Polymorphisms in the inosine triphosphatase (ITPA) gene is associated with anemia induced by peg-interferon (PEG-IFN) plus ribavirin (RBV) treatment for patients with chronic hepatitis C virus (HCV) infection. However, the effect of ITPA polymorphism on sofosbuvir plus RBV treatment is unknown.Two hundred and forty-four patients with chronic HCV genotype 2 infection without decompensated liver cirrhosis were treated with sofosbuvir plus RBV for 12 weeks. The effects of ITPA polymorphism on hemoglobin levels and RBV dose reduction and treatment response were analyzed. ITPA (rs1127354) was genotyped using the Invader assay. Multivariate regression analysis was performed to identify factors associated with sustained virological response (SVR).Overall, SVR12 was achieved in 231 (94.7%) patients, based on intention to treat analysis. During the therapy, reduction of hemoglobin levels was significantly greater in ITPA genotype CC patients than CA/AA patients. Therefore, the cumulative proportion of patients with RBV dose reduction was significantly higher and total dose of RBV was significantly lower in patients with CC genotype compared to CA/AA genotypes. SVR12 rates were similar between ITPA genotypes CC and CA/AA (94.7 and 94.4%, respectively, P = 0.933). Multivariate logistic regression analysis identified FIB4 index <3.25 (odds ratio [OR], 9.388 for ≥3.25; P = 0.005) and low body weight (OR, 1.059, for high body weight; P = 0.017) as independent predictors for SVR12.ITPA polymorphism influences hemoglobin levels and incidence of RBV dose reduction during sofosbuvir plus RBV therapy. However, ITPA genotype CC patients can expect a curative effect equivalent to CA/AA patients for chronic HCV genotype 2 infection.

Wisteria floribunda agglutinin positive human Mac-2-binding protein glycosylation isomer (M2BPGi) is a novel serum glycomarker for liver fibrosis. However, it is not known whether or not M2BPGi reflects only liver fibrosis. We measured serum M2BPGi levels in patients with acute liver injury.Fifty-one patients with acute liver injury were enrolled. M2BPGi levels were measured at the initial visit and at achievement of recovery. The relationship between M2BPGi values at the initial visit and clinical outcomes was analyzed.Serum M2BPGi levels at the initial visit were elevated in 47 of 51 acute liver injury patients (8.33 ± 7.56 cutoff index). M2BPGi values were associated with prothrombin activity (r = −0.600, P = 0.001), total bilirubin level (r = 0.588, P = 0.001), and Model for End-Stage Liver Disease score (r = 0.490, P = 0.001) but not with aspartate aminotransferase (r = −0.070) and alanine aminotransferase (r = −0.073) levels. M2BPGi values at the initial visit were significantly higher in patients with acute liver failure (diagnosed by prothrombin activity of 40% or less; P < 0.001), subsequent development of hepatic coma (P = 0.036), and subsequent requirement of liver transplant (P = 0.014), and in a patient who died (P = 0.045). M2BPGi values decreased after aminotransferase level normalization in patients who recovered from acute liver injury; however, M2BPGi level was not a predictive factor for recovery with medical therapy.Serum M2BPGi values increased in patients with acute liver injury and decreased following recovery. The marker seems to reflect not only liver fibrosis but also other factors, such as liver inflammation, liver damage, and hepatocyte regeneration.

