12th International HIV Drug Resistance Workshop 10–14 June 2003, Cabo del Sol, Los Cabos, Mexico

BACKGROUND: Elvucitabine is an L nucleoside analogue designed to improve on anti-HBV and anti-HIV potency of lamivudine while maintaining the improved safety profile with respect to mitochondrial toxicity over D-nucleoside analogues. In vitro, elvucitabine has demonstrated potent activity against a wide range of HIV-1 variants, including all clinic subtypes and mutant strains carrying common mutations resistant to nucleosides, non-nucleosides and protease inhibitors. This study was designed to evaluate in vitro resistance induction with elvucitabine.

METHODS: CEM-SS cells were infected with HIV-1_LAI and were passaged at increasing concentrations of elvucitabine. The phenotypes and genotypes of viruses collected during each passage were determined at the end of induction. The mutations were back-cloned into the wild-type HIV-1_LAI backbone and the genotypes and phenotypes associated with the mutants were identified.

RESULTS: After 159 days encompassing seven passages in elvucitabine concentrations ranging from 0.05 to 2.0 µM, variants of HIV_LAI with reduced susceptibility were isolated. The EC₅₀ of the resultant variant was 0.2 µM, ~10-fold increase over the wild-type. Genotypic sequencing of the variant isolated from each sequential passage revealed that two mutations in the reverse transcriptase (RT) had emerged simultaneously, M184I and D237E. The mutation at the 184 locus has been described after exposure to lamivudine, but is usually rapidly replaced in the presence of that drug with M184V. No ‘switch’ to M184V could be detected in the presence of elvucitabine. The contribution of the two mutations to viral resistance was confirmed after they were introduced into the wild-type backbone and the resulting virus was tested for its susceptibility to elvucitabine. Preliminary cross resistance study indicated that the mutant was cross resistant to lamivudine, as expected, but not to other nucleoside inhibitors tested. The role of each mutation in the generation of elvucitabine resistance was examined with respect to growth fitness and resistance after they were introduced back into the wild type backbone individually. A computational model was developed to explain the observed phenomenon that the mutation at 184 confers only moderate resistance to elvucitabine but total resistance to lamivudine.

CONCLUSIONS: An in vitro resistance induction study was performed with elvucitabine. The resulting variant with reduced susceptibility carried mutations at amino acid 184 (M to I) and 237 (D to E). The D237E mutation has not been described previously and its role in the generation of resistance variant is under investigation. The double mutation conferred moderate resistance to elvucitabine (approximately 10-fold shift in EC₅₀). A computational model is proposed to explain the phenomenon.

PRESENTING AUTHOR: WG Rice

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2003-07-08
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