Identification Of Dendritic Cells Stimulating Antigen Of Chicken Coccidian And The Immunoprotective Effect Of Their Nano-subunit Vaccine

Chicken coccidiosis is a parasitic protozoa disease caused by one or more species of Eimeria infecting the intestines of chickens and is one of the most important diseases that seriously harm the poultry industry.Among the seven recognized species of Eimeria,E.tenella,E.necatrix,E.maxima,E.acervulina are more harmful to the poultry industry.As a new strategy for the prevention and control of chicken coccidiosis,subunit vaccines have been extensively studied due to their safety and easy mass production,etc.However,subunit vaccines mainly have the problem of weak immunogenicity,and their immune protection effects need to be improved.Dendritic cells are the most powerful antigen-presenting cells,can active innate immune response and initiate adaptive immune response,and play an important role in the immune system.PLGA and chitosan are biodegradable nanomaterials,which can protect the antigen,sustain the release of the antigen,target dendritic cells and enhance the immune response.This study aims to screen Eimeria recombinant antigens that have stimulatory effects on dendritic cells,and use them to prepare PLGA and chitosan nano-subunit vaccines to evaluate their immune protection against Eimeria infection,and optimize the immune procedure of the nano-subunit vaccine with better immune protection effect.This study has reference value for improving the immunogenicity of subunit vaccines and developing nano-subunit vaccines to prevent chicken coccidiosis.1.Screening of dendritic cells stimulating antigens of chicken coccidiaProkaryotic expression system and affinity chromatography method were used to expresse and purify seven recombinant antigens of four species of Eimeria,as E.tenella,E.necatrix,E.maxima,E.acervulina.The recombinant antigens of Eimeria were incubated with chicken splenic dendritic cells respectively.The binding of recombinant antigen to splenic dendritic cells were observed by immunofluorescence.The morphological changes of dendritic cells after stimulation by recombinant antigen were observed under an optical microscope.The expression of surface molecules of dendritic cells stimulated by recombinant antigens were detected by flow cytometry.The concentration of cytokine secretion of splenic dendritic cells was detected by ELISA method.In this way,the ability of seven recombinant antigens of Eimeria to stimulate the maturation of splenic dendritic cells was evaluated.Affinity chromatography purification obtained two E.tenella recombinant antigens,r Et SO7 and r Et TA4;one E.necatrix recombinant antigen,r NPmz19;two E.maxima recombinant antigens,r Em MIC3 and r Em MIC7;two E.acervulina recombinant antigens,namely r Ea3-1E and r Ea MIF.The purification effect as analyzed by SDS-PAGE showed that r Et SO7,r Et TA4,r Em MIC3,r Em MIC7,r Ea3-1E,r Ea MIF and r NPmz19 had a single band with sizes of 44 k Da,29 k Da,91 k Da,34 k Da,22.3 k Da,14k Da and 55 k Da,respectively.Immunofluorescence test showed that all the seven recombinant antigens of Eimeria can bind to splenic dendritic cells,and even some of them have been taken up by dendritic cells.The results of optical microscope observation tell that splenic dendritic cells stimulated by recombinant antigens of Eimeria for 48 hours showed the typical dendritic morphology of mature dendritic cells and dense cytoplasm.Flow cytometry results demonstrated that splenic dendritic cells that we obtain were CD45~+cells,namely interdigitating DC.Compared with the unstimulated control group,splenic dendritic cells incubated with r Et SO7,r Et TA4,r Em MIC3,r Em MIC7,r Ea3-1E,r Ea MIF and r NPmz19 for 24 hours,respectively,can up-regulate the surface costimulatory molecules CD80 and CD86,as well as molecules MHCⅡand CD1.1.ELISA results showed that the seven recombinant antigens of Eimeria could significantly promote the up-regulation of cytokine IL-12B expression in splenic dendritic cells,but have no significant effect on the expression of cytokine IFN-γ,IL-1βand IL-10.In conclusion,the recombinant antigen of Eimeria r Et SO7,r Et TA4,r Em MIC3,r Em MIC7,r Ea3-1E,r Ea MIF and r NPmz19 all have a stimulating effect on splenic dendritic cells and could stimulate the maturation of dendritic cells.2.Preparation nano-subunit vaccines of dendritic cells stimulating antigens of chicken coccidiaSafe and biodegradable PLGA and chitosan nanomaterials were used to load dendritic cells stimulating antigens of Eimeria r Et SO7,r Et TA4,r Em MIC3,r Em MIC7,r Ea3-1E,r Ea MIF and r NPmz19,and prepare them into nano-subunit vaccines.In this study,seven PLGA nano-subunit vaccines and three chitosan nano-subunit vaccines were successfully made.Seven PLGA nano-subunit