Hepatitis C virus (HCV) infection is a significant public health problem with over 170,000,000 chronic carriers and infection rates increasing worldwide. Chronic HCV infection is one of the leading causes of hepatocellular carcinoma which was estimated to result in ∼10,000 deaths in the United States in the year 2011. Current treatment options for HCV infection are limited to PEG-ylated interferon alpha (IFN-α), the nucleoside ribavirin and the recently approved HCV protease inhibitors telaprevir and boceprevir. Although showing significantly improved efficacy over the previous therapies, treatment with protease inhibitors has been shown to result in the rapid emergence of drug-resistant virus. Here we report the activity of two proteins, originally isolated from natural product extracts, which demonstrate low or sub-nanomolar in vitro activity against both genotype I and genotype II HCV. These proteins inhibit viral infectivity, binding to the HCV envelope glycoproteins E1 and E2 and block viral entry into human hepatocytes. In addition, we demonstrate that the most potent of these agents, the protein griffithsin, is readily bioavailable after subcutaneous injection and shows significant in vivo efficacy in reducing HCV viral titers in a mouse model system with engrafted human hepatocytes. These results indicate that HCV viral entry inhibitors can be an effective component of anti-HCV therapy and that these proteins should be studied further for their therapeutic potential.

Anti-hepatitis C virus (HCV) drug development has been challenged by a lack of experience with inhibitors inclusive of in vitro, animal model, and clinical study. This manuscript outlines activity and correlation across such a spectrum of models and into clinical trials with a novel selective nonstructural protein 5B (NS5B) polymerase inhibitor, HCV796. Enzyme assays yielded median inhibitory concentration ( H epatitis C virus (HCV) infects an estimated 170 million people across the world, 1 with substantial risk of severe chronic liver disease, cirrhosis, and hepatocellular cancer. 2,3 Standard therapy with pegylated interferon and ribavirin clears virus in only half of those treated, is associated with significant treatment-related morbidity, and is cost-prohibitive in much of the world. 4,5 HCV is the leading indication for liver transplantation across the developed world. Despite intensive effort, no new drugs have been approved for therapy in the last decade. More effective and less toxic anti-HCV therapeutics as well as preventive vaccines are greatly needed. A major challenge with development of anti-HCV drugs has been the lack of in vitro, animal model, and clinical experience with inhibitors. Although correlation exists for interferon, there are limited data for molecules with novel mechanisms. The goal of the current work was to delineate the activity of a selective nonstructural protein 5B (NS5B) polymerase inhibitor in vitro by enzyme assay and in the replicon, in vivo in the chimeric mouse model, and in patients infected with HCV.Abbreviations: EC 50 , half maximal effective concentration; hAAT, Human ␣-1 antitrypsin; HCV, hepatitis C virus; IC 50 , median inhibitory concentration; IFN, interferon; NS5B, nonstructural protein 5B; pegylated interferon; RdRp, SCID, severe combined immunodeficiency; uPA, urokinase plasminogen activator. From

Direct acting antivirals (DAAs) have led to a high cure rate in treated patients with chronic hepatitis C virus (HCV) infection but this still leaves a large number of treatment failures secondary to the emergence of resistance-associated variants (RAVs). To increase the barrier to resistance, a complementary strategy is to employ neutralizing human monoclonal antibodies (HMAbs) to prevent acute infection. However, earlier efforts with the selected antibodies led to RAVs in animal and clinical studies. Therefore, we identified a HMAb that is less likely to elicit RAVs for affinity maturation to increase potency and, more importantly, breadth of protection. Selected matured antibodies show improved affinity and neutralization against a panel of diverse HCV isolates. Structural and modeling studies reveal that the affinity matured HMAb mediates virus neutralization in part by inducing conformational change to the targeted epitope and that the maturated light chain is responsible for the improved affinity and breadth of protection. A matured HMAb protected humanized mice when challenged with an infectious HCV human serum inoculum for a prolonged period. However, a single mouse experienced breakthrough infection after 63 days when the serum HMAb concentration dropped by several logs; sequence analysis revealed no viral escape mutation. Conclusions The findings suggest that a single broadly neutralizing antibody can prevent acute HCV infection without inducing RAVs and may complement DAAs to reduce the emergence of RAVs.

