The efficacy of direct-acting antiviral agents against hepatitis C virus (HCV) infection can be compromised by substitutions in the HCV genome that occur before treatment (resistance-associated substitutions [RASs]). We performed a meta-analysis to determine the prevalence of RASs and their effects.

We searched publication databases for studies of HCV RNA substitutions that mediate resistance to direct-acting antiviral agents. Findings from 50 studies of the prevalence of RAS in HCV, from 32 countries, were used in a meta-analysis. We retrieved the HCV RNA sequence from the Los Alamos HCV sequence database to estimate the prevalence of the RASs. The degree of resistance to treatment conferred by each RAS was determined based on fold-change in the 50% effective concentration of the drugs.

Our final analysis included data from 49,744 patients with HCV infection and 12,612 HCV sequences. We estimated the prevalence of 56 RASs that encoded amino acids and 114 specific RASs. The average prevalence of RASs was highest in HCV genotype (GT) 6, followed by HCV GT1a, GT2, GT1b, GT3, and GT4. The highest prevalence of RASs observed encoded Q80K in NS3 to NS4A of HCV GT1a, Y93T in NS5A of GT1a, and C316N in NS5B of GT1b. The greatest number of RASs were observed at D168 in NS3 to NS4A, at Y93 in NS5A, and at C316 in NS5B. The prevalence of RASs and mutation burdens were high in Japan, the United States, Germany, Thailand, and the United Kingdom; low in Russia, Brazil, Egypt, and India; and intermediate in China, Canada, Australia, Spain, and France.

In a meta-analysis, we found evidence for 114 RASs in HCV of different genotypes. Patients with HCV infection should be tested for RASs before treatment is selected, especially in regions with a high prevalence of RASs.

Nowadays, due to the development of potent Direct-Acting Antiviral Agents (DAAs) that specifically target NS3, NS5A and NS5B viral proteins, several new and highly efficacious options to treat chronic Hepatitis C virus (HCV) infection are available. The natural presence of resistance associated substitutions (RASs), as well as their rapid emergence during incomplete drug-pressure, are intrinsic characteristics of HCV that greatly affect treatment outcome and the chances to achieve a virolgical cure. To date, a high number of RASs in NS3, NS5A, and NS5B have been associated in vivo and/or in vitro with reduced susceptibility to DAAs, but no comprehensive RASs list is available. This review thus provides an updated, systematic overview of the role of RASs to currently approved DAAs or in phase II/III of clinical development against HCV-infection, discriminating their impact in different HCV-genotypes and DAAs, providing assistance for a fruitful use of HCV resistance testing in clinical practice.

Hepatitis C virus (HCV) NS3 protease inhibitors have been primarily designed against genotype 1, the one with the lowest response to dual therapy. However, less evidence of their efficacy on non-1 genotypes is available, and any such information is mostly concentrated on genotypes 2-4. This study evaluated HCV protease resistance profiles in the major six HCV genotypes and identified genetic barrier (GB) profiles to each available protease inhibitor across HCV strains from different locations worldwide. We obtained 15 099 HCV sequences from treatment-naïve subjects retrieved at the Los Alamos HCV Sequence Database. The wild-type codons of different HCV genotypes were used to analyse the smallest number of nucleotide substitution steps required for changing that codon to the closest one associated with drug resistance. The 36L and 175L RAVs were found as genetic signatures of genotypes 2-5, while the 80K RAV was found in all genotype 5 sequences. Genotypes 4 and 6 showed a higher GB to RAV mutations conferring resistance to telaprevir, while genotypes 2-5 presented baseline resistance to that drug, carrying the 36L mutation. Genotype 4 had a higher GB to simeprevir resistance, requiring three substitutions to acquire the 155K mutation. Subtype 1b showed a higher GB than subtype 1a to resistance for most PIs, with RAVs at codons 36 and 155. Geographic disparities were also found in frequencies of certain RAVs in genotypes 2 and 3. Under a scenario of unprecedented evolution of anti-HCV direct-acting agents, the genetic composition of the circulating HCV sequences should be evaluated worldwide to choose the most appropriate/feasible therapeutic schemes with the highest genetic barriers to resistance.

