Development Of Adenovirus Vector Live Vaccine Of Chicken Anemia Virus VP1 And VP2

Chicken infectious anemia（CIA）is an immunosuppressive disease caused by Chicken anemia virus（CAV）,characterized by bone marrow failure and lymphoid tissue atrophy in chicks,causing huge economic losses worldwide.At present,there is no specific drug to treat the disease.Vaccine prevention and control of early infection is the key to control CAV.The low titer of CAV in chicken embryos and MSB1 cells makes the production cost of CAV inactivated vaccine very high,and it is difficult to be widely applied in the chicken industry.The risk of its potential pathogenicity and virulence reversion limits the development and application of CAV live attenuated vaccines.Therefore,it is of great significance to develop a CIA vaccine with high safety and good protective effect.The CAV genome mainly encodes three proteins:VP1,VP2 and VP3.The capsid protein VP1 is the main immunogen,and its correct folding requires the assistance of the regulatory protein VP2,and the two proteins coordinate to induce the effective production of neutralizing antibodies.Therefore,the construction of a vector vaccine which co-expresses VP1 and VP2 proteins can provide good immune effects.Recombinant adenovirus vector vaccine has become a better choice for CAV vaccine development due to its higher safety,longer duration of action,and stronger cellular and humoral immune responses.To obtain high titer anti-CAV antibody,in this study,CAV VP1 and VP2 were used as the target genes,and p Adeno X-Zs Green1 was used as the adenovirus vector to construct CAV VP1 and VP2 adenovirus vector live vaccine.The CAV VP1-terminator-promoter-VP2was ligated and cloned into the p MD19-T vector.The synthesized sequence was cloned into the vector p Adeno X-Zs Green1.The constructed plasmids were detected by Xhol and Nhel single enzyme digestion.The results showed that the recombinant adenovirus plasmid was successfully constructed.The recombinant adenovirus rAd5-CAV-VP1-VP2-SDAU-1 strain was obtained by transfecting 293 T cells with linearized Pac I enzyme.The results of fluorescent protein observation and western blot showed that the rAd5-CAV-VP1-VP2-SDAU-1 strain infected 293 T cells could successfully express VP1and VP2 proteins.The titer of recombinant adenovirus was 10¹⁰ IFU/m L based on adenovirus hexon protein.The rAd5-CAV-VP1-VP2-SDAU-1 strain was proved to be stable in vitro by PCR and western blot.The absolute fluorescence quantitative standard curve was constructed to determine that rAd5-CAV-VP1-VP2-SDAU-1 strain could be continuously infected on DF-1 cells.In order to evaluate the effectiveness and protective effect of the vaccine,animal immunogenicity experiments were first performed.The animal protection effect was evaluated by body weight determination,specific antibody detection and cytokines IFN-γ,IL-4 detection after SPF chicken immunization.The results showed that there was no abnormality in the mental state of chickens after immunization,and the body weight test showed that the rAd5-CAV-VP1-VP2-SDAU-1 vaccine had no effect on the growth of chickens.Indirect ELISA was used to detect the level of CAV VP2 specific antibody Ig Y in serum of immunized chickens.The results showed that the level of anti-CAV antibody produced by chickens in the rAd5-CAV-VP1-VP2-SDAU-1 vaccine group was significantly higher than that in the normal group from the 7th day after immunization.Subsequently,ELISA kits were used to detect the expression levels of cytokines IL-4 and IFN-γin different groups of chickens.It was found that 7 days after the first immunization,the levels of IL-4 and IFN-γcytokines in the vaccine group were higher than those in the normal group,indicating that the vaccine can stimulate the secretion of Th2 and Th1 cytokines in chickens.Animal protection experiments were then conducted,80 SPF chickens were randomly divided into 4 groups,20 in each group,namely Mock,vaccine group,vaccine+CAV infection group group,and CAV infection group group.The body weight,specific antibodies,cytokines IFN-γand IL-4,and immune organ index of the 4 groups were measured,and hemogram observation,gross anatomy,and histopathological observation were performed.The results showed that the body weight of chickens in the CAV infection group was significantly lower than that in the other 3 groups.The levels of anti-CAV antibodies in the vaccine group and the vaccine+CAV infection group were significantly higher than those in the CAV infection grou,and the levels of cytokines IFN-γand IL-4showed an upward trend.The positive rate of serum virus in chickens was detected after CAV infection.The results showed that the positive rate of serum virus in the vaccine+CAV infection group was 10%,indicating that the antibody induced by vaccine immunization could effectively neutralize CAV.The results of hemogram observation showed that only the early erythroblasts of chickens in the CAV infection group increased significantly.The gross lesions of chickens in different groups were mainly manifested as slight edema and hemorrhage of glandular stomach,hypoplasia of bursa of Fabricius,small spleen volume,obvious atrophy of thymus,and the lesions were alleviated with the increase of age.There was no significant difference between the vaccine+CAV infection group and the vaccine group and the normal group.Histopathological observation showed that a large number of lymphocytes infiltrated in the lamina propria and deep layers of the glandular stomach in the CAV infection group,as well as mucosal villus rupture and epithelial cell shedding,loss of bursal lymphocytes,and spleen lymphocyte decreased slightly.Thymus atrophy,cortical and medullary lymphocyte loss,bone marrow cells decreased,fat cells increased.The chickens in the vaccine+CAV infection group had less lymphocyte infiltration in the lamina propria and deep layer of the glandular stomach,and there was a slight loss of lymphocytes in the thymic medulla area compared with the normal group.There was no obvious pathological change in the bursa of Fabricius,spleen and bone marrow.There was no obvious pathological change in the normal group and the vaccine group.In summary,CAV VP1 and VP2 adenovirus vector live vaccine can effectively stimulate the body to produce specific humoral immunity and cellular immunity,and effectively protect the body,with a protection rate of 90%.The successful development of the vaccine provides a new technology for the prevention and control of CAV in clinical production,and provides a reference for the prevention and control of other animal diseases.