Combination treatment with HCV protease and NS5A inhibitors is effective against recombinant genotype 1a, 2a and 3a virus

With development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi-FL) recombinants only relying on JFH1 NS3 helicase, NS5B and 3' UTR. With identified adaptive mutations, semi-FL recombinants of genotype(isolate) 1a(TN) and 3a(S52) produced supernatant infectivity titers of ∼4 log(10) FFU/ml in Huh7.5 cells. 1a(TN) adaptive mutations allowed generation of 1a(H77) semi-FL virus. Concentration-response profiles revealed higher efficacy of NS3 protease inhibitor asunaprevir (BMS-650032) and NS5A inhibitor daclatasvir (BMS-790052) against 1a(TN and H77) than 3a(S52) viruses. Asunaprevir had intermediate efficacy against previously developed 2a recombinants J6/JFH1 and J6cc. Daclatasvir had intermediate efficacy against J6/JFH1, while low sensitivity was confirmed against J6cc. Using a cross-titration scheme, infected cultures were treated until viral escape or on-treatment virologic suppression occurred. Compared to single drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in NS3 protease and NS5A domain I reported as genotype 1 resistance mutations. Inhibitors showed synergism at drug concentrations reported in vivo. In summary, semi-FL HCV recombinants, including the most advanced reported genotype 3a culture system, permitted genotype-specific analysis of combination treatment in the context of the complete viral life cycle. Despite differential sensitivity to lead compound NS3 protease and NS5A inhibitors, genotype 1a, 2a and 3a viruses were suppressed by combination treatment with relatively low concentrations.