The treatment of hepatitis C virus (HCV) infection has dramatically improved in recent years with the widespread use of interferon-free combination regimens. Despite the high sustained virological response (SVR) rates (over 90%) obtained with direct-acting antivirals (DAAs), drug resistance has emerged as a potential challenge. The high replication rate of HCV and the low fidelity of its RNA polymerase result in a high degree of genetic variability in the HCV population, which ultimately explains the rapid selection of drug resistance associated variants (RAVs).

Results from clinical trials and real-world experience have both provided important information on the rate and clinical significance of RAVs. They can be present in treatment-naive patients as natural polymorphisms although more frequently they are selected upon treatment failure. In patients engaged in high-risk behaviors, RAVs can be transmitted.

Although DAA failures generally occur in less than 10% of treated chronic hepatitis C patients, selection of drug resistance is the rule in most cases. HCV re-treatment options are available, but first-line therapeutic strategies should be optimized to efficiently prevent DAA failure due to baseline HCV resistance. Considerable progress is being made and next-generation DAAs are coming with pangenotypic activity and higher resistance barrier.

Hepatitis C virus (HCV) drug development has resulted in treatment regimens composed of interferon-free, all-oral combinations of direct-acting antivirals. While the new regimens are potent and highly efficacious, the full clinical impact of HCV drug resistance, its implications for retreatment, and the potential role of baseline resistance testing remain critical research and clinical questions. In this report, we discuss the viral proteins targeted by HCV direct-acting antivirals and summarize clinically relevant resistance data for compounds that have been approved or are currently in phase 3 clinical trials.

This report provides a comprehensive, systematic review of all resistance information available from sponsors' trials as a tool to inform the HCV drug development field.

The presence of resistance-associated variants (RAV) may attenuate the efficacy of direct-acting antivirals (DAA) in combination therapy for hepatitis C. The aim of this study was to characterize the NS3 and NS5A regions of hepatitis C virus (HCV) in naturally occurring RAV.

The NS3 and NS5A regions of HCV were amplified by nested polymerase chain reaction and their nucleotide sequences were determined by direct sequencing in 493 genotype 1b patients naive to DAA-based therapies. The effect of baseline RAV on response to pegylated interferon and ribavirin therapy was analyzed in 65 patients after stratification by interleukin (IL)-28B genotype.

The incidence of RAV was 7.9% in NS3 (V36I/L, 1.2%; T54S, 2.8%; Q80K/R, 3.0%; A156S, 0.2%; and D168E/T, 2.4%) and 20.2% in NS5A (L31I/M, 2.2%; and Y93H, 19.0%). The incidence in interferon experienced and naive patients was similar. The incidence of Y93H in NS5A was significantly higher in the IL-28B TT genotype (rs8099917) than non-TT (27.1% vs 9.5%, P < 0.001). The virological response to pegylated interferon plus ribavirin therapy was not affected by the presence of RAV in IL-28B TT genotype.

RAV, especially Y93H in the NS5A region, were highly prevalent in DAA naive patients with genotype 1b HCV in Japan and were linked to IL-28B TT genotype. Interferon-based therapy could be an alternative for patients with RAV because these variants did not attenuate the response to that therapy. The analysis of RAV may impact the selection of the optimal treatment strategy.

