Mina Nakagawa

Genome-wide association of IL28B with response to pegylated interferon-α and ribavirin therapy for chronic hepatitis C

The recommended treatment for patients with chronic hepatitis C, pegylated interferon-α (PEG-IFN-α) plus ribavirin (RBV), does not provide sustained virologic response (SVR) in all patients. We report a genome-wide association study (GWAS) to null virological response (NVR) in the treatment of patients with hepatitis C virus (HCV) genotype 1 within a Japanese population. We found two SNPs near the gene IL28B on chromosome 19 to be strongly associated with NVR (rs12980275, P = 1.93 × 10−13, and rs8099917, 3.11 × 10−15). We replicated these associations in an independent cohort (combined P values, 2.84 × 10−27 (OR = 17.7; 95% CI = 10.0–31.3) and 2.68 × 10−32 (OR = 27.1; 95% CI = 14.6–50.3), respectively). Compared to NVR, these SNPs were also associated with SVR (rs12980275, P = 3.99 × 10−24, and rs8099917, P = 1.11 × 10−27). In further fine mapping of the region, seven SNPs (rs8105790, rs11881222, rs8103142, rs28416813, rs4803219, rs8099917 and rs7248668) located in the IL28B region showed the most significant associations (P = 5.52 × 10−28–2.68 × 10−32; OR = 22.3–27.1). Real-time quantitative PCR assays in peripheral blood mononuclear cells showed lower IL28B expression levels in individuals carrying the minor alleles (P = 0.015).

α-fetoprotein levels after interferon therapy and risk of hepatocarcinogenesis in chronic hepatitis C.

The effects of interferon (IFN) treatment and the post‐IFN treatment α‐fetoprotein (AFP) levels on risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C (CHC) are unknown. To determine the relationship between AFP and alanine transaminase (ALT) levels and HCC risk, a cohort consisting of 1,818 patients histologically proven to have CHC treated with IFN were studied. Cumulative incidence and HCC risk were analyzed over a mean follow‐up period of 6.1 years using the Kaplan‐Meier method and Cox proportional hazard analysis. HCC developed in 179 study subjects. According to multivariate analysis, older age, male gender, advanced fibrosis, severe steatosis, lower serum albumin levels, non sustained virological response (non‐SVR), and higher post‐IFN treatment ALT or AFP levels were identified as independent factors significantly associated with HCC development. Cutoff values for ALT and AFP for prediction of future HCC were determined as 40 IU/L and 6.0 ng/mL, respectively, and negative predictive values of these cutoffs were high at 0.960 in each value. The cumulative incidence of HCC was significantly lower in patients whose post‐IFN treatment ALT and AFP levels were suppressed to less than the cutoff values even in non‐SVR patients. This suppressive effect was also found in patients whose post‐IFN treatment ALT and AFP levels were reduced to less than the cutoff values despite abnormal pretreatment levels. Conclusion: Post‐IFN treatment ALT and AFP levels are significantly associated with hepatocarcinogenesis. Measurement of these values is useful for predicting future HCC risk after IFN treatment. Suppression of these values after IFN therapy reduces HCC risk even in patients without HCV eradication. (Hepatology 2013;58:1253–1262)

Synergistic Inhibition of Intracellular Hepatitis C Virus Replication by Combination of Ribavirin and Interferon-α

Treatment of hepatitis C virus (HCV) infection with interferon (IFN)- alpha and ribavirin combination therapy results in superior clinical antiviral responses than does monotherapy with IFN. To explore the virological basis of the effects of combination therapy, we analyzed the effects of IFN- alpha and ribavirin, singly and in combination, on intracellular HCV replication by use of an HCV replicon system. A new replicon that expressed a selectable chimeric reporter protein comprising firefly luciferase and neomycin phosphotransferase was constructed. The replicon was highly sensitive to IFN-alpha (50% inhibitory concentration [IC(50)], 0.5 U/mL). Therapy with ribavirin showed weak suppression of HCV replication at a lower concentration (IC(50), 126 mu mol/L). The nucleotide sequence diversity of the replicon was increased significantly by therapy with ribavirin, suggesting that error-prone HCV replication was induced by the drug. Importantly, use of a clinically achievable concentration of ribavirin (approximately 10 mu mol/L) in combination with IFN showed strong synergistic inhibitory effects on HCV replication. Our results suggest that the direct effects of ribavirin on the genetic stability of the HCV subgenome and its synergistic action combined, with IFN-alpha, may explain the improved clinical responses to combination therapy.

