Development of an intra- and intergenotypic HCV cell culture method to phenotype and assess antiviral susceptibilities and resistance development of HCV NS3 protease genes from HCV genotypes 1-6

摘要

The development of specific antiviral drugs directly targeting the hepatitis C virusud(HCV) is clinically important, as the current standard interferon/ribavirin combinationudtreatment is only partially effective, expensive and often associated with severeudside effects. Inhibitors of the NS3 protease (PI) therefore represent a promising alternativeudor additional therapy. To date, the development and in vitro evaluation of PIs isudrestricted to the genotype 1/2 based replicon and the genotype 2a full length viral celludculture system. However, proteases of the different HCV genotypes vary substantiallyudin their amino acid sequence and secondary structure and require separate evaluationudof their efficacy before they go into clinical trials.udTo address this issue, a panel of intra- and intergenotypic recombinants based on the recombinantudinfectious clone Jc1 (pFK JFH1/J6/C-846) was developed in this work. Theudviability of these recombinants was assessed in the Huh7.5 cell culture system, whereudreplicating viruses were detected by HCV-NS5A immunostaining. Intergenotypic recombinantsudcontaining genotype 1a, 1b, 3a, 4a and 6a derived proteases were replicationuddefective, whereas the recombinant with genotype 5a derived protease replicatedudefficiently after acquiring cell culture adaptive mutations. The replacement ofudnot only the NS3 protease gene region, but also its cofactor NS4A, allowed the generationudof replication competent intra- and intergenotypic recombinants for all 6 majorudgenotypes. Replacing the NS3 protease of the recombinants with that of patientderivedudproteases also generated replicating recombinants, greatly expanding the paneludof intergenotypic recombinants available for phenotyping and PI evaluation. However,udintra- and intergenotypic recombinants showed substantial differences in theirudreplication kinetics, which may be influenced by naturally occurring polymorphismudbetween genotypes and the differential requirement of adaptive/attenuating cell cultureudmutations. Genotype 1a recombinants replicated very poorly, which may be dueudto incompatibilities between the type 1a NS3/4A protease and the type 2a backbone.ud50% inhibitory concentrations (IC50) of different PIs were measured using Foci FormingudUnits/ml (FFU/ml) reductions and replication inhibition assays. The different recombinantsudshowed consistent, genotype-associated differences in their susceptibilityudto the PI BILN 2061, with genotypes 2a, 3a and 5a derived recombinants showingudapproximately 100-fold lower susceptibility than genotype 1b, 4a and 6a derived recombinants.udThese observations are consistent with major differences in response ratesudfound in recent treatment trials of genotype 1, 2 and 3 infected patients. Differencesudin susceptibility were also observed for VX-950, with genotype 1b, 2a and 6a derivedudrecombinants being twice as susceptible than genotype 3a, 4a and 5a derivedudrecombinants. Passaging the intra- and intergenotypic recombinants under increasingudconcentrations of PI allowed the identification of PI resistance mutations. Resistanceudmutations to BILN 2061 mapped to the previously identified positions 156 and 168udwithin the NS3 protease, with a great diversity of amino acid substitutions observedudwithin each genotype. Reintroduction of the identified resistance mutations into theudoriginal recombinant viruses conferred increased resistance towards BILN 2061 andudsome mutations also affected replication kinetics of the recombinants. The developedudsystem will be of major value for the phenotypic characterisation of naturally occurringudand treatment induced resistance mutations within all 6 major HCV genotypes towardsuddifferent PIs. This will allow treatment response predictions for newly developed PIsudbefore they enter clinical trials and the development of individually tailored antiviraludtreatment regimes.