Hepatitis C virus (HCV) remains a major medical problem. Antiviral treatment is only partially effective and a vaccine does not exist. Development of more effective therapies has been hampered by the lack of a suitable small animal model. While xenotransplantation of immunodeficient mice with human hepatocytes has shown promise, these models are subject to important challenges. Building on the previous observation that CD81 and occludin (OCLN) comprise the minimal human factors required to render mouse cells permissive to HCV entry in vitro, we attempted murine humanization via a genetic approach. Here we show that expression of two human genes is sufficient to allow HCV infection of fully immunocompetent inbred mice. We establish a precedent for applying mouse genetics to dissect viral entry and validate the role of SCARB1 for HCV uptake. We demonstrate that HCV can be blocked by passive immunization, as well as show that a recombinant vaccinia virus (rVV) vector induces humoral immunity and confers partial protection against heterologous challenge. This system recapitulates a portion of the HCV life cycle in an immunocompetent rodent for the first time, opening opportunities for studying viral pathogenesis and immunity and comprising an effective platform for testing HCV entry inhibitors in vivo.

Human hematolymphoid mice have become valuable tools for the study of human hematopoiesis and uniquely human pathogens in vivo. Recent improvements in xenorecipient strains allow for longterm reconstitution with a human immune system. However, certain hematopoietic lineages, for example, the myeloid lineage, are underrepresented, possibly because of the limited cross-reactivity of murine and human cytokines. Therefore, we created a nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-␥-null (NOD-SCID IL2R␥ null ) mouse strain that expressed human stem cell factor, granulocyte-macrophage colonystimulating factor, and interleukin-3, termed NSG-SGM3. Transplantation of CD34 ؉ human hematopoietic stem cells into NSG-SGM3 mice led to robust human hematopoietic reconstitution in blood, spleen, bone marrow, and liver. Human myeloid cell frequencies, specifically, myeloid dendritic cells, were elevated in the bone marrow of humanized NSG-SGM3 mice compared with nontransgenic NSG recipients. Most significant, however, was the increase in the CD4 ؉ FoxP3 ؉ regulatory T-cell population in all compartments analyzed. These CD4 ؉ FoxP3 ؉ regulatory T cells were functional, as evidenced by their ability to suppress T-cell proliferation. In conclusion, humanized NSG-SGM3 mice might serve as a useful model to study human regulatory T-cell development in vivo, but this unexpected lineage skewing also highlights the importance of adequate spatiotemporal expression of human cytokines for future xenorecipient strain development. (Blood. 2011;117(11): 3076-3086) IntroductionHumanized mice are amenable small-animal models that have been transplanted with human cells or tissues (and/or equipped with human transgenes). In particular, animals conditioned to support engraftment of human immune cells have emerged as powerful tools for analysis of human hematopoiesis and the study of pathogens with unique human tropism. From the earliest attempts to engraftment of human immune cells in mice in the late 1980s, the field has progressed substantially, and improved, highly immunocompromised xenorecipient strains now allow for high-level engraftment of human immune cells. Currently, the most advanced strains are the nonobese diabetic, severe combined immunodeficiency (NOD-SCID) mouse with either truncated (NOG) or complete (NSG) disruptions in the interleukin-2 (IL-2) receptor common ␥-chain (IL2R␥ null ) and BALB/c Rag2 Ϫ/Ϫ IL2R␥ null (BRG) mice. 1 Injection of human hematopoietic stem cells (HSCs) isolated from human cord blood 2-5 or fetal liver tissue [5][6][7] results in robust engraftment of a human hematolymphoid system. Such human immune system (HIS) mice have opened new opportunities to analyze human immunity in vivo and to study pathogens with unique human tropism, including Epstein-Barr virus, HIV, and dengue virus. 8 However, current humanized mouse models have several shortcomings that must be overcome to advance toward a robust and predictive model for human immune responses. Specifically, the total amount of h...

Hepatitis C virus (HCV) remains a major public health problem, affecting approximately 130 million people worldwide. HCV infection can lead to cirrhosis, hepatocellular carcinoma, and end-stage liver disease, as well as extrahepatic complications such as cryoglobulinemia and lymphoma. Preventative and therapeutic options are severely limited; there is no HCV vaccine available, and nonspecific, IFN-based treatments are frequently ineffective. Development of targeted antivirals has been hampered by the lack of robust HCV cell culture systems that reliably predict human responses. Here, we show the entire HCV life cycle recapitulated in micropatterned cocultures (MPCCs) of primary human hepatocytes and supportive stroma in a multiwell format. MPCCs form polarized cell layers expressing all known HCV entry factors and sustain viral replication for several weeks. When coupled with highly sensitive fluorescence-and luminescence-based reporter systems, MPCCs have potential as a high-throughput platform for simultaneous assessment of in vitro efficacy and toxicity profiles of anti-HCV therapeutics. viral hepatitis | liver | tissue engineering | drug development | infection

Persistent hepatitis C virus (HCV) infection is a primary etiological factor for the development of chronic liver disease, including cirrhosis and cancer. A recent study identified occludin (OCLN), an integral tight junction protein, as one of the key factors for HCV entry into cells. We explored the splicing diversity of OCLN in normal human liver and observed variable expression of alternative splice variants, including two known forms (WT-OCLN and OCLN-ex4del) and six novel forms (OCLN-ex7ext, OCLNex3pdel, OCLN-ex3del, OCLN-ex3-4del, OCLN-ex3p-9pdel, and OCLN-ex3p-7pdel). Recombinant protein isoforms WT-OCLN and OCLN-ex7ext, which retained the HCV-interacting MARVEL domain, were expressed on the cell membrane and were permissive for HCV infection in in vitro infectivity assays. All other forms lacked the MARVEL domain, were expressed in the cytoplasm, and were nonpermissive for HCV infection. Additionally, we observed variable expression of OCLN splicing forms across human tissues and cell lines. Our study suggests that the remarkable natural splicing diversity of OCLN might contribute to HCV tissue tropism and possibly modify the outcome of HCV infection in humans. Genetic factors crucial for regulation of OCLN expression and susceptibility to HCV infection remain to be elucidated.Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver, the fifth most common malignancy worldwide, and the third leading cause of cancer-related death, after cancers of lung and stomach (WHO Mortality Database [http://www.who.int/healthinfo/morttables/en/index.html]). The estimated incidence of new HCC cases is about 500,000 to 1,000,000 annually, with mortality of 600,000 cases per year on a global scale (12,16,17,20,24). Various risk factors for HCC include infection with hepatitis C virus (HCV) or hepatitis B virus (HBV), toxic exposures (alcohol and aflatoxins), metabolic disease (diabetes, nonalcoholic fatty liver disease, and hereditary hemochromatosis), and immune-related conditions such as primary biliary cirrhosis and autoimmune hepatitis (15).The only established in vivo model for the study of HCV infection in an immunocompetent host is the chimpanzee (23). The inability of HCV to infect animals other than humans and chimpanzees has severely hampered efforts in developing a useful small animal model for the disease, specific antiviral therapies, and an effective vaccine against HCV-mediated liver cancer (18,23).In the United States, chronic HCV infection is the major etiological agent of liver cancer. Among individuals infected with HCV, approximately 80% develop chronic HCV infection, of which 20% will progress to cirrhosis, and 1 to 5% will progress to liver cancer (14). Genetic factors might affect the risk of liver cancer by modifying the susceptibility to HCV infection and viral clearance. Recent studies identified occludin (OCLN), an integral tight junction (TJ) protein, as one of the key factors for HCV entry into cells (8,18). HCV infectivity was exclusively mediated by the second extracell...