Role of the envelope genetic context in the development of enfuvirtide resistance in human immunodeficiency virus type 1-infected patients

Abstract

Acquired human immunodeficiency virus type 1(HIV-1) resistance to the fusion inhibitor enfuvirtide (ENF) is primarily associated with mutations within the highly conserved first heptad repeat (HR1) region of gp41. Viral env sequences, however, are remarkably variable, and the envelope genetic background could have an important impact on optimal expression of HR1 mutations. We have examined the genetic evolution of env sequences, ENF susceptibility, and Env replicative capacity in patients failing ENF treatment. Sequential plasma-derived virus populations, obtained from six patients initiating ENF treatment as part of a salvage therapy, were studied using a recombinant phenotypic assay evaluating the entire gp120 and the gp41 ectodomains. Regardless of major differences in the baseline ENF susceptibilities, viral populations with similar phenotypic ENF resistance (50% inhibitory concentration, >3,000 ng/ml) were selected under treatment in four of six patients. As expected, in all patients ENF-resistant viruses harbored one or more HR1 mutations (positions 36, 38, and 43). Interestingly, in five patients the emergence of resistance mutations was not associated with reduced Env replicative capacity. Phylogenetic analysis of env sequences in sequential samples from two patients showed that the HR1 mutations had emerged in the context of env quasi-species that were different from those prevalent at baseline. Thus, the envelope genetic context appears to play a critical role in the selection of HR1 mutations and the expression of ENF resistance, thereby conditioning the evolution of HIV-1 under fusion inhibitor selective pressure.

FIG. 1.

Evaluation of plasma HIV-1 load (log₁₀ RNA copies/ml) (filled squares) and CD4⁺ T-cell counts (cells/μl) (open circles) before, during, and after the treatment including ENF for the six patients studied. HIV-1 RNA levels were measured by a quantitative RT-PCR assay, and CD4⁺ T-cell counts were measured by flow-activated cytometric assay. The chronology of the follow-up is indicated in weeks on the x axis. The beginning of the treatment including ENF is referred to as week 0, and the sampling times preceding ENF introduction are indicated with negative values. Plasma samples analyzed by phenotypic assay and by sequencing are marked in the graph with arrows. The time of treatment including ENF is shaded.

FIG. 2.

Evolution of phenotypic ENF susceptibility. ENF susceptibility of recombinant viruses expressing envelope proteins of plasma virus populations sampled at different time points for each patient was determined in a single-cycle assay. Columns represent mean IC₅₀ values to ENF of at least three independent experiments (bars represent standard errors). The chronology of the follow-up is indicated on the x axis in weeks, and the time of treatment including ENF is shaded. HR1 resistance mutations harbored by each plasma virus population are indicated.

FIG. 3.

Evolution of the Env replicative capacity. The Env replicative capacity of recombinant viruses expressing the primary envelope proteins from plasma samples obtained during and after treatment including ENF was measured in a single-cycle assay. For each patient, Env replicative capacities are expressed as a percentage of that of pre-ENF therapy viruses, obtained at the latest time point available before the beginning of the ENF treatment. Squares represent means of Env replicative capacities of at least three independent infection experiments (standard errors are indicated). The time of treatment including ENF is shaded.

FIG. 4.

Genotypic evolution of the gp41 ectodomain in patients 1 and 2. At the bottom of each panel, the different time points analyzed before, during, and after the treatment including ENF are indicated and identified with a specific color. The week from which the treatment including ENF was stopped is also indicated (at weeks 25 and 12 for patients 1 and 2, respectively). At the indicated times, RNA was extracted from plasma, and the envelope gene was amplified by RT-PCR and cloned, and the gp41 ectodomain was sequenced. The phylogenetic trees obtained by DNAPARS are shown. The presence of ENF resistance mutations located between residues 36 to 45 of the HR1 domain is indicated.