Immunogenicity And Animal Protection Of A Novel Multi-variant Epitope Ensemble Vaccine Against Avian Leukosis Virus Subgroup J

Avian leukosis virus subgroup J(ALV-J)is a subgroup of retrovirus avian leukosis sarcoma virus group.It mainly causes clinical immunosuppression and tumor formation,resulting in chicken growth retardation and egg dropping,which causes serious loss to the breeding industry.J subgroup avian leukosis virus has high genetic instability and variability.Because of the high glycosylation of the surface protein,the virus itself has the mechanism of immune evasion.The traditional attenuated vaccine and inactivated vaccine cannot produce good protection for chickens.Avian leukosis in developed countries mainly adopts elimination program to control virus transmission.This strategy is effective,but greatly increases the cost of breeding.China is a large country of poultry breeding,but the level of aquaculture development and breeding is not high.It cannot rely entirely on the way of elimination program to control diseases due to many conditions.Therefore,it is urgent to develop a new and effective J subgroup of avian leukosis virus vaccine and medicine in China.Epitope vaccine is a subunit vaccine,which is prepared by genetic engineering.Preparation of polypeptide vaccine contains dominant epitope by genetic engineering technology.Although epitope peptide is small,its immunogenicity is strong and stable.Epitope vaccine is stable and safe,which is more consistent with the future development direction of vaccine.Avian leukosis virus is a virus with high variability.For ALV-J,a highly variable virus,recombinant multi epitope vaccine has more extensive antigenic information,which can effectively avoid immune failure caused by virus variation.Based on previous studies in our laboratory,we determined that the epitope of ALV-J was located in HR1 region of gp85 hypervariable region,and the epitope sequence was N-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-C.Searching NCBI published in recent years,the comparison analysis found that ALV-J strain sequence mutation mainly concentrated in three amino acid sites of A,G and H.The comparison of the amino acid sequence of the epitope of 81 ALV-J strains found that the mutation direction changed mainly in 4 directions,and the mutant strain accounted for about 80%of the total strains.Therefore,4 epitopes with different mutation directions were linked by codon of serine(S)and glycine(G).The gene fragment was synthesized by biological company and constructed prokaryotic expression vector,and the target protein was expressed through Escherichia coli BL21.The SDS-PAGE and Western blot showed that the target protein was in accordance with the expected size and could be combined with the carrier label McAb.ELISA experiments showed that ALV-J epitope integrated protein could react with ALV-J positive serum and could not react with negative sera.In order to further verify the immunogenicity of ALV-J epitope integrated protein,animal immunogenicity test was carried out.ALV-J epitope integrated protein combined with complete/incomplete Freund adjuvant immunized 1 day old SPF chickens and continuously monitored the antibody titer.The highest antibody titers reached 1:64000 on 42 days after vaccination,and lasted for 126 days at least.There was no significant difference between the body weight of the chickens inoculated with the ALV-J epitopes and the normal group,and the histopathology of the chickens in the epitope vaccine group and the normal group had no significant changes.The analysis of the changes in the body weight and the histopathology showed that the ALV-J epitope vaccine had no significant influence on the growth of chickens.In order to verify the protective effect of ALV-J epitope integrated protein,animal protection experiments were carried out and compared with recombinant gp85 protein.After immunization with 1 days old SPF chickens,ALV-J virus was inoculated when the antibody titer reached 10~3.The animals did not detoxify through the cloaca cotton swab,and the pathological changes in the ALV-J infection group were serious.The ALV-J epitopes had no obvious pathological changes in the vaccine group,and the liver inflammatory foci in the gp85 vaccine group.The experimental results showed that the ALV-J epitope integrated vaccine had good animal protection effect.Further,we demonstrated that the antibody induced by the variant multi-variant ensemble epitope vaccine recognized and neutralized different ALV-J strains(NX0101,TA1,WS1,BZ1224 and BZ4).It is found that ALV-J epitope integrated vaccine antiserum can neutralize 5 ALV-J strains,and the neutralization efficiency is significantly higher than that of gp85 vaccine.It is proved that ALV-J epitope integrated vaccine antiserum has good virus neutralization effect.The clinical symptoms,growth status,animal detoxification and histopathological changes in animal protection experiments showed that the ALV-J epitope integrated vaccine had good immunogenicity and animal protection effect.Then the clinical symptoms,antibody titer,chicken growth status,animal detoxification,and histopathological changes were monitored continuously.Animal immune and animal protection experiments showed that ALV-J epitope integrated vaccine had good immunogenicity and animal protection effect,which provides good strategy for associated epitope vaccine study and clinical application.