Monachetti A, Castagna A, Bagnarelli P, Hasson H, Lazzarin A, Clementi M, Menzo S; Conference on Retroviruses and Opportunistic Infections (11th : 2004 : San Francisco, Calif.).
Program Abstr Conf Retrovir Oppor Infect 11th 2004 San Franc Calif. 2004 Feb 8-11; 11: abstract no. 660.
Univ. of Ancona, Italy and 2San Raffaele Vita-Salute Univ., Milan, Italy
BACKGROUND: Enfuvirtide (ENF) is the first in a new class of promising antiretrovirals, the fusion inhibitors. Mutations within gp41 associated with resistance to ENF have been described from in vitro and in vivo studies. We describe in vitro changes in phenotypic resistance and fitness of strains evolved in patients after long-term treatment with ENF.METHODS: We studied 12 subjects treated with ENF for 1 to 2 years from the TORO II trial. For each strain we determined the HR region genotype before and after treatment, and tested the phenotypic resistance and replicative capacity with a recombinant method. In a modified NL4-3 replicative molecular clone a portion of the gp41 (spanning the HR1 and HR2 domains) was substituted by a blunt end restriction site. This portion, amplified from plasma or mutated in vitro, was cloned in the restriction site. The recombinant plasmids were transfected onto susceptible cells and p24 antigen was measured after 4 days in the presence of fusion inhibitors in order to calculate IC 50 values. Since the molecular backbone was identical in all clones, a reliable comparison of pre- and post-treatment isolates was possible.RESULTS: ENF treatment selected resistance mutations in 7 of 12 subjects. Most mutations appeared in the described N-HR hot spot (amino acids 36-45), however other mutations appeared also further downstream and in the C-HR region. Serial sampling allowed, in some case, the detection of further mutations after the initial resistance-associated ones. Upon treatment interruption, in 2 subjects, wildtype virus reappeared. A high degree of resistance was invariably displayed by the mutated sequences, conferring a >100-fold increase in resistance compared to the basal sequence of the same subject. None of the mutants displayed significant cross-resistance to T-1249. The described mutations also reduced replication capacity, although such reduction was relevant only in some mutant isolates. In addition, single point mutations were generated in the context of wildtype gp41 in order to gain more insight in their phenotypic role. Mutations at position 38, 42, 43, 45, were associated with a high degree of resistance (primary mutations), while our data suggest that others (at pos. 36, 44, 72, 90, 113) should be accessory mutations.CONCLUSIONS: This study provides new data on the evolution of resistant HIV-1 strains during long-term treatment with ENF and on the influence of mutant residues on their resistance and replication kinetics.
Publication Types:
Keywords:
- Genotype
- HIV Envelope Protein gp41
- HIV-1
- Humans
- In Vitro
- Long-Term Care
- Mutation
- Peptide Fragments
- enfuvirtide
- genetics
- immunology
Other ID:
UI: 102271623
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