Human Fc-Conjugated Receptor Binding Domain-Based Recombinant Subunit Vaccines with Short Linker Induce Potent Neutralizing Antibodies against Multiple SARS-CoV-2 Variants

The coronavirus disease-19 (COVID-19) pandemic has been ongoing since December 2019, with more than 6.3 million deaths reported globally as of August 2022. Despite the success of several SARS-CoV-2 vaccines, the rise in variants, some of which are resistant to the effects of vaccination, highlights the need for a so-called pan-coronavirus (universal) vaccine. Here, we performed an immunogenicity comparison of prototype vaccines containing spike protein receptor-binding domain (RBD) residues 319–541, or spike protein regions S1, S2 and S fused to a histidine-tagged or human IgG1 Fc (hFC) fragment with either a longer (six residues) or shorter (three residues) linker. While all recombinant protein vaccines developed were effective in eliciting humoral immunity, the RBD-hFc vaccine was able to generate a potent neutralizing antibody response as well as a cellular immune response. We then compared the effects of recombinant protein length and linker size on immunogenicity in vivo. We found that a longer recombinant RBD protein (residues 319–583; RBD-Plus-hFc) containing a small alanine linker (AAA) was able to trigger long-lasting, high-titer neutralizing antibodies in mice. Finally, we evaluated cross-neutralization of wild-type and mutant RBD-Plus-hFc vaccines against wild-type, Alpha, Beta, Delta and Omicron SARS-CoV-2 variants. Significantly, at the same antigen dose, wild-type RBD-Plus-hFc immune sera induced broadly neutralizing antibodies against wild-type, Alpha, Beta, Delta and Omicron variants. Taken together, our findings provide valuable information for the continued development of recombinant protein-based SARS-CoV-2 vaccines and a basic foundation for booster vaccinations to avoid reinfection with SARS-CoV-2 variants.