Therapy, Vaccine and Model Development in Viral Hepatitis and Liver Diseases

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

While direct acting antivirals are highly effective in curing HCV infection, there is no vaccine to prevent infection. The primary goal of vaccine development is to prevent HCV infection by blocking attachment and/or entry of HCV to susceptible cells, which are mediated by HCV E1-E2 envelope proteins. Understanding the structure of the E1-E2 heterodimer is important in designing candidate antigens that can induce broadly neutralizing antibodies. Using well-characterized anti-E2 antibodies, the structure of E1-E2 heterodimer has been partially resolved. Here, we aim to develop and characterize E1-specific nanobodies to further elucidate the structure and function of E1. We screened a VHH (variable domain of a heavy-chain only antibody) phage display library against recombinant E1-E2 and counter-screened against recombinant E2c (core ecto-domain of E2). We identified 17 VHH clones that bind to recombinant E1-E2 but not E2c. The VHH clone was then fused to the human Fc sequence and transfected into 293T cells for production. We selected 9 nanobodies for further characterization of their binding affinities and neutralizing activities. In immunoprecipitation, all the nanobodies could pull down E1-E2 but not cE2. Using a biolayer interferometry assay, these nanobodies bind to E1-E2 and a recombinant form of E1 (sfE1, Matos et al., JV 2024) with high affinities. These nanobodies had variable neutralizing activities against HCVcc and HCVpp of various genotypes, with some of them highly potent against all genotypes. Negative-stain transmission electron microscopy of E1-E2 in complex with these nanobodies and previously characterized anti-E2 Fabs showed that the nanobodies bind to regions of E1-E2 distinct from those of the anti-E2 Fabs. In this study, we generated and characterized a panel of E1-specific nanobodies with high affinities and broadly neutralizing activities. These nanobodies would be highly valuable for further studies of E1-E2 in viral entry and design of HCV vaccine candidates. The humanized chimeric liver mouse model has been an important animal model for studying HCV infection. Despite lacking an adaptive immune system, it reproducibly achieves high viremia from various HCV sources. Here we apply this model to investigate in vivo infection dynamics, replication, and the feasibility of delivering full-length HCV RNA using lipid nanoparticles (LNPs) to establish HCV infection. Immune-deficient Fah−/−/Rag2−/−/Il2rg−/− (FRG) mice engrafted with primary human hepatocytes were intravenously injected with either HCV-H77C-infected chimpanzee serum or LNPs encapsulating full-length HCV genotype 1a (H77C) and 2a (J6/JFH1) RNAs. Previous studies have shown that >90% of RNAs in LNPs are delivered to the liver. Blood samples from the mice were monitored for viremia and viral titers. Once viremia plateaued, glecaprevir/pibrentasvir (G/P) was orally administered daily for three weeks to assess the treatment response. Infectivity of H77C-infected chimpanzee serum in humanized FRG mice was determined with serially diluted serum and found comparable to previously established infectious titer in naive chimpanzees (CID). In infected mice, HCV titers typically plateaued about four weeks post-infection with viremia as high as 108 copies/mL. The viremic mouse serum could be passaged to uninfected FRG mice. Inoculation with full-length H77C and J6/JFH1 HCV-LNPs established similar infection courses as chimpanzee serum. Less than 1 ug of HCV-LNPs was sufficient to establish infection. Three weeks of G/P eliminated HCV viremia in both H77C and J6/JFH1-infected mice. Mice with treatment-induced elimination of HCV could be re-infected and successfully re-treated with G/P. We demonstrate the value and utility of this model in characterizing HCV infection dynamics and delivering full-length HCV RNA using LNP technology. We also show its value in assessing antiviral responses and highlight the potential of LNP technology for vaccine development, particularly in the context of developing challenge inoculum for controlled human infection model. Norway rat hepacivirus (NrHV) is a valuable surrogate model for hepatitis C virus (HCV) because of its shared characteristics including genomic structure, hepatotropism, pathology and chronicity. Virus-like particles (VLP), capable of inducing both humoral and cellular immune responses, have demonstrated promise in vaccine development against various viruses including HCV. Here we build on our previously developed HCV-LP platform (Elmowalid et al, PNAS 2007) by engineering host protein sequences (EABR) - known to promote VLP budding - into the NrHV structural proteins. Using an mRNA delivery system, we evaluate this strategy in the NrHV model as a proof-of-concept stage in HCV vaccine development. To enhance both B and T cell responses, we utilize an mRNA platform encoding NrHV core (C) and envelope proteins (E1E2) with or without the EABR sequence. C57BL/6 mice were immunized with the NrHV CE1E2 or CE1E2-EABR mRNA-lipid nanoparticle (LNP) vaccine and challenged with NrHV. Serum viral RNA levels were monitored weekly following challenge. B cell responses were characterized by ELISA and neutralization assay, and T cell responses by ELISPOT and intracellular cytokine staining (ICS) assay. Incorporation of EABR into the NrHV CE1E2 construct enhanced VLP budding and secretion of intact VLP into the medium. Mice vaccinated with the CE1E2-EABR mRNA-LNP followed by NrHV challenge exhibited a rapid viral clearance and reduced liver damage accompanied by the induction of B and T cell responses. The EABR-containing construct demonstrated a superior response to the non-EABR construct in protection and immune parameters. Studies in the chronic NrHV-rat model are ongoing. Our results demonstrate that this combined VLP and mRNA platform can induce B and T cell responses and protect against NrHV infection in an immunocompetent model. This approach offers promise in developing an HCV vaccine capable of inducing protective immune responses targeting both B and T cells.