Immune Response Induced By Oral DNA Vaccination Against Porcine Reproductive And Respiratory Syndrome Virus Delivered By Salmonella Typhimurium

Porcine Reproductive and Respiratory Syndrome, a highly contagious disease with worldwide distribution, can cause severe economic losses to the swine industry. The causative agent of the disease, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), is a member of the Nidovirales, Arteriviridae whose genome is a non-segmented single-strand positive-sense RNA. The GP5 glycoprotein and GP3 and M protein are the major factors of the virus to which the host responses well with protective antibody production. The recombinant plasmids containing the GP5 gene can induce specific immune responses against PRRSV. However, DNA vaccination by injection or gene gun routes is rather inconvenient and costly in field application in scaled animal farms.Recent studies show that attenuated intracellular bacteria can be used for delivery of DNA vaccines. Salmonella typhimurium, one of the invasive bacterial species, can be attenuated without loss of invasiveness and thus used for delivery of eukaryotic expression vectors into host cells in vivo. The recombinant plasmid containing the target gene is released inside the host cells and gain entry into the nucleus, resulting in expression of encoded antigens and subsequent induction of humoral and cellular immune responses. However, there has been no report on genetic vaccination against stock diseases using S.typhimurium as the oral delivery vector for vaccines. The main purpose of this study was to examine the feasibility of a DNA vaccine against PRRSV using attenuated S.typhimurium with aroA mutation as a vector for delivery of the PRRSV GP5 gene.The partial-length DNA of the GP5 gene (GP5') of a virulent PRRSV strain S1 was amplified by PCR and inserted into pcDNA3.1 under the control of the human cytomegalovirus (CMV) immediate early enhancer and promoter. The GP5' gene from the resulting recombinant plasmid pcDNA3-GP5' was sequenced. The gene was 525bp inlength, encoding the GPS protein composed of 175 amino acids. Sequence analysis and its secondary structure prediction showed that the GPS protein had several major hydrophobic regions, two to four potential glycosylation sites and a hydrophobic signal peptide. The GPS' gene of the strain SI showed 99% homology with other strains. RecombinantpcDNA3-GP5 and pcDNA3-GP5' plasmids were transfected into HEK-293A cells withanion lipid transfection reagent. Subsequently the mRNA of interest gene was analyzed by RT-PCR and the expression of target protein by indirect immunofluorescent assay (DFA).The recombinant eukaryotic expression plasmids pEGFP, pcDNA3-GP5 and pcDNA3-GP5' were transformed by electroporation into an attenuated S.typhimwium strain SL7207 (aroA"), resulting in SL7207/ pEGFP, SL7207/ pcDNA3-GP5 and SL7207/ pcDNA3-GP5'. SL7207/ pEGFP was then used to infect the murine peritoneal macrophages in vitro. There was expression of the GFP protein as showed by the fluorescent microscope. SL7207/ pcDNA3-GP5 and SL7207/ pcDNA3-GP5' were used to immune the mice orally, and after 48h the interest genes were detected in small intestine by RT-PCR. The interest protein were detected in murine peritoneal macrophages by indirect immunofluorescent assay. The results showed that the interest gene could be transfected into eukaryotic cells by an attenuated S.typhimurium strain SL7207 with the mutation of aroA and it also could be transcribed and expressed in cells.The efficacy of the PRRSV DNA vaccines SL7207/ pcDNA3-GP5, SL7207/ pcDNA3-GP5' and naked DNA vaccines pcDNA3-GP5, pcDNA3-GP5' were compared. Six to eight-week-old female mice were divided randomly into seven groups. Mice in group I were immunized orally with 108 cfu of SL7207/ pcDNA3-GP5, group II mice with 10* cfu of SL7207/ pcDNA3-GP5', grouplllmice with 10* cfu of SL7207/ pcDNA3.1. Mice in group IV were immunized with 100 ug of naked plasmid pcDNA3-GP5 by intra-dermal injection , group V mice with 100 ug of naked plasmid pcDNA3-GP5', group VI mice with 100 ug of naked plasmid pcDNA3.1. The mice were boosted three with the frequency of ISd with the same at the sam...