| Background:Toxoplasma gondii(T.gondii)is an intracellular parasite with multiple immune escape mechanisms,which has a serious impact on human health and the development of animal husbandry.At present,there is still no ideal drug for toxoplasmosis.Development of a safe and long-lasting vaccine is of great significance and prospect for the prevention and treatment of toxoplasmosis.Breast cancer is the most common tumor and the main cause of cancer-related death in women all over the world.Microorganisms have been considered as potential biological agents for cancer immunotherapy.In addition,there are many similarities between the process of host resistance to T.gondii infection and breast cancer.However,it is unknown whether the ME49Δgra5 strain of T.gondii constructed in this study can both resist T.gondii infection and breast cancer.Therefore,this research explored the effect of the knockout strain as an attenuated vaccine against T.gondii infection and its potential to become an anti-tumor immunotherapeutic agent.Method:The ME49Δgra5 strain was constructed by CRISPR/Cas9 technology,and its functions were identified in vivo and in vitro.Then the immune protection effect of ME49Δgra5 strain was evaluated,including the protection against acute infection of tachyzoites of T.gondii RH,ME49,and VEG wild-type strains,as well as its effectiveness against cysts infection,and its ability to induce host immune response by detecting the serum cytokines levels of mice after immunization.The 4T1 breast tumor model of mice were constructed,and ME49Agra5 tachyzoites were injected into the tumor according to the treatment scheme.The tumor volume was measured and recorded,and the survival of tumor-bearing mice was analyzed.The lung metastasis process of breast cancer in the treatment group and the control group was analyzed by H&E staining and immunohistochemical staining of Ki67.Serum and tumor tissues of mice were collected to detect the level of cytokines.Immunohistochemical staining was used to check the infiltration of immune cells in tumor tissue.The type and proportion of immune cells in spleen were analyzed by flow cytometry.Results:The proliferation of ME49Agra5 strain in vitro was slowed down,and its proliferation in mice was inhibited,which means the virulence was significantly weakened.In addition,there was a serious defect in cyst formation.ME49Δgra5 strain can induce host immune response,stimulate mice to produce higher levels of cytokines to resist acute infection caused by various wild type tachyzoites of T.gondii,and also has good protective effect against oral infection of cysts which can be used as attenuated vaccine.Intratumoral injection of ME49Δgra5 strain inhibited the growth of 4T1 breast cancer in unilateral and bilateral tumor bearing mice,delayed the process of lung metastasis,and increased the levels of IFN-γ and IL-12 in serum and tumor micro environment(TME).After immunotherapy,the number of infiltrating lymphocytes were increased at the injection end of TME in unilateral and bilateral tumor-bearing mice,but there was no significant effect on the distant TME of bilateral tumor-bearing mice.In addition,the proportion of innate and adaptive immune cells in the spleen of unilateral tumor-bearing mice also increased in treatment group.Conclusion:The gra5 gene of T.gondii is critical for its virulence and cyst formation.ME49Δgra5 tachyzoites vaccination induced the generation of proinflammatory factors that protect mice from infection of three types of T.gondii and this protection was long-lasting.Besides,intratumoral injection of ME49Δgra5 strain showed a high efficacy against the growth of the injected and distant 4T1 tumors,as well as lung metastasis.ME49Δgra5 inoculation promoted the production of IFN-γ and IL-12,and increased the innate and adaptive immune cells of the spleen and tumorinfiltrating.Therefore,ME49Δgra5 was shown to be a promising vaccine against T.gondii infection and a potential immunotherapeutic agent against tumors. |
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