CROI 2016 Abstract eBook

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Poster Abstracts

505 Molecular Dynamics of the CD4-Mimetic Resistant HIV-1 Gp120 by MD Simulation Shigeyoshi Harada 1 ; MasaruYokoyama 2 ; Shuzo Matsushita 3 ; Hironori Sato 2 ;Tetsuro Matano 1 ; KazuhisaYoshimura 1 1 Natl Inst of Infectious Diseases, Shinjuku, Japan; 2 Natl Inst of Infectious Diseases, Musashimurayama, Japan; 3 Kumamoto Univ, Kumamoto, Japan

Background: CD4 mimetic small compounds (CD4MCs), such as NBD-556, inhibit the gp120-CD4 interaction and can also induce conformational changes in gp120 by exposing masked epitopes of neutralizing antibodies on the Env protein. Recently, we have reported the resistance induction of the primary KP-5P virus (subtype B, R5) against the three CD4MCs (NBD-556, YYA-021 or JRC-II-191). Resistance against CD4MCs was associated with V255M, T375N/I or M426I substitutions. In this study, we investigated how the mutated positions affect CD4MCs recognition and sensitivity to other CD4MCs. Methods: Infectious KP-5P clones with CD4MC-resistant mutation were constructed. The susceptibility of the infectious clones to the CD4MCs, was determined using the TZM-bl assay. We also simulated the gp120 3D structures by MD simulation model. Results: 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 with V255M or T375I were highly resistant against the CD4MCs (NBD-556, YYA-021 or JRC-II-191), but the M426I single mutated clone had moderately resistance to those, except for YYA-021. On the other hand, the clone with M426I was more resistant to sCD4 than those with V255M and T375I mutations, because the Phe43 residue of sCD4 is located at a shallow position in the cavity compared to the CD4MCs. We also simulated the binding form between the KP-5P gp120 and NBD-556, YYA-021 or JRC-II-191 using an MD simulation method. The results showed that (i) V255Mmutation abolished the interaction of gp120 and CD4MCs except for JRC-II-191, and (ii) M426I mutation disconnected a hydrogen bond between Lys130 and Glu429, thus the NBD-556 or JRC-II-191 binding site shifted different from the usual, but they were still hold in the cavity, while YYA-021 binding was abolished by the M426I mutation. Conclusions: These data gave elucidation of the molecular details governing the interactions between gp120 and CD4MCs, and will assist in synthesizing novel CD4MCs to search for drugs with more potent power to change the tertiary structure of Env for opening the neutralizing epitopes. 506 Background: Doravirine (DOR), currently in Phase 3 clinical trials, is a novel and potent human immunodeficiency type 1 virus (HIV-1) non-nucleoside reverse transcriptase inhibitor (NNRTI). To assess potential clinical efficacy of DOR against prevalent NNRTI-resistant mutants including rilpivirine (RPV)-associated mutants, the inhibitory quotient (IQ) was calculated based on clinical trough concentration at a dose of 100 mg (selected for Phase 3 trial) and the IC 50 determined in the presence of 100% normal human serum (NHS). To evaluate the capability of DOR, efavirenz (EFV), and RPV in suppressing K103N, Y181C, and K103N/Y181C mutants, in vitro resistance selection was performed with the mutant viruses at clinically relevant concentrations of the NNRTIs. Methods: Antiviral assays were performed using laboratory HIV-1 isolates (WT and respective mutants) and MT4-GFP cells. Cell infection was carried out in RPMI 1640 medium in 10% fetal bovine serum and incubated for 20 hours. The infected cells were washed and resuspended in 100% NHS and then added to plates containing the compounds at various concentrations. The inhibitory potency was determined based on the ratio of green cells determined at 48h and 72h. In vitro resistance selection was performed in a 96-well format with prevalent NNRTI-associated mutants using MT4-GFP cells at clinically relevant concentrations of NNRTIs in the presence of 10% NHS. For every passage, viral breakthrough was monitored for GFP positive cells. Results: DOR displayed IQs of 39, 26, and 21 against the K103N, Y181C, and K103N/Y181C mutants, respectively. In contrast, RPV exhibited IQs of 4.6, 1.4, and 0.8, respectively against the same mutant viruses, while EFV showed IQs of 2.5, 60, and 1.9, respectively. In addition, IQs were also determined with RPV-associated mutants such as E138K/M184V, E138K/M184I, E138K and other K101E mutants. The IQs of DOR with the panel of mutants were significantly higher than those of RPV and comparable to that observed for EFV. Results from in vitro resistance selection conducted with K103N, Y181C, and K103N/Y181C mutants indicated that no viral breakthrough was observed in the selection with DOR, whereas viral breakthrough was detected in the selection with EFV against K103N and K103N/Y181C mutants and with RPV against Y181C and K103N/Y181C mutants. Conclusions: DOR may present a higher barrier for viruses to develop resistance compared to EFV and RPV, which should be a valuable addition to current available antivirals for HIV treatment. 