Resistant HCV populations may pre-exist in patients before NS3 protease inhibitor therapy and would likely be selected under specific antiviral pressure. The higher prevalence and lower rate of response to treatment associated with HCV genotype 1 infections has led to drug discovery efforts being focused primarily on enzymes produced by this genotype. Protease inhibitors may also be useful for non-genotype-1-infected patients, notably for non-responders.
Methods
We investigated the prevalence of dominant resistance mutations and polymorphism in 298 HCV pro-tease-inhibitor-naive patients infected with HCV genotypes 1, 2, 3, 4 or 5. Genotype-specific NS3 primers were designed to amplify and sequence the NS3 protease gene.
Results
None of the 233 analysed sequences contained major telaprevir (TVR) or boceprevir (BOC) resistance mutations (R155K/T/M, A156S/V/T and V170A). Some substitutions (V36L, T54S, Q80K/R, D168Q and V170T) linked to low or moderate decreases in HCV sensitivity to protease inhibitors were prevalent according to genotype (between 2% and 100%). Other than genotype signature mutations at positions 36, 80 and 168, the most frequent substitution was T54S (4 genotype 1 and 2 genotype 4 sequences). All genotype 2–5 sequences had the non-genotype-1 signature V36L mutation known to confer low-level resistance to both TVR and BOC.
Conclusions
We have developed an HCV protease NS3 inhibitor resistance genotyping tool suitable for use with HCV genotypes 1–5. Polymorphism data is valuable for interpreting genotypic resistance profiles in cases of failure of anti-HCV NS3 protease treatment.
References
1.
LangeC.M., SarrazinC., ZeuzemS.Review article: specifically targeted anti-viral therapy for hepatitis C - a new era in therapy. Aliment Pharmacol Ther2010; 32: 14–28.
2.
ThompsonA.J., McHutchisonJ.G.Antiviral resistance and specifically targeted therapy for HCV (STAT-C). J Viral Hepat2009; 16: 377–387.
3.
FowellA.J., NashK.L.Telaprevir: a new hope in the treatment of chronic hepatitis C?Adv Ther2010; 27: 512–522.
4.
SusserS., WelschC., WangY.Characterization of resistance to the protease inhibitor boceprevir in hepatitis C virus-infected patients. Hepatology2009; 50: 1709–1718.
5.
LawitzE.J., SulkowskiM.S., JacobsonI.M.Safety, tolerability and antiviral activity of MK-7009, a novel inhibitor of the hepatitis C virus NS3/4A protease, in patients with chronic HCV genotype 1 infection. Hepatology2008; 48Suppl 1: 403A.
6.
MannsM.B., BenhamouY.Safety and antiviral activity of BI201335, a new HCV NS3 protease inhibitor, in treatment-naive patients with chronic hepatitis C genotype 1 infection given as monotherapy and in combination with peginterferon alfa-2a (P) and ribavirin (R). Hepatology2008; 48Suppl 1: 1133A.
7.
MannsM.P., GaneE.J., Rodrigues-TorresM.Early viral response rates in treatment-naive patients with chronic hepatitic C genotype 1 infection treated with MK-7009. Hepatology2009; 50Suppl 4: 332A.
8.
MannsM.R., MorenoC.OPERA-1 trial: interim analysis of safety and antiviral activity of TMC435 in treatment-naive genotype 1 HCV patients. J Hepatol2009; 50Suppl 1: S7.
9.
SeiwertS.D., AndrewsS.W., JiangY.Preclinical characteristics of the hepatitis C virus NS3/4A protease inhibitor ITMN-191 (R7227). Antimicrob Agents Chemother2008; 52: 4432–4441.
10.
HézodeC., ForestierN., DusheikoG.Telaprevir and peginterferon with or without ribavirin for chronic HCV infection. N Engl J Med2009; 360: 1839–1850.
11.
McHutchisonJ.G., EversonG.T., GordonS.C.Telaprevir with peginterferon and ribavirin for chronic hcv genotype 1 Infection. N Engl J Med2009; 360: 1827–1838.
12.
