Ultradeep sequencing study of chronic hepatitis C virus genotype 1 infection in patients treated with daclatasvir, peginterferon, and ribavirin

Abstract

Direct-acting antivirals (DAAs) are either part of the current standard of care or are in advanced clinical development for the treatment of patients chronically infected with hepatitis C virus (HCV) genotype 1, but concern exists with respect to the patients who fail these regimens with emergent drug-resistant variants. In the present study, ultradeep sequencing was performed to analyze resistance to daclatasvir (DCV), which is a highly selective nonstructural protein 5A (NS5A) inhibitor. Eight patients with HCV genotype 1b, who were either treatment naive or prior nonresponders to pegylated interferon plus ribavirin (Rebetol; Schering-Plough) (PEG-IFN/RBV) therapy, were treated with DCV combined with PEG-IFN alpha-2b (Pegintron; Schering-Plough, Kenilworth, NJ) and RBV. To identify the cause of viral breakthrough, the preexistence and emergence of DCV-resistant variants at NS5A amino acids were analyzed by ultradeep sequencing. Sustained virological response (SVR) was achieved in 6 of 8 patients (75%), with viral breakthrough occurring in the other 2 patients (25%). DCV-resistant variant Y93H preexisted as a minor population at higher frequencies (0.1% to 0.5%) in patients who achieved SVR. In patients with viral breakthrough, DCV-resistant variant mixtures emerged at NS5A-31 over time that persisted posttreatment with Y93H. Although enrichment of DCV-resistant variants was detected, the preexistence of a minor population of the variant did not appear to be associated with virologic response in patients treated with DCV/PEG-IFN/RBV. Ultradeep sequencing results shed light on the complexity of DCV-resistant quasispecies emerging over time, suggesting that multiple resistance pathways are possible within a patient who does not rapidly respond to a DCV-containing regimen. (This study has been registered at ClinicalTrials.gov under registration no. NCT01016912.).

Figures

FIG 1

Plasma HCV RNA levels of 6 patients who achieved SVR during combination therapy with daclatasvir, PEG-IFN alpha-2b, and RBV for 24 weeks followed by 24 weeks posttreatment. LLQ, lower limit of quantitation (15 IU/ml).

FIG 2

Clinical course of case 7 with viral breakthrough during combination therapy. (A) Plasma HCV RNA levels. (B) Time course of the amino acid frequency at L31 and Y93 in the NS5A region by ultradeep sequencing. WT, wild type; LLQ, lower limit of quantitation (15 IU/ml).

FIG 3

Clinical course of case 8 with viral breakthrough during combination therapy. (A) Plasma HCV RNA levels. (B) Time course of the amino acid frequency at L31 and Y93 in the NS5A region by ultradeep sequencing. LLQ, lower limit of quantitation (15 IU/ml).