Nano-Vaccine Of Em14-3-3 Delivered By PLGA Induced Effective Immune Protection Against Eimeria Maxima Infection And Multiple Eimeria Species Infections In Chicken

1.Effects of 5 antigens of Eimeria maxima on the functions of Dendritic cells of ChickenEimeria maxima possess integral families of immunogenic constituents that promote differentiation of immune cells during host-parasite interactions.Dendritic cells（DCs）have an irreplaceable role in the modulation of host immunity.However,the selection of superlative antigens with immune stimulatory efficacies on host DCs is lacking.In this study,five recombinant proteins of Eimeria maxima（Em）,including Em14-3-3,rhomboid family domain-containing proteins（ROM）Em ROM1 and Em ROM2,Microneme protein 2（Em MIC2）,and Em8 were identified to stimulate chicken splenic derived dendritic cells in vitro.The cultured populations were incubated with recombinant proteins,and typical morphologies of stimulated DCs were obtained.Dendritic cell-associated markers major histocompatibility complex（MHC）class II,CD86,CD11c,and CD1.1,were showed up-regulatory expressions by flow cytometry assay.Immunofluorescence assay revealed that recombinant proteins could bind with the surface of chicken splenic-derived dendritic cells.Moreover,quantitative real-time PCR（QRT-PCR）results showed that distinct gene expressions of Toll-like receptors and Wnt signaling pathway were up-regulated after the co-incubation of recombinant proteins with DCs.The ELISA results indicated that the DCs produced a significantly higher level of IL-12 and IFN-γsecretions after incubation with recombinant proteins.Whereas TGF-βwas significantly increased with r Em ROM1,r Em ROM2,and r Em MIC2 as compared to control groups,and IL-10 did not show significant alteration.Taken together,these results concluded that among five potential recombinant antigens,r Em14-3-3 could promote immunogenic functions of chicken splenic-derived DCs more efficiently.2.DCs stimulating antigen 14-3-3 delivered by PLGA and Chitosan nanoparticles conferred improved protection in Chickens against Eimeria maximaEimeria maxima（E.maxima）is an intracellular apicomplexan protozoan that seriously impairs the growth and responsible for avian coccidiosis.The purpose of this study to develop a novel approach for recombinant E.maxima（r Em）14-3-3 antigen to elicit enhanced immunogenic protection using Poly（D,L-lactide-co-glycolide）（PLGA）and chitosan（CS）nanoparticles（NPs）against E.maxima challenge.The purified r Em14-3-3 antigen was expressed.The morphologies of synthesized antigen-loaded NPs（PLGA/CS-r Em14-3-3）were visualized by a scanning electron microscope.The chickens were intramuscular immunized with the PLGA-r Em14-3-3 NPs,CS-r Em14-3-3 NPs and only r Em14-3-3.The sera cytokines,Ig Y-antibody level,T-lymphocytes population,and protective efficacies in the chicken against E.maxima challenge were evaluated.The results showed that encapsulated r Em14-3-3 in PLGA and CS NPs presented spherical morphology with a smooth surface.The chickens immunized with only r Em14-3-3 and PLGA/CS-r Em14-3-3 NPs elicited significantly（P<0.05）higher IFN-γcytokine level,stimulated the proportions of CD4⁺/CD3⁺,CD8⁺/CD3⁺T-cells subsets,and provoked sera Ig Y-antibody immune response as compared to control groups（PBS,p ET-32a,PLGA,and CS）.Whereas,PLGA-r Em14-3-3 NPs immunized chickens raised efficient（P<0.05）ratio of CD4⁺/CD3⁺,CD8⁺/CD3⁺T-cells,sera Ig Y-antibody level,and induced higher Th-1 cell-mediated immunity rather than CS-r Em14-3-3 and bare antigen,relatively.The animal challenge experiment results revealed that PLGA-r Em14-3-3 NPs immunized chickens significantly increased the relative