Construction And Immunogenicity Of An Eukaryotic Plasmid Co-expressing PCV2Cap And TGEV S Genes

Porcine Circovirus (PCVs) belong to the family circoviridae and the genuscircovirus, which comprise PCV1and PCV2. PCV1does not induce a disease in swine,while PCV2is etiologically responsible for PMWS. Infection of PCV2is ubiquitous indomestic pigs and PMWS had been diagnosed on all five continents with theprevalence ranging between4to30%. The TGEV is a single-stranded RNA belongsto the order of Nidovirales, family of Coronaviridae. TGE-virus is etiologicalresponsible of porcine transmissible gastroenteritis. Swine of all species and age weresusceptible to TGEV, and the mortality of the infected piglets can be up to100%under3weeks.Therefore, PCV2and TGEV are the devastating diseases in the porcine industryworldwide; the immunization against those diseases had been showed as efficientstrategy for protecting against PCV2or TGEV-infections. However, the availablevaccines against PCV2and TGEV have been shown to be effective in reducing theclinical syndrome and improving production parameters in farms, but these vaccinescannot completely prevent PCV2or TGEV infection and transmission. Moreover,several experimental inactivated vaccine, DNA-based vaccines, live virus vectorrecombinant containing PCV2Cap or TGEV S genes have been used against PCV2orTGEV-infections. Nonetheless, this is the first time to co-express PCV2Cap andTGEV S genes in the same vaccine vector and immunize the pigs against PCV2orTGEV-infection.The goal of the present study was to co-expressing PCV2Cap and TGEV S fusedprotein genes in an eukaryotic vector, and to evaluate the vaccine potential of thisrecombinant DNA-based vaccine in terms of immunogenicity and protection againstPCV2or TGEV challenge in pigs.Thus,702bp of PCV2Cap gene and1830bp of TGEV S gene containing the siteA (immunodominant) were co-expressed in pEGFP-N1eukaryote vector. The Cap andS gene were fused using a flexible linker (Gly4Ser)3. The recombinant vaccines havebeen confirmed by PCR, endonuclease digestion and sequencing. The PCV2Cap, TEGV S and Cap-S recombinant proteins were expressed and identified in PK-15cellsby western blot. The levels of IFN-γ expressed in vitro were assessed by iELISA.Thirty-two pigs were randomly divided in six groups and immunized with500μg ofpEGFP-Cap, pEGFP-S, pEGFP-Cap-S and2ml of PBS in three times at two weeksinterval. At one week after the third immunization, groups were challenged with2x107TCID50of PCV2or TGEV virus. The DNA-vaccines potential in terms ofimmunogenicity and protection efficacy against PCV2or TGEV challenge wasevaluated in pigs by iELISA, NA, T-lymphocytes cytometric, viral load, HE and IHC.The results in this study showed that vaccination of the pigs with the pEGFP-Capor pEGFP-S induced solid protection against PCV2or TGEV-infection by induction ofhighly specific serum IgG antibodies, IgA, IgG1and IgG2a isotypes and cytokines(IFN-γ and IL-10). These DNA-based vaccines have been shown to alleviate the PCV2or TGEV viral load. The co-express Cap and S gene has been shown to enhance thehumoral and cell immune responses against PCV2or TGEV-infection. The groupstreated with the pEGFP-Cap, pEGFP-S and pEGFP-Cap-S showed no perceptiblehistopathological lesion after HE staining and presented only a few positive cells afterIHC compared to PCV2or TGEV inoculated control pigs.In conclusion, the present study demonstrated that the recombinant DNA-vaccines co-expressing PCV2Cap and TGEV S genes can substantially alleviatePCV2or TGEV-infection in pigs.