Protective Effect Against Toxoplasmosis In BALB/c Mice Vaccinated With Toxoplasma Gondii Macrophage Migration Inhibitory Factor

Background:Toxoplasma gondii is an obligate intracellular parasite responsible for toxoplasmosis,which can cause severe disease in the fetus and immunocompromised individuals.Developing an effective vaccine is crucial to control this disease.Macrophage migration inhibitory factor(MIF)has gained substantial attention as a pivotal upstream cytokine to mediate innate and adaptive immune responses.Homologues of MIF have been discovered in many parasitic species,and one homologue of MIF has been isolated from the parasite Toxoplasma gondii.In this study,the recombinant Toxoplasma gondii MIF(rTgMIF)as a protein vaccine was expressed and evaluated by intramuscular injection in BALB/c mice.We divided the mice into different dose groups of vaccines,and all immunizations with purified rTgMIF protein were performed at 0,2,and 4 weeks.The protective efficacy of vaccination was analyzed by antibody assays,cytokine measurements and lymphoproliferative assays,respectively.The results obtained indicated that the rTgMIF vaccine elicited strong humoral and cellular immune responses with high levels of IgG antibody and IFN-γproduction compared to those of the controls,in addition to slight higher levels of IL-4 production.After vaccination,a stronger lymphoproliferative response was also noted.Additionally,the survival time of mice immunized with rTgMIF was longer than that of the mice in control groups after challenge infection with virulent T.gondii RH tachyzoites.Moreover,the number of brain tissue cysts in vaccinated mice was reduced by 62.26%compared with the control group.These findings demonstrated that recombinant TgMIF protein is a potential candidate for vaccine development against toxoplasmosis.Objective:To explore whether the recombinant Toxoplasma gondii MIF(rTgMIF)can be used as a protein vaccine against toxoplasmosis.Methods:The antigenic index of TgMIF were analyzed by bioinformatics,and pET-28a-TgMIF plasmid was transformed into Rosetta host bacteria cells to express the protein.The purified rTgMIF protein was analyzed by Coomassie blue R-250 staining and Western Blot.Six-to eight-week old female BALB/c mice(n=240)were divided into six groups with 40 mice per group at random.We set two negative groups:blank control and PBS,and four immunization groups(protein doses of 2.5,5.0,10.0 or 20.0μg).For the experimental group,all mice in different groups were intramuscularly injected with 2.5,5.0,10.0 or 20.0μg of rTgMIF emulsified in Freund’s adjuvant,respectively.As negative controls,the mice were intramuscularly injected with 100μl sterile PBS added withadjuvant.Differentimmunizationsampleswereemulsifiedwith complete/incomplete Freud’s adjuvant(C/IFA)at a 1:1 ratio before immunization.The first immunization was with Freund’s complete adjuvant,and the next two immunizations were with Freund’s incomplete adjuvant for booster injections.Mice were immunized using the same protocol on days 1,15,and 29.After three immunizations,the level of antigen-specific IgG was evaluated by ELISA.The spleen cells of each group were stimulated with rTgMIF protein,and splenocytes proliferation assay was analyzed by Cell Counting Kit-8.Cell-free supernatants were harvested to measure IL-4 quantification at 24 h and 96 h for IFN-γafter poststimulation,respectively.Two weeks after the last immunization,the best immune group and two control groups were selected to infect T.gondii.30 mice per group were randomly chosen and challenged with 1x10~3 T.gondii RH strain tachyzoites by intraperitoneal injection.Survival time of the challenged mice was recorded within 30 days and their survival rate was calculated for all animals.Meanwhile,three mice per group were orally challenged with 20 tissue cysts of the PRU strain.One months later,the intact brains from each group were collected and calculated to evaluate the protection efficiency of rTgMIF protein.Results:(1)The ORF of TgMIF is 351 bp,encoding a protein of 116 amino acids with a predicted molecular weight of 13 kDa and an isoelectric point of 8.91.DNASTAR was used to analyze the protein of TgMIF for surface probability,antigenic index,and hydrophilic plot,as well as flexible region.The results revealed that most regions of the TgMIF protein were in hydrophilicity plots and flexible regions,and TgMIF exhibited ideal surface probability and an antigenic index that indicated it was a promising prospect for producing vaccines.(2)rTgMIF had a molecular weight of approximately13 kDa.The specificity of prokaryotic expression of rTgMIF was identified by the polyclonal antibody against TgMIF as a band of approximately 13 kDa,which was consistent with the deduced size.These results indicated that rTgMIF had been successfully expressed and purified.(3)The levels of IgG were significantly increased in the sera of mice immunized with rTgMIF compared with the control groups(p<0.001).Moreover,the level of IgG tended to increase along with the continuous injection.The results also indicated that 5μg rTgMIF induced a humoral immune response in the second week,and other doses induced a response in the fourth week.Moreover,a dose of 5μg rTgMIF elicited the maximum level of IgG.(4)Splenic lymphocytes from mice immunized with 5μg or 10μg rTgMIF had a significantly higher proliferative response compared with the blank or PBS control groups.These results suggested that immunization with rTgMIF induced antigen-specific lymphocytes in mice.(5)The rTgMIF vaccine induced high levels of IFN-γand slight higher low IL-4 levels,indicating that a bias toward the Th1-type immune response was induced by rTgMIF vaccines.(6)Mice in the groups of blank control and PBS died from 7 to 10days after the challenge,and there were no survivors in either group.The mice immunized with 5μg rTgMIF(median survival of 11 days)had a significantly prolonged survival rate in comparison to the two control groups(blank control or PBS)(p<0.001,respectively).25%of the immunized mice survived to the 30th day.(7)The number of cysts in mice immunized with rTgMIF(1333.33±352.8)was significantly lower compared with the groups of blank control(4400±916.5)and PBS(3533.33±290.6)(p<0.01).The number of brain cysts in vaccinated mice showed a62.26%reduction compared with the PBS group.Conclusions:Our research demonstrated that rTgMIF successfully triggered a strong and specific immune response,and the vaccinated BALB/c mice had a certain protective effect against acute and chronic T.gondii infection.Therefore,rTgMIF should be considered to be a potential vaccine candidate against toxoplasmosis.