Enhanced Mucosal Immune Responses In Guinea Pigs By Intranasal Delivery Nanoparticles Loaded FMDV DNA Vaccine

Mucosal system is the first barrier to prevent the invasion of pathogenic microorganisms.The transmission of respiratory tract is considered as an important way for the infection of foot-and-mouth disease.Therefore,it is of great significance to prevent the invasion of foot-and-mouth disease virus.In this study,the nanoparticles that loaded with recombinant the eukaryotic expression plasmids were used to intranasal immunization with guinea pigs.In order to enhance the immune effect of vaccine,this study constructed a eukaryotic expression vector pBudCE4.1/P12A3C/IFN(pP12A3C/IFN).The recombinant plasmids were transferred BHK-21cells.Using indirect immunofluorescence and sandwich ELISA to verify its successful expression.In order to ensure the safety and efficiency of nucleic acid vaccine,pP12A3C/IFN-CS/PLGA-NPs,pP12A3C/IFN-Am/MS-NPs were prepared.Simultaneously,CpG oligodeoxy nucleotides(ODNs)were encapsulated in chitosan-coated poly(lactic-co-glycolic acid)nanoparticles(CpG-CS/PLGA-NPs).The encapsulation efficiency of CS/PLGA-NPs for plasmid and CpG was 83.8%and 88.4%,respectively.The adsorption rate of amino modified silica per 20mg/mL was 50ug/mg for plasmids.Scanning electron microscopy showed that the microspheres had good morphology and the particle size was400-600 nm.The results of in vitro experiments showed that the nanoparticles had a slow release effect on plasmids.The prepared nanoparticles were administrated in guinea pigs via intranasal immunization.The mucosal specific s IgA and serum specific IgG antibody against FMDV were detected by ELISA.The study evaluated the efficacy of immunization by neutralization test and MTT method.Results showed that pP12A3C/IFN-CS/PLGA-NPs+CpG-CS/PLGA-NPs and p P12A3C/IFN-CS/PLGA-NPs immune groups showed higher level of mucosal,cellular and humoral immune response than those administered pP12A3C-CS/PLGA-NPs or naked plasmid vaccine alone.But,sIgA antibodies were not detected in feces and saliva samples in all groups.The CD4~+and CD8~+T cells in pP12A3C/IFN-CS/PLGA-NPs+CpG-CS/PLGA-NPs and pP12A3C/IFN-CS/PLGA-NPs immune groups rose to a higher level than others groups.T spleen lymphocyte proliferated significantly in pP12A3C/IFN-CS/PLGA-NPs+CpG-CS/PLGA-NPs and p P12A3C/IFN-CS/PLGA-NPs groups when stimulated by FMDV as a specific antigen.Neutralizing antibody test showed that the serum of guinea pigs can detect neutralizing antibodies to FMDV in the immune group.The result of challenge experiment 42 days post-vaccine revealed 100%protection in pP12A3C/IFN-CS/PLGA-NPs and pP12A3C/IFN-CS/PLGA-NP+CpG-CS/PLGA-NPs immune groups.However,our attempt to use pP12A3C/IFN-Am/MS-NPs to immunize guinea pigs failed to induce immune responses,the preparation process needs to be further improved.The above results show that CpG and IFN-αadjuvant-based FMD vaccines induce protection in guinea pigs.Moreover,CS-coated PLGA NPs presents an effective and safe mucosal immune delivery system for FMDV DNA vaccine.