Opening the door for hepatitis C virus infection in genetically humanized mice

Authors

  • Mirjam B. Zeisel Ph.D., PharmD,

    1. Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 748, Strasbourg, France
    2. Université de Strasbourg, Strasbourg, France
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  • Daniel Da Costa,

    1. Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 748, Strasbourg, France
    2. Université de Strasbourg, Strasbourg, France
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  • Thomas F. Baumert M.D.

    1. Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 748, Strasbourg, France
    2. Université de Strasbourg, Strasbourg, France
    3. Pôle Hépato-digestif, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
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  • Potential conflict of interest: Nothing to report..

Dorner M, Horwitz JA, Robbins JB, Barry WT, Feng Q, Mu K, et al. A genetically humanized mouse model for hepatitis C virus infection. Nature 2011;474:208-211. Available at: www.nature.com (Reprinted with permission.)

Abstract

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. Although 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 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 scavenger receptor type B class I for HCV uptake. We demonstrate that HCV can be blocked by passive immunization, as well as showing that a recombinant vaccinia virus 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.

Comment

Hepatitis C virus (HCV) infection represents a major public health burden. Although a fraction of infected individuals is able to clear acute HCV infection spontaneously, the majority of HCV-infected patients develops chronic infection. A vaccine to prevent HCV infection is not available, and current therapeutic strategies are costly and limited by side effects. Thus, there is an important, unmet medical need for the development of novel preventive and therapeutic strategies against HCV infection.

Research on HCV and development of antivirals has long been hampered by the lack of a small-animal model and robust cell culture systems. The limited tropism of HCV for human and chimpanzee hepatocytes and its poor ability to replicate in vitro prompted scientists to seek alternative models to allow deciphering parts of the viral life cycle. Major breakthroughs were the development of replicons, which for the first time permitted study of robust HCV replication in the human hepatoma cell line Huh7, and of HCV pseudo-particles (HCVpp) that enabled researchers to characterize the first steps of viral entry and to assess viral neutralization (reviewed in Murray and Rice1). The isolation of a defined genotype 2a isolate (Japanese fulminant hepatitis 1 [JFH1]), presenting unique in vitro growth characteristics, and the generation of highly HCV replication-permissive Huh7-derived cell lines finally lead to the first cell culture system that produced infectious viral particles (HCVcc) (reviewed in Murray and Rice1). Attempts to generate robust full-length clones for other genotypes did not succeed in reaching the efficiency of the JFH1 clone. The HCVcc model could, however, be extended to other genotypes by developing intergenotypic chimeras.2, 3

The possibility of transplanting primary human hepatocytes into transgenic immunodeficient mice with hepatocyte-lethal phenotype (uPA-SCID [urokinase plasminogen activator, severe combined immunodeficient] and Fah/Rag2/IL-2rnull mice) and of subsequently infecting these human hepatocytes yielded the first small-animal models for studying HCV infection in vivo.4, 5 Drawbacks of these models are their high mortality, variable robustness of infection, and the absence of a functional immune system that precludes the evaluation of HCV-specific immune responses and immune-based therapies or vaccines (for review, see Barth et al.6). The development of an immunocompetent small-animal model for the study of HCV infection may be achieved by two approaches: either by transplanting both human liver and immune cells in immunodeficient mice or by generating transgenic mice that express human-specific factors to overcome the restriction of HCV infection of mouse cells. Addressing the first strategy, the reconstitution of a human immune system in immunodeficient Rag2−/−/IL-2rnull Balb/c mice in which human hepatocyte progenitor cells were transplanted led to the detection of human immune cells (natural killer, dendritic, and T cells, but no B cells) with low levels of HCV RNA detected in the liver.7 With the goal of developing an immunocompetent transgenic mouse model, a proof-of-concept for the second approach to render mice permissive for HCV has been reported recently by Marcus Dorner and Alexander Ploss from the Rice laboratory at the Center for the Study of Hepatitis C, Rockefeller University, New York, NY.8

