Nobutoshi Komatsu

Potential Relevance of Cytoplasmic Viral Sensors and Related Regulators Involving Innate Immunity in Antiviral Response

BACKGROUND & AIMS Clinical significance of molecules involving innate immunity in treatment response remains unclear. The aim is to elucidate the mechanisms underlying resistance to antiviral therapy and predictive usefulness of gene quantification in chronic hepatitis C (CH-C). METHODS We conducted a human study in 74 CH-C patients treated with pegylated interferon alpha-2b and ribavirin and 5 nonviral control patients. Expression of viral sensors, adaptor molecule, related ubiquitin E3-ligase, and modulators were quantified. RESULTS Hepatic RIG-I, MDA5, LGP2, ISG15, and USP18 in CH-C patients were up-regulated at 2- to 8-fold compared with nonhepatitis C virus patients with a relatively constitutive Cardif. Hepatic RIG-I, MDA5, and LGP2 were significantly up-regulated in nonvirologic responders (NVR) compared with transient (TR) or sustained virologic responders (SVR). Cardif and RNF125 were negatively correlated with RIG-I and significantly suppressed in NVR. Differences among clinical responses in RIG-I/Cardif and RIG-I/RNF125 ratios were conspicuous (NVR/TR/SVR = 1.3:0.6:0.4 and 2.3:1.3:0.8, respectively). Like viral sensors, ISG15 and USP18 were significantly up-regulated in NVR (4-fold and 2.3-fold, respectively). Multivariate and receiver operator characteristic analyses revealed higher RIG-I/Cardif ratio, ISG15, and USP18 predicted NVR. Lower Cardif in NVR was confirmed by its protein level in Western blot. Also, transcriptional responses in peripheral blood mononuclear cells to the therapy were rapid and strong except for Cardif in not only a positive (RIG-I, ISG15, and USP18) but also in a negative regulatory manner (RNF125). CONCLUSIONS NVR may have adopted a different equilibrium in their innate immune response. High RIG-I/Cardif and RIG-I/RNF125 ratios and ISG15 and USP18 are useful in identifying NVR.

Deep sequencing analysis of variants resistant to the non-structural 5A inhibitor daclatasvir in patients with genotype 1b hepatitis C virus infection

Daclatasvir, a non‐structural (NS)5A replication complex inhibitor, is a potent and promising direct antiviral agent (DAA) for hepatitis C virus (HCV), being most effective in genotype 1b infection. Although it is known that genotype 1b viruses with Y93H and/or L31M/V/F mutations have strong resistance to daclatasvir, it is not known whether there are some clinical background conditions that favor the occurrence of HCV carrying those NS5A mutations.

The presence of steatosis and elevation of alanine aminotransferase levels are associated with fibrosis progression in chronic hepatitis C with non-response to interferon therapy

BACKGROUND/AIMS Interferon (IFN) therapy leads to regression of hepatic fibrosis in chronic hepatitis C patients who achieve a sustained virologic response (SVR), while the beneficial effect is limited in those who fail to do so. The aim of the present study was to define factors associated with progression of fibrosis in patients who do not achieve a SVR. METHODS Fibrosis staging scores were compared between paired liver biopsies before and after IFN in 97 chronic hepatitis C patients who failed therapy. The mean interval between biopsies was 5.9 years. Factors associated with progression of fibrosis were analyzed. RESULTS Fibrosis progressed in 23%, remained unchanged in 47% and regressed in 29%. Steatosis and a high average alanine aminotransferase (ALT) between biopsies were independent factors for progression of fibrosis with risk ratios of 5.53 and 4.48, respectively. Incidence and yearly rate of progression of fibrosis was 64% and 0.22+/-0.29 fibrosis units per year in those with both risk factors compared to 8% and -0.04+/-0.17 fibrosis units per year in those negative for both factors. CONCLUSIONS Hepatic steatosis and elevated ALT levels are risk factors for progression of fibrosis in chronic hepatitis C patients who fail to achieve a SVR to IFN therapy and therefore may be therapeutic targets to halt the potentially progressive disease.

Hepatocellular carcinoma risk assessment using gadoxetic acid-enhanced hepatocyte phase magnetic resonance imaging.