Post-transplant hepatitis B virus (HBV) reinfection is one of the major problems facing patients who undergo HBV-related liver transplantation (LT). We analyzed the clinical impact of serum hepatitis B core-related antigen (HBcrAg) on HBV reinfection in post-LT patients with HBV-related liver diseases.Serum hepatitis B surface antigen (HBsAg), HBV DNA, and HBcrAg were measured over time in 32 post-LT patients. Twenty-one out of 32 patients had HCC at LT. The effects of HBcrAg, hepatocellular carcinoma (HCC) recurrence, and HBs gene mutation on HBV reinfection and withdrawal from hepatitis B immune globulin (HBIG) were analyzed.Sixteen out of 32 patients (50 %) were positive for HBcrAg even though only six patients were thought to have experienced HBV reinfection based on reappearance of either HBV DNA or HBsAg during a median follow-up time of 75 months. Three of these six patients who became re-infected with HBV experienced HCC recurrence after LT. The HBV DNA reappearance rate was significantly higher in patients with HCC recurrence after LT (p < 0.001). Two HBV re-infected patients without HCC recurrence had HBs gene mutations G145R and G145A, respectively. Anti-HBs antibody development rate by HB vaccination was similar between HBcrAg-positive and negative patients (p = 0.325).HBV reinfection is more common than is usually considered based on conventional measurement of HBsAg and HBV DNA. HCC recurrence and mutations in the HBV S gene were associated with HBV reinfection after LT.

Daclatasvir and asunaprevir combination therapy has shown a high virological response for chronic genotype 1 hepatitis C virus (HCV)-infected patients. However, the safety and efficacy of the therapy for older patients are unknown.One hundred seventy patients younger than 75 years and 139 patients aged 75 years or older with genotype 1 HCV infection were treated for 24 weeks with daclatasvir plus asunaprevir. Pretreatment drug-resistance-associated variants at NS5A-L31 and NS5A-Y93 were determined by the Invader assay. Virological response and adverse events according to age were analyzed.The sustained virological response (SVR) rate for older patients was similar to that for younger patients (97.1 and 92.4 % respectively). In multivariate regression analysis, prior simeprevir treatment (odds ratio 56.6 for absence; P < 0.001) was identified as a significant independent predictor of SVR. The SVR rate for patients with pretreatment resistance-associated variants (RAVs) at a low population frequency (less than 25 %) was similar to that for patients with no detectable RAVs. The frequency of adverse events was similar between younger and older patients. All 19 very elderly patients (85 years or older) completed the 24 weeks of treatment and achieved SVR.Older patients have a virological response and tolerance of daclatasvir plus asunaprevir therapy similar to those of younger patients. Even though RAVs were detected, virological response similar to that for patients with no detectable RAVs may still be expected for patients with RAVs as long as the population frequency is low.

Interferon and nucleotide/nucleoside analogues are the main treatments for chronic hepatitis B. These drugs effectively reduce serum hepatitis B virus (HBV) DNA titers but fail to sufficiently reduce hepatitis B surface antigen (HBsAg) levels. Following the recent identification of sodium taurocholate cotransporting polypeptide as a receptor for HBV entry, inhibition of HBV entry has become an attractive therapeutic target for chronic hepatitis B treatment. We therefore evaluated the antiviral effects of antibody to HBsAg (anti-HBs) immunoglobulin (HBIG), which can inhibit HBV entry, by in an vivo study and a clinical trial.In the in vivo study, HBV-infected mice were generated from human hepatocyte chimeric mice and treated with HBIG. A clinical trial evaluating HBIG therapy in patients was also performed.In the mouse study, HBV DNA titers were reduced and serum HBsAg titers decreased to undetectable levels following high-dose HBIG injection. On the basis of this result, eight chronic hepatitis B patients, who had received long-term nucleotide analogue treatment, were treated with monthly HBIG injections as an additional treatment. After 1 year of treatment, an HBsAg level reduction of more than 1 log IU/mL was observed in four patients, and three patients became anti-HBs positive. No adverse events occurred during HBIG therapy.These results suggest that monthly HBIG injection might benefit patients with chronic hepatitis B whose HBsAg titer becomes lower following long-term nucleotide/nucleoside analogue treatment.