body weight gain（%）,decreased lesion score,and enhanced oocyst decrease ratio compared to the chickens immunized with CS-r Em14-3-3NPs and only r Em14-3-3.The anticoccidial index of the chickens vaccinated with the PLGA-r Em14-3-3 NPs was（180.1）higher than that of the Cs-r Em14-3-3 NPs（167.4）and bare antigen（165.9）groups.Collectively,our statistics indicated that PLGA NPs might be an effective antigen（r Em14-3-3）delivery system to act as a nano-subunit vaccine that can improve protective efficacies in chicken against E.maxima challenge.3.PLGA nanoparticles-mediated delivery of 14-3-3 elicited protection against challenge with E.tenella,E.acervulina and E.necatrixAvian coccidiosis may often be caused by the co-infection of Eimeria species.The14-3-3 is identified as potential antigen in Eimeria tenella（E.tenella）,E.acervulina and E.maxima.The purified recombinant E.maxima 14-3-3（r Em14-3-3）antigen was synthesized and packaged by Poly（D,L-lactide-co-glycolide）（PLGA）nanoparticles（NPs）as an nano-conjugated antigen delivery system.The r Em14-3-3 and PLGA-loaded r Em14-3-3 NPs were used to evaluate the immune protective efficacy parameters in chicken against E.tenella,E.acervulina,and E.necatrix challenge.The results showed that intramuscular（I/m）immunization with r Em14-3-3 and PLGA-r Em14-3-3 NPs vaccine elicited significantly（P<0.05）higher IFN-γcytokine level,augmented the CD4⁺/CD3⁺,CD8⁺/CD3⁺T-cells population and provoked sera antibody-mediated Ig Y immune response as compared to control groups（PBS,p ET-32a,PLGA）.The chickens immunized with PLGA-r Em14-3-3 NPs were effectively（P<0.05）derived the percentages of CD4⁺/CD8⁺immune cells,sera Ig Y antibody level and induced higher Th-1 cell-mediated immunity rather than bare antigen.The animal challenge experiment results revealed that immunization with PLGA loaded-r Em14-3-3 NPs vaccine significantly increased the relative body weight gain,decreased lesion score,and enhanced oocyst decrease ratio than r Em14-3-3 alone.The anti-coccidial index（ACI）of the chicken-vaccinated with the PLGA-r Em14-3-3 NPs was higher than that of the bare antigen.These findings showed that chicken-immunized with PLGA-r Em14-3-3 NPs as a nano-subunit vaccine could potentiate the effective immunogenic response,boosted protective efficacy against E.tenella,E.acervulina,and induced moderate protection against E.necatrix,and mixed oocysts infection.4.Optimization of Immunization Procedure for 14-3-3 delivered with PLGA nanoparticles and its stabilityThe immunization procedures of Poly（D,L-lactide-co-glycolide）PLGA-loaded recombinant Eimeria maxima 14-3-3 antigen（PLGA-r Em14-3-3 NPs）,including different routes,doses,time of immunization and ages of primary vaccination of chickens,was optimized.The stability of the nano-subunit vaccine,including store time and temperature,were also evaluated.Efficacy of immunization was assessed on the basis of oocyst reduction ratio,lesion score,average body-weight gain and the anti-coccidial index（ACI）.The chickens were randomly divided into corresponding groups（25 per group）.The challenged（positive）,unchallenged（negative）and control（PBS）groups were designed.The results revealed that 50μg was the optimal immune dose,intramuscular was the most efficient route to prompt improved protection.The chickens immunized twice(7^th and 14^thdays)with PLGA-r Em14-3-3 NPs elicited the ACI level>179 than that of chicken immunized only once.The immunizing efficacy of the nano-conjugated subunit vaccine at different time and temperature was very firm.Together,the optimal immunization procedure for PLGA-r Em14-3-3 NPs vaccine is 50μg,by intramascular inoculation,twice injections at 7 and 14 days.