A yeast surface display platform for oral delivery of a SARS-CoV-2 receptor-binding domain dimer inducing mucosal and systemic immunity.

The development of scalable and stable antigen delivery systems remains a central challenge in vaccine biotechnology, particularly for oral immunization targeting mucosal immunity. Here, we report a yeast surface display (YSD)-based platform for the oral delivery of a SARS-CoV-2 receptor-binding domain (RBD) dimer. A head-to-tail tandem RBD was genetically fused to the Aga2p subunit and anchored to the cell wall of Saccharomyces cerevisiae via the Aga1p-Aga2p system, generating the YSD-RBD-dimer construct. Flow cytometry confirmed clear surface expression, with 51.1% dual-positive cells recognizing both HA-tag and RBD epitopes. Under simulated gastric conditions (pH 1.2), the antibody-binding signal associated with surface-displayed RBD-dimer showed 97.57% retention after 2 h, indicating preservation of antibody-accessible RBD-associated epitopes. In vivo fluorescence tracking revealed gastrointestinal transit with signals peaking in Peyer's patches at 6 h and mesenteric lymph nodes at 12 h. Oral immunization elicited both systemic and mucosal immune responses, characterized by elevated serum anti-RBD IgG and fecal secretory IgA. Notably, vaccine-induced sera substantially inhibited RBD-ACE2 binding in a cell-based assay, with an inhibition rate of 81.6% ± 6.4%. Antigen-specific lymphocyte proliferation and transcriptional analysis revealed a mixed Th1- and Th2-associated response, with greater induction of IFN-γ and T-bet than of IL-4, IL-5, IL-13, and GATA-3. No apparent systemic toxicity or histopathological abnormalities were observed, supporting the preliminary safety of the platform. Collectively, this study supports the feasibility of a scalable yeast surface display-based platform for oral delivery of protein antigens and induction of coordinated systemic and mucosal immune responses.