Background Hepatitis C virus (HCV) causes chronic liver disease that often leads to cirrhosis and hepatocellular carcinoma. In animal studies, chimpanzees were protected against chronic infection following experimental challenge with either homologous or heterologous HCV genotype 1a strains which predominates in the USA and Canada. We describe a first in humans clinical trial of this prophylactic HCV vaccine. Methods HCV E1E2 adjuvanted with MF59C.1 (an oil-in-water emulsion) was given at 3 different dosages on day 0 and weeks 4, 24 and 48 in a phase 1, placebo-controlled, dose escalation trial to healthy HCV-negative adults. Results There was no significant difference in the proportion of subjects reporting adverse events across the groups. Following vaccination subjects developed antibodies detectable by ELISA, CD81 neutralization and VSV/HCV pseudotype neutralization. There was no significant difference between vaccine groups in the number of responders and geometric mean titers for each of the three assays. All subjects developed lymphocyte proliferation responses to E1E2 and an inverse response to increasing amounts of antigen was noted. Conclusions The vaccine was safe and generally well tolerated at each of the 3 dosage levels and induced anti-body and lymphoproliferative responses. A larger study to further evaluate safety and immunogenicity is warranted.

The role of autophagy in disease pathogenesis following viral infection is beginning to be elucidated. We have previously reported that hepatitis C virus (HCV) infection in hepatocytes induces autophagy. However, the biological significance of HCV-induced autophagy has not been clarified. Autophagy has recently been identified as a novel component of the innate immune system against viral infection. In this study, we found that knockdown of autophagy-related protein beclin 1 (BCN1) or autophagy-related protein 7 (ATG7) in immortalized human hepatocytes (IHHs) inhibited HCV growth. BCN1-or ATG7-knockdown IHHs, when they were infected with HCV, exhibited increased expression of interferon-b, 2 0 ,5 0 -oligoadenylate synthetase 1, interferon-a, and interferon-a-inducible protein 27 messenger RNAs of the interferon signaling pathways in comparison with infected control IHHs. A subsequent study demonstrated that HCV infection in autophagy-impaired IHHs displayed caspase activation, poly(adenosine diphosphate ribose) polymerase cleavage, and apoptotic cell death. Conclusion: The disruption of autophagy machinery in HCV-infected hepatocytes activates the interferon signaling pathway and induces apoptosis. Together, these results suggest that HCV-induced autophagy impairs the innate immune response. (HEPATOLOGY 2011;53:406-414)

Hepatitis C virus (HCV) induces autophagosome formation in infected human hepatocytes. We have previously reported that HCV exploits autophagic machinery in favor of virus growth and survival in host cells (S. Shrivastava et al., Hepatology 53:406 -414, 2011); however, the mechanisms for autophagy induction is poorly understood. In the present study, we observed that HCV infection transcriptionally upregulates Beclin1, which forms complex with Vps34, the class III phosphatidylinositol 3-kinase, as a first step for autophagy initiation. Although Bcl-2 has an anti-autophagy effect by its association with Beclin1 in nutrient-deprived cells, our studies revealed that HCV-mediated autophagy occurs independent of Beclin1-Bcl-2 dissociation. Mammalian target of rapamycin (mTOR) is a positive regulator of cell growth and is recognized as an inhibitor of autophagy induction. Our results demonstrated that HCV infection enhances phospho-mTOR expression and its downstream target 4EBP1 activation, suggesting that mTOR is not a negative regulator of HCV-induced autophagy. On the other hand, HCV infection in autophagyimpaired cells reduced phospho-mTOR, mTOR, and phospho-4EBP1 expression. Together, these results suggested that HCV induces autophagy by upregulating Beclin1 and activates mTOR signaling pathway, which in turn may promote hepatocyte growth.

Hepatitis C virus (HCV) is a hepatotropic Flavivirus in theHepacivirus genus. The virus genome contains a linear, positive-strand RNA molecule of ϳ9,500 nucleotides. The HCV genome encodes a polyprotein precursor of about 3,000 amino acids, which is cleaved by both viral and host proteases into structural (core, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins. A number of HCV genomes have been cloned, and sequence divergences indicate several genotypes and a series of subtypes for the virus (19). An estimated 200 million people worldwide and 4 million people in the United States are infected with HCV. The majority of the chronically HCV-infected patients develop end-stage liver disease. Currently, the treatment is limited to a combination therapy of ribavirin and alpha interferon (IFN-␣). This combination therapy is expensive and effective in ϳ50% of HCV genotype 1-infected individuals (45), and the reason for this limited efficacy is not clear. Both viral and host factors may play roles that ultimately affect the level and extent of IFNstimulated gene (ISG) expression and function induced during the course of IFN therapy (11,12,27,28). Thus, there is an urgent need to identify novel antivirals or cellular molecules that can be used for therapy. Viral proteins and nucleic acids are detected by pathogen recognition receptors (PRRs) as pathogen-associated molecular patterns (PAMPs). Subsequently, the signal transduction pathways are activated and trigger type I IFN and the production of other cytokines. Type I IFN then augments the expression of several ISGs and subverts virus replication by a variety of mechanisms (29,32). IFN orchestrates a large number of genes of this antiviral response (14). However, many viruses deploy anti-IFN countermeasures, which for HCV are enacted primarily by the viral protein NS3/4A (5). To identify host factors that modify viral replication, we performed pathwayspecific microarray of IFN-related genes. Our results identified 56 genes that were modulated between HCV-infected immortalized human hepatocytes (IHH) and IFN-pretreated, HCVinfected IHH. After validation, we focused on two IFN effector molecules, ISG56 (also known as IFIT1) and IFITM1, in determining antiviral effect in the current study. The overexpression of ISG56 is known to suppress HCV internal ribosome entry site (IRES)-mediated transcription, suggesting its potential as an anti-HCV molecule (43). On the other hand, IFITM family proteins are emerging as antiviral ISGs (2, 13, 24). Our results revealed that the exogenous expression of ISG56 or IFITM1 inhibits HCV replication and growth in infected hepatocytes. MATERIALS AND METHODSCell lines and IFN-␣. The generation of immortalized human hepatocytes (IHH) by the transfection of the HCV core was described previously (30). Huh7, Huh7.5, and IHH were maintained in Dulbecco's modified Eagle's medium (DMEM) (Cambrex, Walkersville, MD) containing 10% fetal bovine serum (FBS), 100 U/ml of penicillin G, and 100 g/ml of streptomycin at 37°C in...