vaccines,namely PLGA-r Et SO7,PLGA-r Et TA4,PLGA-r Em MIC3,PLGA-r Em MIC7,PLGA-r Ea3-1E,PLGA-r Ea MIF and PLGA-r NPmz19,with particle sizes of 160-200 nm,190-260 nm,80-330 nm,187-250 nm,76-262 nm,60-310 nm and 106-282 nm respectively,as measured by scanning electron microscopy.The encapsulation efficacy of above PLGA nano-subunit vaccines were 80.12%,82.40%,90.03%,91.22%,87.4%,89.86%and 82.41%,respectively.The protein-loaded rate of above nano-subunit vaccines were 2.55%,2.00%,2.89%,3.90%,4.10%,3.95%and 3.29%,respectively.Three chitosan nano-subunit vaccines were prepared,namely Chitosan-r Et SO7,Chitosan-r Em MIC3 and Chitosan-r Em MIC7,with particle sizes of 140-430 nm,121-494nm and 60-500 nm,respectively.The encapsulation efficacies of chitosan nano-subunit vaccines were 96.49%,98.29%and 92.38%,respectively.The protein loaded rates of chitosan nano-subunit vaccines were 19.28%,19.58%and 18.64%,respectively.3.Evaluation of the immune protection effect of nano-subunit vaccine of dendritic cells stimulating antigens of chicken coccidia14-day-old chicken were injected intramuscularly with chicken coccidia dendritic cells stimulating antigens r Em MIC3,r Em MIC7,r Et SO7,r Et TA4,r Ea3-1E,r Ea MIF,r NPmz19and their PLGA nano-subunit vaccines(PLGA-r Em MIC3,PLGA-r Em MIC7,PLGA-r Et SO7,PLGA-r Et TA4,PLGA-r NPmz19,PLGA-r Ea3-1E and PLGA-r Ea MIF)and chitosan(Chitosan)nano-subunit vaccines(Chitosan-r Em MIC3,Chitosan-r Em MIC7 and Chitosan-r Et SO7).ELISA method was used to detect the levels of serum antigen-specific antibody Ig Y and cytokines like IFN-γ、IL-4、IL-6、IL-10、IL-17 and TGF-βin chickens,flow cytometry was used to detect the number of CD4~+T cells and CD8~+T cells in chicken splenic lymphocytes,and animal experiments were used to evaluate the immunoprotective effect of PLGA and chitosan nano-subunit vaccines of Eimeria against Eimeria infection in chickens.ELISA results revealed that compared with the PBS group,both PLGA and chitosan nano-subunit vaccines can significantly increase the levels of antigen-specific antibody Ig Y and cytokines IL-6 and IL-17 in serum.Nano-subunit vaccine PLGA-r Em MIC3 and Chitosan-r Em MIC3,PLGA-r Em MIC7 and Chitosan-r Em MIC7,PLGA-r Et SO7 and Chitosan-r Et SO7 could induce higher levels of antigen-specific antibody Ig Y than r Em MIC3,r Em MIC7 and r Et SO7,respectively.Flow cytometry results showed that except for PLGA-r NPmz19,no significantly difference was found in the changes of chicken splenic CD4~+and CD8~+T cells percentage that induced by PLGA and chitosan nano-subunit vaccines when compared with those of recombinant antigens(P>0.05).Animal experiment results showed that PLGA-r Em MIC3 nano-subunit vaccine achieved highest ACI as 184.00,in the nano-subunit vaccines of E.maxima recombinant antigens.PLGA-r Et TA4 nano-subunit vaccine achieved higher ACI as 173.63 in the nano-subunit vaccines of E.tenella recombinant antigens.The ACI of the nano-subunit vaccine PLGA-r NPmz19 against E.necatrix infection is 170.89.PLGA-r Ea3-1E nano-subunit vaccine achieved higher ACI as 176.00 in the nano-subunit vaccines of E.acervulina recombinant antigens.4.Study on the optimal immunization procedure of nano-subunit vaccine of dendritic cells stimulating antigen of chicken coccidiaThe optimal immunization procedure of PLGA-r Et TA4,PLGA-r NPmz19 and PLGA-r Em MIC3 nano-subunit vaccines that with better immune protection effect against E.tenella,E.necatrix and E.maxima infection,respectively is optimized by immunization dose,immunization route,age of primary immunization of chickens and times of immunization.The optimal immunization dose of PLGA nano-subunit vaccine was selected from 25μg,50μg and 100μg.The optimal immunization route of PLGA nano-subunit vaccine was selected from oral administration,intramuscular injection,and oculor and nasal route.The optimal age of primary immunization of PLGA nano-subunit vaccine was selected from 1day of age,7 days of age and 14 days of age.The optimal immunization times of PLGA nano-subunit vaccine was selected from immunization once and twice.Animal experimental results illustrated that the optimal immunization procedure for PLGA-r Em MIC3 nano-subunit vaccine was to immunize chickens with PLGA-r Em MIC3nano-subunit vaccine that loaded with 50μg r Em MIC3 intramuscularly at 14-day-old,and a booster immunization after 7 days.The optimal immunization procedure for PLGA-r Et TA4 nano-subunit vaccine was to immunize chickens with PLGA-r Et TA4nano-subunit vaccine that loaded with 100μg r Et TA4 intramuscularly at the age of 1 day,and a booster vaccination after 7 days.The optimal immunization procedure for PLGA-r NPmz19 nano-subunit vaccine was a single oral immunization of chickens with PLGA-r NPmz19 nano-subunit vaccine that loaded with 25μg r NPmz19.