Construction And Immune Efficiency Evaluation Of Toxoplasma Gondii MORN1/2 Deletion Mutants

Toxoplasma gondii is an obligate intracellular protozoan which can infect almost all warm-blooded animals.Toxoplasma infection may cause severe consequences on pregnant women,including miscarriage and stillbirth in their fetus.Attenuated Vaccines were considered as the most effective way to control the T.gondii transmission.MORN gene family plays important roles in the development of cytoskeleton during the cytokinesis,which could be an ideal candidate gene for attenuated vaccine.The object of this study is to construct T.gondii ΔMORN1 and ΔMORN2 mutants and evaluate their protection efficacy against T.gondii.Firstly,to illustrate the functions of MORN2 gene on T.gondii virulence,we used CRISPR/Cas9 system to knockout the MORN2 gene on T.gondii RH strain(type I).The pyrimethamine resistance gene(DHFR)was integrated into the CRISPR target by homologous recombination.Finally,the MORN2 knockout strain(ΔMORN2)was successfully constructed after drug selection and monoclonal screening.The plaque assays and virulence assay showed the knockout of MORN2 gene had almost no effect on T.gondii growth in vitro and parasite virulence to mice.This study concluded that the knockout of MORN2 gene has no effect on T.gondii RH strain,which is not able to be a candidate gene for attenuated vaccine against T.gondii.Then,we created a T.gondii ΔMORN1 mutant to evaluate its protection efficacy as a live-attenuated vaccine against acute,chronic,and congenital T.gondii infection in mice.IgG levels and cytokines were detected at 28 and 70 days after immunization,respectively.The results indicated ΔMORN1-mediated immunity fully protected mice from death when challenged with wild-type(wt)RH strain,heterologous PYS,TgC7 strains of the Chinese ToxoDB#9 genotype,and T.gondii PRU strain.The cyst burden in the brain was significantly reduced in immunized mice compared to non-immunized mice.In respect to congenital infection,the litter size,survival rate,and body weight(BW)of pups born to ΔMORN1-immunized dams were not different compared to pups born to na?ve control dams.Also,immunized dams infected with type II PRU strain had significantly less cyst burden in the brain compared with dams in infection control group.These findings suggested ΔMORN1 strain is a good attenuated vaccine candidate against T.gondii different strains.In conclusion,we created T.gondii ΔMORN1-2 strains and evaluated their protection efficacy and found ΔMORN1 strain is a good attenuated vaccine against T.gondii infection,which laid the foundation for T.gondii prevention and control.