Evaluation Of The Immunity Of DNA Vaccines And Recombinant Subunit Vaccines Based On Toxoplasma Gondii Calcium-Dependent Protein Kinases

Toxoplasmosis is an important zoonosis having a worldwide distribution. The disease is caused byToxoplasma gondii, an obligate intracellular parasite, which may lead to encephalitis in children,abortion and neonatal loss in pregnant women and in sheep and goats, and cerebral toxoplasmosis oreven death in immunodeficiency individuals. No available chemical treatments could completelyeliminate the parasite in vivo, thus T. gondii vaccine should be an optimal option for controlling thedisease. However, no available one could be used in humans until now.Calcium-dependent protein kinases (CDPKs) are known as key effectors in regulating calciumrelated signaling pathways, which exist in plants, algae and apicomplexan protozoans. In T. gondii,CDPKs are recognized as significant signaling mediators, involved in control a diverse array offunctions including gliding motility, cell invasion, egress and some other developmental processes thatoccur at distinct stages in its complex life.To evaluate whether T. gondii CDPKs could be used as vaccine candidates and provide the baselinedata for commercial vaccines against the parasite, we firstly examined the sequence variation inTgCDPK2genes among13T. gondii strains from different hosts and geographical locations in thepresent study. Results showed that the sequence variation of complete TgCDPK2DNA and cDNA were1.42%and0.66%, respectively, suggesting that the gene cannot be used as a proper marker for studyingthe genetic diversity among different T. gondii genotypes. Furthermore, the family of CDPKs was alsoquite conservative, and the low sequences variation ranging from0to1.40%.We then translated TgCDPK3, TgCDPK5and TgCDPK6gene sequences into amino acidsequences and analyzed the structure and its potential epitopes using multiple bioinformatics approaches.The results showed that all the three proteins contained numbers of α-helixes, β-folds and random coils.A total of17,17and14potential epitopes were predicted in TgCDPK3, TgCDPK5and TgCDPK6proteins, suggesting that these three antigens are potential vaccine candidates.T. gondii CDPK3, CDPK5and CDPK6genes were then amplified and linked into the eukaryoticexpression vector pVAX I to construct the DNA vaccines, respectively. After the third immunization, theprotective efficacy against T. gondii infection in Kunming mice was evaluated. The results showed thatall the three vaccines could develop Th1type response with a high level of specific antibodies andstrong lymphoproliferative responses. The mice immunized with TgCDPK3, TgCDPK5and TgCDPK6genes were prolonged the survival time to13.5d,9d and12.2d compared to the controls which diedwithin7.2days after lethal challenge with the tachyzoites of the virulent T. gondii RH strain. In chronicinfection, the numbers of brain cysts of the mice immunized with TgCDPK3, TgCDPK5and TgCDPK6were reduced significantly when compared with those in control groups (P <0.05), and the rate ofreduction could reach to51.93%,46.81%and71.1%, suggesting that TgCDPK3, TgCDPK5andTgCDPK6are promising vaccine candidates against T. gondii infection.In the end, we amplified the TgROP18and TgCDPK6genes and ligated to pET-30a for prokaryotic expression. The purified ROP18protein and CDPK6protein were mixed and emulsifiedwith206and poly (lactide-co-glycolide)(PLG). The protein vaccines were used to immunize Kunmingmice. At6weeks after the last immunization, the vaccines could induce strong humoral and cellularimmune responses and generate protective immunity against acute and chronic T. gondii infection.Furthermore, protein plus PLG could elicit better protective efficacy compared to the protein plus the206adjuvant. The identification of the three new vaccine candidates and the evaluation of antigensencapsulated in PLG microparticles provide a valuable basis for developing new vaccines against T.gondii for future use in humans and animals.