Modification Of ORF5 Gene Of Porcine Reproductive And Respiratory Syndrome Virus And The Immunogenicity Of The Recombinant Plasmid DNA

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically significant viral diseases in the swine industry. It is characterized by reproductive problems in sows such as poor farrowing rates, premature farrowings, and increased stillbirths, as well as respiratory problems in piglets such as pneumonia and atrophic rhinitis. Current commercial PRRS vaccines, including both live attenuated and killed vaccines, have been wildly used, but they cannot provide effective protection against PRRS.Although the ORF5-encoded GP5 is the most important immunogenic protein, accumulating evidences have demonstrated that incomplete protection conferred by GP5-based vaccines. In this study, ORF5 gene was engineered with the codon usage optimized for mammalian cell expression based on the native ORF5 gene of PRRS V strain SY0608. Additional modifications, i.e., inserting a T-helper cell epitope(GP4-5, N-7 or GP4-3) between the neutralizing epitope and the non-neutralizing decoy epitope, and mutating four potential N-glycosylation sites (N30, N34, N35 and N51) were also included in the modified ORF5 gene. Recombinant pCI-GP5, pCI-SynGP5, pCI-SynGP5/GP4-5, pCI-SynGP5/N-7 and pCI-SynGP5/GP4-3 were constructed and confirmed by restriction enzyme ananlysis and sequencing. To investigate the expression in vitro and authenticity of the modified ORF5 gene, HEK-293A cells were transfected individually with pCI-SynGP5, pCI-SynGP5/GP4-5, pCI-SynGP5/N-7 and pCI-SynGP5/GP4-3, and Western blot was performed at 48h post-transfection. Meanwhile, constructed pCI-GP5, expressing the native ORF5 gene of PRRSV strain SY0608, was used as control. The results showed that GP5-specific protein bands with expected molecular sizes could be detected in lysates of cells transfected with recombinant plasmids, but not in lysates of cells transfected with the empty vector. Furthermore, the expression of GP5 in the cells transfected with pCI-SynGP5, pCI-SynGP5/GP4-5, pCI-SynGP5/N-7 or pCI-GP5/GP4-3 was increased, comparing to that in the cells tranfected with pCI-GP5. It indicated that the modified ORF5 gene exhibit greater expression efficiency in mammalian cells than the native ORF5 did.In order to know the immunity of the recombinant plasmids, five groups of Balb/c mice (10 mice per group) were inoculated intramuscularly three times at 3-week intervals with the recombinant plasmid DNA, ie. pCI-GP5, pCI-SynGP5, pCI-SynGP5/GP4-5, pCI-SynGP5/N-7 and pCI-SynGP5/GP4-3, at 100μg/mouse. Two additional groups were injected with pCI-neo and PBS as control. Six and nine weeks after primary immunization, mice were sacrificed and splenocytes were isolated for detecting IFN-γ, IL-4 and lymphocyte-proliferation. Serum samples were collected at 6 and 9 weeks after primary inoculation for detecting antibody to PRRSV. The results showed that the mice inoculated with recombinant plasmids developed PRRSV-specific antibodies, cellular immune response at 9 weeks post primary inoculation. However, only mice immunized with recombinant plasmids pCI-SynGP5/GP4-5 developed significantly higher titers of neutralizing antibodies to PRRSV and produced stronger lymphocyte proliferation responses compared to the mice immunized with other recombinant plasmids. It was also found that lymphocytes of the mice immunized with pCI-SynGP5/GP4-5 was primed for significant higher levels of PRRSV specific IFN-y responses upon stimulation with purified PRRSV antigen in vitro than that of mice immunized with other recombinant plasmids. It suggested that the modified ORF5 genes by three strategies could enhance the immuno-genicity of ORF5 gene. The plasmid SynGP5/GP4-5 had the highest immunogenicity in all of the plasmids with the modified ORF5.Finally, the recombinant plasmid pCI-SynGP5/GP4-5 was transformed into an attenuated S.typhimurium strain SL3263 by electroporation, resulting in SL-pCI-SynGP5/ GP4-5. Thirty piglets were divided into 6 groups of five. The first group was intramuscularly (IM) immunized with PPRSV vaccine for one time. And the other five groups were immunized twice with pCI-SynGP5/GP4-5 (IM), pCI-neo (IM), SL-pCI-SynGP5/GP4-5 (oral), SL3263 (oral) and PBS (IM). Serum samples were collected at 2 and 4 weeks after primary inoculation for serological tests. PBMC were collected at 4 weeks after primary immunization for IFN-y assay and IL-4 assay. Then, all pigs were challenged with highly pathogenic PRRSV SY0608 strain at 4 weeks after primary vaccination. Protective efficacy against homologous challenge was examined. The results showed that the animals vaccinated with PRRSV vaccine developed specific anti-PRRSV antibody by ELISA and neutralizing assay, and specific IFN-y responses upon stimulation with purified PRRSV antigen in vitro. The recombinant S.typhimurium could induce lower ELISA antibody and specific IFN-γ responses upon stimulation with purified PRRSV antigen in vitro. Meanwhile, neither humoral immune response nor cell-mediated immunity could be detected in other groups. Following challenge with PRRSV, piglets inoculated with PRRSV vaccine and SL-pCI-SynGP5/GP4-5 showed lighter clinical signs, lower viremia and less gross lesion of lungs, comparing with those in other groups. And a low protective efficiency induced by pCI-SynGP5/GP4-5 has been detected. It indicated that SL-pCI-SynGP5/GP4-5 could markedly increase the immune responses and provide protection against virulent PRRSV challenge in pigs.In conclusion, the present study clearly indicated that the modified ORF5 gene exhibited greater expression efficiency in mammalian cells than the native ORF5 did. Mice immunized with recombinant plasmids pCI-SynGP5/GP4-5 developed significantly higher titers of neutralizing antibodies to PRRSV and produced stronger lymphocyte proliferation responses compared to mice immunized with other recombinant plasmids. SL-pCI-Syn GP5/GP4-5 could markedly increase the immune responses and provide protection against virulent PRRSV challenge in pigs. The recombinant S.typhimurium might be an attractive candidate vaccine for preventing and control of highly pathogenic PRRSV infections.