Background:  Enfuvirtide (T-20) is a fusion inhibitor that blocks gp41-mediated fusion of HIV-1. However, the emergence of specific mutations in gp41 conferring resistance to this drug and limiting its efficacy has been identified. The purpose of this study was to examine the sequential accumulation of genetic changes in the gp41 env region in plasma samples from patients receiving long-term T-20 therapy.

Methods:  We studied 15 heavily ART-experienced patients receiving a salvage regimen containing T-20 in whom no virologic suppression occurred during follow-up. Substitutions in HR1 and HR2 of gp41 were analysed by population-based sequencing on plasma samples at baseline and every month during 96 weeks. To investigate sequence evolution, we also analyzed multiple gp41 clones, derived from plasma, in 3 patients at baseline and at week 2, 4, 12, 24, and 96.

Results:  All patients experienced a substantial viral load decrease after starting T20 (from 5.04 log₁₀ to 3.93 log₁₀) at week 4. However, an increase was observed shortly thereafter in all subjects except in 2. Mutations associated with T20 resistance in gp41 (aa 36 to 45) were early observed in all samples from the 15 patients. The mutations in the HR1 domain included G36V/S/D (n = 4) V38A/L/M/E (n = 7), Q39P (n = 1), Q40H/E (n = 3), N42D/T (n = 5), N43D/S (n = 7), L44M/P (n = 2), L45M (n = 3). Additional accumulation of known T20-resistance mutations in HR1 was not detected once they were established, however a complex and variable genotypic patterns in HR2 was observed during follow-up. Sequence analysis of clones revealed that mutations V38A and N43D were already detected at 2 or 4 weeks after the onset of T20 treatment. The V38A mutation appeared in combination with N43D or G36V but always in independent clones, disappearing in subsequent time-points. In all cases several alternating changes in HR2 domain were noted at different time-points during continued treatment.

Conclusions:  Enfuvirtide resistance-associated substitutions in gp41 were selected in all patients receiving T-20 with non-virologic suppression. Double mutants V38A/G36V or V38A/N43D were rapidly established but they never coexisted in the same viral genome. V38A mutation subsequently reverted probably related to replication compromise. Compensatory mutations, in HR2 domain might occur simultaneously or subsequently to the selection of HR1 mutations and this fact could contribute to increase resistance and improve viral fitness.

Keywords: Enfuvirtide; Resistance; gp41