Characterization of multi-DAA resistance using a novel hepatitis C virus genotype 3a infectious culture system

Abstract

Background and Aims: The high HCV infection cure rates achieved with direct-acting antiviral (DAA) treatments could be compromised in the future by the emergence of antiviral resistance. Thus, it is essential to understand the viral determinants that influence DAA resistance, which is most prevalent in genotype 3. We aimed at studying how resistance to protease-, NS5A-, and NS5Binhibitors influences the activities of glecaprevir/pibrentasvir, sofosbuvir/velpatasvir, and sofosbuvir/velpatasvir/voxilaprevir in cell culture, and how the HCV genome adapts to selective pressure by successive rounds of treatment failure.Approach and Results: A previously developed in vivo infectious cDNA clone of strain S52 (genotype 3a) was adapted to efficiently replicate and propagate in human hepatoma cells (Huh7.5) using 31 adaptive substitutions. DAA escape experiments resulted in the selection of S52 variants with decreased drug susceptibility (resistance), which was linked to the emergence of known resistance-associated substitutions (RASs). NS5A-inhibitor resistance was sufficient to promote treatment failure with double-DAA but not triple-DAA regimens. Enhanced viral fitness associated with the selection of sofosbuvir resistance accelerated escape from DAAs. After serial DAA treatment failure, HCV genetic evolution led to a complex genome-wide network of substitutions, some of which coevolved with known RASs. Conclusions: Baseline NS5A-RAS can compromise the efficacy of double-DAA pangenotypic regimens for HCV genotype 3, and enhanced viral fitness can accelerate treatment failure. Persistence of RASs after successive treatment failure is facilitated by the remarkable evolutionary capacity and plasticity of the HCV genome. Proof-of-concept for the potential development of multi-DAA resistance is shown.

Hepatitis C Virus Antiviral Drug Resistance and Salvage Therapy Outcomes Across Australia

“…Genotype 3a is a particular concern because of its reduced susceptibility to DAA therapy, the high prevalence of NS5A RASs, and a recent report of sofosbuvir resistance (S282T RAS in NS5B) circulating in Pakistan [ 38 ]. Indeed, it is possible to induce replication competent, multidrug resistant virus in vitro [ 39 ], reinforcing the need for ongoing vigilance to maintain the efficacy of current treatments as we work toward global elimination of hepatitis C.…”

Therapeutic preparedness: DAA-resistant HCV variants in vitro and in vivo

“…They described the selection of resistant HCV variants and showed that NS5A RASs could compromise the efficacy of double-DAA pangenotypic regimens in GT3a. [3] Successive treatment with the double-DAA regimens G/P and VEL/SOF resulted in the selection of , for example, L31F+Y93H in NS5A, which was associated with crossresistance to other NS5A inhibitors. Consequently, G/P did not clear viral replication from VEL/SOF-treated isolates and vice versa.…”