Native-like soluble E1E2 glycoprotein heterodimers on self-assembling protein nanoparticles for hepatitis C virus vaccine design.

Hepatitis C virus (HCV) is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide. Development of an E1E2-based HCV vaccine has been hindered by the difficulty of producing a soluble E1E2 (sE1E2) antigen that faithfully recapitulates the native virion-associated heterodimer. Guided by cryo-electron microscopy (cryo-EM) structures, we engineer genotype 1a H77 sE1E2 by truncating the E1 and E2 stems (Cut1), deleting a putative fusion peptide-containing region in E1 (Cut2), and stabilizing the heterodimer using diverse scaffolds. All H77 sE1E2.Cut1+2 scaffolds exhibit native-like E1-E2 association and strong binding to the broadly neutralizing antibody (bNAb) AR4A. A genotype 1a HCV-1 sE1E2.Cut1+2 variant scaffolded by a modified SpyTag/SpyCatcher (SPYΔN) is selected for in vitro and in vivo characterization, as well as further construct refinement. The structure of this HCV-1 sE1E2 construct in complex with bNAbs is determined by cryo-EM and negative-stain EM (nsEM), with an nsEM-based strategy established for antibody epitope mapping. HCV-1 sE1E2.Cut1+2.SPYΔN is displayed on self-assembling protein nanoparticles (SApNPs) to enhance immunogenicity. The HCV-1 sE1E2.Cut1+2.SPYΔN heterodimer and SApNPs bearing wildtype or modified glycans are evaluated in mice, alongside E2 core-based immunogens for comparison. Together, these results establish a framework for advancing E1E2-based HCV vaccines toward clinical development.