Document text
Principal Investigator: T. Jake Liang
Organization: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
Fiscal Year: 2024
Award: $2,063,799
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
Despite the development of highly effective HCV treatments, an effective prophylactic vaccine is still lacking. HCV infection is mediated by its envelope glycoproteins E1 and E2 in the entry process with E2 binding to cell receptors and E1 mediating endosomal fusion. The structure of E1E2 has only been partially resolved by the X-ray crystallography of the core domain of E2 protein (E2c) and its complex with various neutralizing antibodies. Structural understanding of the E1E2 heterodimer in its native form can advance the design of candidates for HCV vaccine development. 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 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.
Terms: <Adaptive Immune System><Affinity><Animal Model><Animal Models and Related Studies><Anti-viral Agents><Anti-viral Response><Antibodies><Assay><Binding><Bioassay><Biologic Models><Biological Assay><Biological Models><Blood Sample><Blood Serum><Blood specimen><Cell Body><Cells><Chimp><Chimpanzee><Complex><Development><Encapsulated><Endosomes><Engraftment><Genotype><Glycoproteins><HBV therapy><HBV treatment><HCV><HCV Vaccine><HCV Viremia><HCV infection><HCV therapy><HCV treatment><Hepatic Cells><Hepatic Disorder><Hepatic Parenchymal Cell><Hepatitis B Therapeutic><Hepatitis B Therapy><Hepatitis B Treatment><Hepatitis C><Hepatitis C Therapeutics><Hepatitis C Therapy><Hepatitis C Vaccine><Hepatitis C Viremia><Hepatitis C Virus Treatment><Hepatitis C Virus Viremia><Hepatitis C treatment><Hepatitis C virus><Hepatitis C virus infection><Hepatitis, Viral, Non-A, Non-B, Parenterally-Transmitted><Hepatitus C><Hepatocyte><Human><Immune Precipitation><Immunoprecipitation><In Vitro><Infection><Interferometry><Intravenous><Length><Libraries><Liver><Liver Cells><Liver diseases><Mediating><Mice><Mice Mammals><Model System><Modeling><Modern Man><Molecular><Molecular Interaction><Monitor><Murine><Mus><NAFLD><Negative Staining><Non-Polyadenylated RNA><Oral Administration><Oral Drug Administration><Pan Genus><Pan Species><Phage Display><Preventative vaccine><Preventive vaccine><Process><Production><Prophylactic vaccine><Proteins><RNA><RNA Gene Products><Receptor Cell><Receptosomes><Recombinants><Reproducibility><Ribonucleic Acid><Serum><Single Crystal Diffraction><Source><Structure><Study models><Technology><Transfection><Transmission Electron Microscopy><Viral><Viral hepatitis><Viremia><Virus Replication><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><acquired immune system><anti-viral compound><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-virals><design><designing><develop a vaccine><develop therapy><develop vaccines><development of a vaccine><developmental><hep C><hepatic body system><hepatic disease><hepatic organ system><hepatitis C virus vaccine><hepatitis non A non B><hepatitis virus infection><hepatopathy><hypoimmunity><immune deficiency><immunodeficiency><improved><in vivo><in vivo Model><infection by hepatitis c virus><intervention development><intraoral drug delivery><lipid based nanoparticle><lipid nanoparticle><liver disorder><model development><model developments><model of animal><mouse model><murine model><nano particle delivery><nanobodies><nanobody><nanoparticle delivered><nanoparticle delivery><neutralizing antibody><non A, non B hepatitis><non-A, non-B hepatitis><non-alcohol fatty liver disease><non-alcoholic fatty liver disease><non-alcoholic liver disease><nonalcoholic fatty liver disease><response to therapy><response to treatment><sdAb><single domain antibodies><therapeutic response><therapeutic vaccine><therapy development><therapy response><treatment development><treatment response><treatment responsiveness><treatment vaccines><vaccine candidate><vaccine development><vaccine for the treatment><vaccine for treatment><viraemia><viral multiplication><viral replication><viral sepsis><virus multiplication><virusemia>