Tetsuo Takehara

Daclatasvir Plus Asunaprevir for Chronic HCV Genotype 1b Infection

All‐oral combinations of direct‐acting antivirals may improve efficacy and safety outcomes for patients with hepatitis C virus (HCV) infection, particularly those who are poor candidates for current interferon/ribavirin‐based regimens. In this open‐label, phase 3 study, 135 interferon‐ineligible/intolerant and 87 nonresponder patients with chronic HCV genotype 1b infection were enrolled at 24 centers in Japan. Patients received daclatasvir 60 mg once daily plus asunaprevir 100 mg twice daily for 24 weeks. The primary endpoint was sustained virologic response 24 weeks after treatment (SVR24). This study is registered with ClinicalTrials.gov (NCT01497834). SVR24 was achieved by 87.4% of interferon‐ineligible/intolerant patients and 80.5% of nonresponder (null and partial) patients; rates were similar in cirrhosis (90.9%) and noncirrhosis (84.0%) patients, and in patients with IL28B CC (84.5%) or non‐CC (84.8%) genotypes. Fourteen patients in each group (12.6%) discontinued dual therapy, mainly due to adverse events or lack of efficacy. Nine nonresponder patients received additional treatment with peginterferon/ribavirin per protocol‐defined criteria. The rate of serious adverse events was low (5.9%) and varied among patients. The most common adverse events were nasopharyngitis, increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST), headache, diarrhea, and pyrexia. Conclusion: Interferon‐free, ribavirin‐free all‐oral therapy with daclatasvir and asunaprevir for 24 weeks is well tolerated and can achieve a high rate of SVR in patients with HCV genotype 1b who were ineligible, intolerant, or had not responded to prior interferon‐based therapy. (Hepatology 2014;59:2083–2091)

Quantitative analysis of hepatitis C virus RNA in serum during interferon alfa therapy

BACKGROUND Interferon alfa is effective for controlling disease activity in chronic hepatitis C. However, many responders suffer relapse after cessation of therapy. In the present study, the serum concentration of hepatitis C virus RNA was correlated with a sustained response to interferon therapy. METHODS Fifty-three patients with chronic hepatitis C received a 26-week course of interferon alfa. Hepatitis C virus RNA was quantitated in serum at the beginning and end of treatment using a competitive assay that combined reverse transcription and polymerase chain reaction. RESULTS In long-term responders, whose alanine aminotransferase levels remained within the normal range during the 24 weeks after therapy, the titer of viral RNA (logarithmic transformed copy numbers per milliliter of serum) before therapy (7.1 +/- 1.2) was significantly lower (P < 0.001) than that of short-term responders (8.3 +/- 0.5) who had a relapse within the 24 weeks after therapy and nonresponders (8.1 +/- 0.4). Multivariate multiple logistic regression showed that the titer of viral RNA before therapy was the strongest independent predictor of a sustained response to interferon-alfa therapy (P = 0.002). CONCLUSIONS The titer of hepatitis C virus RNA is the most important factor influencing the sustained response to interferon treatment.

Critical role of MHC class I-related chain A and B expression on IFN-α-stimulated dendritic cells in NK cell activation: Impairment in chronic hepatitis C virus infection

Dendritic cells (DCs) augment effector functions of NK cells, but the underlying mechanisms are not fully understood. Here we show in an in vitro coculture system that human monocyte-derived DCs enhance IFN-γ production, CD69 expression, and K562 cytolytic ability of NK cells when DCs are prestimulated with various maturation stimuli such as IFN-α or LPS. Of interest is the finding that NK cell activation mediated by LPS-stimulated DCs was dependent on IL-12 produced in DC/NK coculture, but that IFN-α-stimulated DC-mediated activation was not. Alternatively, MHC class I-related chain A and B (MICA/B), ligands for NKG2D activating receptor, were found to be induced on DCs upon IFN-α stimulation and to be responsible for the NK activation because mAb-mediated masking of MICA/B as well as inhibition of direct cell-to-cell contact using transwell insert completely abolished DC-dependent NK cell activation by IFN-α. Finally, DCs recovered from chronic hepatitis C virus-infected patients showed defects in the induction of MICA/B and impaired ability to activate NK cells in response to IFN-α stimulation. These findings suggested that MICA/B induction on DCs may be one of the mechanisms by which IFN-α activates NK cells; this impairment might affect IFN-α responsiveness in hepatitis C virus infection.