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. We showed previously that HCV induces autophagy for viral persistence by preventing the innate immune response. Knockdown of autophagy reduces extracellular HCV release, although the precise mechanism remains unknown. In this study, we observed that knockdown of autophagy genes enhances intracellular HCV RNA and accumulates infectious virus particles in cells. Since HCV release is linked with the exosomal pathway, we examined whether autophagy proteins associate with exosomes in HCVinfected cells. We observed an association between HCV and the exosomal marker CD63 in autophagy knockdown cells. Subsequently, we observed that levels of extracellular infectious HCV were significantly lower in exosomes released from autophagy knockdown cells. To understand the mechanism for reduced extracellular infectious HCV in the exosome, we observed that an interferon (IFN)-stimulated BST-2 gene is upregulated in autophagy knockdown cells and associated with the exosome marker CD63, which may inhibit HCV assembly or release. Taken together, our results suggest a novel mechanism involving autophagy and exosome-mediated HCV release from infected hepatocytes. IMPORTANCEAutophagy plays an important role in HCV pathogenesis. Autophagy suppresses the innate immune response and promotes survival of virus-infected hepatocytes. The present study examined the role of autophagy in secretion of infectious HCV from hepatocytes. Autophagy promoted HCV trafficking from late endosomes to lysosomes, thus providing a link with the exosome. Inhibition of HCV-induced autophagy could be used as a strategy to block exosome-mediated virus transmission.

Hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, were used with MF59 adjuvant as a candidate vaccine for a phase I safety and immunogenicity trial. Ten of the 41 vaccinee sera tested displayed a VSV/HCV surrogate pseudotype neutralization titer of ≥1/20, 15 of the 36 sera tested had a neutralization titer of ≥ 1/400 against HIV/HCV pseudotype, and 10 of the 36 sera tested had a neutralization titer of ≥1/20 of cell culture grown HCVgenotype 1a. Neutralizing sera had increased affinity, and displayed >2 fold higher specific activity to well characterized epitopes on E1/E2, especially to the hypervariable region 1 (HVR1) of E2.

Hepatitis C virus (HCV) is a global health burden with an estimated 170–200 million peoples chronically infected worldwide. HCV infection remains as an independent risk factor for chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, and a major reason for liver transplantation. Discovery of direct acting antiviral (DAA) drugs have shown promising results with more than 90% success rate in clearing the HCV RNA in patients, although long-term consequences remain to be evaluated. microRNAs (miRNAs) are important players in establishment of HCV infection and target crucial host cellular factors needed for productive HCV replication and augmented cell growth. Altered expression of miRNAs is involved in the pathogenesis associated with HCV infection by controlling signaling pathways such as immune response, proliferation and apoptosis. miRNA is emerging as a means of communication between various cell types inside the liver. There is likely possibility of developing circulating miRNAs as biomarkers of disease progression and can also serve as diagnostic tool with potential of early therapeutic intervention in HCV associated end stage liver disease. This review focuses on recent studies highlighting the contribution of miRNAs in HCV life cycle and their coordinated regulation in HCV mediated liver disease progression.

Hepatitis C virus (HCV) infection often causes liver diseases, including fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Liver fibrosis is the outcome of the wound healing response to tissue damage caused by chronic HCV infection. This process is characterized by the excessive accumulation of extracellular matrix (ECM) proteins, such as collagen fibers secreted by activated hepatic stellate cells (HSCs). Activation of HSCs from the quiescent stage is mediated by different mechanisms, including pro-inflammatory cytokines and chemokines released from HCV-infected hepatocytes and liver macrophages. HCV infection modulates the expression of different microRNAs that can be transported and delivered to the HSCs via exosomes released from infected cells, also leading to the development of advanced disease pathogenesis. Although recent advancements in direct-acting antiviral (DAA) treatment can efficiently control viremia, there are very few treatment strategies available that can be effective at preventing pathogenesis in advanced liver fibrosis or cirrhosis in patients. Assessment of fibrosis is considered to be the major part of proper patient care and decision making in clinical practice. In this review, we highlighted the current knowledge of molecular mechanisms responsible for the progression of liver fibrosis in chronically HCV-infected patients, and currently available methods for evaluation of fibrosis in patients. A detailed understanding of these aspects at the molecular level may contribute to the development of new therapies targeting HCV-related liver fibrosis.