To investigate whether the patients with hypovascular liver nodules determined on the arterial phase and hypointensity on the hepatocyte phase gadoxetic acid‐enhanced magnetic resonance imaging (hypovascular hypointense nodules) are at increased risk of hepatocarcinogenesis, we assessed subsequent typical hepatocellular carcinoma (HCC) development at any sites of the liver with and without such nodules.

IL-28B (IFN-λ3) and IFN-α synergistically inhibit HCV replication

Genetic variation in the IL‐28B (interleukin‐28B; interferon lambda 3) region has been associated with sustained virological response (SVR) rates in patients with chronic hepatitis C treated with peginterferon‐α and ribavirin. However, the mechanisms by which polymorphisms in the IL‐28B gene region affect host antiviral responses are not well understood. Using the HCV 1b and 2a replicon system, we compared the effects of IFN‐λs and IFN‐α on HCV RNA replication. The anti‐HCV effect of IFN‐λ3 and IFN‐α in combination was also assessed. Changes in gene expression induced by IFN‐λ3 and IFN‐α were compared using cDNA microarray analysis. IFN‐λs at concentrations of 1 ng/mL or more exhibited concentration‐ and time‐dependent HCV inhibition. In combination, IFN‐λ3 and IFN‐α had a synergistic anti‐HCV effect; however, no synergistic enhancement was observed for interferon‐stimulated response element (ISRE) activity or upregulation of interferon‐stimulated genes (ISGs). With respect to the time course of ISG upregulation, the peak of IFN‐λ3‐induced gene expression occurred later and lasted longer than that induced by IFN‐α. In addition, although the genes upregulated by IFN‐α and IFN‐λ3 were similar to microarray analysis, interferon‐stimulated gene expression appeared early and was prolonged by combined administration of these two IFNs. In conclusion, IFN‐α and IFN‐λ3 in combination showed synergistic anti‐HCV activity in vitro. Differences in time‐dependent upregulation of these genes might contribute to the synergistic antiviral activity.

Comprehensive analysis for viral elements and interleukin‐28B polymorphisms in response to pegylated interferon plus ribavirin therapy in hepatitis C virus 1B infection

To comprehensively characterize the contribution of virological factors as well as interleukin‐28B (IL28B) single‐nucleotide polymorphisms (SNPs) in determining treatment responses in pegylated‐interferon plus ribavirin (Peg‐IFN/RBV) therapy for chronic hepatitis C virus (HCV)‐1b infection, we undertook a retrospective cohort analysis for the pretreatment dominant complete HCV open reading frame (ORF) amino‐acid (aa) sequence study in 103 consecutive HCV‐1b Japanese patients. The dominant HCV sequences classified by the response were subjected to systematic sliding‐window comparison analysis to characterize response‐specific viral sequences, along with IL28B SNP analyses (rs8099917). In each comparison of the patients between with and without rapid viral response (RVR), nonearly viral response (nEVR), sustained virological response (SVR), or relapse, the following regions were extracted as most significantly associated with the different responses respectively: nonstructural protein 5A (NS5A) aa.2224‐2248 (P = 1.2E‐07); core aa.70 (P = 4E‐04); NS5A aa.2340‐2382 (P = 7.0E‐08); and NS5A aa.2360‐2377 (P = 1.1E‐05). Those NS5A regions nearly coincided with the interferon (IFN) sensitivity‐determining region (NS5A aa.2209‐2248) and the IFN/RBV resistance‐determining region (NS5A aa.2339‐2379). In a multivariate analysis, the IL28B SNP (odds ratio [OR] = 16.8; P = 0.009) and NS5A aa.2340‐2382 (OR = 13.8; P = 0.0003) were extracted as the two most‐significant independent variables contributing to the final outcome. Conclusion: In Peg‐IFN/RBV therapy, polymorphisms in IL28B, NS5A aa.2224‐2248, core aa.70, and, most important, NS5A aa.2340‐2382 have a tremendous influence on treatment response in association with viral kinetics, resulting in significantly different outcomes in chronic HCV‐1b infection. (HEPATOLOGY 2012;56:1611–1621)

Deep Sequencing and Phylogenetic Analysis of Variants Resistant to Interferon-Based Protease Inhibitor Therapy in Chronic Hepatitis Induced by Genotype 1b Hepatitis C Virus

