Development Of The Epitope Vaccine Of Foot-and-Mouth Disease Based On Flagellin As Presenting Carrier

Foot and mouth disease(FMD)is an acute,highly contagious disease to pigs,cattle,sheep and other domestic or wild cloven-hoofed animals,which is caused by Foot-and-mouth disease virus(FMDV).In view of the key role of vaccination strategies in the prevention and control of foot-and-mouth disease,efforts to develop a more safe and effective FMDV vaccine have become a pressing priority.Genetic engineering epitope vaccine has good safety and is capable of inducing humoral and cellular immune responses,which representing one of the potential vaccine candidates.Bacterial flagellin,as a pathogen-associated molecular pattern(PAMP),can be recognized by Toll-like receptor 5(TLR5),and then in turn activate both innate and adaptive immunity.An increasing evidence of studies suggests that genetically fusing an antigen of interest to flagellin significantly increases the immunogenicity and protective capacity of the antigen.Here,in this study,a truncated VP1(?VP1)protein and two key B cell epitopes from foot-and-mouth disease virus were fused to C-terminal of the flagellin with a common exogenous T cell epitope to construct three flagellin fusion proteins.Specifically,?VP1 and two duplicates of two key B cell epitopes(2×B1B2)were fused separately to the C-terminus of flagellin with a universal exogenous T cell epitope to construct FT(Flagellin-Truncated VP1)and FME(Flagellin-Multiple Epitopes).In addition,the D3 domain of flagellin was replaced by ?VP1 in FME,yielding FTME(Flagellin-Truncated VP1-Multiple Epitopes).Then,the immunogenicity and protective effect of the resulting recombinant proteins were evaluated.The obtained results show that: 1.All three fusion proteins were efficiently expressed in E.coli,in which FT and FME were in the soluble fraction,wereas FTME was in the inclusion body.2.High-purity target proteins were purified by nickel affinity chromatography column.The inclusion bodies of FTME were refolded successfully by rapid dilution method.3.Western blotting and indirect ELISA showed that FT,FME and FTME could be recognized by specific antibodies of foot-and-mouth disease virus,which proved their exposed antigenic epitopes.FTME had the best antigenicity because of its high antigenic epitope density.4.The bioactivity of purified protein after endotoxin removal was determined by TLR5 assay.The results showed that the three recombinant fusion proteins could stimulate the secretion of cytokine TNF-? by RAW246.7/hTLR5 cells,which was similar to natural flagellin(p > 0.05),while the RAW246.7 cells without TLR5 receptor and the VP1 protein without TLR5 stimulating activity could not.5.FT,FME and FTME could elicited the significant and similar FMDV-specific IgG response in BALB/c mice compared with control group(P<0.05),and FTME was the highest.The immunogenicity of FTME was dose-dependent in guinea pigs.6.Different immune route(muscle and subcutaneous injection route)induced similar antibody responses in BALB/c mice(p>0.05).7.No synergistic effect was found between recombinant protein and the designated adjuvants(ISA-206,poly(I·C),MPLA and CpG-ODN).8.Although weaker than commercial inactivated vaccines,all of three fusion proteins could induce significantly enhanced humoral and cellular immune responses and upregulated the levels of some cytokines including TNF-?,IFN-? and IL-12 in guinea pigs.Importantly,the immunized guinea pigs with 30?g/dose of FTME protein provided 80% protection against FMDV challenge.Therefore,the results presented in this study indicated that the fusion protein FTME can be used as a novel promising vaccine candidate for future prevention and control of foot-and-mouth disease.