| 1.Evaluation of the abilities of recombinant Em UCE,Ea GAPGH,Et ADF,Et PHP14,Et PNP,Et HAD and Et AKO to stimulate the maturation and functions of chicken dendritic cells in-vitroDendritic cells play a crucial role in inducing antigen-specific immunity to pathogens.During host-parasite interaction,host immune response to the parasite molecules is considered essential for recognizing novel antigens for control strategies.Therefore,present study evaluated the immunogenic profiles of chicken dendritic cells(DCs),derived from spleens,and their capacity to proliferate and differentiate autologous T lymphocytes in response to recombinant proteins viz.Eimeria maxima ubiquitinconjugating enzyme(r Em UCE),Eimeria acervulina glyceraldehyde-3-phosphate dehydrogenase(r Ea GAPDH),Eimeria tenella actin-depolymerizing factor(r Et ADF),Eimeria tenella 14-k Da phosphohistidine phosphatase(r Et PHP14),Eimeria tenella purine nucleoside phosphorylases(r Et PNP),Eimeria tenella haloacid dehalogenase-like hydrolase(r Et HAD),and Eimeria tenella aldo-keto reductase(r Et AKO).Immunoblot analysis showed that recombinant proteins were successfully recognized by rat sera generated against corresponding protein.The immunofluorescence test confirmed the binding of target proteins on the surface of chicken DCs.Flow cytometric analysis revealed that phenotypes for MHCII,CD1.1,CD11 c,CD80,and CD86 were increased in DCs after r Em UCE,r Ea GAPDH,r Et ADF,r Et PHP14,r Et PNP,r Et HAD and r Et AKO treatment.Moreover,m RNA expressions of DC maturation biomarkers(CCL5,CCR7,and CD83)were upregulated in response to recombinant proteins.Following r Em UCE,r Ea GAPDH,r Et ADF,r Et PHP14 treatment,chicken DCs activated TLR signaling and inhibited Wnt signaling.In contrast,r Et PNP,r Et HAD and r Et AKO treated DCs suppressed TLR signaling and triggered Wnt signaling.In addition,recombinant proteins promoted DCdirected proliferation of autologous na(?)ve T cell in DC/T cell co-incubation system.CD3+/CD4+ T-cells were significantly enhanced when r Em UCE,r Ea GAPDH,r Et ADF and r Et PHP14-treated DCs were co-incubated with T-cells.Cytokine analysis of r Em UCE,r Ea GAPDH,r Et ADF and r Et PHP14-pulsed DCs showed increased levels of IL-12 and IFN-γ,while IL-10 and TGF-β remained unchanged.However,DCs treated with r Et PNP,r Et HAD and r Et AKO revealed that production of IL-10 and TGF-β was improved,whereas IL-12 and IFN-γ were not changed relative to negative controls.Likewise,the coincubation of r Em UCE,r Ea GAPDH,r Et ADF and r Et PHP14 treated DCs with T-cells indicated increased production of IFN-γ but not IL-4.Collectively,r Em UCE,r Ea GAPDH,r Et ADF and r Et PHP14 could polarize chicken DCs to immunogenic phenotype and shift the immune cells towards Th1 response.Our observations provide valuable insight for future research aimed at vaccine development against avian coccidiosis.2.Immune protection of r Em UCE delivered by PLGA and chitosan nanoparticles in chickens against Eimeria maxima challenge infectionEimeria maxima is a protozoan parasite endemic in chickens and is one of the causative agents of avian coccidiosis.We previously reported that recombinant Eimeria maxima ubiquitin-conjugating enzyme(r Em UCE)promotes immunogenic functions of chicken dendritic cells and stimulates Th-type-1 immune response.In the present study,the nano-vaccine was formulated by encapsulating r Em UCE antigen with Poly(D,L-lactideco-glycolide;PLGA)and chitosan(CS)nanoparticles.Em UCE loaded PLGA(PLGAr Em UCE)and r Em UCE loaded CS(CS-r Em UCE)nanoparticles were administered to twoweek old chickens via intramuscular route.The immunogenicity and protective immunity of PLGA-r Em UCE and CS-r Em UCE was evaluated against E.maxima challenge.Chickens injected with empty p ET32 a protein,blank PLGA nanoparticles(NPs),blank CS NPs and PBS were served as control groups.Splenic T lymphocytes subpopulation and cytokine profile of vaccinated birds were determined using flow cytometry and ELISA,respectively.Finally,the protective efficacies of PLGA-r Em UCE and CS-r Em UCE were evaluated by immunization and challenge experiments.Results revealed that both PLGA-r Em UCE and CS-r Em UCE vaccinated groups induced significantly higher proportions of CD4+ and CD8+ T lymphocytes,cytokine