RaneyK.D., SharmaS.D., MoustafaI.M.Hepatitis C virus non-structural protein 3 (HCV NS3): a multifunctional antiviral target. J Biol Chem2010; 285: 22725–22731.
13.
BartelsD.J., ZhouY., ZhangE.Z.Natural prevalence of hepatitis C virus variants with decreased sensitivity to NS3.4A protease inhibitors in treatment-naive subjects. J Infect Dis2008; 198: 800–807.
14.
KuntzenT., TimmJ., BericalA.Naturally occurring dominant resistance mutations to hepatitis C virus protease and polymerase inhibitors in treatment-naive patients. Hepatology2008; 48: 1769–1778.
15.
PawlotskyJ.M.Hepatitis: HCV variability, the immune system and resistance to antiviral drugs. Nat Rev Gastroenterol Hepatol2009; 6: 383–385.
16.
SarrazinC., ZeuzemS.Resistance to direct antiviral agents in patients with hepatitis C virus infection. Gastroenterology2010; 138: 447–462.
17.
AkhavanS., SchnurigerA., LebrayP.Natural variability of NS3 protease in patients infected with genotype 4 hepatitis C virus (HCV): implications for antiviral treatment using specifically targeted antiviral therapy for HCV. J Infect Dis2009; 200: 524–527.
18.
López-LabradorF.X., MoyaA., Gonzalez-CandelasF.Mapping natural polymorphisms of hepatitis C virus NS3/4A protease and antiviral resistance to inhibitors in worldwide isolates. Antivir Ther2008; 13: 481–494.
19.
GermerJ.J., RysP.N., ThorvilsonJ.N.Determination of hepatitis C virus genotype by direct sequence analysis of products generated with the Amplicor HCV test. J Clin Microbiol1999; 37: 2625–2630.
20.
LapercheS., SauneK., DenyP.Unique NS5b hepatitis C virus gene sequence consensus database is essential for standardization of genotype determinations in multicenter epidemiological studies. J Clin Microbiol2006; 44: 614–616.
21.
VerbeeckJ., StanleyM.J., ShiehJ.Evaluation of Versant hepatitis C virus genotype assay (LiPA) 2.0. J Clin Microbiol2008; 46: 1901–1906.
22.
CombetC., GarnierN., CharavayC.euHCVdb: the European hepatitis C virus database. Nucleic Acids Res2007; 35: D363–D366.
23.
KuikenC.The hepatitis C sequence database in Los Alamos. Nucleic Acids Res2008; 36: D512–D516.
24.
ColsonP., BroukN., LemboF.Natural presence of substitution R155K within hepatitis C virus NS3 protease from a treatment-naive chronically infected patient. Hepatology2008; 47: 766–767.
ZhouY., BartelsD.J., HanzelkaB.L.Phenotypic characterization of resistant Val36 variants of hepatitis C virus NS3-4A serine protease. Antimicrob Agents Chemother2008; 52: 110–120.
31.
YiM., TongX., SkeltonA.Mutations conferring resistance to SCH6, a novel hepatitis C virus NS3/4A protease inhibitor. Reduced RNA replication fitness and partial rescue by second-site mutations. J Biol Chem2006; 281: 8205–8215.
32.
BartenschlagerR., LohmannV.Novel cell culture systems for the hepatitis C virus. Antiviral Res2001; 52: 1–17.
33.
BarbatoG., CiceroD.O., NardiM.C.The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism. J Mol Biol1999; 289: 371–384.
34.
YanY., LiY., MunshiS.Complex of NS3 protease and NS4A peptide of BK strain hepatitis C virus: a 2.2 A resolution structure in a hexagonal crystal form. Protein Sci1998; 7: 837–847.
35.
CourcambeckJ., BouzidiM., PerbostR.Resistance of hepatitis C virus to NS3-4A protease inhibitors: mechanisms of drug resistance induced by R155Q, A156T, D168A and D168V mutations. Antivir Ther2006; 11: 847–855.
36.
SarrazinC., KiefferT.L., BartelsD.Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir. Gastroenterology2007; 132: 1767–1777.
37.
TongX., GuoZ., Wright-MinogueJ.Impact of naturally occurring variants of HCV protease on the binding of different classes of protease inhibitors. Biochemistry2006; 45: 1353–1361.
