13th International HIV Drug Resistance Workshop


8–12 June 2004, Tenerife Sur-Costa Adeje, Canary Islands, Spain


IN VITRO SELECTION AND CHARACTERIZATION OF RESISTANCE TO THE NEW HIV PROTEASE INHIBITOR GW640385

Antiviral Therapy 2004; 9:S16

P Yates1, R Hazen3, M St Clair2, L Boone3 and R Elston1
1International Clinical Virology (ICV), GlaxoSmithKline, Stevenage, UK; 2ICV; and 3Virology, Research Triangle Park, NC, USA

BACKGROUND: GW640385 is a new HIV-1 aspartyl protease inhibitor (PI) with potency against multiple PI resistant clinical isolates. In vitro passage of wild-type (HXB2, IC50=0.5 nM) virus with GW640385, was carried out in MT-4 cells at both high and low selection pressure. The aim of this study was to elucidate the impact of individual mutations, selected during in vitro passage, upon GW640385 anti-HIV activity.

METHODS: Viral RNA was extracted from each passage and the protease (PRO)/cleavage sites (CS) (p7/p1–p1/p6) were subjected to population and clonal sequence analysis to confirm linkage between resistance mutations. Site-directed mutant viruses were constructed containing individual or combinations of the selected substitutions to determine the contribution of each mutation to GW640385 resistance.

RESULTS: Low pressure in vitro passage (13 passages up to 5 nM, ~10×IC50) led to the selection of PRO substitutions: Q58E, A71V, V82I and CS R452K. Phenotypic evaluation of individual mutations, or combinations thereof, demonstrated no more than a 2.55-fold reduction in susceptibility to GW640385. High pressure in vitro passage (15 passages up to 120 nM, ~240×IC50) led to the selection of PRO substitutions: L10F, G16E, E21K, A28S, M46I, F53L, A71V and CS L449F, P453T. All the site-directed mutants grew successfully in cell culture except for the A28S variants. Phenotypic evaluation of these individual variants showed that each had minimal impact on GW640385 activity (up to twofold decrease in susceptibility). Viruses containing the A28S mutation, either alone or in combination with other PRO/CS changes, failed to replicate or replicated poorly and could not be phenotypically evaluated. Recombinant virus generated from cloned virus from the original passage experiment also either failed to grow or grew very poorly.

CONCLUSIONS: The in vitro passage of HXB2 in the presence of GW640385 has identified amino acids associated with decreased susceptibility to GW640385. High pressure passage has led to the selection of the A28S protease mutation which was associated with a dramatic reduction in replicative capacity of the virus.

PRESENTING AUTHOR: P Yates

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2004-06-08
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