A genetically humanized mouse model for hepatitis C virus infection

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.

Figure 1. Genetic requirements for HCV entry in vivo

a, Timeline for administration of adenovirus and HCV-CRE. b, HCV entry into Rosa26-Fluc mice 96h post-injection of medium (n=20), 10 adenovirus encoding CD81 and OCLN (n=10) or 10 particles encoding CD81, SCARB1, CLDN and OCLN of the indicated species (n=20 each). Bioluminescence was measured at 72h post-injection of 2×10⁷ 50%-tissue culture infectious doses (TCID₅₀) HCV-CRE. c, Frequency of HCV-infected hepatocytes. Rosa26-GNZ mice were injected with medium or adenovirus encoding human CD81, mKate-SCARB1, Cerulean-CLDN1 and Venus/YFP-OCLN. 72h post-injection of 2×10⁷ TCID₅₀ HCV-CRE, the frequency of infected hepatocytes was determined by flow cytometry. Data are percent infected (eGFP⁺) cells relative to either CD81/OCLN transduced (left axis) or CD81/SCARB1/CLDN1/OCLN transduced hepatocytes (right axis) d. Rosa26-Fluc mice were injected with human (h) and murine (m) entry factors 24h prior to injection of HCV-CRE (n≥4). e, Entry factor mutants reduce infection in vivo. Combined delivery of human (h) and mutant human (h^mut), hCD81/F186L/E188K or hOCLN/mEL2 (n=3). f. mSCARB1 is an essential HCV entry factor in vivo. Rosa26-Fluc mice were crossed with mSCARB1^−/− mice offspring were injected with adenoviruses encoding human CD81, CLDN1, and OCLN, and 24h later with 2×10⁷ TCID₅₀ HCV-CRE (n=5–7). Data represent mean ± standard deviation (SD). Statistical significance was calculated by Kruskal-Wallis one way analysis of variance (* p<0.05, ** p<0.01 and *** p<0.001)

Figure 2. HCV entry into murine hepatocytes in vivo can be blocked by antibodies or passive transfer of vaccine induced antiserum

a–c, Experimental layout of a. CD81-blocking, b, virus pre-neutralization with anti-E2 (3/11) and c, in vivo neutralization with pooled sera from immunized mice. d. Rosa26-Fluc mice were injected with anti-CD81 antibodies (two doses of 1, 10 or 100 µg/animal at 24h prior to and with injection of HCV-CRE, n=4) or isotype control (n=3). e. HCV-CRE was incubated with 5, 50 or 500µg of anti-HCV E2 (clone 3/11) for 1h prior to injection into Rosa26-Fluc mice expressing all four human entry factors (n=3). f. Rosa26-Fluc mice were injected with 200µl pooled serum from wild-type (FVB/nJ) mice immunized with rVV-HCV1 or naïve control (2 doses 24h prior to and with injection of HCV-CRE [n=4]). Data shown are mean ± SD. Statistical significance was calculated by Kruskal-Wallis one way analysis of variance (* p<0.05, ** p<0.01 and *** p<0.001)

Figure 3. Use of genetically humanized mouse model to evaluate vaccines against multiple HCV genotypes

a. Genetic humanization supports HCV entry mediated by structural proteins of various genotypes. Rosa26-Fluc mice expressing all four human entry factors were infected with intergenotypic chimeras (Con1 genotype 1b, Jc1 genotype 2a, ED43 genotype 4a, HK6a genotype 6a and QC69 genotype 7a, 2×10⁷ TCID₅₀/animal, n=3). b. Priming of humoral immune responses with recombinant vaccinia virus (rVV). Rosa26-Fluc mice were injected intraperitoneally with rVV encoding the HCV-1 (genotype 1a) structural genes (10⁷ PFU/animal, n=10) and anti-HCV E2 antibody titers were determined by ELISA. c–f. Protection of rVV-immunized mice against challenge. Immunized mice were challenged with heterologous HCV strains Con1 (1b), Jc1 (2a) and ED43 (4a) and infection was quantified by bioluminescence imaging 72h later (n=5). Data represent mean ± SD.