Nicolas A. Margot

Molecular Mechanisms of Resistance to Human Immunodeficiency Virus Type 1 with Reverse Transcriptase Mutations K65R and K65R+M184V and Their Effects on Enzyme Function and Viral Replication Capacity

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resistance mutations K65R and M184V result in changes in susceptibility to several nucleoside and nucleotide RT inhibitors. K65R-containing viruses showed decreases in susceptibility to tenofovir, didanosine (ddI), abacavir, and (−)-β-d-dioxolane guanosine (DXG; the active metabolite of amdoxovir) but appeared to be fully susceptible to zidovudine and stavudine in vitro. Viruses containing the K65R and M184V mutations showed further decreases in susceptibility to ddI and abacavir but increased susceptibility to tenofovir compared to the susceptibilities of viruses with the K65R mutation. Enzymatic and viral replication analyses were undertaken to elucidate the mechanisms of altered drug susceptibilities and potential fitness defects for the K65R and K65R+M184V mutants. The relative inhibitory capacities (Ki/Km) of the active metabolites of tenofovir, ddI, and DXG were increased for the RT containing the K65R mutation compared to that for the wild-type RT, but the relative inhibitory capacity of abacavir was only minimally increased. For the mutant viruses with the K65R and M184V mutations, the increase in tenofovir susceptibility compared to that of the mutants with K65R correlated with a decrease in the tenofovir inhibitory capacity that was mediated primarily by an increased Km of dATP. The decrease in susceptibility to ddI by mutants with the K65R and M184V mutations correlated with an increase in the inhibitory capacity mediated by an increased Ki. ATP-mediated removal of carbovir as well as small increases in the inhibitory capacity of carbovir appear to contribute to the resistance of mutants with the K65R mutation and the mutants with the K65R and M184V mutations to abacavir. Finally, both the HIV-1 K65R mutant and, more notably, the HIV-1 K65R+M184V double mutant showed reduced replication capacities and reduced RT processivities in vitro, consistent with a potential fitness defect in vivo and the low prevalence of the K65R mutation among isolates from antiretroviral agent-experienced patients.

Genotypic and Phenotypic Predictors of the Magnitude of Response to Tenofovir Disoproxil Fumarate Treatment in Antiretroviral-Experienced Patients

Results from 2 placebo-controlled intensification trials of tenofovir disoproxil fumarate (DF) in treatment-experienced human immunodeficiency type 1 (HIV-1)-infected patients (n=332) were integrated to determine the effects of resistance at baseline on HIV-1 RNA response. In these trials, there was a high prevalence of HIV-1 resistance mutations, with 94% of patients having nucleoside-associated mutations and 71% having thymidine analogue-associated mutations (TAMs). Statistically significant HIV-1 RNA reductions associated with tenofovir DF treatment, relative to placebo (P<.001), were observed for patients without TAMs (n=97) or for patients with 1-2 (n=88) or >or=3 TAMs (n=147). Response to tenofovir DF was reduced among patients with HIV-1 with >or=3 TAMs inclusive of either the M41L or L210W mutation (n=86) or patients who had a preexisting K65R mutation (n=6). Slightly increased treatment responses were observed when the M184V mutation was present. Phenotypic cutoffs were established at 1.4-fold and 4-fold, respectively, for the beginning of reduced response to tenofovir DF and for a strongly reduced response. The results from these controlled clinical trials provide guidance for the use of tenofovir DF for treatment-experienced patients.

Genotypic and phenotypic analyses of HIV-1 in antiretroviral-experienced patients treated with tenofovir DF

Objective To evaluate the virologic responses and mutational profiles in antiretroviral-experienced patients adding tenofovir DF once-daily to their existing regimens. Design Resistance analyses were performed for patients in a phase II placebo-controlled clinical trial of tenofovir DF. Methods HIV-1 reverse transcriptase and protease genes from plasma samples were analyzed genotypically and phenotypically at baseline, week 24, and week 48. Results Of 184 patients, 173 (94%) had baseline HIV-1 expressing one or more nucleoside reverse transcriptase inhibitor-associated resistance mutation. Protease inhibitor and non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations were observed in 57% and 32% of patients, respectively. Compared to placebo, significant reductions in HIV-1 RNA were observed for tenofovir DF-treated patients who had thymidine analog- (TAM), lamivudine- (M184V), NNRTI- or protease inhibitor-associated mutations. Patients with phenotypic susceptibility to tenofovir within 4-fold of wild-type responded durably to tenofovir DF 300 mg therapy with a decline in plasma HIV-1 RNA of ⩾ 0.5 log10 copies/ml; few patients had a more than 4-fold reduced susceptibility to tenofovir at baseline. Four patients (2%) developed the K65R mutation (selected by tenofovir in vitro) and showed 3- to 4-fold reductions in tenofovir susceptibility but no evidence of rebound viremia. Thirty-four percent of patients developed additional TAMs, coincident with concurrent zidovudine or stavudine therapy, but also showed durable HIV-1 reductions. There was no evidence of novel resistance to tenofovir. Conclusions Adding tenofovir DF 300 mg to an existing regimen in patients with ongoing viral replication and a wide range of genotypic resistance patterns resulted in significant and durable HIV-1 RNA reductions. In addition, there was a low incidence of genotypic or phenotypic resistance to tenofovir DF arising during 48 weeks of therapy.