Older patients with chronic hepatitis C have a lower virological response to interferon (IFN) treatment compared to younger patients. The efficacy of telaprevir (TVR) and PEG-IFN plus ribavirin combination therapy and the predictive value of recently identified IFN lambda (IFNL) 4 polymorphisms on the outcome of therapy for older patients have not been addressed.We assessed predictive factors for sustained virological response (SVR) to triple therapy in 226 younger (≤65 years) and 87 older (>65 years) Japanese patients with chronic genotype 1 hepatitis C. IFNL4 polymorphism ss469415590 was analyzed by Invader assay.The SVR rate for older patients was slightly lower than for younger patients (69 vs. 82 %, P = 0.043). In the older group, the SVR rate for patients with the IFNL4 TT/TT genotype was significantly higher than patients with TT/ΔG or ΔG/ΔG genotypes (81.8 and 42.9 %, P = 0.003). In multivariate regression analysis, rapid virological response (OR 36.601, P = 0.002) and IFNL4 TT/TT genotype (OR 19.502, P = 0.009) were identified as significant independent predictors for SVR in older patients. Treatment-related decreases in hemoglobin and increases in serum creatinine were higher in older patients than younger patients. Reduction of initial TVR dose to 1,500 mg per day alleviated these adverse events without compromising SVR rate in older patients.Analysis of IFNL4 polymorphisms is a valuable predictor in older patients receiving TVR triple therapy. 1,500 mg per day is a suitable initial TVR dose for older Japanese patients.

Hepatitis C virus (HCV) infection is a serious health problem leading to cirrhosis, liver failure and hepatocellular carcinoma. The recent introduction of telaprevir, which was approved in November 2011, in combination with peg-interferon and ribavirin is expected to markedly improve the eradication rate of the virus. However, side effects of triple therapy may be severe. In a phase three III clinical trial, 2250 mg of telaprevir, which is the same dosage used in clinical trials in Western countries, was given to Japanese patients. As this dosage is considered to be relatively high for Japanese patients, who typically have lower weight than patients in Western countries, reduction of telaprevir is recommended in the 2012 revision of the guidelines established by the Study Group for the Standardization of Treatment of Viral Hepatitis Including Cirrhosis published by the Ministry of Health, Labour and Welfare of Japan. Other protease inhibitors with fewer side effects are now in clinical trials in Japan. Alternatively, treatment of patients with combination of direct acting antivirals without interferon has been reported. In this review we summarize current treatment options in Japan and discuss how we treat patients with chronic HCV infection.

Treatment for chronic hepatitis B has improved drastically with the use of nucleot(s)ide analogues (NAs). However, NA therapy typically fails to eliminate Hepatitis B virus (HBV) completely, and it is difficult to discontinue these therapies. We previously demonstrated that NA therapy induced immature viral particles, including HBV RNA in sera of chronic hepatitis B patients. In the study reported here, we analyzed the association between HBV RNA titer and the recurrence rate of hepatitis after discontinuation of NA therapy.The study cohort comprised 36 patients who had discontinued NA therapy. Serum HBV DNA or DNA plus RNA levels were measured by real time PCR and statistical analyses were performed using clinical data and HBV markers.At 24 weeks after discontinuation of NA therapy, HBV DNA rebound was observed in 19 of the 36 patients (52.8 %), and alanine aminotransferase (ALT) rebound was observed in 12 of 36 patients (33.3 %). Multivariate statistical analysis was used to identify factors predictive of HBV DNA rebound. The HBV DNA + RNA titer following 3 months of treatment was significantly associated with HBV DNA rebound [P = 0.043, odds ratio (OR) 9.474, 95 % confidence interval (CI) 1.069–83.957)]. Absence of hepatitis B e antigen (HBeAg) at the end of treatment was significantly associated with ALT rebound (P = 0.003, OR 13.500, 95 % CI 2.473–73.705). In HBeAg-positive patients, the HBV DNA + RNA titer after 3 months of treatment was marginally associated with ALT rebound (P = 0.050, OR 8.032, 95 % CI 0.997–64.683).Monitoring of serum HBV DNA + RNA levels may be a useful method for predicting re-activation of chronic hepatitis B after discontinuation of NA therapy.