Ledipasvir and sofosbuvir fixed-dose combination with and without ribavirin for 12 weeks in treatment-naive and previously treated Japanese patients with genotype 1 hepatitis C: an open-label, randomised, phase 3 trial

BACKGROUND Compared with other countries, patients with chronic hepatitis C infection in Japan tend to be older, have more advanced liver disease, and are more likely to have been previously treated for hepatitis C. We aimed to assess the efficacy and safety of an all-oral, fixed-dose combination of the hepatitis C virus NS5A inhibitor ledipasvir and the NS5B nucleotide polymerase inhibitor sofosbuvir with and without ribavirin for 12 weeks in treatment-naive and previously treated Japanese patients with chronic genotype 1 hepatitis C virus infection. METHODS In this randomised, open-label study, we enrolled patients from 19 clinical Japanese centres. Patients were randomly assigned (1:1) to receive either ledipasvir (90 mg) and sofosbuvir (400 mg) or ledipasvir, sofosbuvir, and ribavirin (dosed according to the Japanese Copegus product label-ie, patients ≤60 kg received 600 mg daily, patients >60 kg to ≤80 kg received 800 mg daily, and patients >80 kg received 1000 mg daily) orally once daily for 12 weeks. After completion or early discontinuation of treatment, patients were followed up off-treatment for 24 weeks. Eligible patients were at least 20 years of age with chronic genotype 1 hepatitis C virus infection with serum hepatitis C virus RNA concentrations of at least 5 log10 IU/mL, creatinine clearance of at least 1·0 mL/s, and a platelet count of at least 50 × 10(9) per L. An interactive web response system was used to manage patient randomisation and treatment assignment. Randomisation was stratified by the presence or absence of cirrhosis for treatment-naive patients and stratified by presence or absence of cirrhosis and by previous treatment category (relapser or breakthrough, non-responder, or interferon-intolerant) for previously treated patients. Within each strata, patients were sequentially assigned to either treatment with ledipasvir-sofosbuvir or ledipasvir-sofosbuvir plus ribavirin in a 1:1 ratio with block size of 4. The primary endpoint was sustained virological response 12 weeks after completion of treatment (SVR12) assessed in all patients who were randomly assigned and received at least one dose of study drug; safety outcomes were assessed in all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, number NCT01975675. FINDINGS Between Oct 15, 2013 and Dec 13, 2013, 341 patients were randomly assigned to treatment groups and received at least one dose of study treatment. SVR12 was achieved in all 171 (100%) patients (83 of 83 treatment naive and 88 of 88 treatment experienced) receiving ledipasvir-sofosbuvir (95% CI 98-100) and 167 (98%) of 170 patients (80 of 83 treatment naive and 87 of 87 treatment experienced) receiving ledipasvir-sofosbuvir plus ribavirin (95% CI 95-100). Of the 76 patients with baseline NS5A resistant variants, 75 (99%) achieved SVR12. Two (1·2%) of 170 patients in the ledipasvir-sofosbuvir plus ribavirin group discontinued treatment because of adverse events. The most common adverse events were nasopharyngitis (50 [29·2%] of 171), headache (12 [7·0%] of 171), and malaise (nine [5·3%] of 171) in patients receiving ledipasvir-sofosbuvir; and nasopharyngitis (40 [23·5%] of 170), anaemia (23 [13·5%] of 170), and headache in those receiving ledipasvir-sofosbuvir and ribavirin (15 [8·8%] of 170). INTERPRETATION Although existing regimens for the treatment of hepatitis C virus are effective for many patients, medical needs remain unmet, particularly in Japan where the population with hepatitis C virus genotype 1 is generally older and treatment-experienced, with advanced liver disease. The efficacy, tolerability, and absence of drug-drug interactions of ledipasvir-sofosbuvir suggest that it could be an important option for treatment of genotype 1 hepatitis C virus in Japanese patients. FUNDING Gilead Sciences.

