Efficacy Of Salmonella Flagellin As A Potential Adjuvant For Newcastle Disease Vaccines

Newcastle disease (ND) is a highly contagious, acute and potent infectious disease caused by Newcastle disease virus (NDV). The disease has considerable impact on the global poultry industry. Although both live attenuated and inactivated vaccines are used to prevent and control chickens against ND, but the outbreaks of ND in commercial poultry flocks around the world indicate that routine vaccinations are not effective enough to induce protective immunity. Therefore, it’s necessary to develop the new vaccines. In this study, F gene of NDV F48E8strains was generated in Pichia pastoris expression system, then mixed with flagellin (FliC) extracted from the surface of Salmonella Typhimurium. The efficiency and mechanism of FliC as an adjuvant was evaluated in murine model. The capacity of FliC as an adjuvant for low virulent NDV vaccine was further evaluated by challenged with F48E8in SPF chickens. The results laid the promising foundation for flagellin as an adjuvant of ND vaccines.1. Study of flagellin as an adjuvant of NDV subunit vaccineF gene of NDV F48E8was amplified by PCR, and then cloned into pPICZaA vector, the correct recombinant plasmid was transferred through electroporation into Pichia pastoris GS115to construct recombinant strains GS115(pPICZaA-F). After induced by a final concentration of0.5%methanol for3days at28℃,250rpm, the protein was analyzed by SDS-PAGE and Western blotting assay. SDS-PAGE results showed that F protein mainly presented in the intracellular, and it’s molecular weight was72KD. Western blotting results indicated that the F protein had good immunoreactivity. In this study, FliC was extracted from the surface of ATCC14028s(pTrc99a-fliC-WT). C3H/HeJ mice were immunized with FliC intraperitoneally to detect the relative mRNA expression levels of TLR5and different cytokines in spleen at different time points by qRT-PCR. The results showed that the mRNA levels of TLR5were raised117and293-fold (p<0.05) after injection of FliC1h and6h. After injection with FliC, the expression levels of pro-inflammatory cytokines IL-6, TNF-α, IL-1β and IFN-γ, IL-12, IL-8were up-regulated in varying degrees at different time points, and IL-10was no significant increase(p>0.05). Next, we evaluated the efficiency and mechanism of FliC as an adjuvant. C3H/HeJ mice were co-administrated with FliC and F protein, ten days after boost immunization, the titer of IgG2a was significantly higher than IgG1in F+FliC group (p<0.01). At the same time, the spleen lymphocytes in each group were stimulated with F protein, the results showed that the level of IFN-γ was significantly higher than that of IL-4in F+FliC group(p<0.001), and level of IFN-γ in F+FliC group was significantly higher than in F protein group (p<0.01). It indicated that FliC induced a Thl-based immune response, and was consistent with the results of IgG subtypes. By detecting dynamic changes of antibody titers within6weeks after the boost immunization, we found that the IgG titers of F+FliC and F+Al(OH)3groups were up to the highest in the21th day. And the IgG titer of F+FliC group was significantly higher than that of F protein group (p<0.01). The results also showed that the antibodies could sustain a month or so. It indicated that FliC can promote the innate immune responses and the adaptive immune responses.2. Efficacy of flagellin as an adjuvant of low virulent NDV vaccineIn this study,1-week-old SPF chickens were co-administrated intranasally with FliC and low virulent NDV vaccine (LaSota strain). It was found that flagellin could significantly enhance the levels of NDV-specific IgG antibodies and HI antibodies (p<0.05). After challenged with F48E8, the PBS group all died in the6th days, while the LaSota+FliC and LaSota vaccine groups all survived. So, we established real time RT-PCR method based on F gene of F48E8for detecting virus shedding of each group after challenge. The results showed that the virus load in LaSota+FliC group was significantly lower than in LaSota vaccine group at cloacal samples in3th day post challenge and oral samples in5th day post challenge (p <0.05). The virus titer of LaSota+FliC group was also slightly lower than that of the LaSota vaccine group at the rest of each time point, both at the oral and cloacal samples,indicating that FliC suppress the virus replication and shedding in the upper respiratory and intestinal tracts. All of these results demonstrated that flagellin as an adjuvant of LaSota vaccine can enhance the humoral immune responses of chicken, and the efficiency of vaccine protection, contributing to the strong resistance against to NDV attacks. This study laid the foundation for the further research of flagellin as an adjuvant of vaccines.