The IL28B locus attracted the attention of HCV researchers after a series of genome-wide association studies independently identified a strong association between common IL28B polymorphisms and the outcome of PEG-IFN-α plus ribavirin combination therapy in patients chronically infected with HCV genotype 1. This association was subsequently replicated for other HCV genotypes and has been linked to spontaneous eradication of HCV, development of steatosis and biochemical changes (such as altered levels of γ-glutamyl transpeptidase and LDL). Despite the introduction of direct-acting antiviral drugs, IL28B genetics are likely to play a part in patient selection and treatment decisions—moving towards a personalized approach to therapy. In HCV-infected patients with the so-called favourable IL28B genotype (rs12979860 CC; associated with better treatment response), hepatic expression levels of IL28B and interferon-stimulated genes seem to be reduced at baseline, but are induced more strongly after IFN-α administration, perhaps resulting in more effective elimination of the virus. Clarification of the mechanisms underlying these biological phenomena will lead to improved understanding of the antiviral effects of IFN-λ and, ideally, to the development of better therapies against HCV infection. This Review summarizes current understanding of the role of IL28B in HCV infection and response to therapy.

Several single nucleotide polymorphisms (SNPs) within the interleukin 28B (IL28B) locus are associated with sustained viral response in chronic hepatitis C (HCV) patients who were treated with pegylated interferon (PEG-IFN) plus ribavirin (RBV) combination therapy. Recently, an association between γ-GTP level and IL28B genotype was identified. In this study, the relationship between IL28B genotype and liver steatosis was analyzed.One hundred fifty-three patients who underwent liver biopsy before PEG-IFN plus RBV combination therapy were enrolled. The level of liver steatosis was measured using a BIOREVO BZ-9000 microscope, and the proportion of fatty change and clear cell change were calculated using Dynamic cell count BZ-H1C software. IL28B SNP genotype (rs8099917) was determined using the Invader Assay.Vesicular change was significantly associated with body mass index (BMI), HCV RNA titer, serum aspartate aminotransferase, γ-GTP, IL28B genotype and liver fibrosis level (P < 0.05). Clear cell change was significantly associated with serum aspartate aminotransferase, γ-GTP and IL28B genotype by univariate logistic regression analysis (P < 0.05). Under multiple logistic regression, IL28B genotype (ORadj = 8.158; 95% CI 2.412–27.589), liver fibrosis (ORadj = 2.541; 95% CI 1.040–6.207) and BMI (ORadj = 1.147; 95% CI 1.011–1.301) were significant independent factors for vesicular change and IL28B genotype (ORadj = 3.000; 95% CI 1.282–7.019) for clear cell change.In this study, a new quantitative method to objectively evaluate hepatic steatosis was described. IL28B genotypes were significantly associated with both vesicular and clear cell changes of livers in chronic hepatitis C patients.

Treatment of genotype 1b chronic hepatitis C virus (HCV) infection has been improved by extending peg-interferon plus ribavirin combination therapy to 72 weeks, but predictive factors are needed to identify those patients who are likely to respond to long-term therapy.We analyzed amino acid (aa) substitutions in the core protein and the interferon sensitivity determining region (ISDR) of nonstructural protein (NS) 5A in 840 genotype 1b chronic hepatitis C patients with high viral load. We used logistic regression and classification and regression tree (CART) analysis to identify predictive factors for sustained virological response (SVR) for patients undergoing 72 weeks of treatment.When patients were separately analyzed by treatment duration using multivariate logistic regression, several factors, including sex, age, viral load, and core aa70 and ISDR substitutions (P = 0.0003, P = 0.02, P = 0.01, P = 0.0001, and P = 0.0004, respectively) were significant predictive factors for SVR with 48 weeks of treatment, whereas age, previous interferon treatment history, and ISDR substitutions (P = 0.03, P = 0.01, and P = 0.02, respectively) were the only significant predictive factors with 72 weeks of treatment. Using CART analysis, a decision tree was generated that identified age, cholesterol, sex, treatment length, and aa70 and ISDR substitutions as the most important predictive factors. The CART model had a sensitivity of 69.2% and specificity of 60%, with a positive predictive value of 68.4%.Complementary statistical and data mining approaches were used to identify a subgroup of patients likely to benefit from 72 weeks of therapy.

