UNLABELLED: Hepatitis C virus (HCV) infection is a global health problem for which no vaccine is available. HCV has a highly heterogeneous RNA genome and can be classified into seven genotypes. Due to the high genetic and resultant antigenic variation among the genotypes, inducing antibodies capable of neutralizing most of the HCV genotypes by experimental vaccination has been challenging. Previous efforts focused on priming humoral immune responses with recombinant HCV envelope E2 protein produced in mammalian cells. Here, we report that a soluble form of HCV E2 (sE2) produced in insect cells possesses different glycosylation patterns and is more immunogenic, as evidenced by the induction of higher titers of broadly neutralizing antibodies (bNAbs) against cell culture-derived HCV (HCVcc) harboring structural proteins from a diverse array of HCV genotypes. We affirm that continuous and discontinuous epitopes of well-characterized bNAbs are conserved, suggesting that sE2 produced in insect cells is properly folded. In a genetically humanized mouse model, active immunization with sE2 efficiently protected against challenge with a heterologous HCV genotype. These data not only demonstrate that sE2 is a promising HCV vaccine candidate, but also highlight the importance of glycosylation patterns in developing subunit viral vaccines.

IMPORTANCE: A prophylactic vaccine with high efficacy and low cost is urgently needed for global control of HCV infection. Induction of broadly neutralizing antibodies against most HCV genotypes has been challenging due to the antigenic diversity of the HCV genome. Here, we refined a high-yield subunit HCV vaccine that elicited broadly neutralizing antibody responses in preclinical trials. We found that soluble HCV E2 protein (sE2) produced in insect cells is distinctly glycosylated and is more immunogenic than sE2 produced in mammalian cells, suggesting that glycosylation patterns should be taken into consideration in efforts to generate antibody-based recombinant vaccines against HCV. We further showed that sE2 vaccination confers protection against HCV infection in a genetically humanized mouse model. Thus, our work identified a promising broadly protective HCV vaccine candidate that should be considered for further preclinical and clinical development.