Reduced Numbers and Impaired Ability of Myeloid and Plasmacytoid Dendritic Cells to Polarize T Helper Cells in Chronic Hepatitis C Virus Infection

Hepatitis C virus (HCV) infection induces a wide range of chronic liver injuries. The mechanism by which HCV evades the immune surveillance system remains obscure. Blood dendritic cells (DCs) consist of myeloid and plasmacytoid subsets that play distinct roles in the regulation of antivirus immune responses; however, their roles in the pathogenesis of HCV infection are yet to be determined. We compared the numbers and functions of myeloid and plasmacytoid DCs between 43 patients with chronic hepatitis and 26 age-matched healthy volunteers. Absolute numbers of myeloid DCs, plasmacytoid DCs, and DC progenitors in the periphery were significantly lower in patients with chronic hepatitis than in healthy volunteers. Myeloid and plasmacytoid DCs from the patients had impaired abilities to stimulate allogeneic CD4 T cells and to produce interleukin (IL)-12 p70 and interferon- alpha , compared with those from healthy volunteers. After exposure to naive CD4 T cells, myeloid DCs from the patients were less able to drive the T helper type 1 response, whereas myeloid and plasmacytoid DCs from the patients primed more IL-10-producing cells than did those from healthy volunteers. In conclusion, in chronic HCV infection, both types of blood DCs are reduced and have an impaired ability to polarize T helper cells.

Expression and role of MICA and MICB in human hepatocellular carcinomas and their regulation by retinoic acid

Natural killer (NK) cells are important effector cells for the first line of defense against tumor, but the mechanisms by which they recognize and kill human hepatocellular carcinoma (HCC) remains to be elucidated. Distant MHC class I homologs MICA and MICB are recently identified human ligands for NK cell activating receptor NKG2D. In our present study, MICA or MICB transcript was detected in 6 of 10 human hepatocellular carcinoma tissues, but not in the surrounding non‐cancerous tissues. Immunohistochemical analysis showed that MICA/B were expressed in the tumor cells of the cancerous tissues. Huh7 and HepG2 hepatoma cells, but not Hep3B cells, substantially expressed MICA/B on their cell surface. MICA/B expressed on hepatoma cells contributed to their NK sensitivity, because Huh7 and HepG2 were less susceptible to NK cytolysis when MAb against MICA/B was added during the cytolysis assay. Of interest is the finding that retinoic acid upregulated expression of MICA/B in Huh7 and HepG2 cells. Retinoic acid‐treated hepatoma cells induced IFNγ production from cocultured NK cells and rendered themselves more susceptible to NK cells. This was clearly dependent on upregulation of MICA/B, because both the enhanced IFNγ production and NK cytolysis were completely abolished by MAb‐mediated masking of MICA/B. These results suggest that MICA/B, expressed on a subset of human HCCs, may play an important role in their susceptibility to NK cells. Furthermore, retinoic acid can function as a modulator of MICA/B expression and thereby further activate NK cells.

Negative Regulation of NK Cell Activities by Inhibitory Receptor CD94/NKG2A Leads to Altered NK Cell-Induced Modulation of Dendritic Cell Functions in Chronic Hepatitis C Virus Infection

NK cells are potent activators of dendritic cells (DCs), but it remains obscure how third-party cells affect the ability of NK cells to modulate DC functions. We show here that NK cells derived from healthy donors (N-NK), when cocultured with human liver epithelial cells, induced maturation as well as activation of DCs, such as increased migratory capacity as well as T cell stimulatory activity. In contrast, NK cells from chronic hepatitis C virus-infected donors (HCV-NK) were not capable of activating DCs under the same conditions. In comparison to N-NK, HCV-NK showed higher expression of CD94/NKG2A and produced IL-10 and TGFβ when cultured with hepatic cells, most of which express HLA-E, a ligand for CD94/NKG2A. Blockade of NKG2A restored the ability of HCV-NK to activate DCs, which appeared to result from the reduced NK cell production of IL-10 and TGFβ. The blockade also endowed HCV-NK with an ability to drive DCs to generate Th1-polarized CD4+ T cells. These findings show that NK cell modulation of DCs is regulated by third-party cells through NK receptor and its ligand interaction. Aberrant expression of NK receptors may have an impact on the magnitude and direction of DC activation of T cells under pathological conditions, such as chronic viral infection.