To standardize diagnosis, treatment, and management of chronic hepatitis B, updated treatment guidelines in Japan have been formulated by a research team of the Japanese Ministry of Health, Labor and Welfare (MHLW)’s study project “Research on standardization of treatment for viral liver diseases including liver cirrhosis.” The guidelines recommend first that chronic hepatitis B patients characterized by ALT levels of at least 31 IU/l, with HBe antigen (HBeAg)-positive patients having HBV DNA levels of at least 5 log copies/ml and HBeAg-negative patients with HBV DNA levels of at least 4 log copies/ml, be targeted for treatment. Patients indicated for treatment intervention, for whom are suggested such basic treatments as entecavir therapy, long-term interferon (IFN) therapy, and sequential therapy, are divided into age groups comprising young patients (less than 35 years of age) and middle-aged/elderly patients (35 years and older), HBeAg-positive and HBe-negative patients, and patients with HBV DNA levels of at least 7 log copies/ml and those of less than 7 log copies/ml. As for switching to entecavir for chronic hepatitis B patients currently undergoing lamivudine therapy, the guidelines suggest specific therapies for different groups, dividing patients into those undergoing lamivudine therapy for less than 3 years and those treated for 3 years or longer, as well as those having HBV DNA levels of less than 1.8 log copies/ml and those of 1.8 log copies/ml or more. Formulated on the basis of consideration of chronic hepatitis B patient age, ALT level, HBeAg status, HBV DNA level, and liver disease severity, these guidelines are considered useful for the understanding and implementation of chronic hepatitis B infection diagnosis and treatment, which in recent years have advanced markedly and grown ever more diverse.

Recent studies reported finding hepatitis E virus (HEV) in sera of domestic animals in Japan and also that HEV is a causative agent of non-A, non-B, non-C acute hepatitis and fulminant hepatitis. To clarify the incidence and severity of the disease caused by HEV in Hiroshima, Japan, we studied 97 patients with acute sporadic hepatitis. Stored serum samples were analysed for markers of hepatitis viruses. Only one patient, who developed fulminant hepatitis that was indistinguishable from acute exacerbation of autoimmune hepatitis (AIH), and died of liver failure, was positive for IgM and IgG antibodies against HEV by enzyme-linked immunosorbent assay and for HEV RNA by reverse transcription-polymerase chain reaction. Nucleotide sequence analyses of cDNA showed that the isolate, HA-Hir1, belongs to genotype III of HEV. A careful study of the life style of the patient failed to identify the infectious route. Although the incidence was not high, we considered that HEV was an important causative agent of severe hepatitis and the clinical features of acute hepatitis E were sometimes indistinguishable from those of acute exacerbation of AIH.

Background & AimsSingle nucleotide polymorphisms within the interferon lambda 4 (IFNL4) locus are strongly associated with spontaneous clearance of hepatitis C virus (HCV) infection and early viral response to interferon therapy. Interaction between host genotype and amino acid substitutions might also influence the risk of antiviral resistance in interferon-free direct acting antiviral (DAA) therapies.MethodsThe relationship between IFNL4 genotype and HCV substitutions was analyzed in 929 patients with chronic HCV genotype 1b infection. Ultra-deep sequencing and quasispecies reconstruction was performed on the N-terminal region of NS5A in 57 patients.ResultsIFNL4 genotype was strongly associated with HCV NS5A Y93 and core protein substitutions, and the number and diversity of predicted quasispecies was marginally greater in IFNL4 TT/TT patients compared to TT/ΔG, ΔG/ΔG patients. RNA secondary structure prediction of the NS5A region suggests that variable sites are more likely to occupy unpaired, high entropy positions.ConclusionsHCV infection is proposed to induce a more efficient antiviral response in individuals with the IFNL4 TT/TT genotype that results either in viral clearance or selection for viral adaptations. The association between IFNL4 TT/TT genotype and Y93 substitutions may impact the risk of antiviral resistance in NS5A inhibitors in DAA therapy.

