Single self-cleaving mRNA vaccine expressing multiple viral structural proteins elicits robust immune responses and protects nursing piglets against PDCoV infection.

As an emerging swine enteric coronavirus, porcine deltacoronavirus (PDCoV) poses a severe threat to the global swine industry and has demonstrated potential for cross-species transmission. Therefore, the development of safe and effective vaccines is a top priority for the future prevention and control of PDCoV. In this study, we first designed and prepared an mRNA vaccine, S2P-mRNA-LNP, that expresses the full-length S2P (E855P, V856P) protein of PDCoV. Animal experiments demonstrated that S2P-mRNA-LNP induced significantly stronger humoral and cellular immune responses in mice than did an inactivated vaccine. Then, we introduced the P2A self-cleaving peptide into the S2P-mRNA-LNP design, generating SMN-mRNA-LNP, an LNP-encapsulated mRNA vaccine that allows the simultaneous expression of three major structural proteins of PDCoV (S, M, and N) from a single mRNA. The immunization of piglets demonstrated that both S2P-mRNA-LNP and SMN-mRNA-LNP induced robust humoral immune responses. Notably, SMN-mRNA-LNP conferred significantly superior active immune protection in piglets (5/5) than did S2P-mRNA-LNP (4/5). Further immunization experiments in pregnant sows showed that piglets born to SMN-mRNA-LNP-vaccinated sows acquired high levels of IgG, IgA, and neutralizing antibodies through the ingestion of colostrum, conferring complete passive protection (5/5). The protective efficacy of SMN-mRNA-LNP was markedly superior to that of the inactivated vaccine. In conclusion, these findings demonstrate that SMN-mRNA-LNP is a novel and highly efficacious candidate vaccine against PDCoV. In addition, the design strategy of single mRNA-LNP simultaneously delivering multiple viral antigens in this study provides a new idea for the development of porcine intestinal coronavirus mRNA vaccine in the future.IMPORTANCEIn this study, we designed and developed a novel mRNA vaccine, SMN-mRNA-LNP, capable of expressing the three major structural proteins (S, M, and N) of PDCoV from a single mRNA. This vaccine conferred superior active immune protection on piglets to that conferred by S2P-mRNA-LNP expressing only the S protein. Furthermore, following the immunization of pregnant sows with SMN-mRNA-LNP, their colostrum showed remarkably high IgA antibody titers reaching 1∶10^5.4, representing a 25-fold increase over that in the inactivated vaccine group. By suckling, newborn piglets acquired significantly greater passive immunity, which ultimately conferred complete protection against PDCoV challenge.