remains partly unclear what determines the human specificity of HCV infection. Presumably, the presence of appropriate entry receptors is essential, and this may explain why HCV is unable to infect nonhuman hepatocytes. However, using mice with chimeric human livers, we show in this study that the presence of human hepatocytes, and therefore human entry receptors, is not sufficient for HCV infection. In successfully transplanted SCID/Alb-uPA mice, infection with HCV is reliable only when ϳ70 -80% of the liver consists of human hepatocytes. We show that chimeric mice, which are hard to infect with HCV, have significant groups of human hepatocytes that are readily infected with hepatitis B virus. Thus it is unlikely that the lack of infection with HCV can simply be attributed to low hepatocyte numbers. We investigated whether the humanization of lipoprotein profiles is positively associated with infection success. We show that the lipoprotein profiles of chimeric mice become more human-like at high levels of engraftment of human hepatocytes. This and expression of markers of human lipoprotein biosynthesis, human apolipoprotein B (ApoB) and cholesterol ester transfer protein (CETP), show a strong positive correlation with successful infection. Association of HCV in the blood of chimeric mice to ApoB-containing lipoproteins is comparable to association of HCV in patient serum and provides further support for a critical role for ApoB-containing lipoproteins in the infectious cycle of HCV. Our data suggest that the weakest link in the HCV infection chain does not appear to be the presence of human hepatocytes per se. We believe that HCV infection also depends on the presence of sufficient levels of human lipoproteins. apolipoprotein B; low-density lipoprotein; xenotransplantation; immunodeficient mouse; Flavivirus HEPATITIS C VIRUS (HCV) is a small enveloped, positive-strand RNA virus of the family of Flaviviridae and the only member of the genus Hepacivirus. Another genera in the family are Flavivirus, such as West Nile and dengue virus, and Pestivirus, such as bovine diarrhea virus. These viruses have a positivestrand RNA genome in common. Their genome consists of a single open reading frame, encoding a polyprotein that is cleaved co-and posttranslationally (11). Most Flaviviruses can infect a large range of hosts (11), but tropism of HCV infection is limited to humans and chimpanzees, and it remains unclear why other species are not susceptible to HCV infection. Possibly, the lack of entry receptors on the surface of liver cells of other species prevents infection, and recent evidence may support that idea: expression of human occludin, together with human CD81, allows for infection of mouse cells with hepatitis C pseudoparticles (18). However, replication of HCV in, for example, mouse cells is inefficient (23, 28), and there is no evidence that assembly and release of virions occur in murine hepatocytes. It is therefore likely that several other proteins or processes contribute to the tight association of HCV infe...

Human hepatocyte transplantation is an alternative treatment for acute liver failure and liver diseases involving enzyme deficiencies. Although it has been successfully applied in selected recipients, both isolation and transplantation outcomes have the potential to be improved by better donor selection. This study assessed the impact of various donor variables on isolation outcomes (yield and viability) and posttransplant engraftment, using the SCID/Alb-uPA (severe combined immunodeficient/urokinase type plasminogen activator under the control of an albumin promoter) human liver chimeric mouse model. Human hepatocytes were obtained from 90 human liver donor specimens and were transplanted into 3942 mice. Multivariate analysis revealed improved viability with younger donors (P ¼ 0.038) as well as with shorter warm ischemic time (P ¼ 0.012). Hepatocyte engraftment, assessed by the posttransplant level of serum human a1-antitrypsin, was improved with shorter warm ischemia time. Hepatocytes isolated from older donors (!60 years) had lower viability and posttransplant engraftment (P 0.01). In conclusion, the selection of young donors (<60 years) and rapid liver specimen retrieval, allowing for shorter warm ischemia time, are key determinants for the success of both the isolation of high viability human hepatocytes and their subsequent posttransplantation capacity for engraftment and expansion. Liver Transpl 16:974-982,

