Construction And Immunological Evaluation Of DNA Vaccine Expressing GP3 And GP5 Genes Of Highly Pathogenic PRRSV

Porcine Reproductive and Respiratory Syndrome Virus?PRRSV?causes porcine reproductive and respiratory syndrome,which is characterized by poor reproductive health and increased mortality in susceptible pigs,especially in piglets and respiratory diseases.It is an important disease in pork producing countries worldwide,leading to sow reproductive failure and respiratory disease in young pigs,which is essential for the occurrence of regional diseases and chronic economic losses.The GP5 protein has a molecular weight of approximately 25 kDa and contains a neutralizing antigenic epitope.It is generally believed that the production of neutralizing antibodies is closely related to it.GP3 is a highly glycosylated structural protein with a molecular weight of approximately 28 kDa and is generally considered to be a neutralizing activity that may affect the protein.DNA vaccines have shown good applicative prospects in many fields,which can cause humoral and cellular immune responses in the host,to achieve the purpose of preventing and treating diseases.In this study,GD strains GP3 and GP5 genes of highly pathogenic porcine reproductive and respiratory syndrome virus of the American type were studied as the research object,and the recombinant DNA vaccine plasmid pVAX-N35HP was constructed with pVAX1 as the vector,and the constructed plasmid was identified.The correct DNA vaccine plasmid pVAX-N35HP was used in combination with adjuvants A1,A2,A3,A4,A8 and groups were named pVAX-N35HP+A1,pVAX-N35HP+A2,pVAX-N35HP+A3,pVAX-N35HP+A4 and pVAX-N35HP+A8.Evaluation the immune effects of combined immunized mice and healthy piglets.During mice experiment,serum was collected weekly to detect neutralizing antibodies,GP3 and GP5 antibodies,IL-4 and IFN-?levels;after spleen lymphocytes were isolated,the percentage of T lymphocyte subsets was detected.The results showed that the neutralizing antibody titer of pVAX-N35HP+A3 group was the highest,reaching 1:16;the antibody titer of commercial inactivated vaccine was the same,and the IL-4 level of pVAX-N35HP+A3 group was 165.74 pg/?l,higher than the commercial vaccine group;IFN-?level reached 342.67 pg/?l,higher than the commercial vaccine group.Percentage of lymphocyte subsets showed that the highest percentage of CD3⁺CD4⁺T lymphocytes in the pVAX-N35HP+A1 group was24.37%,which was slightly lower than the percentage of commercially vaccine group;the pVAX-N35HP group had the highest CD3⁺CD8⁺percentage of T lymphocytes reached 45.21%,which was higher than the percentage of commercial vaccine group.Results illustrate that pVAX-N35HP+A1,pVAX-N35HP+A2 and pVAX-N35HP+A3could induce strong cellular immunity and humoral immunity in mice.Piglets were immunized with pVAX-N35HP+A1,pVAX-N35HP+A2 and pVAX-N35HP+A3 and evaluate the immune effect.Serums were collected from piglets every week,and the serum was used in detection of neutralizing antibodies and cytokines.The lymphocytes were aseptically isolated for detection of T cell subsets at35 days.Piglets were challenged by PRRSV GD,2 x 10⁵ TCID₅₀.After PRRSV GD challenge,rectal temperature and health status of the piglets were observed.Pigs were executed according to animal welfare 14 days later.Heart,liver,spleen,lung,kidney,inguinal lymph nodes and submandibular lymph nodes were used for viral load detection.Pathological specimen sections were made by lungs.Results showed that the pVAX-N35HP+A1 group and the pVAX-N35HP+A3 group had the highest neutralizing antibody titer of 1:18,which was lower than the commercial vaccine group but the statistical difference was not significant?p>0.05?;pVAX-N35HP+A1had the highest IFN-?level,reaching 227.37 pg/?l,which was 1.22 times that of pVAX-N35HP group,but the statistical difference was not significant?p>0.05?,which was also higher than that of commercial vaccine group?p<0.05?;pVAX-N35HP+A3 group has the highest IL-4 level,which was 136.77 pg/?l,which was higher than the commercial vaccine group,and the statistical difference was extremely significant?p<0.01?.The percentage of CD3⁺CD4⁺T lymphocytes in the pVAX-N35HP+A1 group was 36.16%,which was similar to the commercial vaccine group.There was no statistical difference?p>0.05?.The percentage of CD3⁺CD8⁺T lymphocytes in the pVAX-N35HP+A3 group reached 62.37%,slightly higher than the commercial vaccine group,there was no difference in statistics?p>0.05?.Body temperature and health status of each immunized group were better than the negative control group.Viral load in the tissues was measured 14 days after PRRSV challenge.The results of lung test showed that the pVAX-N35HP+A1 group is the lowest group with 2.14×10³ copies/g,which was lower than the commercial vaccine group.pVAX-N35HP group,pVAX-N35HP+A2 group and pVAX-N35HP+A3 group were higher than the commercial vaccine group but in the same order of magnitude.The results of submandibular lymph node examination showed that the pVAX-N35HP+A1 group is the lowest with 7.25×10³ copies/g,which was lower than the commercial vaccine group.The results of inguinal lymph node examination showed that the pVAX-N35HP+A1 group is the lowest with 8.70×10³ copies/g,which was lower than the commercial vaccine group.Pathological specimen sections of the lung showed that each immunized group was better than that of the negative control group.pVAX-N35HP+A3 can significantly improve the cellular and humoral immunity levels in piglets.It shows good protective effect and it can be used as a vaccine to prevent and protect piglets from highly pathogenic porcine reproductive and respiratory syndrome attack.