38.
KukoljG., BenhamouY., MannsM.P.BI 201335, a potent hcv ns3 protease inhibitor, in treatment-naive and -experienced chronic hcv genotype-1 infection: genotypic and phenotypic analysis of the ns3 protease domain. J Hepatol2009; 50Suppl 1: S347.
39.
LuL., Pilot-MatiasT.J., StewartK.D.Mutations conferring resistance to a potent hepatitis C virus serine protease inhibitor in vitro. Antimicrob Agents Chemother2004; 48: 2260–2266.
40.
LenzO., VerbinnenT., LinT.I.In vitro resistance profile of the hepatitis C virus NS3/4A protease inhibitor TMC435. Antimicrob Agents Chemother2010; 54: 1878–1887.
41.
QiuP., SanforenzoV., CurryS.Identification of HCV protease inhibitor resistance mutations by selection pressure-based method. Nucleic Acids Res2009; 37: e74.
42.
YangW., ZhaoY., FabryckiJ.Selection of replicon variants resistant to ACH-806, a novel hepatitis C virus inhibitor with no cross-resistance to NS3 protease and NS5B polymerase inhibitors. Antimicrob Agents Chemother2008; 52: 2043–2052.
43.
FarciP., ShimodaA., CoianaA.The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science2000; 288: 339–344.
44.
GaudieriS., RauchA., PfafferottK.Hepatitis C virus drug resistance and immune-driven adaptations: relevance to new antiviral therapy. Hepatology2009; 49: 1069–1082.
45.
CuberoM., EstebanJ.I., OteroT.Naturally occurring NS3-protease-inhibitor resistant mutant A156T in the liver of an untreated chronic hepatitis C patient. Virology2008; 370: 237–245.
46.
BenhamouY., MoussalliJ., RatziuV.Results of a proof of concept study (C210) of Telaprevir monotherapy and in combination with peginterferon alpha-2A and ribavirin in treatment-naïve genotype 4 HCV patients. J Hepatol2009; 50Suppl 1: S6.
47.
FosterG.R., HezodeC., BronowickiJ.P.Activity of telaprevir alone or in combination with peginterferon Alfa-2a and ribavirin in treatment-naive, genotype 2 and 3, hepatitis C patients: final results of study C209.45th Annual Meeting of The European Association for the Study of the Liver. 14–18 April 2010, Vienna, Austria. Abstract 57.
48.
ValletS., GouriouS., NousbaumJ.B.Genetic heterogeneity of the NS3 protease gene in hepatitis C virus genotype 1 from untreated infected patients. J Med Virol2005; 75: 528–537.
49.
Ceccherini-SilbersteinF., Cozzi-LepriA., RuizL.Impact of HIV-1 reverse transcriptase polymorphism F214L on virological response to thymidine analogue-based regimens in antiretroviral therapy (ART)-naive and ART-experienced patients. J Infect Dis2007; 196: 1180–1190.
50.
ValletS., GouriouS., NkontchouG.Is hepatitis C virus NS3 protease quasispecies heterogeneity predictive of progression from cirrhosis to hepatocellular carcinoma?J Viral Hepat2007; 14: 96–106.
51.
Rodriguez-FriasM., CuberoJ.I., EstebanM.Detection of clinically relevant minor HCV and HBV antiviral-resistant mutants in treatment-naive patients using ultra-deep pyrosequencing.44th Annual Meeting of the European Association for the Study of the Liver. 30 October–3 November 2009, Boston, MA, USA. Abstract 447.
52.
ZhouY., MuhU., HanzelkaB.L.Phenotypic and structural analyses of hepatitis C virus NS3 protease Arg155 variants: sensitivity to telaprevir (VX-950) and interferon alpha. J Biol Chem2007; 282: 22619–22628.
53.
Legrand-AbravanelF., HenquellC., Le Guillou-GuillemetteH.Naturally occurring substitutions conferring resistance to hepatitis C virus polymerase inhibitors in treatment-naive patients infected with genotypes 1-5. Antivir Ther2009; 14: 723–730.
