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Background:  Reductions in enfuvirtide (ENF) susceptibility are conferred by various amino acid substitutions at positions 36 to 45 located within the ENF binding site (heptad repeat 1) of gp41. Here we describe the rapid selection of a virus variant bearing a rare single mutation that confers high level resistance to ENF. The subject was a highly treatment-experienced, ENF-naïve, 52-year-old male with AIDS and multi-drug resistant HIV. Based on the results of a combined phenotype­genotype resistance assay he initiated a salvage regimen of tenofovir (TDF), lamivudine (3TC), saquinavir/ritonavir (SQV/r), and ENF. The pre-therapy HIV-1 RNA was 20,000 copies/mL and CD4 T-cell count was 15/mm³. Response to therapy was poor with a paradoxical rise in viral load and lack of CD4 T-cell count improvement.

Methods: Baseline and on-treatment ENF susceptibilities were measured using a single cycle HIV-1 env pseudovirion infectivity assay. We determined gp160 env sequences at multiple time points by population and clonal methods using chain terminator reaction chemistry.

Results:  ENF susceptibilities (fold-change in IC₅₀ compared to the JRCSF reference) at baseline and days 9 and 30 were 0.4, 7.6, and 400, respectively. High-level ENF resistance was maintained through month 27 of ENF therapy and the virus remained R5-tropic throughout this time. Population genotyping of the day-9 virus revealed a fairly common G36D mutation along with a rare V38E mutation, both as mixtures with the wild-type sequence. On day 30, and all subsequent time points, only the V38E mutation was detected. Phylogenetic analysis of the baseline and on-teatment gp160 sequences confirmed the relatedness of all samples.  Clonal analysis of the day 9 sample identified viruses with single G36D and V38E mutations. No double mutant was observed in the 48 clones analyzed.  Reductions in infectivity of the on-treatment viruses were not observed.

Conclusions:  Rapid selection of high-level ENF resistance may occur in clinical practice, albeit uncommon. Phenotypic resistance was associated with the emergence of a virus variant bearing the V38E mutation within the ENF binding site. This rare mutation (in combination with the N42S polymorphism) was recently reported to be associated with high-level ENF resistance (395-fold) and reduced fitness in the absence of ENF. The rapid outgrowth and maintenance of the V38E mutant on ENF therapy is consistent with a high selective advantage of this mutation.

Keywords: enfuvirtide; resistance; phenotype