![]() ![]() Neutralizing antibodies (nAbs) induced during natural infection have been mapped onto both E1 and E2. While it is known that glycoproteins E1 and E2 form heterodimers on the surface of a virion, how they mediate viral membrane fusion and cell entry remain elusive 13, 14, 15, 16. HCV entry is mediated by the interactions between viral envelope glycoproteins and cell surface receptors such as CD81, scavenger receptor class B type I (SR-BI) and other host entry factors 11, 12. However, due to the poor fidelity of the viral RNA polymerase 9 and the high replication rate (10 12 virions/day), HCV exhibits extraordinary genetic diversity with over 30% sequence difference between genotypes 10, posing a significant challenge to the development of a broadly effective vaccine. Although small-molecule drugs are available for treatment 5, a prophylactic vaccine will offer a cost-effective means to prevent HCV transmission and to eventually eradicate the virus 6, 7, 8. Hepatitis C is a global health burden with approximately 180 million chronic carriers of hepatitis C virus (HCV) worldwide 1, 2, 3, 4. Our study thus demonstrates the utility of a multi-scale scaffolding strategy in epitope vaccine design and provides promising HCV immunogens for further assessment in vivo. We then investigated a “multivalent scaffolding” approach by displaying 24 copies of an epitope scaffold on a self-assembling nanoparticle, which markedly increased the avidity of antibody binding. We experimentally confirmed that three E1 and five E2 epitope scaffolds bound to their respective neutralizing antibodies, but with different kinetics. For each antigenic site, ten scaffolds were selected for computational design and the resulting epitope scaffolds were analyzed using structure-scoring functions and molecular dynamics simulation. We first combined six structural alignment algorithms in a “scaffolding meta-server” to search for diverse scaffolds that can structurally accommodate the HCV epitopes. In this study, we designed and characterized epitope vaccine antigens for the antigenic sites of HCV envelope glycoproteins E1 (residues 314–324) and E2 (residues 412–423), for which neutralizing antibody-bound structures are available. Scaffolding, by grafting an epitope onto a heterologous protein scaffold, offers a possible solution to epitope vaccine design. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required.Development of a prophylactic vaccine against hepatitis C virus (HCV) has been hampered by the extraordinary viral diversity and the poor host immune response. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. This epitope-scaffold was well folded as assessed by circular dichroism and isothermal titration calorimetry, and its crystal structure (determined in complex with motavizumab to 1.9 Å resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Two of the epitope-scaffolds could be purified, and one epitope-scaffold based on a Staphylococcus aureus protein A domain bound motavizumab with kinetic and thermodynamic properties consistent with the free epitope-scaffold being stabilized in a conformation that closely resembled the motavizumab-bound state. ![]() Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a potent neutralizing antibody that binds to a helix-loop-helix motif in the RSV fusion glycoprotein. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed.
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