Chronic hepatitis C virus infection represents a serious global public health problem, typically resulting in fibrosis, cirrhosis, and ultimately hepatocellular carcinoma. Based on our previous discovery of lead compound 2 (Liu et al. J Med Chem 54:5747-5768, 2011), 35 new quinoxalinone derivatives were explored in this study. Outline of the structure-activity relationships (SARs) revealed that compound BH6870 (36) showed high anti-HCV potency ([Formula: see text]) and a good cell safety index (SI [Formula: see text]). SARs analysis indicated that quinoxalin-2(1H)-one containing a 4-aryl-substituted thiazol-2-amine moiety was optimal for antiviral activity. Introducing a hydrogen-bond acceptor (such as ester or amide group) at the C-3 position of quinoxalin-2(1H)-one was beneficial for the antiviral potency, and especially, N,N-disubstituted amide was far superior to N-monosubstituted amide. Incorporation of more than one halogen (fluorine or chlorine atom) or a strong electron-withdrawing group on the benzene ring of the thiazole-phenyl moiety might reduce electron atmosphere density further and resulted in a dramatical loss of activity. The NH-group of the lactam moiety was clearly required for anti-HCV activity. Design and synthesis of quinoxalin-2(1H)-one derivatives as new non-nucleoside small-molecule HCV inhibitors. BH6870 (36), showing higher antiviral potency and a good cell safety index, was identified.

Vaniprevir (MK-7009) is a hepatitis C virus (HCV) non-structural 3/4a protease inhibitor which significantly increases virologic response rates in HCV genotype (GT) 1-infected patients when added to peginterferon and ribavirin (PR).

This was a phase II, multicenter, double-blind, randomized, dose-ranging study in Japanese patients with HCV GT1 infection and previous relapse. Patients received twice daily vaniprevir 100, 300, or 600 mg, or placebo plus PR for 4 weeks then PR alone for 2 weeks. Further treatment with PR was continued up to a maximum of 72 weeks. The primary endpoint was rapid virologic response (RVR; undetectable HCV RNA at treatment week 4).

Ninety patients completed 4 weeks of vaniprevir/placebo plus PR. Rates of RVR were significantly higher with vaniprevir compared with placebo (86, 95, and 76 % in the vaniprevir 100-, 300-, and 600-mg arms versus 20 % with control; p<0.001 for all comparisons). Rates of SVR, an exploratory analysis, in the vaniprevir 100-, 300-, 600-mg, and control arms were 95, 100, 100, and 72 %, respectively. No patient had virologic breakthrough or non-response while receiving vaniprevir. There were no serious adverse events (AEs) or discontinuations due to an AE during vaniprevir treatment. Diarrhea and nausea were more common with vaniprevir 600 mg than control or lower vaniprevir doses.

The addition of vaniprevir to PR was associated with an increase in RVR and SVR. Combined with a generally safe and well-tolerated profile, these data supported the further evaluation of vaniprevir in Japanese patients with HCV GT1 infection (#NCT00880763).

Virologic failure following treatment of hepatitis C virus (HCV) genotype 1 with direct-acting antiviral agents is often accompanied by the emergence of resistant variants. MK-5172 is an investigational once-daily protease inhibitor. We analyzed variants in treatment-naive noncirrhotic patients with virologic failure on MK-5172 (100-800 mg/day) plus pegylated interferon alfa/ribavirin (peg-IFN/RBV) during a phase 2 trial.

Population and selective clonal sequencing were performed at baseline and at virologic failure in the 4 MK-5172 dosing arms. MK-5172 activity was determined using a mutant replicon assay.

Six of 266 (2.3%) MK-5172 recipients satisfied prespecified criteria for virologic failure, all with genotype 1a infection. Five patients with virologic failure were in the MK-5172 100-mg arm, including 4 patients with low plasma MK-5172 levels documented during triple therapy. Variants associated with >4-fold loss of potency were detected in 3 of the 4 patients with genotype 1a breakthrough while on MK-5172. The fifth patient had undetectable HCV-RNA levels at the end of triple therapy but subsequently broke through during the peg-IFN/RBV tail 16 weeks after completion of MK-5172. Three patients had D168 variants at virologic failure, including 2 with the D168A variant associated with a 95-fold loss of potency. The sole apparent relapse was actually a genotype 3a reinfection in the MK-5172 200-mg group.

Virologic failure occurred uncommonly (6/266 [2.3%]) in MK-5172/peg-IFN/RBV recipients. The most prevalent treatment-emergent variants were detected at the D168 locus. D168A variants conferring approximately 2-log reduction in MK-5172 susceptibility emerged in 2 of the 4 evaluable patients with genotype 1a breakthrough. Clinical Trials Registration. NCT01353911.