Pre-treatment prediction of response to pegylated-interferon plus ribavirin for chronic hepatitis C using genetic polymorphism in IL28B and viral factors

BACKGROUND & AIMS Pegylated interferon and ribavirin (PEG-IFN/RBV) therapy for chronic hepatitis C virus (HCV) genotype 1 infection is effective in 50% of patients. Recent studies revealed an association between the IL28B genotype and treatment response. We aimed to develop a model for the pre-treatment prediction of response using host and viral factors. METHODS Data were collected from 496 patients with HCV genotype 1 treated with PEG-IFN/RBV at five hospitals and universities in Japan. IL28B genotype and mutations in the core and IFN sensitivity determining region (ISDR) of HCV were analyzed to predict response to therapy. The decision model was generated by data mining analysis. RESULTS The IL28B polymorphism correlated with early virological response and predicted null virological response (NVR) (odds ratio=20.83, p<0.0001) and sustained virological response (SVR) (odds ratio=7.41, p<0.0001) independent of other covariates. Mutations in the ISDR predicted relapse and SVR independent of IL28B. The decision model revealed that patients with the minor IL28B allele and low platelet counts had the highest NVR (84%) and lowest SVR (7%), whereas those with the major IL28B allele and mutations in the ISDR or high platelet counts had the lowest NVR (0-17%) and highest SVR (61-90%). The model had high reproducibility and predicted SVR with 78% specificity and 70% sensitivity. CONCLUSIONS The IL28B polymorphism and mutations in the ISDR of HCV were significant pre-treatment predictors of response to PEG-IFN/RBV. The decision model, including these host and viral factors may support selection of optimum treatment strategy for individual patients.

Antiviral effects of the interferon‐induced protein guanylate binding protein 1 and its interaction with the hepatitis C virus NS5B protein

Interferons (IFNs) and the interferon‐stimulated genes (ISGs) play a central role in antiviral responses against hepatitis C virus (HCV) infection. We have reported previously that ISGs, including guanylate binding protein 1 (GBP‐1), interferon alpha inducible protein (IFI)‐6‐16, and IFI‐27, inhibit HCV subgenomic replication. In this study we investigated the effects of these ISGs against HCV in cell culture and their direct molecular interaction with viral proteins. HCV replication and virus production were suppressed significantly by overexpression of GBP‐1, IFI‐6‐16, or IFI‐27. Knockdown of the individual ISGs enhanced HCV RNA replication markedly. A two‐hybrid panel of molecular interaction of the ISGs with HCV proteins showed that GBP‐1 bound HCV‐NS5B directly. A protein truncation assay showed that the guanine binding domain of GBP‐1 and the finger domain of NS5B were involved in the interaction. Binding of NS5B with GBP‐1 inhibited its guanosine triphosphatase GTPase activity, which is essential for its antiviral effect. Taken together, interferon‐induced GBP‐1 showed antiviral activity against HCV replication. Conclusion: Binding of the HCV‐NS5B protein to GBP‐1 countered the antiviral effect by inhibition of its GTPase activity. These mechanisms may contribute to resistance to innate, IFN‐mediated antiviral defense and to the clinical persistence of HCV infection. (HEPATOLOGY 2009.)

Expressional screening of interferon-stimulated genes for antiviral activity against hepatitis C virus replication

Summary.  Type‐I interferons (IFNs) and the interferon‐stimulated genes (ISGs) play a major role in antivirus responses against hepatitis C virus (HCV) infection. In this study, we studied expression profiles of ISGs in cells supporting subgenomic HCV replication (Huh7/Rep), and screened their activities to suppress HCV replication. Real‐time PCR analyses showed that the expression levels of 23 ISGs were significantly lower in Huh7/Rep than naive Huh7 cells due to transcriptional suppression of the interferon‐stimulated response element (ISRE). Furthermore, the expression level of ISGs was also decreased in the cured Huh7 cells in which replicon had been eliminated (cHuh7), indicating adaptation of the cells to support HCV replication by downregulating ISGs. On the other hand, expression of HCV replicon was significantly suppressed by overexpression of several ISGs including PKR, MxA, IRF‐9, GBP‐1, IFI‐6‐16, IFI‐27, 25OAS and IRF‐1. Knock down of GBP‐1, IFI‐6‐16 and IFI‐27 by short hairpin RNA resulted in increase of HCV replication. Thus, we conclude that downregulation of ISG expression is required in the host cells supporting HCV replication and that several ISGs directly suppress HCV replication. The search for ISGs that regulate HCV replication may help to elucidate the cellular antiviral defence mechanisms against HCV infection.

