Construction of subunit vaccine by fusing Salmonella flagellin with neutralizing epitopes of porcine epidemic diarrhea virus

IntroductionTo address practical application and production demands, this study aims to develop a Porcine epidemic diarrhea (PED) subunit vaccine with high expression efficiency and immunogenicity, using molecular biology and structural biology approaches.MethodsBased on the established immunostimulatory properties of flagellin, a recombinant fusion protein was constructed by linking the Salmonella choleraesuis flagellin mutant FliCL3A to the core neutralizing epitope (COE) of the spike protein of porcine epidemic diarrhea virus (PEDV). The target protein was expressed using a CHO-S cell expression system and subsequently formulated into a subunit vaccine with adjuvant 201. Immunogenicity was systematically evaluated in a mouse model by measuring serum levels of PEDV-specific IgG, IgA, and IgM antibodies, as well as the cytokines IFN-γ and IL-4.ResultsStructural prediction of the FliCL3A-COE fusion protein by AlphaFold2 indicated that the protein can spontaneously assemble into a bouquet-like multimeric structure. Following condition optimization, a CHO-S cell expression system with a transfection efficiency of approximately 80% was successfully established. Post-immunization ELISA results demonstrated that the level of PEDV-specific IgG increased progressively with the number of immunizations and reached a peak after the second booster immunization; IgA levels peaked after the first booster immunization but declined following the second booster; IgM levels reached their maximum after the primary immunization and decreased significantly with subsequent boosters. The expression trends of IFN-γ and IL-4 were consistent, both continuously increasing with each booster immunization. Compared with the commercial vaccine, the FliCL3A-COE subunit vaccine induced lower levels of IFN-γ; the levels of IgG, IgA, IgM, and IL-4 showed no significant difference from those induced by the commercial vaccine.ConclusionThe FliCL3A-COE fusion protein induces PEDV-specific antibodies and elicits detectable immune responses in mice. Its immunogenicity profile in terms of antibody induction is generally similar to that of a commercial vaccine, although IFN-γ responses were lower. These findings indicate that the fusion protein has certain immunostimulatory properties, but further optimization may be needed to improve its immune potency.