ATP-Dependent Removal of Nucleoside Reverse Transcriptase Inhibitors by Human Immunodeficiency Virus Type 1 Reverse Transcriptase

ABSTRACT Removal of nucleoside chain terminator inhibitors mediated by human immunodeficiency virus (HIV) reverse transcriptase (RT) using ATP as an acceptor molecule has been proposed as a novel mechanism of HIV resistance. Recombinant wild-type and mutant HIV type 1 (HIV-1) RT enzymes with thymidine analog resistance mutations D67N, K70R, and T215Y were analyzed for their ability to remove eight nucleoside reverse transcriptase inhibitors in the presence of physiological concentrations of ATP. The order for the rate of removal of the eight inhibitors by the mutant RT enzyme was zidovudine (AZT) > stavudine (d4T) ≫ zalcitabine (ddC) > abacavir > amdoxovir (DAPD) > lamivudine (3TC) > didanosine (ddI) > tenofovir. Thymidine analogs AZT and d4T were the most significantly removed by the mutant enzyme, suggesting that removal of these inhibitors by the ATP-dependent removal mechanism contributes to the AZT and d4T resistance observed in patients with HIV expressing thymidine analog resistance mutations. ATP-dependent removal of tenofovir was 22- to 35-fold less efficient than removal of d4T and AZT, respectively. The addition of ATP and the next complementary deoxynucleoside triphosphate caused a reduction of ATP-mediated removal of d4T, ddC, and DAPD, while AZT and abacavir removal was unaffected. The reduction of d4T, ddC, and DAPD removal in the presence of the deoxynucleoside triphosphate could explain the minor changes in susceptibility to these drugs observed in conventional in vitro phenotypic assays using cells that have higher deoxynucleoside triphosphate pools. The minimal removal of abacavir, ddC, DAPD, 3TC, ddI, and tenofovir is consistent with the minor changes in susceptibility to these drugs observed for HIV mutants with thymidine analog resistance mutations.

ANTIVIRAL ACTIVITY OF TENOFOVIR (PMPA) AGAINST NUCLEOSIDE-RESISTANT CLINICAL HIV SAMPLES

The presence of the lamivudine-associated M184V RT mutation increases tenofovir susceptibility in multiple HIV genotypes. Tenofovir is uniquely active against multinucleoside-resistant HIV expressing the Q151M mutation, but shows reduced susceptibility to the T69S insertion mutations. HIV with common forms of zidovudine and lamivudine resistance are susceptible to tenofovir, corroborating phase II clinical results demonstrating the activity of tenofovir DF in treatment-experienced patients.

Impact of Primary Elvitegravir Resistance-Associated Mutations in HIV-1 Integrase on Drug Susceptibility and Viral Replication Fitness

ABSTRACT Elvitegravir (EVG) is an effective HIV-1 integrase (IN) strand transfer inhibitor (INSTI) in advanced clinical development. Primary INSTI resistance-associated mutations (RAMs) at six IN positions have been identified in HIV-1-infected patients failing EVG-containing regimens in clinical studies: T66I/A/K, E92Q/G, T97A, S147G, Q148R/H/K, and N155H. In this study, the effect of these primary IN mutations, alone and in combination, on susceptibility to the INSTIs EVG, raltegravir (RAL), and dolutegravir (DTG); IN enzyme activities; and viral replication fitness was characterized. Recombinant viruses containing the six most common mutations exhibited a range of reduced EVG susceptibility: 92-fold for Q148R, 30-fold for N155H, 26-fold for E92Q, 10-fold for T66I, 4-fold for S147G, and 2-fold for T97A. Less commonly observed primary IN mutations also showed a range of reduced EVG susceptibilities: 40- to 94-fold for T66K and Q148K and 5- to 10-fold for T66A, E92G, and Q148H. Some primary IN mutations exhibited broad cross-resistance between EVG and RAL (T66K, E92Q, Q148R/H/K, and N155H), while others retained susceptibility to RAL (T66I/A, E92G, T97A, and S147G). Dual combinations of primary IN mutations further reduced INSTI susceptibility, replication capacity, and viral fitness relative to either mutation alone. Susceptibility to DTG was retained by single primary IN mutations but reduced by dual mutation combinations with Q148R. Primary EVG RAMs also diminished IN enzymatic activities, concordant with their structural proximity to the active site. Greater reductions in viral fitness of dual mutation combinations may explain why some primary INSTI RAMs do not readily coexist on the same HIV-1 genome but rather establish independent pathways of resistance to EVG.

