Immunoenhancement Of Cytokines To PCV2-Cap Protein And Immunogenicity Identification Of The Co-expressed Cap And FMDV-Vp1Proteins

Porcine circovirus type2(PCV2) has been recognized as the primary causative agent ofpostweaning multisystemic wasting syndrome (PMWS), which is extensively prevalent and results intremendous economic losses in the swine industry worldwide. PCV2contains two major open readingframes (ORFs): ORF1encodes two viral replication-associated proteins, Rep and Rep’, and ORF2encodes viral structural capsid (Cap) protein. The Cap protein is the major immunogenic protein thathas the ability to elicit PCV2neutralizing antibodies and protective immunity against PCV2infection;therefore, it comprises the desired target antigen for the development of genetically engineered vaccinesagainst PCV2infection. It has been shown that cytokines, such as IFN-γ, IL-2, and GM-CSF, couldexert an immunoadjuvant effect on enhancing the immunogenicity of a certain vaccine, therebysubstantially enhancing protective efficacies of vaccines. In this study, PCV2novel subunit vaccine wasdeveloped via the combination of cytokine with PCV2-Cap protein, and it was investigated that whetherporcine IFN-γ (PoIFN-γ), porcine IL-2(PoIL-2), and porcine GM-CSF (PoGM-CSF), as immuneadjuvants, could enhance the protective immunity to the Cap protein-based PCV2subunit vaccine.To investigate the immunoenhancement of PoIFN-γ on the Cap protein-based PCV2subunitvaccine, PCV2-Cap protein, PoIFN-, and the fusion protein (Cap-PoIFN-) of PCV2-Cap protein andPoIFN-were expressed using baculovirus expression system, and three PCV2subunit vaccines(Cap-PoIFN-γ vaccine, PCV2-Cap+PoIFN-γ vaccine, and PCV2-Cap vaccine) were generated based onthe three target proteins, and the protective immunity to the three PCV2subunit vaccines was confirmedin mice. IPMA and the blocking ELISA were performed to detect, respectively, PCV2-specific IPMAantibodies and neutralizing antibodies in serum samples following vaccination and challenge, and theresults indicate that the PCV2-Cap+PoIFN-γ vaccine produced higher antibody titers compared to theCap-PoIFN-γ and PCV2-Cap vaccines. The results from lymphocyte proliferation assay suggest that thePCV2-Cap+PoIFN-γ vaccine induced significantly higher proliferative responses compared to theCap-PoIFN-γ and PCV2-Cap vaccines. Following challenge, a significantly lower PCV2viral load oflungs in immunized mice was present in the PCV2-Cap+PoIFN-γ group compared to the Cap-PoIFN-γand PCV2-Cap groups, indicating that the PCV2-Cap+PoIFN-γ vaccine produced stronger protectiveimmunity compared to the Cap-PoIFN-γ and PCV2-Cap vaccines. In this study, PoIFN-γ has beendemonstrated to essentially enhance the protective immunity to the Cap protein-based PCV2subunitvaccine when co-administrated with PCV2-Cap protein.To demonstrate the immunoenhancement of PoIL-2and PoGM-CSF on the Cap protein-basedPCV2subunit vaccine, PoIL-2, PoGM-CSF, and the fusion protein (Cap-PoIL-2or Cap-PoGM-CSF) ofPCV2-Cap protein and PoIL-2or PoGM-CSF were expressed using baculovirus expression system, andfive PCV2subunit vaccines (Cap-PoIL-2vaccine, PCV2-Cap+PoIL-2vaccine, Cap-PoGM-CSFvaccine, PCV2-Cap+PoGM-CSF vaccine, and PCV2-Cap vaccine) were generated, and the protectiveimmunity to the five PCV2subunit vaccines was confirmed in mice. IPMA was implemented to detectPCV2-specific antibodies in serum samples following vaccination and challenge, and the results suggest that the PCV2-Cap+PoIL-2, Cap-PoGM-CSF, PCV2-Cap+PoGM-CSF, and PCV2-Cap vaccinesproduced the same levels of PCV2antibodies, but higher than the Cap-PoIL-2vaccine. The results fromlymphocyte proliferation assay and cytokine platinum ELISA show that the PCV2-Cap+PoIL-2,Cap-PoGM-CSF, and PCV2-Cap+PoGM-CSF vaccines produced significantly higher proliferativeresponses and induced higher levels of IFN-γ and IL-2secreted by splenic lymphocytes of immunizedmice compared to the Cap-PoIL-2and PCV2-Cap vaccines. Following challenge, a significantly lowerPCV2viral load of lungs in immunized mice was observed in the PCV2-Cap+PoIL-2, Cap-PoGM-CSF,and PCV2-Cap+PoGM-CSF groups compared to the Cap-PoIL-2and PCV2-Cap groups, indicating thatthe PCV2-Cap+PoIL-2, Cap-PoGM-CSF, and PCV2-Cap+PoGM-CSF vaccines elicited greaterprotective immunity compared to the Cap-PoIL-2and PCV2-Cap vaccines. In this study, PoIL-2andPoGM-CSF have been shown to substantially heighten Th1-type immune response to the Capprotein-based PCV2subunit vaccine when co-administrated with PCV2-Cap protein or delivered as aCap-PoGM-CSF fusion protein, thereby enhancing protective immunity against PCV2infection.Foot and mouth disease virus (FMDV) has been regarded as the major causative agent affectingcloven hoofed animals, which causes acute, pyretic, and highly contagious infectious diseases andbrings about enormous economic losses in the pig industry worldwide. FMDV consists of four structuralproteins Vp1, Vp2, Vp3, and Vp4, in which Vp1is the main immunogenic protein capable of inducingneutralizing antibodies. In recent years, PCV2and FMDV infections are extremely severe in pigs inChina, and the control of the two viral diseases mainly depend on the inactivated vaccines; however, thetruth is the difficulty to obtain high titers of PCV2inactivated vaccine virus and the risk of FMDVdissemination due to the incomplete viral inactivation of FMDV inactivated vaccine. It has been shownthat the subunit vaccines based on recombinant proteins exhibit desirable protective efficacy againstPCV2and FMDV infections; therefore, it is of vital importance to develop the bivalent subunit vaccineagainst the two viral diseases simultaneously. In this study, PCV2-Cap protein, FMDV-Vp1protein,Cap-Dual-Vp1dual-expressed products and the fusion protein (Cap-Linker-Vp1) of PCV2-Cap andFMDV-Vp1proteins were expressed in the baculovirus system, and four PCV2and FMDV bivalentsubunit vaccines (Cap-Dual-Vp1-A vaccine, Cap-Dual-Vp1-B vaccine, Cap-Linker-Vp1vaccine, andPCV2-Cap+FMDV-Vp1vaccine) were generated, and the immunogenicity of the four vaccines wasconfirmed in mice. The results show that the four bivalent subunit vaccines produced high levels ofPCV2-specific antibodies and anti-FMDV-Vp1antibodies, and there was no significant difference inantibody titers induced by the four vaccines. Taking production costs into consideration, it is a feasiblestrategy to develop the bivalent subunit vaccine composed of a mixture of PCV2-Cap and FMDV-Vp1proteins against PCV2and FMDV infections due to the lower efficiency of dual expression and fusionexpression of PCV2-Cap and FMDV-Vp1proteins. This study lays the foundation for the furtherdevelopment of the bivalent subunit vaccine for the control of both PCV2and FMDV infections.