Construction And Immunological Study On Recombinant Adenovirus Vaccines Against EU-type PRRSV

Procine reproductive and respiratory syndrome diseases caused by procine reproductive and respiratory syndrome virus(PRRSV)which was first reported in late of 1995 in China and became epidemic in pig farms within several years. The CH1 a strains and its virant strains of NA-type PRRSV were dominant genotype in China now. While in recent years, the EU-type strain of PRRSV have outbreak in Asia such as China,South Korea and other countries. In Beijing and Shanghai and other cities of China have reported the EU-type PRRSV which caused significantly economic losses to the pig industry in the early of 1990 in European. The disease was founded in China which brought new challenge to prevent its transmission. In this study, the deficient adenovirus as vectors were used to package the recombinant adenoviruse expressing the ORF3 and ORF5 genesof EU-type PRRSV LV strain. Then, the constructed recombinant adenoviruse were immunized with mice and swine to analysis the immunological level of animals.The genes of ORF3 and ORF5 of LV strain and IL-18 of swine were used to constructe the shuttle plasmids of pacad-EU-ORF3, pacad-EU-ORF5, pacad-EU-ORF3-ORF5, pacad-IL-18 and pacad-EU-ORF3-ORF5-IL-18. Then the recombinant adenovirus shuttle plasmids and the plasmids of pacad 9.2-100 vector were linearized by restriction enzyme of PacI. The linearized plasmid were co-transfected into HEK293 cells for packing the recombinant adenovirus of rAd-EU-ORF3, rAd-EU-ORF5, rAd-EU-ORF3-ORF5, r Ad-EU-ORF3-ORF5-IL18 and rAd-IL18. The Western Blot and IFA were used to identify the recombinant adenovirus expressing the protein of GP3 and GP5. All results showed that the recombinant adenoviruses could expressed the GP3 protein and GP5 protein. The 2A genes of FMDV as linker were used to connect the different genes. In order to identify the efficiency of 2A, we packed the recombinant adenovirus rAd-EU-ORF3-ORF5-EGFP, and detected the expression of green fluorescent protein. The result showed the rAd-EU-ORF3-ORF5-EGFP infected with HEK293 cells expressing green fluorescent gene.Mice were immunized with candidate recombinant adenovirus vaccine rAd-EU-ORF3, rAd-EU-ORF5, rAd-EU-ORF3-ORF5, rAd-EU-ORF3-ORF5-IL-18 and rAd-IL-18. The results showed that all immunized mice can produce humoral immune and cellular immune response. In which co-expressed GP3-GP5 groups reach the highest level of immune response. Immunized with rAd-EU-ORF3-ORF5-IL-18 the specific antibody level was 1.29 times and 1.10 times than groups immunized with rAd-EU-ORF3 and rAd-EU-ORF5 at 35 dpi. The neutralizing antibody showed that the highest titers was 1:16(P<0.05) in group immunized with rAd-EU-ORF3-ORF5-IL-18 at 35 dpi. The T lymphocyte proliferation levels in mice at 35 dpi immunized with recombinant adenovirus vaccines were significant higher than 14 dpi. Also the recombinant adenovirus vaccines can promote the CD3+CD4+, CD3+CD8+ T lymphocyte proliferation. the level of CD4+ and CD8+ immunized with rAd EU-ORF3-ORF5 were 2.3 times and 1.4 times than ad-wt group,and immunized with rAd-EU-ORF3-ORF5-IL-18 were1.6 times and 1.5 times than ad-wt group. The residue tests showed recombinant adenovirus vaccines persist as long as 14 d in mice, suggesting that recombinant adenovirus vaccine can provide longer immune response.Pigs were immunized with recombinant adenovirus vaccine rAd-EU-ORF3, rAd-EU-ORF5, rAd-EU-ORF3-ORF5, rAd-EU-ORF3-ORF5-IL-18, rAd-IL-18 and rAd-EU-ORF3-ORF5-IL-18+Quil A. The results showed all immunized groups with higher level antibody at 35 dpi. Group immunized with rAd-EU-ORF3-ORF5-IL-18+Quil A, the GP3 and GP5 specific antibody level was1.13 and 1.15 times than group of rAd-EU-ORF3-ORF5-IL-18 without adjuvant. Neutralizing antibodies revealed that most groups of neutralizing antibody titers reached 1:16 or more(P <0.05), which can effectively prevent the spread of virus in population. The result of lymphocyte proliferation and T cell subsets indicated that recombinant adenovirus vaccine can effectively stimulate the proliferation of lymphocytes, the level of CD3+CD4+ and CD3+CD8+ were significantly higher than immunized with ad-wt(P <0.05). Showed that when PRRSV virus invade the body again, T lymphocyte produce an immune response rapidly. All immunized animals were challenged with PRRSV LV strain at 35 dpi, and detection the viral load in main organs at 49 dpi. The result showed that the viral load in groups immunized with rAd-EU-ORF3-ORF5-IL-18 and rAd-EU-ORF3-ORF5-IL-18+Quil A only 10% of ad-wt group. Indicates the recombinant adenvorius vaccines can reduce the viral load in blood and tissues, decreased the incidence of viremia.Overall,the candidate recombinant adenovirus vaccines can improve humoral and cellular immunity in mice and pig, provide better protection. Through the study of recombinant adenovirus vaccine, lay the foundation for further development of the European type PRRSV vaccine.