title: Combination treatment with hepatitis C virus protease and NS5A inhibitors is effective against recombinant genotype 1a, 2a, and 3a viruses.
abstract: With the 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 relying only on the JFH1 NS3 helicase, NS5B, and the 3' untranslated region. With identified adaptive mutations, semi-FL recombinants of genotypes(isolates) 1a(TN) and 3a(S52) produced supernatant infectivity titers of ~4 log(10) focus-forming units/ml in Huh7.5 cells. Genotype 1a(TN) adaptive mutations allowed generation of 1a(H77) semi-FL virus. Concentration-response profiles revealed the higher efficacy of the NS3 protease inhibitor asunaprevir (BMS-650032) and the 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 the NS3 protease and NS5A domain I reported to be 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 infectious 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.
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Denmark.
authors.forename: Judith M
authors.initials: JM
authors.lastname: Gottwein
authors.forename: Sanne B
authors.initials: SB
authors.lastname: Jensen
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Li
authors.forename: Lubna
authors.initials: L
authors.lastname: Ghanem
authors.forename: Troels K H
authors.initials: TK
authors.lastname: Scheel
authors.forename: Stéphanie B N
authors.initials: SB
authors.lastname: Serre
authors.forename: Lotte
authors.initials: L
authors.lastname: Mikkelsen
authors.forename: Jens
authors.initials: J
authors.lastname: Bukh
mesh_terms: D020413:3' Untranslated Regions; D019943:Amino Acid Substitution / Q000187:drug effects; D002219:Carbamates; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D024882:Drug Resistance, Viral / Q000187:drug effects; D004359:Drug Therapy, Combination; D060005:Genotyping Techniques; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D022781:Hepatocytes / Q000187:drug effects / Q000821:virology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology*; D009154:Mutation / Q000187:drug effects*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011759:Pyrrolidines; D011995:Recombination, Genetic; D013449:Sulfonamides / Q000494:pharmacology*; D014633:Valine / Q000031:analogs & derivatives; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D020413:3' Untranslated Regions; D002219:Carbamates; D007093:Imidazoles; D007546:Isoquinolines; D011480:Protease Inhibitors; D011759:Pyrrolidines; D013449:Sulfonamides; D014633:Valine; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C549273:daclatasvir; C571889:asunaprevir