Studies On The Protective Immunity Elicited By The Transgenic Eimeria Parasites Expressing The Immunodominant Antigens Of Heterologous Pathogens

Vaccination is one of the efficient means for animal diseases control.Vaccine delivery is critical in antigen discovery and vaccine efficacy and safety.With the establishment of the reverse genetic system in Eimeria parasite and the detection system of specific immune responses to recombinant Eimeria,Eimeria parasite showed its potential use as a novel vaccine delivery vehicle.In this study,we focused on analyzing the critical factors of efficient protective immunity elicited by the transgenic Eimeira parasite.In detail,we optimized the Eimeria vector both from the antigen factors,including the location,the expression level and the expression pattern of the exogenous antigens,and from the host factors to improve the protection provided by the recombinant Eimeria.Our results will support for the use of Eimeria parasite as a vaccine delivery vector with the detailed data.To investigate the impact of the exogenous antigens' expresson level on the immune responses,we used the following methods:optimizing the regulatory elements of the exogenous gene,co-expressing double or multiple exogenous genes mediated by P2A and increasing target genes copy number to improve the expression level of the model antigens of enhanced yellow fluorescent protein(EYFP)or the exceullar domain of M2 protein(M2e)of the H9N2 avian influenza virus.Our results showed that promoter is the key factor in controlling the expression of the exogenous genes.The promoter from the surface antigen 13 of E.tenella,P2A mediated double exogenous genes expression and multiple copies of M2e efficiently enhaced the expression level of EYFP or M2e.Moreover,transgenic Eimeria experessing high level exogenous antigen elicited enhanced immune responses.Our results indicated that the exogenous antigens'expression level is critical to trigger an effective immune response.To reveal the factors from the host that influenced the immune responses,we conducted the transgenic Eimeria lines expressing chicken interleukin 2(ChIL-2)or profilin(the ligands of the immune receptor).Our results showed that transgenic Eimeria parasite expressing ChIL-2(EmiChIL-2)elicited a higher cellular immunity compared with the wild type parasite.In consistent with ChIL-2,profilin derived from E.maxima(EmPro)helped improving the immunity of transgenic E.tenella.In addition,transgenic E.tenella expressing EmPro(Et-EmPro)altered the gut microbiome of chickens which Bacillus are the dominant microbiota after infection.Both EmiChIL-2 and Et-EmPro immunized birds exhibited better immune resistance against subsequent wild type parasite infection.Our results indicated that the factors from the host such as cytokines and immune receptor ligand are also critical to induce an effective immunity.To analyze the protective immunity provided by the transgenic parasites against heterogeneous pathogens infection,immunodominant antigens from Toxoplasma gondii(surface antigen 1,TgASG1)and E.maxima(immune mapped protein 1,EmIMP1 or apical memberance antigen,EmAMA1)were delivered by E.tenella and the protection was assessed.We found that transgenic E.tenella expressing TgSAG1(Et-TgSAGl)stimulated TgSAGl-specific immunities in chickens and protected chickens against T.gondii infection,and that Et-TgSAG1 elicited a Th-1 dominant immune response in mice.Et-TgSAG1 immmunized mice showed a prolonged survival after infection with T.gondii RH strain.Transgenic E.tenella expressing EmIMPI(Et-EmIMPI)also elicited protective immunity against E.maxima infection in chickens.The protective immunity against E.maxima infection was enhanced when the chickens immunized with Et-EmIMPI and Et-EmAMA1.Our results indicated that Eimeria parasite as vaccine delivery vehicle efficiently carrying and relsesing heterogeneous pathogens' antigens to the host immune system and trigger an effective protective immunity.Taken together,the exogenious antigen and Eimeria specific immunitis elicited by the transgenic Eimeria were significantly improved after optimizing the key factors from the antigens or the host.Eimeria parasite carrying the heterogeneous immunodominant antigens efficiently trigger an effective immune response and provided partial protection against heterogeneous pathogens infection.These data are encouraging for development of next generation coccidiosis vaccine strains and also demonstrated that Eimeria parasites used as a novel vaccine delivery vehicle is practical and feasible.