CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

589 Enhanced Neutralization of HIV-1With Fusion Inhibitor Resistant Mutations Muntasir Alam 1 ;Takeo Kuwata 1 ; Kristel P. Ramirez 1 ;Yasuhiro Maruta 1 ; KazukiTanaka 1 ; Kazuya Shimura 2 ; Shinya Oishi 2 ; Nobutaka Fujii 2 ; Masao Matsuoka 2 ; Shuzo Matsushita 1 1 Kumamoto University, Kumamoto, Japan; 2 Kyoto University, Kyoto, Japan Background: Fusion inhibitors bind to gp41 heptad repeat 1 (HR1), inhibiting the formation of six-helix bundle structure and subsequently blocking the entry of HIV-1 inside host cell. Several fusion inhibitors with improved potency and stability, such as C34, SC34 and SC34EK, have been developed and shown higher genetic barrier for resistance induction than Enfuvirtide or T-20, the only FDA approved fusion inhibitor. Here we investigated the effect of mutations to escape from these novel fusion inhibitors on sensitivities against neutralizing antibodies. Methods: HIV-1 JR-FL Env glycoprotein gp160 bearing T-20 resistant mutants (V38A, Q40H and N43D) and C34, SC34, SC34EK resistant mutations (selected by in vitro) in gp41 were constructed. In gp41 mutations are distributed among several functional domains including heptad repeat 1 and 2, MPER, immunodominant region, transmembrane domain and cytoplasmic domain. Pseudoviruses expressing these Env were used in neutralization assay with monoclonal antibodies (MAbs) against CD4 binding site (b12, VRC01, 49G2, 82D5, 42F9), V3 loop (1C10, KD247, 16G6), CD4-induced site (916B2, 4E9C), gp41 membrane proximal external region (4E10, 2F5, 10E8) and gp41 (2E8S34). IC 50 value was calculated from neutralization curves and compared with that of wild type virus. Results: Pseudoviruses expressing resistant mutant envelopes showed reduced level of infectivity when compared to wild type. Our overall neutralization data indicated that fusion inhibitor resistant mutations in gp41 did not reduce neutralization potency of antibodies tested, rather increased the potency of some antibodies. T-20 resistant mutant N43D showed enhanced neutralization by 4E10 (3.5 × ), 10E8 (12 × ) and also by anti-V3 antibodies, 1C10 (3.1x) and KD247 (4.3x). SC34EK-resistant virus was significantly sensitive to anti-MPER antibodies (4E10; 22.8x, 2F5; 20.6x, 10E8; 12.4x). MAb 49G2 and 42F9, which fail to neutralize the wild type JR-FL pseudovirus, neutralized C34 and SC34 resistant mutants. Anti-V3 MAbs neutralized C34, SC34 and SC34EK resistant mutants with 3 to 10 fold lower IC 50 values than that of wild type. Conclusions: Our data indicate that, fusion inhibitor resistant mutations in gp41 had impact on major epitopes on gp120 and gp41, enhancing sensitivities to antibodies. Therefore, next generation fusion inhibitors and MAbs could be a potential combination for future regimen of combined antiretroviral therapy. 590 Mutations at the Bottom of the Phe43 Cavity Are Responsible for Cross-Resistance to NBD Analogues Background: CD4 mimic small compounds (CD4MCs), NBD-556 and its analogues, inhibit the gp120-CD4 interaction and can also induce conformational changes in the gp120 architecture thereby exposing masked epitopes of neutralizing antibodies on the Env protein. Recently, some groups have reported novel potent NBD analogues. We designed and synthesized five new NBD analogues (YIR compounds). In this study, we characterized CD4MC-resistant viruses induced by in vitro selection to these novel NBD analogues. Methods: Resistant variants were induced by five CD4MCs (NBD-556, YYA-021, HAR1-71, JRC-II-191 or HAR-431) using the primary KP-5P virus (subtype B, R5) in PM1 cells. We constructed infectious KP-5P clones with CD4MC-resistant mutation following in vitro selection. The susceptibility of the infectious clones to the novel CD4MCs (YIR-327, YIR-329, YIR-438, YIR-501, YIR-504, DMJ-I-228 and DMJ-II-121) and other entry inhibitors (Maraviroc, Cenicriviroc, RPR103611 and IC9564) was tested in a TZM-bl assay. Results: Resistance against five NBD analogues, NBD-556, YYA-021, HAR-171, JRC-II-191 and HAR-431, was associated with V255M, T375I, or M426I substitutions that line the Phe43 cavity of gp120. Two of three mutated residues, V255M