A combination of decreased NRTI incorporation and decreased excision determines the resistance profile of HIV-1 K65R RT.

Objective:To determine the mechanisms of resistance of K65R mutant reverse transcriptase (RT) to the currently approved nucleoside and nucleotide RT inhibitors (NRTI). Methods:Susceptibilities of K65R mutant HIV-1 to NRTI were determined in cell culture. The Ki/Km values were measured to determine the relative binding or incorporation of the NRTI, and ATP-mediated excision of incorporated NRTI was measured to determine NRTI stability as chain terminators. Results:K65R HIV-1 had decreased susceptibility to most NRTI, but increased susceptibility to zidovudine (ZDV). Ki/Km values were increased 2- to 13-fold for K65R compared to wild-type RT for all NRTI, indicating decreased binding or incorporation. However, K65R also showed decreased excision of all NRTI compared to wild-type, indicating greater stability once incorporated. At physiological nucleotide concentrations, excision of ZDV, carbovir (the active metabolite of abacavir; ABC), stavudine (d4T), and tenofovir was further decreased, while excision of didanosine (ddI), zalcitabine (ddC), lamivudine (3TC), and emtricitabine (FTC) was unchanged. The decreased binding or incorporation of ZDV by K65R appeared counteracted by decreased excision resulting in overall increased susceptibility to ZDV in cell culture. For ABC, tenofovir, and d4T, despite having decreased excision, decreased binding or incorporation resulted in reduced susceptibilities to K65R. For ddI, ddC, 3TC, and FTC, decreased binding or incorporation by K65R appeared responsible for the decreased susceptibilities in cell culture. Conclusions:NRTI resistance in cells can consist of both altered binding or incorporation and altered excision of the NRTI. For K65R, the combination of these opposing mechanisms results in decreased susceptibility to most NRTI but increased susceptibility to ZDV.

Low level of the K103N HIV-1 above a threshold is associated with virological failure in treatment-naive individuals undergoing efavirenz-containing therapy.

Background:Study GS-01-934 was a randomized open-label phase III study comparing efavirenz and tenofovir/emtricitabine to efavirenz and zidovudine/lamivudine in treatment-naive HIV-1-infected individuals. Through 144 weeks, 50 of 487 participants without baseline nonnucleoside reverse transcriptase inhibitor resistance by population sequencing (efavirenz/tenofovir/emtricitabine, n = 19; efavirenz/zidovudine/lamivudine, n = 31) experienced virologic failure (>400 copies/ml). Here, we analyzed whether the presence of low levels of K103N at baseline correlated with virologic failure. Methods:Available baseline plasma samples (n = 485) were amplified and tested for K103N using an allele-specific PCR (AS-PCR) assay with a lower detection cut-off of 0.5%. Results:Sixteen of 476 (3.4%) evaluable participants had low-level K103N at baseline by AS-PCR (0.8–15%). The abundance of the K103N subpopulation at baseline distinguished individuals with virologic failure from those who responded durably to efavirenz-containing therapy. Among six participants with at least 2000 K103N copies/ml before treatment, five experienced virologic failure, compared with only one virologic failure among 10 who had less than 2000 K103N copies/ml (P = 0.008). Multivariate logistic regression analysis showed that K103N viral load at least 2000 copies/ml increased the risk of virologic failure with an odds ratio of 47.4 (95% confidence interval 5.2–429.2, P = 0.0006). Conclusion:The presence of K103N mutant virus in plasma above 2000 copies/ml prior to therapy in treatment-naive individuals correlated with increased risk of virologic failure of these efavirenz-containing triple-drug regimens.

