ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global pandemic. The virus is rapidly evolving, characterized by the emergence of several major variants. Stable prefusion spike protein (Pre) is the immunogen in current vaccines but is limited in protecting against different variants. Here, the immune responses induced by the relatively conserved stem subunit (S2) of spike protein versus Pre are investigated. Pre generates the most robust neutralization responses against SARS-CoV-2 variants in vesicular stomatitis virus pseudovirus-based assessment but elicits less antibody-dependent cellular cytotoxicity (ADCC) activity than S2. By contrast, S2 induces the most balanced immunoglobulin G (IgG) antibodies with potent and broad ADCC activity although produces weaker neutralization. The immunogenicity of S2 and Pre improves by incorporating the two proteins into double-layered protein nanoparticles. The resulting protein nanoparticles Pre/S2 elicit higher neutralizing antibodies than Pre alone, and stronger ADCC than S2 alone. Moreover, nanoparticles produce more potent and balanced serum IgG antibodies than the corresponding soluble protein mixture, and the immune responses are sustained for at least four months after the immunization. Thus, the double-layered protein nanoparticles have the potential to be developed into broader SARS-CoV-2 vaccines with excellent safety profiles.