Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. The development of much needed specific antiviral therapies and an effective vaccine has been hampered by the lack of a convenient small animal model. The determinants restricting HCV tropism to human and chimpanzee hosts are unknown. Replication of the viral RNA has been demonstrated in mouse cells1,2, but these cells are not infectable with either lentiviral particles bearing HCV glycoproteins (HCVpp)3 or HCV produced in cell culture (HCVcc)(unpublished data), suggesting a block at the level of entry. Through an iterative cDNA library screening approach we have identified human occludin (OCLN) as an essential HCV cell entry factor that is able to render murine cells infectable with HCVpp. Similarly, OCLN is required for HCV-susceptibility of human cells, since its overexpression in uninfectable cells specifically enhanced HCVpp uptake while its silencing in permissive cells impaired both HCVpp and HCVcc infection. In addition to OCLN, HCVpp infection of murine cells required expression of the previously identified HCV entry factors, CD814, scavenger receptor class B type I (SR-BI)5, and claudin-1 (CLDN1)6. While the mouse versions of SR-BI and CLDN1 function at least as well as the human proteins for promoting HCV entry; both OCLN and CD81, however, must be of human origin to allow efficient infection. The species-specific determinants of OCLN were mapped to its second extracellular loop. The identification of OCLN as a new HCV entry factor further highlights the importance of the tight junction complex in the viral entry process and provides a major advance towards efforts to develop small animal models for HCV.

The mammalian liver possesses a remarkable regenerative ability. Two modes of damage response have been described: (1) The ''oval cell'' response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. (2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). While the oval cell response has been captured in vitro by growing organoids from cholangiocytes, the hepatocyte proliferative response has not been recapitulated in culture. Here, we describe the establishment of a long-term 3D organoid culture system for mouse and human primary hepatocytes. Organoids can be established from single hepatocytes and grown for multiple months, while retaining key morphological, functional and gene expression features. Transcriptional profiles of the organoids resemble those of proliferating hepatocytes after PHx. Human hepatocyte organoids proliferate extensively after engraftment into mice and thus recapitulate the proliferative damage-response of hepatocytes.

Summary Hepatitis C virus uniquely requires the liver specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (Ago) during HCV infection showed robust Ago binding on the HCV 5′UTR, at known and predicted miR-122 sites. On the human transcriptome, we observed reduced Ago binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 “sponge” effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. We describe a quantitative mathematical model of HCV induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV.

Several viral vector-based gene therapy drugs have now received marketing approval. A much larger number of additional viral vectors are in various stages of clinical trials for the treatment of genetic and acquired diseases, with many more in pre-clinical testing. Efficiency of gene transfer and ability to provide long-term therapy make these vector systems very attractive. In fact, viral vector gene therapy has been able to treat or even cure diseases for which there had been no or only suboptimal treatments. However, innate and adaptive immune responses to these vectors and their transgene products constitute substantial hurdles to clinical development and wider use in patients. This review provides an overview of the type of immune responses that have been documented in animal models and in humans who received gene transfer with one of three widely tested vector systems, namely adenoviral, lentiviral, or adeno-associated viral vectors. Particular emphasis is given to mechanisms leading to immune responses, efforts to reduce vector immunogenicity, and potential solutions to the problems. At the same time, we point out gaps in our knowledge that should to be filled and problems that need to be addressed going forward.