Molecular Mechanisms of Tenofovir Resistance Conferred by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Containing a Diserine Insertion after Residue 69 and Multiple Thymidine Analog-Associated Mutations

ABSTRACT Two amino acids inserted between residues 69 and 70 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are rare mutations that may develop in viruses containing multiple thymidine analog (zidovudine [AZT], stavudine)-associated mutations and that confer high-level resistance to all currently approved chain-terminating nucleoside and nucleotide RT inhibitors (NRTIs). The two known mechanisms of resistance to NRTIs are decreased incorporation and increased excision. The mechanism used by RT insertion mutants has not been described for tenofovir (TFV), a recently approved agent in this class. A patient-derived HIV-1 strain (strain FS-SSS) that contained an insertion mutation in a background of additional resistance mutations M41L, L74V, L210W, and T215Y was obtained. A second virus (strain FS) was derived from FS-SSS. In strain FS the insertion and T69S were reverted but the other resistance mutations were retained. The FS virus showed strong resistance to AZT but low-level changes in susceptibilities to other NRTIs and TFV. The FS-SSS virus showed reduced susceptibilities to all NRTIs including TFV. Steady-state kinetics demonstrated that the relative binding or incorporation of TFV was slightly decreased for FS-SSS RT compared to those for wild-type RT. However, significant ATP-mediated excision of TFV was detected for both mutant RT enzymes and followed the order FS-SSS RT > FS RT > wild-type RT. The presence of physiological concentrations of the +1 nucleotide inhibited TFV excision by the wild-type RT and slightly inhibited excision by the FS RT, whereas the level of excision by the FS-SSS RT remained high. Computer modeling suggests that the increased mobility of the β3-β4 loop may contribute to the high-level and broad NRTI resistance caused by the T69 insertion mutation.

Extended treatment with tenofovir disoproxil fumarate in treatment-experienced HIV-1-infected patients: genotypic, phenotypic, and rebound analyses.

Objective: To study the potential development of genotypic and phenotypic resistance to tenofovir disoproxil fumarate (tenofovir DF) when used as a part of a 96‐week HIV‐1 treatment regimen for antiretroviral treatment‐experienced HIV‐infected patients. Design and Methods: Clinical trial GS‐98‐902 was a placebo‐controlled, 48‐week phase 2 study of three doses of tenofovir DF when added to stable antiretroviral therapy for 189 treatment‐experienced HIV‐infected patients (mean of 4.6 years of prior antiretroviral treatment; 94% had nucleoside reverse transcriptase [RT] inhibitor [NRTI]‐associated mutations). There was a statistically significant reduction in the mean HIV‐1 RNA level at week 24 (average change in HIV‐1 RNA level of ‐0.58 log10 through week 24) with 300 mg of tenofovir DF once daily that was durable through week 48 (average change in HIV‐1 RNA level of ‐0.62 log10 through week 48). At week 48, 135 patients enrolled in an open‐label, 48‐week extension phase with 300 mg of tenofovir DF once daily added to their antiretroviral therapy. Genotypic analysis of plasma HIV‐1 was performed for all patients after 96 weeks of study or upon early termination. Phenotypic analyses were performed at week 96 for patients with increases in HIV‐1 RNA levels of ≥0.5 log10 from week 48 to week 96. Results: Genotypic results were obtained for 96 of 135 patients. NRTI‐associated mutations developed in 41 (30%) of 135 patients from week 48 to week 96. Those mutations were primarily thymidine analog‐associated mutations (33/41 patients) and developed while patients were receiving either stavudine or zidovudine. Two patients (1.5%) developed the K65R RT mutation (selected by tenofovir in vitro) but maintained HIV‐1 suppression (‐0.39 log10). These 96‐week results were analogous to the 48‐week results, in which 33% (n = 63) and 2.1% (n = 4) of patients developed thymidine analog‐associated mutations or the K65R mutation, respectively. Although most patients maintained HIV‐1 RNA suppression, an analysis of patients with increases in HIV‐1 RNA levels of ≥0.5 log10 (n = 21) from week 48 to week 96 was performed. For eight of 21 patients, development of primary protease inhibitorassociated or non‐NRTI‐associated resistance mutations was likely responsible for the HIV‐1 RNA rebound. The remaining patients developed either no mutation (n = 3) or a new NRTI‐associated mutation (n = 10) and were analyzed phenotypically. No phenotypic changes for tenofovir were observed in these analyses. In addition, no new mutations potentially associated with tenofovir DF therapy were identified. Overall, patients had a similar reduction in HIV‐1 RNA levels at week 96 and at week 48 compared with baseline (‐0.55 and ‐0.60 log10, respectively). Conclusions: Adding tenofovir DF (300 mg) to existing antiretroviral therapy for highly treatment‐experienced patients with preexisting resistance mutations showed significant and durable reductions in HIV‐1 RNA levels through week 96. Through 96 weeks of tenofovir DF therapy, 48 weeks of which included suboptimal doses of tenofovir DF, there was infrequent development of RT mutations associated with tenofovir DF therapy (K65R mutation, 3%), consistent with the durability of the observed HIV‐1 RNA responses.