507 The Effect of E157Q in HIV-1 Integrase on R263K-Mediated Dolutegravir Resistance Kaitlin Anstett 1 ;Vincent Cutillas 2 ; Robert Fusco 1 ;Thibault Mesplede 2 ; Bonnie A. Spira 2 ; Mark A.Wainberg 2 1 McGill Univ, Montreal, QC, Canada; 2 McGill Univ AIDS Cntr, Montreal, QC, Canada Background: The integrase strand transfer inhibitor (INSTI) dolutegravir (DTG) has a high genetic barrier to resistance, which has only been selected thus far in treatment- experienced patients and in tissue culture. The novel R263K substitution in integrase (IN) is the predominant mechanism of DTG resistance in INSTI-naïve patients. However in INSTI-experienced patients, resistance emerges through the accumulation of resistance substitutions for other drugs of this class. E157Q can be selected after treatment with raltegravir (RAL), and is a polymorphism present in the circulating virus as well. We have also previously identified E157 as an interactor with the IN DNA binding inhibitor FZ41. As it was recently reported that a patient failed RAL and subsequently DTG with the E157Q substitution, we investigated the effects of this substitution on the emergence of R263K, its effects on enzyme biochemical function, and viral infectivity and drug resistance. Methods: E157Q and R263K were introduced into the pET15b IN protein expression vector and pNL4.3 viral vector by site-directed mutagenesis. Strand transfer and DNA binding activities were measured with fluorescence-based biochemical assays using purified recombinant IN proteins. Viral infectivity and drug resistance were measured through the infection of TZM-bl cells and observation of luciferase production. Results: R263K decreased IN strand transfer, DNA binding activities, and NL4.3 infectivity by ~20%when compared to WT. Neither biochemical function nor infectivity showed a significant decrease fromWT when the E157Q mutant was evaluated, and the presence of this substitution in the R263K background partially restored the enzymatic and infectious defects conferred by the latter mutation. Despite this restoration, neither virus with E157Q displayed increased resistance to FZ41, although susceptibility to DTG was modestly decreased. Conclusions: DTG is arguably one of the best current therapies for HIV infection, displaying a high genetic barrier for resistance and very few treatment failures to date. However, we show that the E157Q substitution is able to restore the defects in enzyme function and viral infectivity that are conferred by the DTG resistance mutation R263K. As position 157 in IN is polymorphic, its presence at the initiation of DTG therapy is possible, which could lead to the selection of a replicatively competent, DTG-resistant virus. This could have important consequences for the clinical care of HIV-positive individuals. 508 Drug Resistance Mutations in HIV-2 Patients Failing Raltegravir and Dolutegravir Silvia Requena 1 ; Carmen de Mendoza 2 ;Teresa Cabezas 3 ; Rosa García 4 ; Maria Jose Amengual 5 ; Ana Belen Lozano 3 ; Juan Manuel Fernandez 3 ;Vicente Soriano 6 ; AnaTreviño 7 ; for the Spanish HIV-2 Study Group 1 Puerta de Hierro Rsr Inst, Majadahonda, Spain; 2 La Paz Univ Hosp, Madrid, Spain; 3 Hosp de Poniente, Almería, Spain; 4 Hosp Universitario Fundación Jiménez Díaz, Madrid, Spain; 5 Corporación Sanitaria Parc Taulí, Barcelona, Spain; 6 Hosp La Paz, Madrid, Spain; 7 Hosp Universitario Puerta de Hierro de Madrid, Madrid, Spain Background: A broader extent of replacements at the integrase of HIV-2 compared to HIV-1 might enable greater cross-resistance between raltegravir (RAL) and dolutegravir (DGV) in HIV-2 patients ( Smith et al. Retrovirology 2015;12:10 ). Studies assessing drug resistance mutations in HIV-2 patients that fail on RAL are scarce. No studies have tested in HIV-2 the virological response prior HIV-2 RAL failures. Suppress NNRTI-Resistant Mutants by Doravirine at Clinically Relevant Concentrations Ming-Tain Lai ; Nancy Sachs; MeiZhen Feng; Min Xu; Jay Grobler;Wade Blair; Mike Miller; Daria J. Hazuda Merck & Co, Inc, West Point, PA, USA

Poster Abstracts

196

CROI 2016