Evaluations Of The Immune Response And "Prime-boost" Efficacy Of The Recombinant Plasmid Of Ubiquitin Fusioned With Viral Structural Proteins Of IBV

Infectious bronchitis(IB)is a high contagious upper-respiratory tract disease of chickens that is caused by the Gammacoronavirus genus group,family Coronaviridae-infectious bronchitis virus(IBV)and it can lead to severe economic losses in poultry industry worldwide.However,it can cause latent infection,and may become a source of mutation and restructuring of the virus which was due to high degree of variability of IBV,the frequency of deletion and insertion mutations of the RNA genome,or even recombination between different strains.Although attenuated live vaccines and inactivated vaccines have played a critical role in control of the prevalence of disease,the failure of immunization is still a problem.Thus,there is an urgent need for developing new delivery methods and strategy that are able to confer potent protection.The experimental chickens were divided into 7 groups,and immunization groups were made with the following strategy:?pVAX1-Ub-linker-N+pVAX1-Ub-linker-N?pVAX1-U'b-linker-S1+pVAX1-Ub-linker-S1?pVAX1-U b-linker-N-S1+pVAX1-Ub-linker-N-S1?pVAX1-Ub-linker-N-S1+inactivated vaccine ?H120+pVAX1-Ub-linker-N-S1?H120+inactivated vaccine ?PBS.The diluted plasmids were administered to the 14-day-old chickens by intramuscularly injection.After two weeks later,the chickens were boosted by DNA vaccine.The attenuated live vaccine was administered by intranasal and eye,and the inactivated vaccine was administered by subcutaneous injection.Two weeks after boosting,chickens were challenged by virulent IBV strains GX-YL5,GX-YL9,GX-NN7 and GX-C.After that,the percentage of CD4+ and CD8+ subgroups of peripheral blood T lymphocytes,the specific IgG and were measured to detect the immune response of different groups before and after 14d and 28d immunizations.The results of specific antibody show that there was a significant difference(P<0.05)in ELISA antibody levels elicited by pVAX1-Ub-linker-N-S1+pVAX1-Ub-linker-N-S1?pVAX1-Ub-linker-N-S1 +inactivated vaccine and H120+ PVAX1-Ub-linker-N-S 1 immunization groups than pVAX1-Ub-linker-N+ pVAX1-Ub-linker-N and PVAX1-Ub-linker-S1+pV AX 1-Ub-linker-S1 immunization groups since 14-day after first inoculation.There was a significant difference(P<0.05)in ELISA antibody levels elicited by pVAX1-Ub-linker-S1+pVAX1-Ub-linker-S1 than PVAX1-Ub-linker-N+pVAX1-Ub-linker-N vaccination group.The percentage of CD4+,CD8+ T-lymphocytes significantly higher(P<0.05)was observed from chickens immunized with PVAX1-Ub-linker-N-S 1+pVAX1-Ub-linker-N-S1?pVAX1-Ub-linker-N-S 1 + inactivated vaccine and H120 + pVAX1-Ub-linker-N-S1 immunization groups at the 14 and 28 days after the first vaccination.The percentage of CD4+,CD8+ T-lymphocytes was higher in pVAX1-Ub-linker-N + pVAX1-Ub-linker-N than in pVAX1-Ub-linker-S1+pVAX1-Ub-linker-S1,but there was no significant difference(P>0.05)between pVAX1-Ub-linker-N-S 1+ pVAX1-Ub-linker-N-S1 and pVAX1-Ub-linker-N-S1+inactivated vaccine group.For the study of the proposal of"Prime-boost",two of the four groups can induct much higher immunological effects compared with the two another one.Priming with DNA vaccine and boosting with inactivated vaccine could confer protection of chickens against homologous and heterologous IBV strains.The protection rate against heterologous and homologous IBV strains of pVAX1-Ub-linker-N + pVAX1-Ub-linker-N was between 50%-66.7%,The protection rate against heterologous and homologous IBV strains of pVAX1-Ub-linker-S1+pVAX1-Ub-linker-S1 was between 50%-58.3%,The protection rate against heterologous and homologous IBV strains of pVAXl-Ub-linker-N-S 1+pVAX1-Ub-linker-N-S1 was between 58.3%-83.3%,The protection rate against heterologous and homologous IBV strains of pVAX1-Ub-linker-N-S1+inactivated vaccine was between 66.7%-91,7%,The protection rate against heterologous and homologous IBV strains of H120 + pVAX1-Ub-linker-N-S1 was between 50%-66.7%.The results of this experiment showed that co-expression of Ub-N-S1 can elicit more effective immune responses than Ub-N and Ub-S1 and improve higher protection rate against heterologous and homologous IBV strains.The sequential approach of priming with DNA vaccine and boosting with the inactivated IBV vaccine shows the most potent immunological advantage and could be a potential vaccination strategy to provide protection against homologous or heterologous IBV infection.Therefore,the results could provide solid scientific ground to further study of ubiquitin fusioned IBV main structural protein bioactivities and the theoretical basis to the discovery of new type DNA vaccine to viral disease.