Rapid emergence of telaprevir resistant hepatitis C virus strain from wildtype clone in vivo

Abstract

Telaprevir is a potent inhibitor of hepatitis C virus (HCV) NS3-4A protease. However, the emergence of drug-resistant strains during therapy is a serious problem, and the susceptibility of resistant strains to interferon (IFN), as well as the details of the emergence of mutant strains in vivo, is not known. We previously established an infectious model of HCV using human hepatocyte chimeric mice. Using this system we investigated the biological properties and mode of emergence of mutants by ultra-deep sequencing technology. Chimeric mice were injected with serum samples obtained from a patient who had developed viral breakthrough during telaprevir monotherapy with strong selection for resistance mutations (A156F [92.6%]). Mice infected with the resistant strain (A156F [99.9%]) developed only low-level viremia and the virus was successfully eliminated with interferon therapy. As observed in patients, telaprevir monotherapy in viremic mice resulted in breakthrough, with selection for mutations that confer resistance to telaprevir (e.g., a high frequency of V36A [52.2%]). Mice were injected intrahepatically with HCV genotype 1b clone KT-9 with or without an introduced resistance mutation, A156S, in the NS3 region, and treated with telaprevir. Mice infected with the A156S strain developed lower-level viremia compared to the wildtype strain but showed strong resistance to telaprevir treatment. Although mice injected with wildtype HCV showed a rapid decline in viremia at the beginning of therapy, a high frequency (11%) of telaprevir-resistant NS3 V36A variants emerged 2 weeks after the start of treatment. Conclusion: Using deep sequencing technology and a genetically engineered HCV infection system, we showed that the rapid emergence of telaprevir-resistant HCV was induced by mutation from the wildtype strain of HCV in vivo. (HEPATOLOGY 2011;54:781-788)

Increased Replicative Fitness Can Lead to Decreased Drug Sensitivity of Hepatitis C Virus

“…In the case of RNA viruses, high mutation rates during genome replication provide viral populations with an ample reservoir of phenotypic variants, including mutants that can escape selective constraints. Resistance to a single drug that targets a viral protein develops at a rate that depends on the genetic barrier (number and types of mutations needed to acquire resistance) and the phenotypic barrier (fitness cost) imposed by the resistance mutations (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). When drug resistance mutations do not entail a significant fitness cost-either because the mutations per se do not critically affect viral functions or because compensatory mutations are acquired-they may reach detectable levels despite no prior exposure of the viral population to the drug (1,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27).…”

Deep Sequencing and Phylogenetic Analysis of Variants Resistant to Interferon-Based Protease Inhibitor Therapy in Chronic Hepatitis Induced by Genotype 1b Hepatitis C Virus

“…Due to the recent development of deep sequencing techniques using next-generation sequencers (15)(16)(17)(18), detailed analysis of quasispecies has become possible. Several deep sequencing studies have been undertaken to disclose the origin of DAA-resistant variants through analyzing DAA-resistant variant populations over time (10,11,16,19,20). On the other hand, previous investigations tended to focus on hot spots for specific mutations but lacked the phylogenetic analysis that is needed to determine the origins of certain viral populations.…”

Analysis of Hepatitis C Virus Intrahost Diversity across the Coding Region by Ultradeep Pyrosequencing

“…While many of these compounds have more powerful antiviral activity than first-generation protease inhibitors, their utility is limited by the development of viral mutations conferring cross-resistance (13). Resistance mutations differ depending on the specific drug used and the HCV subtype, though mutations conferring resistance to all currently approved drugs have already been described (10,14,28). Furthermore, uncommon variants of the viral quasispecies with reduced susceptibility to DAAs can occur naturally even before treatment begins.…”

“…While targeted sequencing has been used to analyze differences in HCV variability in HCV-monoinfected and HIV-HCVcoinfected subjects (32), as well as to determine antiviral resistance mutations against protease inhibitors (14,26), no study has employed second-generation sequencing techniques to examine HCV subtype 1a heterogeneity across the entire coding region. Here we combined pyrosequencing with a transposon-based fragmentation method to perform genomewide ultradeep sequencing of four HCV-1a genomes, allowing analysis of viral sequence heterogeneity and identification of minor variants conferring preexisting HCV-specific drug resistance.…”