production(IFN-γ,IL-17)and specific-Em UCE Ig Y antibody levels than that of control groups.In comparative analysis,it was found that PLGA-r Em UCE group exhibited significantly increased levels of CD4+ and CD8+ T lymphocytes,IFN-γ,IL-17 and specific-Em UCE Ig Y antibody compared to the CSr Em UCE group.Moreover,PLGA-r Em UCE and CS-r Em UCE immunized chickens improved body weight gain,decreased the oocyst output,and alleviated enteric lesions significantly as compared to control groups.Meanwhile,chickens vaccinated with PLGA-r Em UCE revealed anti-coccidial index(ACI)higher than that of CS-r Em UCE group.These results indicate that r Em UCE encapsulated PLGA nanoparticles could improve the host immune response and enhance protective efficacy against E.maxima infection.Our results suggested that r Em UCE is an immunogenic molecule and its protective efficacy could be improved following conjugation with PLGA NPs.3.Assessment of protective efficacy of nanovaccine PLGA-r Em UCE against Eimeria tenella,Eimeria maxima and Eimeria acervulina infection in chickensCoccidiosis is a common intestinal disease of poultry birds mainly caused by multiple species of the apicomplexan protozoan,Eimeria,and is one of the most economically devastating diseases for the poultry industry worldwide.Host immunity to Eimeria infection,however,is relatively species-specific.The ability to immunize chickens against different species of Eimeria using a single vaccine will have a major beneficial impact on commercial poultry production.In the previous study,Eimeria maxima ubiquitin-conjugating enzyme(Em UCE)was identified as one of the immunogenic common antigen in E.tenella,E.acervulina and E.maxima.The aim of the present study was to evaluate the immunogenicity and protective efficacy of r Em UCE conjugated with Poly(D,L-lactide-co-glycolide;PLGA)nanoparticles(PLGA-r Em UCE)against infection with three species of Eimeria both individually and simultaneously.The immunogenicity and protective efficacy of PLGA-r Em UCE was analyzed.PLGA-r Em UCE-mediated changes of T cell subpopulations were assessed using flow cytometry.Cytokine profile and anti-r Em UCE antibody Ig Y were evaluated by ELISA.The protective efficacy of PLGAr Em UCE was estimated by immunization and challenge experiments.Results revealed that PLGA-r Em UCE immunized group significantly improved the percentage of CD4+ and CD8+ T cells and produced a strong Ig Y response compared to that of control groups.Similarly,PLGA-r Em UCE inoculated chickens promoted the secretion of Th1-type(IFN-γ)and Th17-type(IL-17)cytokines.Moreover,chickens immunized with PLGA-r Em UCE and challenged with Eimeria oocysts had increased body weight gains,diminished fecal oocyst shedding and lessened intestinal lesion scores compared with the controls.In conclusion,the r Em UCE,common antigen among avian Eimeria species,encapsulated in PLGA nanoparticles induced significant humoral and cellular immune response and effective protection against E.tenella,E.acervulina,E.maxima,and mixed infection of the three Eimeria species.4.Optimization of immunization procedure of nanovaccine PLGA-r Em UCE against E.maxima infection in chickensThe efficacy of vaccines is determined by various factors,including immunization schedule,age of animals,immunization dose and route of vaccine administration.Therefore,in the present study,the immunization procedure of nano-vaccine [recombinant Eimeria maxima ubiquitin-conjugating enzyme conjugated with Poly(D,L-lactide-co-glycolide;PLGA)nanoparticles(PLGA-r Em UCE)],including route,dose,time of immunization and age of primary immunization of chicken,was optimized against E.maxima challenge infection.The effects of the protective immunity against challenge infection were assessed according to average body weight gain,oocyst output,lesion score and the anti-coccidial index(ACI).Chickens were randomly distributed into corresponding groups(30/group).The challenged and unchallenged control groups were designed.The results indicated that intramuscular immunization was the most efficient route to elicit immune response and 100 μg was the optimal immune dose.Moreover,the single immunization dose at the age of 14 days was optimal. |
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