APOBEC3 proteins function as part of innate antiviral immunity and induce G to A hypermutation in retroviruses and hepatitis B virus (HBV) genomes. Whether APOBEC3 proteins affect viruses that replicate without a reverse transcription step is unknown. TT virus (TTV), known to present in serum of healthy individuals and HBV carriers, has a single-stranded circular DNA genome and replicates without reverse transcription. In this study, we examined 67 blood samples obtained from healthy individuals and HBV carriers and observed G to A hypermutation of genomes of TTV in both healthy individuals and HBV carriers. During ALT flare-up in HBV carriers, G to A hypermutation of HBV increased, but TTV genomes significantly decreased in number and hypermutated TTV genomes became undetectable. Our results show that hypermutated TTV exist in healthy individuals and HBV carriers and that TTV genomes were susceptible to immune reaction directed to HBV by interacting with APOBEC3 proteins.

Advances in understanding the hepatitis C virus (HCV) life cycle and the urgent need to find complementary direct-acting antiviral (DAA) therapies has led to substantial advancements in treating chronic hepatitis C. The introduction of telaprevir and boceprevir in 2011 increased the sustained virological response (SVR) rate from approximately 50% to > 70%, but this therapy further restricted patient eligibility and is only approved for treating HCV genotype 1 infection. Interferon has long remained the backbone of HCV therapy and helps prevent viral breakthrough. However, interferon has limited effectiveness and is associated with severe adverse effects and toxicity, especially among cirrhotic patients. Moving to interferon-free therapies should greatly improve SVR rates and offer new treatments for other HCV genotypes and for ineligible patients or patients failing to respond to prior therapies. However, without the relative safety of interferon to suppress viral escape, vigilance will be required to select appropriate therapies and monitor resistance. Several DAAs are currently undergoing clinical trials and will soon undergo the approval process. Goals of future HCV clinical research will be to identify combinations of DAAs with high genetic barriers, investigate optimal treatment doses and durations, and determine the role of ribavirin in DAA therapies.

•We compared tenofovir disoproxil fumarate (TDF) susceptibilities between HBV genotype A and C.•N236T substitution in HBV RT region is significantly associated with TDF susceptibility.•Genotypic differences in TDF susceptibility were observed between genotypes A and C in vitro and in vivo.Background & aimsTenofovir disoproxil fumarate (TDF) has been approved for chronic hepatitis B treatment, and favorable susceptibility of hepatitis B virus (HBV) has been indicated. However, differences in TDF susceptibility among HBV genotypes and drug-resistant strains are unclear. In this study, TDF susceptibilities between genotypes A and C were evaluated in vitro and in vivo using several drug-resistant HBV clones.MethodsHBV expression plasmids were constructed from sera of HBV carriers, and drug-resistant substitutions were introduced by site-directed mutagenesis. TDF susceptibility was evaluated by changes of core-associated HBV replication intermediates in vitro or by change of serum HBV DNA in human hepatocyte chimeric mice carrying each HBV clone in vivo.ResultsTDF susceptibilities of lamivudine-resistant clones (rtL180M/M204V) and lamivudine plus entecavir-resistant clones (rtL180M/S202G/M204V) were similar to wild type clones in vitro. However, lamivudine plus adefovir-resistant clones (rtA181T/N236T) acquired tolerance to TDF, and the rtN236T mutation was considered to be a causal substitution for TDF resistance. Furthermore, genotypic differences in TDF susceptibility were also observed between genotypes A and C in vitro, and the differences could be confirmed in vivo (p = 0.023).ConclusionsThe present study indicates that TDF susceptibility varies among HBV genotypes and drug-resistant HBV clones.

