| dc.description.abstract | The development of specific antiviral drugs directly targeting the hepatitis C virus
(HCV) is clinically important, as the current standard interferon/ribavirin combination
treatment is only partially effective, expensive and often associated with severe
side effects. Inhibitors of the NS3 protease (PI) therefore represent a promising alternative
or additional therapy. To date, the development and in vitro evaluation of PIs is
restricted to the genotype 1/2 based replicon and the genotype 2a full length viral cell
culture system. However, proteases of the different HCV genotypes vary substantially
in their amino acid sequence and secondary structure and require separate evaluation
of their efficacy before they go into clinical trials.
To address this issue, a panel of intra- and intergenotypic recombinants based on the recombinant
infectious clone Jc1 (pFK JFH1/J6/C-846) was developed in this work. The
viability of these recombinants was assessed in the Huh7.5 cell culture system, where
replicating viruses were detected by HCV-NS5A immunostaining. Intergenotypic recombinants
containing genotype 1a, 1b, 3a, 4a and 6a derived proteases were replication
defective, whereas the recombinant with genotype 5a derived protease replicated
efficiently after acquiring cell culture adaptive mutations. The replacement of
not only the NS3 protease gene region, but also its cofactor NS4A, allowed the generation
of replication competent intra- and intergenotypic recombinants for all 6 major
genotypes. Replacing the NS3 protease of the recombinants with that of patientderived
proteases also generated replicating recombinants, greatly expanding the panel
of intergenotypic recombinants available for phenotyping and PI evaluation. However,
intra- and intergenotypic recombinants showed substantial differences in their
replication kinetics, which may be influenced by naturally occurring polymorphism
between genotypes and the differential requirement of adaptive/attenuating cell culture
mutations. Genotype 1a recombinants replicated very poorly, which may be due
to incompatibilities between the type 1a NS3/4A protease and the type 2a backbone.
50% inhibitory concentrations (IC50) of different PIs were measured using Foci Forming
Units/ml (FFU/ml) reductions and replication inhibition assays. The different recombinants
showed consistent, genotype-associated differences in their susceptibility
to the PI BILN 2061, with genotypes 2a, 3a and 5a derived recombinants showing
approximately 100-fold lower susceptibility than genotype 1b, 4a and 6a derived recombinants.
These observations are consistent with major differences in response rates
found in recent treatment trials of genotype 1, 2 and 3 infected patients. Differences
in susceptibility were also observed for VX-950, with genotype 1b, 2a and 6a derived
recombinants being twice as susceptible than genotype 3a, 4a and 5a derived
recombinants. Passaging the intra- and intergenotypic recombinants under increasing
concentrations of PI allowed the identification of PI resistance mutations. Resistance
mutations to BILN 2061 mapped to the previously identified positions 156 and 168
within the NS3 protease, with a great diversity of amino acid substitutions observed
within each genotype. Reintroduction of the identified resistance mutations into the
original recombinant viruses conferred increased resistance towards BILN 2061 and
some mutations also affected replication kinetics of the recombinants. The developed
system will be of major value for the phenotypic characterisation of naturally occurring
and treatment induced resistance mutations within all 6 major HCV genotypes towards
different PIs. This will allow treatment response predictions for newly developed PIs
before they enter clinical trials and the development of individually tailored antiviral
treatment regimes. | en |