and T375I, are located at the bottom of the Phe43 cavity, while M426I is at the edge of the cavity. Clones V255M or T375I were highly resistant against the five NBD analogues. These mutated clones were cross-resistant to all our novel CD4MCs, YIR-327, YIR-329, YIR-438, YIR-501 and YIR-504, and also to DMJ-I-228, DMJ-II-121. On the other hand, the clone with M426I was more resistant than those with V255M and T375I mutations to sCD4 but not as resistant to the CD4MCs tested, because the Phe43 residue of sCD4 is located at a shallow position in the cavity compared to the CD4MCs. However, the mutated clones retained wild type sensitivity to other entry inhibitors (Maraviroc, Cenicriviroc, RPR103611 and IC9564). These results suggest that two mutations at the bottom of Phe43 cavity are critically important not only for binding but also broad resistance to CD4MCs. Conclusions: The mutations V255M and T375I at the bottom of Phe43 cavity can induce broad and potent cross-resistance to NBD analogues. These data provide additional knowledge for synthesizing novel NBD analogues with a high genetic barrier. 591 SIV mac239 Integrase as a Model of HIV Drug Resistance Against Integrase Inhibitors s in the nanomolar range, and integrase (IN) mutant viruses displayed similar resistance profiles to HIV. A long-acting form of a new INSTI termed S/GSK-1265744, a DTG analogue, was shown to protect macaques against repeated vaginal and rectal exposures of SHIV. These studies show that nonhuman primates can be utilized to investigate the potential role of IN strand transfer inhibitors (INSTIs) in HIV therapy, pathogenesis and transmission. Our objectives were to observe whether HIV and SIV share similar resistance pathways under INSTI pressure in selections and cell-free assays and to test the effects of HIV-1 IN resistance mutations on SIV IN activity. Methods: Tissue culture selections were performed in rhesus macaques peripheral blood mononuclear cells (PBMCs) infected with SIV mac239 viruses in the presence of RAL, EVG and DTG. Viral RNA was extracted from cell culture fluids and sequenced for any changes in the integrase coding region. To elucidate the molecular mechanism of resistance, SIVmac239 IN protein was cloned into a bacterial expression vector, pET15b, and resistance mutations were introduced by site-directed mutagenesis. Purified recombinant SIVmac239 WT, G118R, Y143R, Q148R, N155H, or R263K IN enzymes were obtained and strand transfer activities assessed using cell-free based assays. Results: After 22 weeks of DTG pressure, an R263K mutation was noted in rhesus macaque PBMCs infected with SIV mac239 . Our analysis of IN activity showed that resistance mutations in SIV recapitulate the effects observed in HIV-1. In particular, G118R and G140S/Q148R substitutions decreased target DNA affinity (~5.5 and 2-fold) and enzyme viruses treated with DTG led to the emergence of R263K, which is similar to the unique pattern of DTG resistance in HIV in participants in the SAILING study. This study further confirms that the same mutations associated with drug resistance in HIV exhibit similar profiles in SIV. Shigeyoshi Harada 1 ;Yu Irahara 2 ; Samatchaya Boonchawalit 1 ; Mai Goryo 1 ; HirokazuTamamura 2 ;Tetsuro Matano 1 ; Shuzo Matsushita 3 ; KazuhisaYoshimura 1 1 National Institute of Infectious Diseases, Shinjuku, Japan; 2 Tokyo Medical and Dental University, Chiyoda, Japan; 3 Kumamoto University, Kumamoto, Japan Said Hassounah 1 ;Thibault Mesplede 1 ; Maureen Oliveira 1 ; Peter K. Quashie 1 ; Daniela Moisi 1 ; Paul A. Sandstrom 2 ; Mark A.Wainberg 1 ; Bluma Brenner 1 1 McGill University, Montréal, Canada; 2 National HIV and Retrovirology Laboratory, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada Background: We previously showed that SIV mac239 is susceptible to raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG) with IC 50 efficiency to 20% and 40% of WT levels, respectively. G140S/Q148R negatively impacted strand transfer activity (70% of WT levels). Conclusions: This study supports the use of nonhuman primate model to study HIV pathogenesis, therapy and transmission. SIV mac239

Poster Abstracts

376

CROI 2015