Hepatitis C Virus Replication Fitness As a Determinant of Antiviral Therapy Outcome

Background and Aims: Hepatitis C virus (HCV) infections were previously treated with interferon (IFN) but today direct acting antivirals (DAAs) with cure rates >95% are available. DAA treatment failure is primarily attributed to resistance associated mutations (RAMs), often imposing a fitness cost. Interferon treatment outcome was shown to be associated with the interferon sensitivity determining region (ISDR), which is part of the replication enhancing domain (ReED) in non-structural protein (NS) 5A. We found that accumulation of mutations in the ReED was indicative of elevated viral genome replication fitness. This study investigates the impact of HCV replication fitness on antiviral treatment outcomes. Methods: We utilized chimeric HCV subgenomic replicons containing RAMs and ReED sequences from patients after interferon treatment or DAA failure to assess replication fitness of patient isolates in presence and absence of inhibitors. Results: Replication fitness did not impact on IFN sensitivity in cell culture but resulted in higher remaining antigen levels for highly replicating variants at a given IFN concentration. Furthermore, we identified ReED variants substantially increasing HCV replication in several patients who failed DAA therapy across different genotypes. High replicator ReEDs rescued the fitness loss caused by RAMs like Y93C/H (NS5A) and S282T (NS5B). While high replication fitness did not intrinsically increase drug sensitivity (IC50), it allowed the virus to sustain robust replication despite antiviral pressure. Conclusions: Elevated replication fitness might support interferon treatment due to increased antigen presentation, facilitating adaptive immune responses. Furthermore, ReED mediated increase in replication fitness could contribute to DAA treatment failure by preserving higher replication upon treatment and compensating for RAM associated fitness costs. Thus, patients failing DAA treatment should be monitored for RAMs and ReED mutations. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, 519777725, 272983813 – TRR 179 Deutsches Zentrum für Infektionsforschung (DZIF), DZIF academy stipend, TTU Hepatitis 05.821