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This research aims to develop a yeast system for producing SARS-CoV-2 RBD and evaluate its immunogenic potential.

June 13, 2026Microbial Cell Factories0 citationsOpen Access

Engineering Komagataella phaffii for secreted and surface-displayed production of SARS-CoV-2 receptor-binding domain and its immunogenicity evaluation

KDKostyantyn Dmytruk ODOlena Dmytruk MSMarta Semkiv

Key Points

This research aims to develop a yeast system for producing SARS-CoV-2 RBD and evaluate its immunogenic potential.
Engineered K. phaffii strains for RBD production in secreted and surface-displayed formats.
Used glycine–serine linkers to improve secretion efficiency, yielding 50 mg/L of RBD.
Conducted immunogenicity studies in mice with both purified protein and whole-cell yeast displaying RBD.
Achieved strong humoral immune response after intraperitoneal administration of purified RBD.
Sera from immunized mice showed potent inhibition of spike protein binding to the ACE2 receptor.
Oral administration of K. phaffii displaying RBD also elicited robust immune responses.

Abstract

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.

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