Background:  The fusion inhibitor enfuvirtide (T-20) is a synthetic 36-amino acid peptide corresponding to residues 127-162 of gp41.  It blocks HIV-1 entry by interacting with the HR1 domain of gp41 and preventing formation of the fusion-active conformation.  Mutations between amino acids 36-45 in HR1 are associated with reduced drug susceptibility, however it is possible that other areas of the viral envelope may play a role. Since HR1 interacts with HR2 in a 6 helices bundle structure, we have assessed whether HR2 mutations co-evolve with HR1 mutations in a cohort of patients receiving long-term T-20 therapy.

Methods:  We studied 17 highly ART-experienced patients receiving T-20, in whom virological rebound, or non-suppression, was identified during follow up. We analyzed 74 samples, obtained as late as day 624 of T-20 therapy. HR1 and HR2 regions of the gp41 gene were sequenced from plasma virus.  

Results:  We identified HR1 and HR2 sequence changes over time in relation to baseline sequences. Mutations in HR1 (aa region 36-45) were noted in all cases, including previously unreported changes N42Q/H and N43Q. These mutations often changed over time, demonstrating continual selective pressure. In addition to a range of HR2 sequence changes at polymorphic sites, 6/17 (35%) patients developed an S138A substitution in the HR2 domain, at least 120 days after starting T-20, and, also subsequent to the first emergence of HR1 mutations. In most, but not all cases, S138A accompanied HR-1 mutations at position 43.  Of note, samples available from three patients demonstrated the loss of both HR1 and S138A HR2 mutations following cessation of therapy.

Conclusions:  Continual evolution of HR1, in the amino acid 36-45 domain, is observed during long term T-20 therapy. A possible compensatory mutation, S138A, in the HR2 domain of gp41 was observed in one third of patients. This is a highly conserved residue in most HIV-1 subtypes. Further structural and phenotypic studies are  required to evaluate these mutational changes (both in the HR1 and HR2 domains) with respect to their precise role in virus fusion and development of T-20 resistance.

Keywords: T-20; gp41; resistance