Autocrine/Paracrine IL-15 That Is Required for Type I IFN-Mediated Dendritic Cell Expression of MHC Class I-Related Chain A and B Is Impaired in Hepatitis C Virus Infection

We previously reported that monocyte-derived dendritic cells activate resting NK cells by expressing MHC class I-related chain A and B (MICA/B), ligands for NKG2D, in response to IFN-α, but the MICA/B expression was severely impaired in patients with chronic hepatitis C virus (HCV) infection. In the present study, we examined induction of MICA/B on DCs by various innate cytokines and found that DCs from either healthy donors or HCV-infected individuals, upon IL-15 stimulation, express MICA/B and can activate NK cells, which is solely dependent on MICA/B-NKG2D interaction. Of interest is the finding that IL-15- and type I IFN-mediated induction of MICA/B in healthy donors is completely inhibited when DCs are incubated in the presence of anti-IFN-α/βR or anti-IL-15Rα, respectively, suggesting interdependent roles of these cytokines in MICA/B expression. Indeed, DCs produced IL-15 in response to type I IFN, whereas they directly produced IFN-β, in response to IL-15, which was followed by the production of IFN-α. In HCV-infected individuals, type I IFN-mediated production of IL-15 was virtually absent, but IL-15-mediated production of type I IFN was not compromised, which is consistent with the distinct ability of these cytokines to induce MICA/B in these patients. The present study demonstrates that IL-15 and type I IFN lead to DC expression of MICA/B and subsequent DC activation of NK cells, which is critically dependent on each other’s autocrine/paracrine effect, and suggests that impaired IL-15 production is one of the mechanisms of the aberrant response of DC to type I IFN in HCV-infected patients.

Antiviral therapy for chronic hepatitis C: past, present, and future

Antiviral therapy for chronic hepatitis C has dramatically advanced since the discovery of the hepatitis C virus (HCV) in 1989 and the introduction of interferon (IFN) monotherapy in the early 1990s. The current standard therapy uses a combination of pegylated IFN and ribavirin. The duration of therapy and response to therapy are HCV genotype-specific. Genotype 1 patients require 48 weeks of the combination therapy for 50% successful viral elimination, while genotype 2 patients require 24 weeks of therapy for 80% or 90% viral elimination. Early viral kinetics after the initiation of therapy is a useful predictor of the sustained virologic response (SVR), which is formally determined at 24 weeks after completion of the treatment. For example, an early virologic response, which is determined by a 2-log reduction of HCV RNA or viral elimination at 12 weeks after the initiation of therapy, is a strong negative predictor of SVR in genotype 1 patients. In contrast, a rapid virologic response of HCV RNA-negative at 4 weeks after the initiation of therapy identifies genotype 2 “super-responders,” who may require a shorter period of therapy. Adherence to therapy is one of the most important factors for successful viral clearance. Hematopoietic growth factors such as epoetin and granulocyte-colony stimulating factor help reduce therapy-mediated cytopenia and improve patient compliance, thereby leading to better viral clearance. New types of anti-HCV agents such as HCV protease and polymerase inhibitors are needed for those patients that do not respond to combination therapy.

Cytoplasmic STAT3 represses autophagy by inhibiting PKR activity.

In a screen designed to identify novel inducers of autophagy, we discovered that STAT3 inhibitors potently stimulate the autophagic flux. Accordingly, genetic inhibition of STAT3 stimulated autophagy in vitro and in vivo, while overexpression of STAT3 variants, encompassing wild-type, nonphosphorylatable, and extranuclear STAT3, inhibited starvation-induced autophagy. The SH2 domain of STAT3 was found to interact with the catalytic domain of the eIF2α kinase 2 EIF2AK2, best known as protein kinase R (PKR). Pharmacological and genetic inhibition of STAT3 stimulated the activating phosphorylation of PKR and consequent eIF2α hyperphosphorylation. Moreover, PKR depletion inhibited autophagy as initiated by chemical STAT3 inhibitors or free fatty acids like palmitate. STAT3-targeting chemicals and palmitate caused the disruption of inhibitory STAT3-PKR interactions, followed by PKR-dependent eIF2α phosphorylation, which facilitates autophagy induction. These results unravel an unsuspected mechanism of autophagy control that involves STAT3 and PKR as interacting partners.