Cytopathic effects are currently believed to contribute to hepatitis C virus (HCV)-induced liver injury and are readily observed in Huh7.5 cells infected with the JFH-1 HCV strain, manifesting as apoptosis highly correlated with growth arrest. Reactive oxygen species, which are induced by HCV infection, have recently emerged as activators of AMP-activated protein kinase. The net effect is ATP conservation via on/off switching of metabolic pathways that produce/consume ATP. Depending on the scenario, this can have either pro-survival or pro-apoptotic effects. We demonstrate reactive oxygen species-mediated activation of AMP-activated kinase in Huh7.5 cells during HCV (JFH-1)-induced growth arrest. Metabolic labeling experiments provided direct evidence that lipid synthesis is attenuated, and ␤-oxidation is enhanced in these cells. A striking increase in nuclear peroxisome proliferator-activated receptor ␣, which plays a dominant role in the expression of ␤-oxidation genes after ligand-induced activation, was also observed, and we provide evidence that peroxisome proliferator-activated receptor ␣ is constitutively activated in these cells. The combination of attenuated lipid synthesis and enhanced ␤-oxidation is not conducive to lipid accumulation, yet cellular lipids still accumulated during this stage of infection. Notably, the serum in the culture media was the only available source for polyunsaturated fatty acids, which were elevated (2-fold) in the infected cells, implicating altered lipid import/export pathways in these cells. This study also provided the first in vivo evidence for enhanced ␤-oxidation during HCV infection because HCV-infected SCID/Alb-uPA mice accumulated higher plasma ketones while fasting than did control mice. Overall, this study highlights the reprogramming of hepatocellular lipid metabolism and bioenergetics during HCV infection, which are predicted to impact both the HCV life cycle and pathogenesis.With ϳ200 million people infected worldwide, hepatitis C virus (HCV) 2 is a global health problem and a major cause of viral hepatitis. Persistent infection occurs in ϳ70% of infected patients leading to inflammation, insulin resistance, steatosis, fibrosis, and hepatocellular carcinoma (1). Current direct-acting antivirals are predicted to be a cure for most patients, but the high cost of this treatment means that the pathological consequences of persistent HCV infection will remain a concern.Although the pathology associated with chronic HCV infection was initially thought to be due to HCV-specific immune responses (2), the current opinion is that direct cytopathic effects in virally infected cells also contribute to HCV-associated liver injury (3, 4). The cellular mechanisms by which HCV replication might mediate liver injury are unclear, but there is no doubt that oxidative/nitrosative stress in HCV-infected cells plays an important role in the initiation and progression of liver damage (3, 5, 6). Oxidative/nitrosative stress essentially arises when the production of reactive oxygen (...

1. Immunocompromised mice with humanized livers were developed in the mid-1990s to allow the study of human hepatotropic viruses, which normally replicate only in higher primates. The production of the uPA/SCID mouse was the vanguard of these models and remains the most widely worked upon model for an ever increasing range of applications. 2. Since toxicology is conducted in laboratory animal species with the implicit intent of predicting the outcome of accidental, or intentional, human exposure, the potential for using an in vivo model with a humanised metabolism opens up the possibility of better predicting the human response following exposure to drugs and industrial chemicals. Chimeric humanised mice provide the tool for bridging between the non-clinical laboratory safety and metabolism studies, carried out in rodent and non-rodent species, and the first in man clinical trials. 3. Chimeric mice carrying a human liver have now been validated against a wide range of different drugs and chemical classes, and have been shown to clearly differentiate metabolically from the recipient mouse, and to show metabolic pathways more similar to those expected from human liver. 4. This review critically appraises the available animal models carrying human livers and where future developments would improve the existing systems.

No abstract