Background:  HIV-1 genotypic profiles resistance to enfuvirtide (ENF), obtained by bulk sequencing after ENF-discontinuation, showed in few studies a rapid reversion to wild-type viruses, within the 3 months following drug interruption. The kinetics of disappearance from plasma of ENF-resistant viral populations after ENF-discontinuation for virological failure were evaluated quantitatively, by molecular cloning analysis.

Methods:  Plasma samples from 7 patients, who received ENF for at least 3 months (median, 6.4 months; range 3 to 14), were assessed to evaluate quantitatively the kinetics of disappearance of viral resistant populations harbouring mutations clustering in a sequence (amino acids 36 to 45) of the HR-1 gp41 env gene region. Molecular cloning of polymerase chain reaction (PCR) products was performed at 2 time-points following ENF-discontinuation:  the last time-point with ENF-resistance mutations detectable by bulk sequencing; and the first time-point at which ENF-resistance mutations were no more detectable by bulk sequencing. At least, 20 to 30 clones were analyzed at each point.

Results:  Genotypic profiles of ENF resistance at ENF discontinuation were as follow:  V38A in 3 patients; V38A+N42T+N43D in 1 patient; N43D in 2 patients; and N43K in 1 patient. Three patients had completely achieved genotype reversion from mutated to wild type profile at time of molecular cloning. In these 3 cases, the total disappearance of resistant clones was observed at 1 month (A38V), 3 months (K43N), and 9 months (A38V+T42N+D43N). For the 4 remaining patients, drug-resistant variants persisted in a proportion of 5% (N43K), 8% (V38A), 24% (V38A), and 88% (N43D) at 6, 3, 5, and 6 months after ENF-discontinuation, respectively. No association was observed between ENF-resistance mutation and the kinetics of mutation disappearance. The persistence of ENF-resistant minority viral populations correlated with the duration of ENF therapy (r = –0.84, p = 0.02).

Conclusions:  Clonal analysis approach of the kinetics of disappearance of ENF-resistance mutations after drug discontinuation showed that a high proportion of resistant clones persist only in patients who had received ENF for long periods (>6 months). Further investigations are needed to determine whether HIV-1 resistance to ENF generated during long-term treatment could facilitate persistence of resistant variants.