Immunogenicity evaluation of an Alum-adjuvanted recombinant prefusion RBD-Fd SARS-CoV-2 protein subunit produced in Glycoengineered Pichia pastoris

The global coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has accelerated vaccine development worldwide. While whole inactivated virus vaccines have reduced the incidence of severe disease and mortality, their effectiveness against emerging variants is limited. mRNA vaccines offer broader protection but face challenges in cost, production, and storage. Protein subunit vaccines targeting the viral spike (S) protein present a promising alternative due to their safety and scalability. In this study, we developed a recombinant protein subunit using the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, fused with a foldon domain (RBD-Fd) to promote trimer formation. The protein was expressed in Pichia pastoris GlycoSwitch® to achieve human-like glycosylation and was formulated with aluminium hydroxide (Alhydrogel®/Alum) to enhance immunogenicity. The resulting prototype protein subunit was evaluated in mice via subcutaneous injection at doses of 5 μg or 10 μg. Results showed that the alum-adjuvanted RBD-Fd formulation induced a strong antibody response following two doses at both concentrations. However, it generated only a partial T cell response with CD8? T cell activation but no corresponding CD4? response. These findings highlight the potential of prefusion RBD-based protein subunit and support further optimization to enhance cellular immunity.