Regulation of Hepatitis C Virus Replication by Interferon Regulatory Factor 1

ABSTRACT Cellular antiviral responses are mediated partly by the expression of interferon-stimulated genes, triggered by viral genomes, their transcripts and replicative intermediates. Persistent replication of a hepatitis C virus (HCV) replicon suggests that the replicon does not elicit cellular innate antiviral responses. In the present study, we investigated regulatory factors of the interferon-mediated antiviral system in cells expressing an HCV replicon. Luciferase reporter assays revealed that the baseline activity of the interferon-stimulated response element (ISRE) was significantly lower in cells harboring the replicon than in naive cells. Among the proteins involved in the IFN/Jak/STAT pathway and in ISRE activity, the expression level of interferon regulatory factor 1 (IRF-1) was found to be significantly lower in cells harboring the replicon. Transfection of an IRF-1 expression construct into cells harboring the replicon caused an increase of ISRE activity, accompanied by suppression of expression of the HCV replicon. Moreover, in cured Huh7 cells from which the HCV replicon had been eliminated, the expression levels of IRF-1 and ISRE activity also were suppressed, demonstrating that the decrease of IRF-1 is attributable, not to active suppression by the viral proteins, but to adaptation of cells that enables replication of the HCV subgenome. The high permissiveness of the cured cells for the replicon was abolished by transgenic supplementation of IRF-1 expression. Taken together, IRF-1 is one of the key host factors that regulate intracellular HCV replication through modulation of interferon-stimulated-gene-mediated antiviral responses.

Hepatitis C virus NS4B protein targets STING and abrogates RIG‐I–mediated type I interferon‐dependent innate immunity

Hepatitis C virus (HCV) infection blocks cellular interferon (IFN)‐mediated antiviral signaling through cleavage of Cardif by HCV‐NS3/4A serine protease. Like NS3/4A, NS4B protein strongly blocks IFN‐β production signaling mediated by retinoic acid–inducible gene I (RIG‐I); however, the underlying molecular mechanisms are not well understood. Recently, the stimulator of interferon genes (STING) was identified as an activator of RIG‐I signaling. STING possesses a structural homology domain with flaviviral NS4B, which suggests a direct protein‐protein interaction. In the present study, we investigated the molecular mechanisms by which NS4B targets RIG‐I–induced and STING‐mediated IFN‐β production signaling. IFN‐β promoter reporter assay showed that IFN‐β promoter activation induced by RIG‐I or Cardif was significantly suppressed by both NS4B and NS3/4A, whereas STING‐induced IFN‐β activation was suppressed by NS4B but not by NS3/4A, suggesting that NS4B had a distinct point of interaction. Immunostaining showed that STING colocalized with NS4B in the endoplasmic reticulum. Immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays demonstrated that NS4B specifically bound STING. Intriguingly, NS4B expression blocked the protein interaction between STING and Cardif, which is required for robust IFN‐β activation. NS4B truncation assays showed that its N terminus, containing the STING homology domain, was necessary for the suppression of IFN‐β promoter activation. NS4B suppressed residual IFN‐β activation by an NS3/4A‐cleaved Cardif (Cardif1‐508), suggesting that NS3/4A and NS4B may cooperate in the blockade of IFN‐β production. Conclusion: NS4B suppresses RIG‐I–mediated IFN‐β production signaling through a direct protein interaction with STING. Disruption of that interaction may restore cellular antiviral responses and may constitute a novel therapeutic strategy for the eradication of HCV. (HEPATOLOGY 2013)

Diabetes mellitus may be associated with hepatocarcinogenesis in patients with chronic hepatitis C.

It is unknown whether diabetes mellitus is a risk factor of the hepatocarcinogenesis in patients with chronic hepatitis C. Three hundred eleven anti-hepatitis C virus (HCV) -positive patients who had undergone liver biopsies were studied. Patients with histologically proven cirrhosis or withdrawing within 12 months were excluded. Thus, the remaining 279 patients were followed-up for 65.9 ± 29.4 months until the occurrence of hepatocellular carcinoma (HCC). During the observation period, HCC developed in 13 patients. Diabetes, age, sex, habitual alcohol intake, history of blood transfusion, serum α-fetoprotein level, histological findings, HCV genotype, viral load, and interferon therapy were assessed as potential risk factors. The Cox proportional hazard model identified that diabetes mellitus, histological staging, and age were independently associated with the occurrence of HCC. With multivariate analysis, only diabetes mellitus and age were associated with the occurrence of HCC. Diabetes mellitus may be associated with hepatocarcinogenesis in patients with chronic hepatitis C.

ITPA gene variant protects against anemia induced by pegylated interferon-α and ribavirin therapy for Japanese patients with chronic hepatitis C.

Aim:  Host genetic variants leading to inosine triphosphatase (ITPA) deficiency, a condition not thought to be clinically important, protect against hemolytic anemia in chronic hepatitis C patients receiving ribavirin. In this study, we evaluated the clinical significance of ITPA variants in Japanese hepatitis C patients who were treated with pegylated interferon plus ribavirin.