Persistence versus Reversion of 3TC Resistance in HIV-1 Determine the Rate of Emergence of NVP Resistance

Abstract

When HIV-1 is exposed to lamivudine (3TC) at inhibitory concentrations, resistant variants carrying the reverse transcriptase (RT) substitution M184V emerge rapidly. This substitution confers high-level 3TC resistance and increased RT fidelity. We established a novel in vitro system to study the effect of starting nevirapine (NVP) in 3TC-resistant/NNRTI-naïve clinical isolates, and the impact of maintaining versus dropping 3TC pressure in this setting. Because M184V mutant HIV-1 seems hypersusceptible to adefovir (ADV), we also tested the effect of ADV pressure on the same isolates. We draw four conclusions from our experiments simulating combination therapy in vitro. (1) The presence of low-dose (1 μM) 3TC prevented reversal to wild-type from an M184V mutant background. (2) Adding low-dose 3TC in the presence of NVP delayed the selection of NVP-associated mutations. (3) The presence of ADV, in addition to NVP, led to more rapid reversal to wild-type at position 184 than NVP alone. (4) ADV plus NVP selected for greater numbers of mutations than NVP alone. Inference about the “selection of mutation” is based on two statistical models, one at the viral level, more telling, and the other at the level of predominance of mutation within a population. Multidrug pressure experiments lend understanding to mechanisms of HIV resistance as they bear upon new treatment strategies.

In Vitro HIV-1 Evolution in Response to Triple Reverse Transcriptase Inhibitors & In Silico Phenotypic Analysis

“…As described previously [19], clinical isolates were derived from frozen samples. The primary clinical isolates were derived from 4 individuals who had previously received NRTI and protease inhibitors, but who had never been exposed to NNRTIs.…”

“…The serial combination passage experiments with isolates #1–#5 were conducted as follows [19]: (A) no drugs were added to the media, (B) 1 µM 3 TC and 2 µM ADV were added and maintained, (C) NVP was added and concentrations were doubled with each passage (0.01 µM NVP during the first passage up to 20.48 µM during the last passage), (D) 2 µM ADV and increasing concentrations of NVP were added, (E) 1 µM 3 TC and increasing concentrations of NVP were added and (F) 1 µM 3 TC and 2 µM ADV and increasing concentrations of NVP were added to the medium, see Figure 1. Isolates #2 and #3 were derived from the same individual but were run independently in experimental set-ups C, D & E. For each experimental set-up (A–F), 12 single-passage experiments were run, in total 5* 3* 12+4* 3* 12 = 324 single-passage experiments with a median duration of 21 days, respectively.…”

“…The final concentration was 2048-fold (20.48 µM), below reported cytotoxic levels [23]. Cultures were passaged as previously described [19]. Viral growth was monitored using a p24 antigen assay (Abbott Laboratories, Chicago, IL).…”

“…Population-based sequencing of amplified cDNA from viral RNA was performed as described previously [19], [24]. cDNA was obtained using Superscript-One-Step RT-PCR reagent (Life Technologies, Gaithersburg, MD).…”

“…To understand drug resistance during combination antiviral drug treatments, an in vitro assay [19] was established in stimulated PBMC infected with virus isolates from 4 NRTI-experienced (but NNRTI-naive) patients. Mutations were selected by passage in different combinations and concentrations of ADV, 3 TC and NVP and viral fitness and resistance were estimated on the basis of a stochastic model of viral growth.…”