•We report 2 patients who achieved viral responses with little effect on the blood levels of cyclosporine and tacrolimus using Simeplevir plus pegylated-interferon and ribavirin (PEG-IFN/RBV) treatment.•Serum hepatitis C virus (HCV) RNA became undetectable by TaqMan polymerase chain reaction (PCR) test after 4 weeks of treatment in both patients, and no remarkable fluctuation in blood concentration was observed either in cyclosporine or tacrolimus during the 12 weeks of Simeplevir treatment.•Both patients achieved sustained virological response 12 weeks after the end of treatment.We previously reported our data on telaprevir (TVR) used in combination with pegylated-interferon and ribavirin (PEG-IFN/RBV) for the treatment of recurrent hepatitis C virus (HCV) genotype 1 infection after liver transplantation (LT). TVR substantially increases the blood levels of immunosuppressive agents such as cyclosporine and tacrolimus for drug-drug interactions. On the other hand, the effect of simeprevir (SMV) on the blood levels of these immunosuppressive agents is unclear. We report 2 patients who achieved viral responses with little effect on the blood levels of cyclosporine and tacrolimus using SMV plus PEG-IFN/RBV treatment. The first was a 71-year-old woman with HCV-related liver cirrhosis and hepatocellular carcinoma who failed to respond to PEG-IFN/RBV after living donor LT. She was treated with 40 mg/d of cyclosporine, and received SMV plus PEG-IFN/RBV treatment. The second was a 65-year-old man with HCV-related liver cirrhosis who failed to respond to PEG-IFN/RBV after living donor LT. He was treated with 3 mg/d of tacrolimus, and received SMV plus PEG-IFN/RBV treatment. Serum HCV RNA became undetectable using TaqMan polymerase chain reaction (PCR) test after 4 weeks of treatment in both patients, and no remarkable fluctuation in blood concentration was observed either in cyclosporine or tacrolimus during the 12 weeks of SMV treatment. Completion of 12-week SMV triple therapy was followed by PEG-IFNα2b plus RBV, and both patients achieved sustained virological response 12 weeks after the end of treatment. SMV plus PEG-IFNRBV treatment showed a remarkable viral response with little effect on blood levels of immunosuppressive agents for recurrent HCV genotype 1 infection after LT.

•Mice with high HCV titer developed viral breakthrough by vaniprevir monotherapy.•DAA combination therapy succeeded in eradicating　HCV　regardless of viral titer.•DAA combination therapy eliminates HCV via a non-ISG-mediated mechanism.We previously reported that interferon (IFN)-free direct-acting antiviral combination treatment succeeded in eradicating genotype 1b hepatitis C virus (HCV) in human hepatocyte chimeric mice. In this study, we examined the effect of vaniprevir (MK7009, NS3/4A protease inhibitor) and BMS-788329 (NS5A inhibitor) combination treatment on HCV genotype 1b and the expression of IFN-stimulated genes (ISGs) using a subgenomic replicon system and the same animal model. Combination treatment with vaniprevir and BMS-788329 significantly reduced HCV replication compared to vaniprevir monotherapy in HCV replicon cells (Huh7/Rep-Feo cells). HCV genotype 1b-infected human hepatocyte chimeric mice were treated with vaniprevir alone or in combination with BMS-788329 for four weeks. Vaniprevir monotherapy reduced serum HCV RNA titers in mice, but viral breakthrough was observed in mice with high HCV titers. Ultra-deep sequence analysis revealed a predominant replacement by drug-resistant substitutions at 168 in HCV NS3 region in these mice. Conversely, in mice with low HCV titers, HCV was eradicated by vaniprevir monotherapy without viral breakthrough. In contrast to monotherapy, combination treatment with vaniprevir and BMS-788329 succeeded in completely eradicating HCV regardless of serum viral titer. IFN-alpha treatment significantly increased ISG expression; however, vaniprevir and BMS-788329 combination treatment caused no increase in ISG expression both in cultured cells and in mouse livers. Therefore, combination treatment with vaniprevir and BMS-788329 eliminated HCV via a non-ISG-mediated mechanism. This oral treatment might offer an alternative DAA combination therapy for patients with chronic hepatitis C.