Construction Of Oral COVID-19 Vaccine Based On Saccharomyces Cerevisiae Surface Display Technology And Analyses Of Its Immune Respones

Objective:COVID-19 oral candidate vaccine was generated based on Saccharomyces cerevisiae(S.cerevisiae)surface display technology,and its expression efficiency in vitro and immune responses levels were analyzed.Methods:The S gene(Gen Bank:MN908947)of SARS-Co V-2 was used as a template,and the target gene RBD was amplified by PCR.The RBD gene and expression plasmid p YD1 were digested with NheⅠ/Eco RⅠ,and then the recombinant plasmid p YD1-RBD was obtained after ligation by T4 DNA ligase.The p YD1-RBD was transformed into competent E.coli DH5α.PCR,double endonuclease digestion analysis and sequencing were used to determined the positive clone E.coli DH5α/p YD1-RBD.Further,the recombinant plasmid p YD1-RBD was extracted from E.coli DH5α/p YD1-RBD,and then transformed into competent S.cerevisiae EBY100.The positive clone EBY100/p YD1-RBD was screened by tryptophan auxotrophic(Trp~-)medium and determined by PCR and sequencing.Galactose was used as an inducer,Western blot,immunofluorescence microscope and flow cytometry were performed for detecting the expression analyses of EBY100/p YD1-RBD in vitro at 72h post-induction.SPF BALB/c mice was used as an animal model of oral immunization for investigating the levels of immune responses induced by EBY100/p YD1-RBD.The humoral immune response,mucosal immune response and cellular immune response in mice orally adminisitrated with EBY100/p YD1-RBD were measured by ELISA.Neutralizing antibodies of the vaccinated mice were detected by SARS-Co V-2 pseudovirus.Results:EBY100/p YD1-RBD was successfully generated by conventional molecular biology methods.The expression of specific RBD protein in EBY100/p YD1-RBD was analyzed by Western blot,and the molecular weight of RBD protein was approximately 32k Da.The expression location and expression efficiency of RBD protein were further analyzed by immunofluorescence microscope and flow cytometry.The RBD protein was expressed on the surface of S.cerevisiae,the expression efficiency was approximately 45.1%.Oral administration of EBY100/p YD1-RBD in BALB/c mice could induce specific serum Ig G and secretory Ig A.Furthermore,the expression levels of cytokines were analyzed by flow cytometry.The secretion level of IFN-γmediated by CD4~+T and CD8~+T cells is higher than that of IL-4,suggesting that EBY100/p YD1-RBD could elicite significant cell immune responses and the mixed Th1/Th2 responses and Th1 bias.The results of neutralization assay indicate that mice orally vaccinated with EBY100/p YD1-RBD can produce the significant levels of neutralizing antibodies.Conclusion:S.cerevisiae surface display technology provides a reliable strategy for the development of more safe and effective COVID-19 oral vaccine candidates,which also provides a new research idea and strategy for the development of other highly infectiou virus or bacterial oral vaccines.