Study On The Effect And Mechanism Of New Tuberculosis Vaccine Strain On T Cell Immune Memory

Objective:This study intends to take the new tuberculosis vaccine strain（B/R strain）constructed and identified by the research group as the research object.Compare the effects of BCG strain,H37Ra strain and B/R strain on T cell immune memory,explore the anti-tuberculosis immune protection effect and mechanism of B/R strain,and provide theoretical basis for B/R strain as a new tuberculosis vaccine strain.Methods:1.Preparation of vaccine strains:BCG,H37Ra and B/R strains were inoculated on Lowenstein-Jensen and cultured for 3 weeks to prepare 1?10⁷CFU/m L bacterial suspension.2.Animal grouping and immunization:Animals were divided into PBS group,BCG group,H37Ra group and B/R strain group.C57BL/6 mice（1?10⁶CFU/100u L/mouse）were immunized subcutaneously with bacterial suspensions of corresponding vaccine strains,and an equal volume of PBS was injected into the PBS group as a negative control.3.Effects of B/R strain on the establishment and maintenance of T cells immune memory after immunizing mice:After 8,12 and 16 weeks of immunization,mice serum and spleen lymphocytes were collected.ELISA method was used to detect the levels of IL-2 and IFN-γin the serum of immunized mice;Flow cytometry was used to detect the levels of effector memory lymphocytes(T_EM)and central memory lymphocytes(T_CM)in CD4⁺and CD8⁺T cells of spleens of immunized mice.4.Evaluate the effect of B/R strain on memory T cells after specific antigen stimulation:At 8,12,and 16 weeks after immunization of mice,mouse spleen lymphocytes were stimulated and cultured with PPD in vitro,and cell supernatants and cells were collected.ELISA method to detect the level of antigen-specific Th1/Th2 cytokines secreted by mouse splenic lymphocytes;Flow cytometry was used to detect the levels of T_EMand T_CMin CD4⁺and CD8⁺T cells after PPD stimulation;CFSE labeling was used to detect the proliferation ability of CD4⁺and CD8⁺T lymphocytes.5.Evaluation of the short-term immune protection effect of B/R strain:HE staining was used to judge the pathological damage degree of lung and spleen in mice;Colony forming unit（CFU）in organs and tissues was used to analyze the clearance of Mycobacterium tuberculosis after vaccination.Results:1.Effects of B/R strain on the establishment and maintenance of T cell immune memory in mice:After 8 weeks of immunization,compared with BCG group,serum IL-2 and IFN-γlevels in group B/R were higher in group B/R（P（27）0.05）,and there is no statistical difference with H37Ra group.After 12 weeks of immunization,the levels of IL-2 and IFN-γin serum of mice in B/R group were not significantly different from those in BCG group and H37Ra group.After 16 weeks of immunization,the serum IFN-γlevel of the B/R group was higher than that of the BCG（P（27）0.05）,and there is no statistical difference with H37Ra group;The serum IL-2 level of the B/R group was not statistically different from that of the BCG and H37Ra groups.The number of T_CMand T_EMin CD₄⁺and CD₈⁺T cells stimulated by B/R group was significantly higher than PBS group at 8,12 and 16 weeks after immunization（P（27）0.05）,except that CD₈⁺T_CMdecreased at 12 weeks.Compared with BCG group,B/R group can maintain a higher level of T_EMwith prolonged immune time（P（27）0.05）.2.Effects of B/R strains on T cell immune memory in mice after specific antigen stimulation:In the early period of 8 weeks after immunization,there was no significant difference in the levels of antigen-specific Th1 cytokines,IFN-γand IL-2secreted by splenic lymphocytes in each vaccine group.At 12 weeks,the level of antigen-specific IFN-γsecreted by the B/R strain group was higher than that of the BCG group（P<0.05）.In this time,the IL-2 level was slightly higher than that of the BCG group,but the difference was not statistically significant.With the extension of immunization time,IFN-γand IL-2 secreted by B/R strain were significantly higher than BCG group and H37Ra group at 16 weeks（P<0.05）.There was no statistical difference in the content of Th2 cytokine IL-4 between the four groups at each time period.After 8 weeks of immunization,the T_CMand T_EMlevels of CD4⁺and CD8⁺in B/R group were not significantly different from those in BCG group,but CD4⁺T_EMwas lower than that in H37Ra group（P<0.05）.At 12 weeks,the levels of CD4⁺T_CM,T_EMand CD8⁺T_CMin B/R strain group had no difference from BCG group and H37Ra group,the T_EMof CD8⁺was higher than BCG group and H37Ra group（P<0.05）.At 16 weeks,CD4⁺,CD8⁺T_CMand T_EMlevels in B/R group were significantly higher than BCG group with the prolongation of time（P<0.05）;The proliferation rate of CD4⁺and CD8⁺T cells in B/R strain group was significantly higher than that in BCG group and H37Ra group at 16 weeks（P<0.05）.3.Short-term immune protection effect of B/R strain immunized mice:Compared with PBS group,the pathological damage degree of lung and spleen in B/R strain group was significantly reduced,which was equivalent to the pathological changes in BCG group.The colony count in the lung and spleen organs were significantly reduced compared with the PBS group,and there was no statistical difference in CFU count among the vaccine groups.Conclusion:1.After immunizing mice with B/R strain,a higher level of T cell immune memory was established,mainly T_EM.2.B/R strain plays an immune protection role by awakening higher level memory T cells and producing immune response mainly of Th1 type,and their immune memory could be maintained for at least 4 months.3.B/R strain inherits the advantages of parental BCG in preventing primary infection,and has better immune protection effect,which is a promising tuberculosis vaccine.