Building on their previous report that mouse cells may be rendered permissive for HCV entry in vitro upon expression of human orthologues of the HCV entry factors scavenger receptor class B type I (SR-BI), claudin-1 (CLDN1), CD81, and occludin (OCLN)—the latter two representing the species-specific factors9—Dorner and colleagues first expressed the four human HCV entry factors in mice using recombinant adenoviruses.8 Although approximately 18%-25% of murine hepatocytes coexpressed human CD81 and OCLN and 5% of the cells coexpressed the four human HCV entry factors in vivo, only transient low-level HCV RNA could be detected in both the serum and the liver of infected animals, and no significant bioluminescent signals were obtained after infection of mice with luciferase-reporter HCV. This may partly be due to the restriction of HCV replication and assembly in mouse hepatocytes and/or from strong murine innate antiviral immune responses. This low level of infection thus hampers the study of HCV infection in these humanized mice. To overcome this hurdle, the authors next developed more sensitive reporter model systems to monitor single-cycle HCV infection in vivo. Bicistronic HCV genomes expressing cyclization recombination (CRE) recombinase activating either a loxP-flanked luciferase reporter or a nuclear-localized green fluorescent protein (GFP)/β-galactosidase reporter in transgenic mice allowed study of single-cycle HCVcc infection in mouse hepatocytes by assessing live animal bioluminescent signals or by quantifying GFP-positive hepatocytes, through use of flow cytometry after isolation of murine hepatocytes (Fig. 1).8 Of note, expression of CD81 and CLDN1 mutants known to reduce HCV infection in vitro, as well as absence of SR-BI expression, also reduced HCV infection of murine hepatocytes in vivo. These data suggest that the HCV entry process in vivo resembles the pathway uncovered in vitro. Furthermore, the impact of this mouse model for the study of entry inhibitors and vaccines could be elegantly demonstrated by assessing the efficiency of anti-CD81 and anti-E2 antibodies in reducing HCV infection. It is worth noting that immunization of mice with a recombinant vaccinia virus vector expressing HCV proteins leads to robust anti-E2 antibody titers and decreased susceptibility to heterologous HCV challenge. Finally, the mice were susceptible to infection with recombinant viruses expressing structural proteins from genotypes 1b, 2a, 4a, 6a, and 7a.

Figure 1.

Immunocompetent mouse model to study single-cycle HCV infection described by Dorner et al.8 Gt(ROSA)26Sortm1(Luc)Kaelin mice (Rosa26-Fluc) contain in the Gt(ROSA)26Sor locus, a LoxP-flanked STOP cassette restricting firefly luciferase expression. Adenoviruses encoding human CD81, OCLN, CLDN-1, and SR-BI were injected intravenously into Gt(ROSA)26Sortm1(Luc)Kaelin mice 24 hours prior to intravenous challenge with recombinant bicistronic HCVcc expressing CRE protein (HCV-CRE). Upon HCV entry into mouse hepatocytes, CRE recombinase activates the luciferase reporter, leading to bioluminescence signals that can be monitored using a live animal bioluminescence imaging device. In an alternative approach, CRE expression leads to activation of a nuclear-localized GFP/β-galactosidase reporter (Rosa26-GNZ) that allows one to assess HCV permissivity of murine hepatocytes by quantifying GFP-positive murine hepatocytes, through use of flow cytometry.

In conclusion, this sensitive, genetically humanized mouse model allows for the first time the study of virus–host interactions during the early stages of HCV infection in the presence of a functional immune system in a small animal. Thus, this model will be very useful for the in vivo investigation of HCV entry and antibody-mediated neutralization, as well as the preclinical development of entry inhibitors and B cell vaccines. The preclinical evaluation of entry inhibitors is of particular interest for the prevention of liver graft infection.10, 11 However, the lack of robust HCV replication in mouse hepatocytes and the need for CRE-expressing recombinant HCVcc currently precludes the study of infection using patient-derived HCV and the investigation of virus–host interactions during replication and particle production.

Nevertheless, despite these limitations, this study is a breakthrough toward the development of an immunocompetent HCV mouse model, because it opens the door for a genetically humanized mouse model for the study of the complete life cycle of HCV infection. A recent study has shown that mouse hepatoma cell lines are able to produce infectious virions following expression of mouse or human apolipoprotein E,12 which means the identification of species-specific restriction factors for viral replication will most likely be the final step required to produce humanized transgenic mice that are able to recapitulate the entire viral life cycle.

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