title: Differential efficacy of protease inhibitors against HCV genotypes 2a, 3a, 5a, and 6a NS3/4A protease recombinant viruses.
abstract: OBJECTIVE
  The hepatitis C virus (HCV) genotype influences efficacy of interferon (IFN)-based therapy. HCV protease inhibitors are being licensed for treatment of genotype 1 infection. Because there are limited or no data on efficacy against HCV genotypes 2-7, we aimed at developing recombinant infectious cell culture systems expressing genotype-specific nonstructural (NS) protein 3 protease (NS3P).
  
  
  METHODS
  Viability of J6/JFH1-based recombinants with genotypes 1-7 NS3P/NS4A was evaluated in Huh7.5 human hepatoma cells. Adaptive mutations were identified in reverse genetic studies. Efficacy of lead compound linear protease inhibitors VX-950 (telaprevir) and SCH503034 (boceprevir) and macrocyclic inhibitors TMC435350, ITMN-191 (danoprevir), and MK-7009 (vaniprevir) was determined in high-throughput infection assays.
  
  
  RESULTS
  For genotype(isolate) 2a(J6), 3a(S52), 5a(SA13), and 6a(HK6a), we developed culture systems producing supernatant infectivity titers of 3.5-4.0 log₁₀ focus forming units/mL. Against 2a(J6), 5a(SA13), and 6a(HK6a), all inhibitors showed similar efficacy; macrocyclic inhibitors had ~10-fold greater potency than linear inhibitors. However, compared with 2a recombinant J6/JFH1, efficacy against 3a(S52) was 16- to 70-fold lower for macrocyclic inhibitors and 2- to 7-fold lower for linear inhibitors. Testing of additional genotype 2a and 3a isolates showed that these differences were genotype specific. The resistance of 3a isolates was similar to J6/JFH1 with engineered resistance mutations originally observed for genotype 1 patients. In contrast, we found similar efficacy of NS5A inhibitor BMS-790052 and interferon-alfa2.
  
  
  CONCLUSIONS
  Novel HCV culture systems with genotype specific NS3P/NS4A revealed similar efficacy of protease inhibitors against genotypes 2a, 5a, and 6a and comparatively low but varying efficacy against genotype 3a isolates. These systems will facilitate genotype-specific studies of HCV protease inhibitors and of viral resistance.
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre and Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Judith M
authors.initials: JM
authors.lastname: Gottwein
authors.forename: Troels K H
authors.initials: TK
authors.lastname: Scheel
authors.forename: Tanja B
authors.initials: TB
authors.lastname: Jensen
authors.forename: Lubna
authors.initials: L
authors.lastname: Ghanem
authors.forename: Jens
authors.initials: J
authors.lastname: Bukh
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000473:pathology / Q000821:virology*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D003521:Cyclopropanes; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D006526:Hepatitis C / Q000188:drug therapy; D006801:Humans; D007211:Indoles / Q000494:pharmacology / Q000627:therapeutic use; D047908:Intracellular Signaling Peptides and Proteins; D054833:Isoindoles; D007769:Lactams / Q000494:pharmacology / Q000627:therapeutic use; D047029:Lactams, Macrocyclic; D007930:Leucine / Q000031:analogs & derivatives; D008113:Liver Neoplasms / Q000473:pathology / Q000821:virology*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology / Q000627:therapeutic use; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use; D011480:Protease Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D013449:Sulfonamides / Q000494:pharmacology / Q000627:therapeutic use; D016896:Treatment Outcome; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D002352:Carrier Proteins; D003521:Cyclopropanes; D007211:Indoles; D047908:Intracellular Signaling Peptides and Proteins; D054833:Isoindoles; D007769:Lactams; D047029:Lactams, Macrocyclic; D007930:Leucine; D009842:Oligopeptides; D011392:Proline; D011480:Protease Inhibitors; D013449:Sulfonamides; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus; C486464:telaprevir; C512204:N-(3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl)-3-(2-((((1,1-dimethylethyl)amino)carbonyl)amino)-3,3-dimethyl-1-oxobutyl)-6,6-dimethyl-3-azabicyclo(3.1.0)hexan-2-carboxamide; C553752:danoprevir; C540393:vaniprevir