15th International HIV Drug Resistance Workshop 13-17 June 2006, Sitges, Spain

HIGH GENOTYPIC AND PHENOTYPIC RESOLUTION OF HCV NS3/4A PROTEASE QUASISPECIES: MUTATIONS ASSOCIATED WITH DRUG RESISTANCE IN TREATMENT-NAÏVE PATIENTS

Antivir Ther. 2006, 11:S14 (abstract no. 12)

S Franco, B Clotet and MA Martínez
Fundació irsiCaixa, Universitat Autònoma de Barcelona, Hospital Universitari Germans Trias i Pujol, Badalona, Spain

BACKGROUND: The HCV NS3/4A protease, that cleaves the viral polyprotein and cellular factors responsible of the innate antiviral response, is the target for the development of new antiviral drugs.

METHODS: We analysed the quasispecies structure of the HCV NS3/4A protease (genotype 1b) from 3 patients (A, B and C) with an HCV plasma load of 2.364, 109.000 and 1300000 IU/ml, respectively. The three patients, two coinfected (A and B) with HCV-HIV and one HCV monoinfected C, had already not received HCV treatment. We obtained and sequenced bidirectionally 100 clones per sample patient. In addition, the catalytic efficiencies of the different proteases were analysed with a genetic screening based on the bacteriophage lambda developed in our laboratory.

RESULTS: The mean intrasample DNA genetic distances were significantly (P<0.0001) different between the three patients (A, 0.7%; B, 1.1% and C, 1.2%). The nucleotide diversity or Shannon entropy (Sn) was higher in patients B (0.92) and C (0.96) than in A (0.60). When we analysed the amino acid diversity, we obtained a higher Sn in patient B (0.61) than in other patients (0.46 and 0.38, from A and C, respectively). These results were in concordance with the higher ratio of ds/dn obtained in patient C (17.06) compared with patients A (5.6) and B (6.75). Of note, within the patient B variant spectrum was possible to identify 47 different amino acid sequences, while patient A and C only showed 37 and 32, respectively. Interestingly, 2.6% of protease variants presented resistance mutations to protease inhibitors SCH6, SCH 503034 and VX-950. Importantly, we detected protease activity in near 65% of the proteases analysed. Finally, protease variants with similar catalytic efficiencies than their respective master proteases were detected in 52.6%.

CONCLUSIONS: Our findings revealed the presence of protease resistance mutations in HCV therapy naïve patients. Although a high number of protease variants were identified in the three patients studied, 52.6% of these variants displayed a catalytic efficiency comparable to that showed by the master proteases. Different selective forces are acting in different infected individuals and affect the HCV protease amino acid diversification.

2006-06-13
12

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