constructed 7 SIV IN mutants (E92Q, T97A, G118R, Y143R, N155H, R263K, and G140S/Q148H) by site-directed mutagenesis (SDM). Mutations conferring resistance to INSTIs, some of which had not been investigated until now, were introduced into wild-type (WT) HIV-1 pNL4.3 by SDM (Y143R, N155H, R263K, R263K/M50I, R263K/H51Y, R263K/ E138K, and G140S/Q148H). Single-cycle infection assays (in TZM-bl reporter cells) were used to quantify the sensitivities of WT and IN variants of SIV and HIV-1 against BIC. Both cabotegravir (CTG) and dolutegravir (DTG) were included as controls. Results: BIC showed comparable activity against SIV and HIV-1 in a single cycle infection with EC50s in the low nanomolar range (~2-3 nM). Amino acid changes E92Q, T97A, Y143R, N155H in SIV did not have major impact on BIC susceptibility (≤3-fold increase in EC50) whereas G118R and R263K in SIV conferred ~13-fold and ~ 6-fold increases in EC50, respectively. HIV-1 IN mutants Y143R, N155H, R263K, R263K/M50I, R263K/E138K, and G140S/Q148H remained susceptible to BIC (≤3-fold increase in EC50). However, R263K/H51Y conferred low resistance to BIC (~8-fold). BIC exhibited a comparable resistance profile to CTG and DTG against SIV and HIV-1 IN mutants and displayed an improved resistance profile compared to RAL and EVG. Conclusion: Altogether, our results show that the same mutations that are associated with drug resistance in HIV exhibit similar profiles in SIV. The resistance profiles of BIC and DTG in SIV are also similar. Our data provide an important reference point for interpretation of additional mutational patterns that may emerge in HIV-1. 499 A HIGH-LEVEL RESISTANCE TO INTEGRASE INHIBITORS SELECTED OUTSIDE INTEGRASE HIV-1 GENE Background: Dolutegravir (DTG), a strand-transfer integrase inhibitor, seems to have, as in vitro and in vivo, a higher genetic barrier to resistance than raltegravir (RAL) and elvitegravir (EVG). Most in vitro experiments for selecting resistance to DTG were conducted increasing progressively DTG concentrations and showed difficulties for the virus to select mutations in the integrase gene under DTG pressure. In this study, we tried to select resistance to DTG using, from the beginning, high-level DTG concentrations. Methods: In vitro resistance selection was performed by infecting 2.106 MT4 cells with the HIV-1 Laï virus, using an equivalent of 1200 ng of p24 antigen. Twenty-four hours post-infection, cells were washed and DTG was added (500 nM), this concentration maintained all along the in vitro selection during three months. Phenotypic susceptibility assays were performed in HeLa-P4 cells in the presence of increasing concentrations of DTG, up to 500nM at 48 h post-infection. RNA virus extracts from supernatants, collected during selection, were used for genotypic analysis. Site-directed mutagenesis experiments were performed using pNL43 plasmid. Results: After three months of culture, we detected highly resistant viruses to DTG. Phenotypic analyses showed that viruses had a constant viral replication level in the presence of DTG, up to 500 nM (IC95 > 500nM). These results were confirmed using phenotypic PBMC assays. Analyses of different samples throughout the whole selection steps during three months did not show any mutation selection in the integrase gene compared to the baseline virus. The whole HIV-1 genome sequencing found the emergence of five mutations, all located in the nef region. The A9053C mutation situated six nucleotides upstream the 3’PPT motif (located between nucleotides 9069 to 9083, numbered relative to HxB2 sequence) and the four other changes clustered in the 3’ end of the 3’PPT motif, inside the G-tract. The motif is represented below, including the mutations in brackets 5’AAAAGAAAAG(G9079C)(G9080A)G(G9082T)(G9083deleted)3’. Site directed mutagenesis experiments introducing all mutations/deletion described above in NL43 HIV-1 showed a high-level of resistance to DTG but also to RAL and EVG, comparing to the wildtype. Conclusion: Mutations selected in vitro by DTG and located outside the integrase gene can confer themselves very high-level of resistance to all strand-transfer integrase inhibitors. 500LB EMERGENCE OF INTEGRASE RESISTANCE MUTATIONS DURING INITIAL THERAPY WITH TDF/FTC/DTG Jennifer A. Fulcher 1 , Yushen Du 1 , Ren Sun 1 , Raphael J. Landovitz 2 1 Univ of California Los Angeles, Los Angeles, CA, USA, 2 UCLA Cntr for Clinical AIDS Rsr & Educ, Los Angeles, CA, USA Background: Dolutegravir (DTG) with two NRTIs has emerged as a preferred regimen for initial treatment of HIV-infected individuals due to high efficacy, tolerability, and previously unreported INI drug resistance when used as initial therapy. We present genotypic information during a period of virologic failure in a 46 year old treatment naive man who initiated tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) plus DTG with a high viral load. Methods: Longitudinal patient samples (3 time points) covering an eight day period of increasing viral load were used for deep sequencing analysis of integrase (IN) genotypes. Plasma RNA was isolated and reverse transcribed, then target regions of IN pre-amplified using nested PCR. Paired end sequencing was performed using Illumina HiSeq 2000. Population sequencing was performed by standard clinical genotype assay (Quest Diagnostics) at the start of antiretroviral therapy and time of virologic inflection. Isabelle Malet 1 , Frédéric Subra 2 , Charlotte Charpentier 3 , Gilles Collin 4 , Diane Descamps 4 , Vincent Calvez 1 , Anne-Geneviève Marcelin 1 , Olivier Delelis 2 1 Pitie-Salpetriere Hosp, Paris, France, 2 Ecole Normale Supérieure de Cachan, Cachan, France, 3 INSERM, Paris, France, 4 Bichat Hosp, Paris, France

Poster and Themed Discussion Abstracts

CROI 2017 208