Evaluation Of Heterologous Prime-boost Strategy In Vector Vaccine Of The E Protein From Duck Tembusu Virus

Duck Tembusu virus(DTMUV)spreads rapidly which causes massive economic losses in the Chinese waterfowl industry.This study explored the immune responses of heterologous prime-boost strategy in vector vaccines of the E protein from DTMUV for the first time,with the aim of improving the immune effect of the DTMUV vaccines.The research contents were summarized as follows:1.Analysis of antibody levels in ducklings after immunizationThe ducklings(n=170)were randomly divided into five groups(34/group,each group took10 of them to challenge),the group(ads/ads group)one was priming and boosting with the recombinant adenovirus vaccine strain;group two(SE/SE group)was priming and boosting with salmonella vector vaccine strain;group three(SE/ads group)was priming with the salmonella vector vaccine strain and boosting with recombinant adenovirus vaccine strain;group four(WF/WF group)was priming and boosting the positive vaccine;group five(PBS/PBS group)was priming and boosting with PBS.The ducklings were primed at 3-dayold and boosted at 15-day-old,and each group of the ducklings(n=3)were randomly selected and euthanized at 3(days post prime injection,dpi),10 dpi,17 dpi,24 dpi,31 dpi,38 dpi,48 dpi,60 dpi,and collected their serum and spleen.Neutralization assay and enzyme-linked immunosorbent assay were used to detect the antibody titers in the collecting serum.The results showed that the testing groups all could produce specific neutralizing antibodies.The levels of neutralizing antibody in SE/ads group reached the peak at 38 dpi(about 1:123),and was higher than the ads/ads and SE/SE groups after 17 dpi,and higher than WF/WF group after 31 dpi.As for the Ig G levels in serum,SE/ads group reached the peak at 38 dpi(OD value was about 1.43)and was higher than ads/ads and SE/SE groups after 17 dpi,and higher than WF/WF group after31 dpi.Compared with the groups of homologous prime-boost strategy,the group of heterologous prime-boost strategy could induce higher antibody levels in the ducklings.2.Analysis of the INF-γ,IL-2,IL-4 and IL-10 changes in ducklingsThe cytokine changes in the spleen after immunization was detected,and the results showed that the expression levels of IFN-γ and IL-2 in SE/ads group was low in the early stage(3 dpi-31 dpi),but the expression level of IFN-γ and IL-2 was higher than those in the ads/ads,SE/SE,and WF/WF groups in the later stage(after 31 dpi),and maintained a high level in a long time.The expression levels of IFN-γ and IL-2 in ads/ads group decreased slowly after peaking at 38 dpi and 48 dpi.The expression levels of IFN-γ and IL-2 in the SE/SE group reached the peak at 31 dpi and 38 dpi,but the levels were generally lower than ads/ads,SE/ads and WF/WF groups.As for the expression levels of IL-4 and IL-10,the SE/ads group increased rapidly from 10 dpi to 31 dpi,and decreased after the peak at 31 dpi,and was lower than SE/SE after 38 dpi;the expression levels of IL-4 and IL-10 in SE/SE group continued increasing until the peak at 48 dpi;the expression levels of IL-4 and IL-10 in the ads/ads groups were lower than those in the SE/ads and SE/SE groups,and increased slowly after immunization.3.Immune protective test of ducklings after challenging stimulated by prime-boost strategyThe challenge test found that there were some lesions in tissues of ads/ads and SE/SE groups,no significant lesions in the tissues of SE/ads and WF/WF groups;and pericardial effusion with pericardial adhesions,liver embrittlement,splenomegaly,and kidney bleeding were found in PBS/PBS group.Histopathological detection found that,in the ads/ads group,there were slight myocardial fiber rupture and lymphocytic infiltration in heart,slight hepatocyte vacuolation,increasing reticulocyte and unclear boundary between red and white pulp in liver;slight myocardial fiber rupture and lymphocytic infiltration in heart,hepatocyte vacuolation and lymphocyte cell infiltration in liver,increasing reticulocyte and unclear boundary between red and white pulp in kidney,the detaching basement membranes of renal epithelial cells,vascular sleeve phenomenon in brain were founded in the SE/SE group;all tissues in the SE/ads group and WF/WF groups found no obvious pathological changes;Myocardial fiber rupture,edema and severe lymphocytic infiltration in heart,severe hepatocyte vacuolation,hepatocyte necrosis and massive lymphocyte infiltration in liver,increasing reticulocyte and unclear boundary between red and white pulp in spleen,bleeding and necrotic in the epithelial cells in kidney and vascular sleeve in brain were founded in PBS/PBS group.The results showed that the viral loads could be detected in the five tissues(heart,liver,spleen,kidney,and brain)of each group,and the viral loads of the negative control group were significantly higher than that of the other four groups(P<0.05),indicating that the testing groups all could inhibit the virus replication in vivo to a certain extent.The virus loads of SE/ads group in heart were significantly lower than ads/ads group(P<0.05),and were significantly lower than SE/SE group in heart,liver,spleen and brain(P<0.05),and had no significant with WF/WF group in the five tissues(P>0.05).It showed that the heterologous prime-boost immunization strategy could better eliminate DTMUV in ducks after challenge than the homologous prime-boost strategy.