Identification of the NS5B S282T resistant variant and two novel amino acid substitutions that affect replication capacity in hepatitis C virus-infected patients treated with mericitabine and danoprevir

Abstract

Baseline and posttreatment samples from hepatitis C virus (HCV) genotype (GT) 1-infected patients who received a combination of danoprevir and mericitabine from a phase II clinical study (INFORM-SVR) were analyzed. In addition to resistance monitoring, sequencing and phenotypic assays were combined with statistical analysis to identify potential novel amino acid substitutions associated with treatment outcome. The NS5B S282T substitution associated with mericitabine resistance was identified in 2/30 viral breakthrough patients and was replaced by wild-type viruses after cessation of drug treatment (during follow-up). The NS3 R155K substitution associated with danoprevir resistance was also observed in these 2 patients. All 69 GT 1a-infected patients who experienced viral breakthrough on treatment or relapsed during follow-up (relapsers) developed NS3 R155K. Among GT 1b-infected patients, substitutions at the danoprevir resistance locus NS3 D168 were observed in 15/20 subjects, whereas substitutions at the danoprevir resistance locus NS3 R155 were observed in 5/20 subjects. Interestingly, the baseline polymorphism NS5B Q47H was more prevalent in GT 1a-infected patients who achieved a sustained virologic response at follow-up week 24 (SVR24) than in non-SVR24 patients (2/13 versus 0/72), and a postbaseline NS3 S122G substitution was more prevalent in GT 1a-infected patients with viral breakthrough than in relapsers (4/22 versus 0/47). Neither substitution conferred resistance to danoprevir or mericitabine, but the substitutions reduced (NS5B Q47H) or improved (NS3 S122G) replication capacity by 2- to 4-fold. The NS5B S282T mericitabine-resistant variant was rare and did not persist once drug was discontinued. NS5B Q47H and NS3 S122G are two newly identified substitutions that affected replication capacity and were enriched in distinct treatment response groups. (This study has been registered at ClinicalTrials.gov under registration no. NCT01278134.).

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

UDPS analysis of persistence of NS5B S282T and NS3 R155K substitutions after cessation of drug treatment (during follow-up). The percentage of NS5B S282T and NS3 R155K determined by UDPS is plotted over study days for patients 1 (A) and 2 (B). FUWK, follow-up week; PDISC, premature discontinuation; VL, viral load. The on-treatment period is shaded. The reversion of S282T to the wild type occurred in both patients after the cessation of drug treatment (during follow-up). The LOD by UDPS was 0.5% (see Materials and Methods for details).

FIG 2

Biphasic DNV dose-response curve obtained by NS3 phenotyping of the patient 1 follow-up week 12 sample. The percentage of replicon inhibition is plotted against the log10 DNV concentration, and the data were fitted using the biphasic program in GraphPad Prism (version 5) for Windows (GraphPad Software, San Diego, CA). The arrows indicate EC50s calculated for the first and second phases. One representative of 4 independent experiments is shown.

FIG 3

Replication capacity of NS3 mutant replicons under treatment with various concentrations of DNV. Replicon cells transfected with NS3 mutant constructs were dosed with 0.5% DMSO as a control or DNV, as indicated. Replication capacity was determined as the luciferase signal at 96 h posttransfection divided by that at 4 h posttransfection. The replication capacity of mutant replicons is expressed as a fraction of that of the GT 1a H77 reference replicon, which is set to a value of 1. Error bars represent standard deviations from 3 to 8 experiments. WT, wild type.

FIG 4

Replication capacity of NS5B mutant replicons under treatment with various concentrations of DNV and MCB. Replicon cells transfected with NS5B mutant constructs (with or without NS3 R155K) were dosed with 0.5% DMSO as a control or DNV and MCB, as indicated. Replication capacity was determined as the luciferase signal at 96 h posttransfection divided by that at 4 h posttransfection. The replication capacity of mutant replicons is expressed as a fraction of that of the GT 1a H77 reference replicon, which is set to a value of 1. Error bars represent standard deviations from 3 to 8 experiments.