Hepatitis B virus (HBV) chronically infects 400 million people worldwide and is a leading driver of end-stage liver disease and liver cancer. Research into the biology and treatment of HBV requires an in vitro cell-culture system that supports the infection of human hepatocytes, and accurately recapitulates virus-host interactions. Here, we report that micropatterned cocultures of primary human hepatocytes with stromal cells (MPCCs) reliably support productive HBV infection, and infection can be enhanced by blocking elements of the hepatocyte innate immune response associated with the induction of IFN-stimulated genes. MPCCs maintain prolonged, productive infection and represent a facile platform for studying virus-host interactions and for developing antiviral interventions. Hepatocytes obtained from different human donors vary dramatically in their permissiveness to HBV infection, suggesting that factors-such as divergence in genetic susceptibility to infection-may influence infection in vitro. To establish a complementary, renewable system on an isogenic background in which candidate genetics can be interrogated, we show that inducible pluripotent stem cells differentiated into hepatocyte-like cells (iHeps) support HBV infection that can also be enhanced by blocking interferon-stimulated gene induction. Notably, the emergence of the capacity to support HBV transcriptional activity and initial permissiveness for infection are marked by distinct stages of iHep differentiation, suggesting that infection of iHeps can be used both to study HBV, and conversely to assess the degree of iHep differentiation. Our work demonstrates the utility of these infectious systems for studying HBV biology and the virus' interactions with host hepatocyte genetics and physiology.HBV persistence | innate immunity | viral hepatitis H epatitis B virus (HBV) is a small 3.2-kb DNA virus that selectively infects hepatocytes in the human liver (1). The global disease burden is large, with ∼400 million people chronically infected worldwide, of whom about one-third will develop severe HBV-related complications, such as cirrhosis and liver cancer. Lifelong treatment is often required because of the stable nature of viral episomal DNA, known as covalently closed circular DNA (cccDNA), which maintains basal levels in infected cell nuclei even upon nucleos(t)ide inhibitor treatment. To date, HBV research has been hampered by a distinct lack of robust infectious model systems that both support productive HBV infection and accurately mimic virus-host interactions. Recently, the bile acid pump sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a receptor for both HBV and hepatitis D virus (2), and overexpression of NTCP in hepatoma cell lines renders them susceptible to HBV infection. However, hepatoma cells are known to be defective in many cellular pathways implicated in the innate immune response (3, 4), metabolism (5), and cell proliferation (6), which may contribute to published contradictory evidence regarding the e...

The cytokine macrophage-migration inhibitory factor (MIF) is secreted by a number of cell types upon induction by lipopolysaccharide (LPS). Because colitis is dependent on interplay between the mucosal immune system and intestinal bacteria, we investigated the role of MIF in experimental colitis. MIF-deficient mice failed to develop disease, but reconstitution of MIF-deficient mice with wild-type innate immune cells restored colitis. In addition, established colitis could be treated with anti-MIF immunoglobulins. Thus, murine colitis is dependent on continuous MIF production by the innate immune system. Because we found increased plasma MIF concentrations in patients with Crohn's disease, these data suggested that MIF is a new target for intervention in Crohn's disease.

Hepatitis C virus (HCV) establishes a chronic infection in the majority of exposed individuals and can cause cirrhosis and hepatocellular carcinoma. The role of antibodies directed against HCV in disease progression is poorly understood. Neutralizing antibodies (nAbs) can prevent HCV infection in vitro and in animal models. However, the effects of nAbs on an established HCV infection are unclear. Here, we demonstrate that three broadly nAbs, AR3A, AR3B and AR4A, delivered with adeno-associated viral (AAV) vectors can confer protection against viral challenge in humanized mice. Furthermore, we provide evidence that nAbs can abrogate an ongoing HCV infection in primary hepatocyte cultures and in a human liver chimeric mouse model. These results showcase a novel therapeutic approach to interfere with HCV infection exploiting a previously unappreciated need for HCV to continuously infect new hepatocytes in order to sustain chronicity.

The requirements for interleukin (IL)-12/signal transducer and activator of transcription (Stat)-4 signaling and induction of T cell–specific interferon (IFN)-γ expression in the development of T helper cell (Th)1–type pathology were examined in two different models of experimental colitis. In each model, abnormal reconstitution of the T cell compartment in immunodeficient mice by adoptive cell transfer leads to a wasting syndrome and inflammation of the colon, induced by IFN-γ and tumor necrosis factor (TNF)-α–producing T cells. We show here that treatment with anti–IL-12 antibodies in one of the models, or reconstitution with T cells from Stat-4–deficient (Stat-4null) mice in both models resulted in a milder disease in the majority of recipient animals, compared with those that were left untreated or that had been reconstituted with wt cells. Protected mice in each group also harbored lower frequencies of IFN-γ–producing T cells than did diseased mice, suggesting that effects on wasting and colitis resulted from the attenuation of IFN-γ expression by T cells. To test whether the development of pathogenic T cells in the two colitis models was directly dependent on T cell–specific IFN-γ expression, IFN-γnull donors were used for T cell reconstitution in each system. Surprisingly, large numbers of IFN-γnull–reconstituted mice developed wasting and colitis, which in many cases was of comparable severity to that seen in animals reconstituted with wt cells. Furthermore, T cells from these animals expressed TNF-α, demonstrating that they had retained the ability to produce another proinflammatory cytokine. Taken together, these results demonstrate that in some forms of chronic experimental colitis the development of pathogenic T cells is influenced predominantly, though not exclusively, by IL-12 via the actions of Stat-4 proteins. Furthermore, our data suggest that in the models of colitis studied here the effects of IL-12/Stat-4 or other Th1 promoting pathways are not limited to the induction of IFN-γ gene expression in T lymphocytes.