Direct-acting antivirals (DAAs) are predicted to transform hepatitis C therapy, yet little is known about the prevalence of naturally occurring resistance mutations in recently acquired HCV. This study aimed to determine the prevalence and frequency of drug resistance mutations in the viral quasispecies among HIV-positive and -negative individuals with recent HCV.

The NS3 protease, NS5A and NS5B polymerase genes were amplified from 50 genotype 1a participants of the Australian Trial in Acute Hepatitis C. Amino acid variations at sites known to be associated with possible drug resistance were analysed by ultra-deep pyrosequencing.

A total of 12% of individuals harboured dominant resistance mutations, while 36% demonstrated non-dominant resistant variants below that detectable by bulk sequencing (that is, <20%) but above a threshold of 1%. Resistance variants (<1%) were observed at most sites associated with DAA resistance from all classes, with the exception of sofosbuvir.

Dominant resistant mutations were uncommonly observed in the setting of recent HCV. However, low-level mutations to all DAA classes were observed by deep sequencing at the majority of sites and in most individuals. The significance of these variants and impact on future treatment options remains to be determined. Clinicaltrials.gov NCT00192569.

HIV coinfection accelerates disease progression in chronic hepatitis C and reduces sustained antiviral responses (SVR) to interferon-based therapy. New direct-acting antivirals (DAAs) promise higher SVR rates, but the selection of preexisting resistance-associated variants (RAVs) may lead to virologic breakthrough or relapse. Thus, pretreatment frequencies of RAVs are likely determinants of treatment outcome but typically are below levels at which the viral sequence can be accurately resolved. Moreover, it is not known how HIV coinfection influences RAV frequency. We adopted an accurate high-throughput sequencing strategy to compare nucleotide diversity in HCV NS3 protease-coding sequences in 20 monoinfected and 20 coinfected subjects with well-controlled HIV infection. Differences in mean pairwise nucleotide diversity (π), Tajima's D statistic, and Shannon entropy index suggested that the genetic diversity of HCV is reduced in coinfection. Among coinfected subjects, diversity correlated positively with increases in CD4(+) T cells on antiretroviral therapy, suggesting T cell responses are important determinants of diversity. At a median sequencing depth of 0.084%, preexisting RAVs were readily identified. Q80K, which negatively impacts clinical responses to simeprevir, was encoded by more than 99% of viral RNAs in 17 of the 40 subjects. RAVs other than Q80K were identified in 39 of 40 subjects, mostly at frequencies near 0.1%. RAV frequency did not differ significantly between monoinfected and coinfected subjects. We conclude that HCV genetic diversity is reduced in patients with well-controlled HIV infection, likely reflecting impaired T cell immunity. However, RAV frequency is not increased and should not adversely influence the outcome of DAA therapy.

Recently direct-acting antiviral agents, such as hepatitis C virus (HCV) non-structural 3/4A (NS3/4A) protease inhibitors (PI), have been introduced, and triple therapy regimens that include PI with conventional pegylated interferon α and ribavirin have significantly improved the sustained virological response (SVR) rate, up to 80% for both treatment-naïve and treatment-experienced patients with HCV genotype 1. We here report for the first time a case of the successful treatment of HCV genotype 1 infection with a first generation PI drug (telaprevir) based triple therapy after treatment failure with a second generation PI drug (vaniprevir) based triple therapy. A 67-year-old treatment-naïve Japanese man with HCV genotype 1b infection took part in a phase III clinical trial of vaniprevir-based triple therapy. His serum HCV RNA had become undetectable at week 2 and SVR was highly expected, but HCV RNA reappeared at week 4 after vaniprevir treatment. Polymerase chain reaction direct sequence of the HCV NS3/4A gene at week 8 after vaniprevir treatment showed the emergence of a vaniprevir-resistance mutation (D168V), the probable reason for the treatment failure. Six months later, retreatment with telaprevir-based triple therapy was started. Although the dosages of telaprevir and ribavirin had to be reduced due to severe anemia, the patient achieved an SVR. This case shows the value of repeating PI-based triple therapy with a different drug, a process that would reduce the chance of drug resistant mutation.

