Antiviral Activity and Resistance Analysis of NS3/4A Protease Inhibitor Grazoprevir and NS5A Inhibitor Elbasvir in Hepatitis C Virus GT4 Replicons

Abstract

Although genotype 4 (GT4)-infected patients represent a minor overall percentage of the global hepatitis C virus (HCV)-infected population, the high prevalence of the genotype in specific geographic regions coupled with substantial sequence diversity makes it an important genotype to study for antiviral drug discovery and development. We evaluated two direct-acting antiviral agents-grazoprevir, an HCV NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor-in GT4 replicons prior to clinical studies in this genotype. Following a bioinformatics analysis of available GT4 sequences, a set of replicons bearing representative GT4 clinical isolates was generated. For grazoprevir, the 50% effective concentration (EC 50 ) against the replicon bearing the reference GT4a (ED43) NS3 protease and NS4A was 0.7 nM. The median EC 50 for grazoprevir against chimeric replicons encoding NS3/4A sequences from GT4 clinical isolates was 0.2 nM (range, 0.11 to 0.33 nM; n ϭ 5). The difficulty in establishing replicons bearing NS3/4A resistance-associated substitutions was substantially overcome with the identification of a G162R adaptive substitution in NS3. Single NS3 substitutions D168A/V identified from de novo resistance selection studies reduced grazoprevir antiviral activity by 137-and 47-fold, respectively, in the background of the G162R replicon. For elbasvir, the EC 50 against the replicon bearing the reference full-length GT4a (ED43) NS5A gene was 0.0002 nM. The median EC 50 for elbasvir against chimeric replicons bearing clinical isolates from GT4 was 0.0007 nM (range, 0.0002 to 34 nM; n ϭ 14). De novo resistance selection studies in GT4 demonstrated a high propensity to suppress the emergence of amino acid substitutions that confer high-potency reductions to elbasvir. Phenotypic characterization of the NS5A amino acid substitutions identified (L30F, L30S, M31V, and Y93H) indicated that they conferred 15-, 4-, 2.5-, and 7.5-fold potency losses, respectively, to elbasvir. The activity profiles of grazoprevir and elbasvir supported the testing of the direct-acting antivirals in clinical studies.KEYWORDS HCV, genotype 4, resistance, antiviral, elbasvir, grazoprevir, NS5A, NS3/4A, antiviral agents, drug resistance mechanisms, hepatitis C virus H epatitis C virus (HCV) is a leading cause of chronic liver disease, with an estimated 170 million people infected globally (1, 2). The World Health Organization estimates that more than 350,000 people die every year from hepatitis C-related liver diseases (3). Approximately 20% of individuals chronically infected with HCV can be expected to develop liver cirrhosis and, of these, 6% will decompensate to end-stage liver disease, with an additional 4% developing hepatocellular carcinoma. A number of treatment options are now available with combinations of interferon-free direct-acting

Resistance characterization of ledipasvir and velpatasvir in hepatitis C virus genotype 4

“…The present study indicates that the most prevalent subtypes of GT4 HCV, 4a and 4d, respond very well to LDV/SOF treatment, with 25 of 25 and 10 of 10 patients achieving SVR12 following 12 weeks of treatment (Table S1) GT4b (3.6-34 nmol/L) compared to other subtypes of GT4 (range: 0.0002-0.072 nmol/L for subtypes 4a/d/f/g/m/o/q) has also been reported. 24,25 Although the number of patients is small for some subtypes of GT4 in this study, the results are in agreement with the observation from Halfon et al 26 This suggests that specific triple and quadruple substitutions may affect LDV susceptibility differentially depending on the HCV GT4 subtype. This observation is supported by the difference in EC 50 values for patient isolates from different subtypes exhibiting the same NS5A…”

“…This indicates that the reported substitution combinations are the sole contributor to LDV in vitro resistance in the GT4b isolates. Interestingly, a significantly higher median EC 50 for elbasvir against GT4b (3.6‐34 nmol/L) compared to other subtypes of GT4 (range: 0.0002‐0.072 nmol/L for subtypes 4a/d/f/g/m/o/q) has also been reported …”

Treatment of hepatitis C virus infection with direct‐acting antiviral agents: 100% cure?

“…Elbasvir (EBV), an NS5A inhibitor and Grazoprevir (GZR), an NS3/4A protease inhibitor, have demonstrated high in vitro potency against HCV genotype 1 and 4 replicons, as well as resistance‐associated substitutions (RASs) that confer resistance to first‐generation protease inhibitors and RASs related to treatment failures on DCV and LDV …”

HCV genotype 4, 5 and 6: Distribution of viral subtypes and sustained virologic response rates in clinical trials of approved direct‐acting antiviral regimens

“…Cell culture studies demonstrated antiviral activity of each drug component against each approved GT, with data generally demonstrating consistent activity across several representative subtypes of GT 4 and 6. Although it is not feasible to capture all of the genetic diversity of GT 4 and 6 subtypes in cell culture studies and clinical trials, antiviral activity generally has been demonstrated, at minimum, across the most common representative subtypes of GT 4 and 6…”

“…Cell culture studies demonstrated antiviral activity of each drug component against each approved GT, with data generally demonstrating consistent activity across several representative subtypes of GT 4 and 6. Although it is not feasible to capture all of the genetic diversity of GT 4 and 6 subtypes in cell culture studies and clinical trials, antiviral activity generally has been demonstrated, at minimum, across the most common representative subtypes of GT 4 and 6 [10][11][12][13][14][15][16][17][18][19][20][21] To describe characteristics of less common GTs and relevant subgroups represented in clinical trials, we conducted an analysis of DAA clinical trials evaluating FDA-approved regimens for treatment of HCV GT 4, 5 or 6 infection. Specifically, the objectives of our analyses were to describe the representation and geographic distribution of GT 4 and 6 viral subtypes in clinical trials and to summarize the treatment outcomes for GT 4, 5 and 6 with FDA-approved DAA regimens.…”