Development of a self-assembling multimeric Bann-RBD fusion protein in Pichia pastoris as a potential COVID-19 vaccine candidate.

The development of an affordable and accessible vaccine platform is essential for achieving global and long-term protection against COVID-19 and other emerging viral diseases. In this study, we developed a multimeric fusion protein comprising the SARS-CoV-2 receptor-binding domain (RBD) and the β-annulus (Bann) from the tomato bushy stunt virus (TBSV) as a potential subunit vaccine candidate. Molecular modeling of Bann-RBD revealed a 60-mer structure with the RBD displayed on its outer surface. The Bann-RBD gene was constructed and overexpressed in Pichia pastoris X-33. SDS-PAGE analysis of the purified Bann-RBD showed a protein band at 45 kDa, corresponding to monomeric glycosylated Bann-RBD. Peptide mapping analysis using LC-MS/MS confirmed that the expressed Bann-RBD was consistent with the designed protein fusion. The Bann-RBD protein was observed to spontaneously self-assemble into spherical nanocapsids with a diameter of approximately 50 nm. Antigenicity studies demonstrated that the purified Bann-RBD was strongly recognised by monoclonal human anti-SARS-CoV-2 spike-S1 IgG antibodies. Immunogenicity studies revealed that Bann-RBD elicited a robust humoral immune response in BALB/c mice, generating potent neutralising antibodies. Collectively, these findings suggest that the recombinant Bann-RBD produced in Pichia pastoris X-33 is a promising candidate for the development of a COVID-19 vaccine.