HIV-1 drug resistance in the iPrEx preexposure prophylaxis trial

Abstract

Background: The iPrEx study demonstrated that combination oral emtricitabine and tenofovir disoproxil fumarate (FTC/TDF) as preexposure prophylaxis (PrEP) protects against HIV acquisition in men who have sex with men and transgender women. Selection for drug resistance could offset PrEP benefits.

Methods: Phenotypic and genotypic clinical resistance assays characterized major drug resistant mutations. Minor variants with FTC/TDF mutations K65R, K70E, M184V/I were measured using 454 deep sequencing and a novel allele-specific polymerase chain reaction (AS-PCR) diagnostic tolerant to sequence heterogeneity.

Results: Control of primer-binding site heterogeneity resulted in improved accuracy of minor variant measurements by AS-PCR. Of the 48 on-study infections randomized to FTC/TDF, none showed FTC/TDF mutations by clinical assays despite detectable drug levels in 8 participants. Two randomized to FTC/TDF had minor variant M184I detected at 0.53% by AS-PCR or 0.75% by deep sequencing, only 1 of which had low but detectable drug levels. Among those with acute infection at randomization to FTC/TDF, M184V or I mutations that were predominant at seroconversion waned to background levels within 24 weeks after discontinuing drug.

Conclusions: Drug resistance was rare in iPrEx on-study FTC/TDF-randomized seroconverters, and only as low-frequency minor variants. FTC resistance among those initiating PrEP with acute infection waned rapidly after drug discontinuation. Clinical Trials Registration.NCT00458393.

Keywords: 454 deep sequencing; AS-PCR; FTC/TDF; HIV-1; PrEP; drug resistance; minor variant; preexposure prophylaxis.

Figure 1.

Genotype, phenotype, and minor variant drug resistance in iPrEx seroconverters by randomization arm. Genotype and phenotype results from a subset of iPrEx participants were described previously in the interim analysis report [3] and are included here for a cumulative assessment. Of the 131 participants with incident infection (48 FTC/TDF, 83 placebo), drug resistance assays were performed on specimens collected at first evidence of seroconversion (n = 128) or if unavailable, the HIV RNA-positive visit prior to (n = 2) or following (n = 1) seroconversion. Minor variant drug resistance testing by qMVA was performed on all participants at 1 or more drug resistance mutations. The number of mutation sites ineligible for testing based on preestablished criteria regarding cure primer 3′ end match with viral target sequence among the 131 samples possible are as follows: K65R: n = 2 (1.5%); K70E: n = 11 (8.4%); M184V: n = 5 (3.8%); and M184I: n = 4 (3.0%). Plasma virus from 1 participant was not available for analysis by 454 sequencing (FTC/TDF arm). Abbreviations: 3TC, lamivudine; BCO, biological cut-off; FC IC₅₀, fold change half maximal inhibitory concentration; FTC, emtricitabine; HIV, human immunodeficiency virus; MAX, maximal FC IC₅₀; PCR, polymerase chain reaction; qMVA, quantitative minor variant assay; TDF, tenofovir disoproxil fumarate; ZDV, zidovudine.

Figure 2.

Schematic of the qMVA for low-level drug resistance. A, FTC/TDF selected mutations measured by the qMVA. DNA amplicons spanning TDF-associated mutations K65R, K70E, and FTC-associated mutations M184V/I are generated by nested RT-PCR from plasma HIV-1 virions, representing the predominant and minor quasispecies. The example shows a hypothetical viral mixture at rt 65 with 99% WT (consensus B codon AAA, Lys) and 1.0% Mut (consensus B codon AGA, Arg) where the mutant sequence is undetectable by population sequencing. B, Cure PCR step. The cure PCR step is performed prior to the AS-PCR step to normalize possible sequence heterogeneity in the AS-PCR primer target sites that can reduce PCR efficiency and result in inaccurate quantification of the minor variant population. The example portrays a virus that differs from the discriminatory AS-PCR primer at 2 sites upstream from the SNP conferring resistance. A low-stringency PCR with limited cycle number is performed on the mixed WT (99%) and Mut (1%) DNA amplicons, using a consensus sequence-based primer pair covering the AS-PCR target sites adjacent to but not including the SNP conferring resistance. When paired with another consensus primer, AS-PCR target amplicons are generated representing the original WT:Mut SNP mixtures, but with AS-PCR target sequences normalized to HIV-1 consensus sequences. C, AS-PCR reaction. Amplicons generated by the cure PCR are diluted appropriately and duplicate PCR reactions are performed with primers specific for the WT or Mut SNP. The discriminatory capability of the AS-PCR primers is enhanced by incorporating a 3′, -2 base mismatch into the AS-PCR primer [36]. D, Determining percent mutant by ΔCt. The percent minor variant in a mixture of WT:Mut amplicons is determined by ΔCt measurements from real-time PCR using WT- and Mut-specific discriminatory primers. When extrapolated by linear regression against a 6-point standard curve run simultaneously (0.1% to 24.3% Mut input in WT background), the percent minor variant (65R) can be derived. Abbreviations: AS-PCR, allele-specific polymerase chain reaction; FTC, emtricitabine; HIV, human immunodeficiency virus; Mut, mutant; PCR, polymerase chain reaction; qMVA, quantitative minor variant assay; RT-PCR, reverse-transcriptase PCR; SNP, single nucleotide polymorphism; TDF, tenofovir disoproxil fumarate; WT, wild-type.

Figure 3.

Longitudinal measurements of FTC-associated drug resistance over time. HIV-1 Log₁₀ viral load vs the relative proportion of FTC-associated drug resistance mutations M184V (A) and M184I (B) measured by the qMVA (blue, solid lines) and 454 deep sequencing (black, dashed lines) are shown for each of 2 iPrEx participants with unrecognized infection at baseline, and randomized to FTC/TDF. The dashed green line represents the lowest measured BCO value between the qMVA and 454 sequencing assays (0.5%), above which values are considered positive. Shaded areas represent the potential FTC/TDF exposure window from the entry/randomization visit to first evidence of seroconversion, when study drug was terminated. Abbreviations: BCO, biological cut-off; Cps, copies; FTC, emtricitabine; HIV, human immunodeficiency virus; qMVA, quantitative minor variant assay; TDF, tenofovir disoproxil fumarate.

Figure 4.

Plasma and PBMC drug levels present in the estimated infection window in iPrEx seroconverters. The blood plasma FTC, TFV (A) and cellular FTC-TP, TFV-DP (B) levels are shown for all 8 of the 48 iPrEx participants randomized to the FTC/TDF arm, with incident infection showing any detectable drug at study visits within 90 days prior to SC, or at first evidence of SC. Individuals are differentiated by color. Seven participants had measurable drug either before (triangles), or at the SC visit (circles), but not at both. One participant (orange symbol) had detectable drug at multiple time points. The open symbol represents the single participant with minor variant drug resistance at the SC visit (M184I by qMVA, 0.53%). To the right of (B), the estimated dosing frequency corresponding to TFV-TP concentration is shown (median, IQR) as reported from the STRAND trial [8]. BLQ values for TFV, FTC = 10 ng/mL; TFV-DP = 2.5 fmol/10⁶ PBMCs; FTC-TP = 0.1 pmol/10⁶ PBMCs. Abbreviations: BLQ, below the limit of quantitation; FTC, emtricitabine; FTC-TP, FTC triphosphate FTC; IQR, interquartile range; PBMC, peripheral blood mononuclear cell; SC, seroconversion; TDF, tenofovir disoproxil fumarate; TFV, tenofovir; TFV-DP, TFV diphospate.