An all-oral therapy for treating hepatitis C (HCV) is now available for the most common genotypes although interferon-based therapy is still recommended for interferon-eligible patients with HCV genotype 1. For most patients, a prior history of treatment or presence of cirrhosis does not appear to significantly impact eradication rates with direct acting antivirals (DAA). The promising results from the numerous clinical trials of DAA's for HCV together with their tolerability represent a tremendous advance in our management of this disease, particularly as treatment is often well-tolerated, associated with few side effects and significantly shortened in duration. We will review several of the peer-reviewed landmark studies of DAA's for the treatment HCV and conclude with an expert commentary and five year view.

Hepatitis C virus (HCV) is a causative agent of chronic hepatitis. Although the standard therapy for HCV-infected patients consists of pegylated interferon plus ribavirin, this treatment is associated with serious side effects and high costs, and fails in some patients infected with specific HCV genotypes. To address this problem, we are developing small-molecule inhibitors of cyclin-dependent kinases (CDKs) as novel anti-HCV drug candidates. Previous data showed that HCV replication is inhibited by retinoblastoma protein, which is itself inactivated by CDK-mediated phosphorylation. Here, we report that CDK inhibitors suppress HCV replication in vitro and in vivo, and that CDK4 is required for efficient HCV replication. These findings shed light on the development of novel anti-HCV drugs that target host factors.

The combination of vaniprevir (a NS3/4A protease inhibitor) with peginterferon and ribavirin was shown to increase rates of sustained virologic response (SVR) significantly, compared with peginterferon and ribavirin alone, in treatment-experienced patients with chronic hepatitis C virus (HCV) genotype 1 infection without cirrhosis. We performed a blinded, randomized, controlled trial of the effects of vaniprevir with peginterferon and ribavirin in patients with cirrhosis who did not respond to prior therapy with peginterferon and ribavirin.

Treatment-experienced patients (88% white and 35% prior null responders) with HCV genotype 1 infection and compensated cirrhosis were assigned randomly to groups given vaniprevir (600 mg twice daily) with peginterferon and ribavirin for 24 weeks (n = 16), vaniprevir (600 mg twice daily) for 24 weeks with peginterferon and ribavirin for 48 weeks (n = 14), vaniprevir (300 mg twice daily) with peginterferon and ribavirin for 48 weeks (n = 15), vaniprevir (600 mg twice daily) with peginterferon and ribavirin for 48 weeks (n = 15), or placebo with peginterferon and ribavirin for 48 weeks (n = 14, control). Cirrhosis was documented by liver biopsy (84%) or noninvasive methods (16%). Before randomization, participants were stratified based on their historical response to peginterferon and ribavirin.

In the primary analysis, SVR rates among patients in the respective vaniprevir groups were 9 of 15 (60.0%), 9 of 13 (69.2%), 8 of 15 (53.3%), and 10 of 13 (76.9%), compared with 2 of 14 (14.3%) in the control group (pairwise P values ≤ .016). Cirrhotic patients with null or partial responses to prior therapy achieved SVR less often than patients with prior breakthrough or relapse, although 42.1% of prior null responders in the vaniprevir groups achieved SVRs. Patients in the vaniprevir groups more frequently experienced mild-moderate nausea, vomiting, and diarrhea than controls; 5% developed grade 2 anemia compared with none in the control group (no patient developed grade 3 or 4 anemia). Among patients in the vaniprevir groups who experienced virologic failure, resistance-associated variants were detected predominantly at positions 155, 156, and 168 in the HCV protease gene.

In a controlled phase 2B trial, vaniprevir with peginterferon and ribavirin significantly increased rates of SVR among treatment-experienced patients with chronic HCV genotype 1 infection, compared with re-treatment with peginterferon and ribavirin alone. Vaniprevir generally was well tolerated for up to 48 weeks in patients with compensated cirrhosis. ClinicalTrials.gov number, NCT00704405.