Project Summary Hepatitis C virus (HCV) infects 1-2% of the world population and poses a significant threat to public health. Recent studies have identified a panel of broadly neutralizing antibodies (bnAbs) and their genetic preferences. The crystal structures of various HCV E2 constructs in complex with bnAbs and non-nAbs provide a structural basis for rational vaccine design. In Project 2 of this P01 proposal, we will combine structural optimization of HCV antigens, nanoparticle engineering, in vivo assessment, and next-generation sequencing (NGS) of B-cell repertoires to assist in rational design of HCV vaccines that can elicit a bnAb response. The Specific Aims in this project are: (1) To develop vaccine antigens to target multidonor class antibody responses. The major virus neutralizing site, the E2 neutralizing face (E2 NF), is a well-known multidonor class antibody target on HCV. E2 NF is conformationally flexible and is surrounded and protected by immunodominant variable loops and N-glycans. The antigenic surface at and around E2 NF will be optimized by computational modeling and mutagenesis scanning to “lock” E2 NF into desired neutralizing conformations. A rigid E2 NF, together with modifications to improve protein folding and to minimize the decoy effect of immunodominant epitopes, should improve the immunogenicity of vaccine antigens and as a result elicit multidonor class bnAbs to the conserved neutralizing epitopes. (2) To develop E1E2-based vaccine antigens. In addition to E2 NF, the native E1E2 complex also present other conserved bnAb epitopes. We will apply scanning mutagenesis to the interface of E1 and E2 ectodomains and design novel E1 and E2 fusion constructs to improve the stability and production of the soluble E1E2 complex for the elicitation of bnAbs targeting the quaternary structure of the complex. (3) To develop self-assembling antigen-presenting nanoparticle vaccines. The engineered E2 and E1E2 complex will be fused with the scaffold proteins of different nanoparticle platforms to identify a platform optimal for the multimeric display of HCV antigens to the immune system. Based on our preliminary data, this strategy will greatly improve the immunogenicity of vaccine antigens and elicit a rapid and potent nAb response. (4) To evaluate immunogenicity and antibody responses of vaccine candidates in the mouse and non-human primate (NHP) rhesus macaque models. The engineered E2, E1E2 antigens and nanoparticles will first be studied in mice to confirm their immunogenicity in vivo and their ability to elicit antibodies that can bind and neutralize HCV. The antigens and nanoparticles with the best properties in vitro and in mice will be studied further using the NHP model. We have recently shown that rhesus macaques react to E1E2 immunization in a manner highly reminiscent to that used by humans and develop bnAbs against E2 NF with genetic similarity to human bnAbs. Here, we will immunize NHPs, identify bnAbs, and perform r...