A Genetically Engineered Bivalent Vaccine Coexpressing a Molecular Adjuvant against Classical Swine Fever and Porcine Epidemic Diarrhea.

Classical swine fever (CSF) and porcine epidemic diarrhea (PED) are highly contagious viral diseases that pose a significant threat to piglets and cause substantial economic losses in the global swine industry. Therefore, the development of a bivalent vaccine capable of targeting both CSF and PED simultaneously is crucial. In this study, we genetically engineered a recombinant classical swine fever virus (rCSFV) expressing the antigenic domains of the porcine epidemic diarrhea virus (PEDV) based on the modified infectious cDNA clone of the vaccine strain C-strain. The S1N and COE domains of PEDV were inserted into C-strain cDNA clone harboring the mutated 136th residue of N ^pro and substituted 3'UTR to generate the recombinant chimeric virus vC/SM3'UTR _N -S1NCOE. To improve the efficacy of the vaccine, we introduced the tissue plasminogen activator signal (tPAs) and CARD domain of the signaling molecule VISA into vC/SM3'UTR _N -S1NCOE to obtain vC/SM3'UTR _N -tPAsS1NCOE and vC/SM3'UTR _N -CARD/tPAsS1NCOE, respectively. We characterized three vaccine candidates in vitro and investigated their immune responses in rabbits and pigs. The N ^pro _D136N mutant exhibited normal autoprotease activity and mitigated the inhibition of IFN-β induction. The introduction of tPAs and the CARD domain led to the secretory expression of the S1NCOE protein and upregulated IFN-β induction in infected cells. Immunization with recombinant CSFVs expressing secretory S1NCOE resulted in a significantly increased in PEDV-specific antibody production, and coexpression of the CARD domain of VISA upregulated the PEDV-specific IFN-γ level in the serum of vaccinated animals. Notably, vaccination with vC/SM3'UTR _N -CARD/tPAsS1NCOE conferred protection against virulent CSFV and PEDV challenge in pigs. Collectively, these findings demonstrate that the engineered vC/SM3'UTR _N -CARD/tPAsS1NCOE is a promising bivalent vaccine candidate against both CSFV and PEDV infections.