ABSTRACT Because of recent advances in deep sequencing technology, detailed analysis of hepatitis C virus (HCV) quasispecies and their dynamic changes in response to direct antiviral agents (DAAs) became possible, although the role of quasispecies is not fully understood. In this study, to clarify the evolution of viral quasispecies and the origin of drug-resistant mutations induced by interferon (IFN)-based protease inhibitor therapy, the nonstructural-3 (NS3) region of genotype 1b HCV in 34 chronic hepatitis patients treated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) was subjected to a deep sequencing study coupled with phylogenetic analysis. Twenty-six patients (76.5%) achieved a sustained viral response (SVR), while 8 patients did not (non-SVR; 23.5%). When the complexity of the quasispecies was expressed as the mutation frequency or Shannon entropy value, a significant decrease in the IFNL3 (rs8099917) TT group and a marginal decrease in the SVR group were found soon (12 h) after the introduction of treatment, whereas there was no decrease in the non-SVR group and no significant decrease in mutation frequency in the IFNL3 TG/GG group. In the analysis of viral quasispecies composition in non-SVR patients, major populations greatly changed, accompanied by the appearance of resistance, and the compositions were unlikely to return to the pretreatment composition even after the end of therapy. Clinically TVR-resistant variants were observed in 5 non-SVR patients (5/8, 62.5%), all of which were suspected to have acquired resistance by mutations through phylogenetic analysis. In conclusion, results of the study have important implications for treatment response and outcome in interferon-based protease inhibitor therapy. IMPORTANCE In the host, hepatitis C virus (HCV) consists of a variety of populations (quasispecies), and it is supposed that dynamic changes in quasispecies are closely related to pathogenesis, although this is poorly understood. In this study, recently developed deep sequencing technology was introduced, and changes in quasispecies associated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) triple therapy and their clinical significance were investigated extensively by phylogenetic tree analysis. Through this study, the associations among treatment response, changes in viral quasispecies complexity in the early stage of treatment, changes in the quasispecies composition, and origin of TVR-resistant variant HCV were elucidated.

Liver stiffness measurement for risk assessment of hepatocellular carcinoma

Liver fibrosis is a risk factor for hepatocellular carcinoma (HCC), but at what fibrotic stage the risk for HCC is increased has been poorly investigated quantitatively. This study aimed to determine the appropriate cut‐off value of liver stiffness for HCC concurrence by FibroScan, and its clinical significance in hepatitis B virus (HBV), hepatitis C virus (HCV) and non‐B, non‐C (NBNC) liver disease.

Semiannual Imaging Surveillance Is Associated with Better Survivalin Patients with Non-B, Non-C Hepatocellular Carcinoma

Since it remains elusive whether and how the imaging surveillance affects the survival in patients with non-B, non-C hepatocellular carcinoma (NBNC-HCC), we conducted this retrospective study which investigated the association between the semiannual surveillance prior to HCC diagnosis and the survival in patients with the initial diagnosis of HCC induced by hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infections (N = 141) and non-B, non-C etiology (N = 30). It was demonstrated that surveillance was less frequently performed in the NBNC-HCC patients compared to that in HCC patients with HBV and/or HCV infections (B/C-HCC patients), and the survival was unfavorable in NBNC-HCC patients. On the other hand, the survival of NBNC-HCC patients with semiannual surveillance was significantly favorable than those patients without semiannual surveillance, and the survival was similar between B/C-HCCs and NBNC-HCCs with semiannual surveillance. In conclusion, though NBNC-HCC patients compared to B/C-HCC patients had poorer prognosis overall, these NBNC-HCC patients with semiannual surveillance had a better survival almost equivalent to the survival of B/C-HCC patients with semiannual surveillance, demonstrating the clinical utility of the semiannual imaging surveillance program for NBNC-HCCs.

HBV patients with low HBsAg and high HBcrAg titers have a high risk of HBV-related HCC: HBsAg and HBcrAg in HCC risk assessment

Although the viral markers hepatitis B surface antigen (HBsAg) and hepatitis B core‐related antigen (HbcrAg) could reflect intrahepatic hepatitis B virus (HBV) replication activity and constitute important biomarkers for hepatocellular carcinoma (HCC), the value of using these two markers in combination for assessing HCC risk has not been clarified in detail.