Oral yeast-displayed SARS-CoV-2 spike protein vaccine enhanced by trimerization and flagellin peptide

The global COVID-19 pandemic has underscored the need for innovative vaccine strategies, including oral vaccines, to improve accessibility and enhance immunity. In this study, we explored a novel oral vaccine approach by using yeast surface display to express the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein on Pichia pastoris using SED1 as a carrier protein. The constructs included a fibritin foldon domain from bacteriophage T4 for trimerization and truncated flagellin from Salmonella typhimurium (FliC) as an adjuvant to boost immunogenicity. Three recombinant yeast constructs were designed and evaluated: construct I (RBD-SED1), construct II (RBD-foldon-SED1), and construct III (FliC-RBD-foldon-SED1). Structural integrity, proper folding, and trimerization (in construct III) were modeled and confirmed using ColabFold. These constructs were successfully expressed on the yeast surface and confirmed through fluorescence microscopy, flow cytometry, and ELISA. Oral immunization of BALB/c mice with the recombinant yeast significantly induced spike protein-specific IgG antibodies in serum and IgA in fecal samples, indicating strong humoral and mucosal immune responses. Notably, construct III induced the highest immune response. These findings suggest that yeast-based oral vaccines expressing trimerized SARS-CoV-2 spike protein RBD, combined with a peptide fragment from flagellin, as an immunomodulatory adjuvant, can elicit robust immunity, offering a promising alternative for future COVID-19 vaccination strategies.