Vaccination With Recombinant Toxoplasma Gondii CDPK3 Induces Protective Immunity Against Experimental Toxoplasmosis

Background: Toxoplasma gondii,a ubiquitous and obligate intracellular pathogen,belonging to the phylum Apicomplexa,is capable of infecting a broad range of warm-blooded hosts including birds and mammals nearly worldwide.Acute infection with Toxoplasma gondii can cause congenital defects such as hydrocephalus,epilepsy and even stillbirth.Chronic infection is the major cause of progressive blindness in some countries and can cause neurological disorders in patients with weakened immune function.Preventive measures for toxoplasmosis are currently lacking and as such,development of novel vaccines is of urgent need.Calcium-dependent protein kinase(CDPK)mainly exists in calcium signals of plants and some apicomplexan parasites but not in animals or fungi.The kinase domain of CDPK is a typical Ser/Thr kinase that controls various functions in the life cycle of the apicomplexan,including sliding motion,cell invasion,egress,and some other developmental processes that occur at different stages of its complex life.Based on these characteristics of CDPK3,in this study,recombinant Toxoplasma gondii CDPK3 protein(rTgCDPK3)will be prepared,and purified rTgCDPK3 protein will be used to immunize BALB/c mice to evaluate the possibility of the protein as a vaccine.Objective: To investigate the possibility of T.gondii calcium-dependent protein kinase 3(TgCDPK3)protein as a candidate vaccine for toxoplasmosis,and provide a new direction for the development of toxoplasmosis vaccine.Methods: The biochemical information technology was used to analyze the physicochemical properties and epitopes of TgCDPK3.The recombinant plasmid p ET28a-TgCDPK3 was transferred into E.coli BL21 host bacteria cells to induce and express the recombinant TgCDPK3 protein.Coomassie brilliant blue R-250 staining and Western blotting were used to identify the purified rTgCDPk3 protein.210 female BALB/c mice of appropriate age were purchased and randomly divided into 6 groups,namely the blank group,the PBS group,2.5 ?g,5.0 ?g,10.0 ?g,and 20.0 ?g.The purified rTgCDPK3 was emulsified with adjuvant and immunized mice through intramuscular injection.The mice were immunized every two weeks for a total of three immunizations.After the immunization was completed,the levels of mouse serum-specific antibodies IgG,IgG1 and IgG2 a were detected by ELISA.Then the spleen lymphocytes of the last immunized mice were stimulated with rTgCDPK3 protein,and the proliferation of lymphocytes was detected by the CCK8 test.The culture supernatants were collected to measure the activities of IL-2 and IL-4 at 24 h;IL-10 activity at 72 h and IFN-? activitiey at 96 h,respectively.Two weeks after the last immunization,each group randomly selected 20 mice to intraperitoneally inject 1 × 103 RH tachyzoites.The survival status of the challenged mice within 30 days was recorded and the survival rate was calculated and analyzed on the software.To evaluate the protection efficiency of the vaccine against chronic toxoplasmosis,six mice from each group were randomly selected to challenge 20 PRU tissue cysts.Two months after post-infection,the intact brains from each group were collected and homogenized in 1 ml of PBS.The homogenized brain cysts were calculated under an optical microscope and the size of the tissue cysts was observed.Results:(1)Analysis of biological information software DNAStar show that TgCDPK3 protein not only has ideal surface antigen and antigen index,but also has more than one B cell epitope,and has good hydrophilicity and flexibility,indicating that it has a good prospect as a candidate vaccine forToxoplasma gondii.(2)The detection of Coomassie blue staining and Western blotting: Coomassie blue staining revealed that an expected 59 KDa band was obtained at a specific location.Western blotting showed that the rabbit polyclonal CDPK3 antibody could only specifically recognize the TgCDPK3 protein and not recognize another purified recombinant Tg ROP18 protein,which proved that successful induction and purification of rTgCDPK3 protein has been completed.(3)Detection of mouse serum-specific antibodies IgG,IgG1 and IgG2a: The levels of IgG,IgG1 and IgG2 a in the serum of rTgCDPK3 immunized mice were significantly higher than those in the blank group and the PBS group(p <0.001),the level of IgG,IgG1 and IgG2 a gradually increased with the increase of the number of immunizations in mice.In addition,IgG2 a was significantly higher than IgG1,indicating that rTgCDPK3 induced Th1-biased immune responses.(4)Splenocytes proliferation assay: The splenocytes of mice immunized with 5.0?g,10.0?g and 20.0?g rTgCDPK3 had a significant proliferation response,while there was no significant difference between the control group and the PBS group.(5)Detection of cytokines: The secretion of IFN-? increased in splenocytes of mice immunized with rTgCDPK3(p <0.001).There was no significant change in the levels of IL-2,IL-4 and IL-10 between different groups.(6)Protective activity in rTgCDPK3 immunized mice: The mice in the blank group died within 9 days after being infected with RH tachyzoites.The survival time of rTgCDPK3 immunized mice was significantly prolonged and the brain cyst counts in each immune group were significantly reduced(p <0.001).Conclusions: Our research showed that rTgCDPK3 exhibited a mixed Th1/Th2 response with a predominance of IgG2a(Th1)over IgG1(Th2),and BALB/c mice immunized with the rTgCDPK3 protein were able to resist acute and chronic Toxoplasma infection,suggesting that the recombinant rTgCDPK3 protein is a prospective candidate for the development of anti-Toxoplasma vaccine.