Abstract Background The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein represents a key antigen for vaccine development due to its critical role in the ACE2 receptor recognition. Yeast-based expression systems, particularly Komagataella phaffii , offer scalable and cost-effective platforms for recombinant protein production. In addition to secretion, cell surface display provides an alternative strategy enabling direct antigen delivery, including applications via mucosal routes. Results In this study, we engineered K. phaffii strains for the production of SARS-CoV-2 RBD in two formats: as a secreted recombinant protein and as a cell surface-displayed antigen using a Sag1 anchoring system. Incorporation of glycine–serine linkers enhanced secretion efficiency, yielding up to 50 mg/L of RBD, with minimal intracellular retention. Western blot analysis indicated the presence of glycosylated forms of RBD, and the recombinant protein was subsequently purified for further characterization. Surface localization of RBD was validated by immunofluorescence microscopy and quantitative fluorescence measurements. Immunogenicity studies in mice demonstrated that intraperitoneal administration of purified RBD elicited a strong humoral immune response. Importantly, sera from immunized animals efficiently inhibited spike protein binding to the Ace2 receptor, indicating potent neutralizing activity. Comparable results were obtained following oral administration of K. phaffii cells displaying RBD on their surface, demonstrating the feasibility of a whole-cell yeast-based vaccine approach. Conclusions A yeast-based system enabling both secretion and surface display of SARS-CoV-2 RBD has been developed. Both delivery strategies, intraperitoneal administration of the purified protein and oral administration of recombinant yeast cells, induced robust and functionally relevant immune responses in mice. These findings highlight the potential of K. phaffii as a versatile system for the development of cost-effective subunit and oral vaccine candidates.
Dmytruk et al. (2026) studied this question.