| Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV) is a severe infectious disease in swine herds characterized by reproductive failure in sows and respiratory illness in young pigs. Genomic analysis indicated that PRRSV has two major genotypes, the European represented by strain LV and the North American represented by strain VR-2332. As PRRSV naturally infects macrophages and traditional strategy of vaccine development doesn't work very well to the disease, therefore, development of safe and effective vaccines with genetic engineering technology would be a promising approach to protection from the disease.Based on the roles of different structural proteins of PRRSV in eluting immune responses, a series of double-expressing plasmids were constructed on a mammalian expression vector pIRESlweo. These DNA constructs include pIRESorf5/4, pIRESorf6/5 and pIRESorf7/5, which contain PRRSV ORF5 and ORF4, ORF6 and ORF5, ORF5 and ORF7, respectively, and pIRESorf5/IFN , pIRESorf7/IFN , pIRESorf5/IL-2 and pIRESorf7/IL-2, which contain ORF5 or ORF7 of PRRSV and gene encoding for porcine IFNy or IL-2, respectively.The expressions of these recombinant plasmids were first detected in vitro by indirect immunofluorescence test and in BALB/c mice by ELISA and Western Blotting assay.Thirty PRRSV-negative piglets were divided into ten groups. One of the ten groups was set as mock control and the other groups were inoculated with pIRESorf5/4, pIRESorf6/5, pIRESorf7/5, pIRESorf5/IFN , pIRESorf7/IFN , pIRESorf5/IL-2, pIRESorf7/IL-2, inactivated vaccines or pIRESlneo, respectively. The humoral immune responses of the pigs were tested by ELISA and Western Blotting (WB), with recombinant antigens of rGP4 (ORF4), rtGP5 (truncated ORF5), rM (ORF6) and rN (ORF7) expressed in E. col. The antibodies to N protein could be detected by ELISAand WB in most of piglets inoculated with the plasmids containing ORF7 after the second inoculation. In contrast, the specific antibodies induced by ORF4, ORF5 or ORF6 appeared later and the titers of ELISA antibodies were lower too. After the fourth inoculation, the specific antibodies could be detected by WB in all piglets inoculated with recombinant plasmids. But antibody responses in some piglets of these groups were not detectable by ELISA over the study period.Usually, most of PRRSV infections are subclinical. In the study, respiratory problems and blue ear were not observed on all the pigs after challenged with PRRSV strain CH-1a. But the rectal temperatures of the pigs in the negative control groups (plERS1neo-immunized group and mock group) were higher than the pigs immunized with the DNA vaccines. The highest temperature of a mock pig reached 40.6 .The percentages of CD4+, CD8+ or CD4+/CD8+ T cells in peripheral blood mononuclear cells (PBMC) were measured. Much difference of the T cell subpopulations was observed among individuals after immunization. The percentages of CD4+, CDS+ or CD4+/CD8+ T cells of the animals immunized with DNA vaccines increased at different extents compared to the pIRES1neo-immunized or mock-immunized animals. After challenged with PRRSV, the CD4+ and CD8+ T cells of the pigs immunized with pIRESorf5/4, pIRESorf6/5, pIRESorf7/5, pIRESorf7/IFNy, pIRESorf5/IL-2 or pIRES1neo increased, but CDS+ T cells decreased soon after. These results differ from the previously reported.Though the clinical respiratory illness did not appear, the pathological changes were observed in lungs from the most of the challenged animals. Portions of the lungs were tan in color and partly collapsed, with occasional anteroventral areas of congestion and consolidation. Two of three pigs immunized with pIRESorf5/IFN , one of three pigs with pIRESorf5/IL-2, one of three pigs with pIRESorf7/IL-2 and one of two pigs with inactivated vaccines didn't have gross lesions in lungs.Detection of PRRS virions in the main organs of the challenged animals showed that, all recombinant plasmids and the inactivated vaccine failed to prevent the occurring of v...
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