Characterization of Hepatitis C virus resistance from a multiple-dose clinical trial of the novel NS5A inhibitor GS-5885

Abstract

GS-5885 is a novel hepatitis C virus (HCV) NS5A inhibitor. In a 3-day monotherapy study in treatment-naive genotype 1a (GT1a) and GT1b HCV-infected subjects, median viral load reductions ranged from 2.3 to 3.3 log10 HCV RNA IU/ml across dosing cohorts (1, 3, 10, 30, or 90 mg once daily). Here, we report viral sequencing and phenotypic analysis of clinical isolates from this study. Detection of baseline NS5A amino acid substitutions at positions 28, 30, 31, or 93 in GT1a was associated with a reduced treatment response. In the GT1b cohort, Y93H was detected in 100% of subjects at day 4 or 14. In the Gt1a cohort, population sequencing detected NS5A resistance-associated mutations at day 4 or 14 for 3/10 subjects at the 1-mg dose and for all subjects dosed at ≥3 mg. A subset of mutants that confer a low level of reduced susceptibility to GS-5885 was not detected by population sequencing at the 30- and 90-mg doses. Subject-derived M28T, Q30R, L31M, and Y93C mutations all conferred >30-fold reductions in GS-5885 and daclatasvir susceptibilities in vitro. Site-directed NS5A mutants also showed reduced susceptibility to GS-5885. However, all NS5A mutants tested remained fully susceptible to other classes of direct-acting antivirals (DAAs), interferon alpha, and ribavirin. Importantly, the nonoverlapping resistance profile and high potency of GS-5885 support its further development with other direct-acting antivirals for the treatment of chronic HCV. (This study has been registered at ClinicalTrials.gov under registration number NCT01193478.).

Fig 1

Maximal reduction in HCV RNA log₁₀ IU/ml by cohort. The maximal change from baseline in HCV RNA is plotted for each individual subject receiving active GS-5885 in study GS-US-256-0102 (median indicated). NS5A was population sequenced for all subjects at baseline. Resistance-associated mutations detected at baseline by population sequencing are labeled and enclosed by a square. Circled subjects had resistance-associated mutations detected as minority variants by deep sequencing at baseline but not detected by population sequencing. The percentage of resistance variants present at baseline is indicated for the circled patients.

Fig 2

Clonal frequency of single-substitution resistance-associated mutations in genotype 1a-infected patients at day 4 dosed at ≥3 mg of GS-5885. TOPO TA cloning and subsequent sequencing of inserts were performed from amplicons used in population-sequencing reactions. “Wildtype” indicates no mutations at NS5A positions 24, 25, 28, 30, 31, 58, 62, and 93 compared to the 1a-H77 reference strain. K24E, K24N, and K24R mutations were combined into one category. For all graphs, n = number of patients in the dosing cohort from which clonal sequences were derived.