Study On The Immune Protection Efficacy And Immunological Memory Of The New Tuberculosis Vaccine Strain

Tuberculosis,caused by Mycobacterium tuberculosis as a pathogen infection,is an infectious disease with a major public health threat worldwide.Nowadays,the prevalence of tuberculosis in the world shows a high trend,and the incidence of drug-resistant tuberculosis is also increasing.At present,the only vaccine for tuberculosis prevention in clinical practice:BCG,has been available for a hundred years.The disadvantages of its unstable protective effect and poor preventive effect are gradually emerging.In order to effectively prevent and control the epidemic of tuberculosis,it is one of the main research directions to develop a more effective new tuberculosis vaccine.Objective:In this study,followed the previous findings of the research group:a new type of Mycobacterium tuberculosis fusion strain（B/R strain）,the inactivated B/R strain was prepared by heat inactivation method.Then,the inactivated B/R strain,as a tuberculosis vaccine,will be investigated the effect of immune protection and immune memory on anti-tuberculosis in mice.Methods:（1）The B/R strain and BCG strain with good growth status were taken to prepare the bacterial suspension,and the inactivated B/R strain was prepared by heat inactivation.Healthy female C57BL/6mice aged 6-8 weeks were selected and divided into experimental groups according to the random number table method:BCG strain group,B/R strain group and inactivated B/R strain group,and PBS group as control.According to the grouping,the vaccine strains of each group were inoculated by subcutaneous injection.（2）The serum of mice in each group was gathered at week 3 after immunization.ELISA method was used to test the serum antibody Ig G level of mice in each group.The serum of the inactivated B/R strain group was collected at week 3,6 and 9 after immunization.The secretion level of antibodies Ig G1 and Ig G2a in inactivated B/R strain group were detected by ELISA.（3）The serum and lymphocytes came from spleen of mice in each group were gathered at week 9,12and 15 after immunization.The levels of IL-2 and IFN-γin serum of mice in each group were detected by ELISA.Flow cytometry was selected to detect the number of effector memory lymphocytes(T_EM)and central memory lymphocytes(T_CM)in CD4⁺and CD8⁺T cells of spleen lymphocytes in each group.（4）The spleen lymphocytes in each mice group were gathered at week 9,12 and 15 after immunization,and stimulated in vitro with tuberculosis specific antigen PPD.The levels of IL-2 and IFN-γsecreted by spleen lymphocytes in each group were detected by ELISA.At the same time,flow cytometry was used to detect the number of T_EM and T_CM in CD4⁺and CD8⁺T cells of mouse spleen lymphocytes after stimulation.（5）At week 8 after immunization,mice were infected with high-concentration BCG strains by tail vein injection.4 weeks later,the lung and spleen tissues of mice were taken for pathological examination to observe the pathological changes of lung tissues.The clearance ability of mice in each group to BCG strain infection was evaluated by counting the colony forming unit（CFU）of organ tissues.Results:（1）The concentration of vaccine strain suspension in each experimental group was stable.The B/R strain after heat inactivation had no colony growth.（2）At the week 3 after immunization,the serum Ig G level of mice in PBS group was significantly lower than that in each experimental group（P<0.05）,and there was no significant difference between the experimental groups.At week 9,the secretion level of Ig G2a in the inactivated B/R strain group was significantly higher than that of Ig G1（P<0.05）,and Ig G2a/Ig G1>1.（3）At week 9,12 and 15 after immunization,the levels of IL-2（P<0.05）and IFN-γ（P<0.05）in serum of mice in each experimental group were higher than those in PBS control group.There was no significant difference between the inactivated B/R strain group and the B/R strain group.At week 9,the serum IFN-γlevel in the inactivated B/R strain group was higher than that in the BCG strain group（P<0.05）.At week 12,the serum IL-2 level in the inactivated B/R strain group was higher than that in the BCG strain group（P<0.05）.At week 15,the serum IFN-γlevel in the B/R strain group was higher than that in the BCG strain group（P<0.05）.（4）The results of T_EM and T_CM in CD4⁺and CD8⁺T cells of spleen lymphocytes in each group were detected by flow cytometry at week 9,12 and 15 after immunization with vaccine strains.The results showed that the number of T_CM and T_EM in CD4⁺and CD8⁺T cells of spleen lymphocytes in each experimental group was higher than that in PBS group（P<0.05）.There was no significant difference between the inactivated B/R strain group and the B/R strain group.（5）The spleen lymphocytes of mice in each group were stimulated by PPD at week 9,12 and 15 after immunization.The levels of cytokines IL-2 and IFN-γsecreted by spleen lymphocytes of mice in each group were detected by ELISA.The results showed that the levels of cytokines IL-2（P<0.05）and IFN-γ（P<0.05）in the cell culture supernatant of spleen lymphocytes of mice in each experimental group were higher than those in PBS group（P<0.05）.There was no significant difference in the levels of cytokines IL-2 and IFN-γin the cell culture supernatant between the inactivated B/R strain group and the B/R strain group.The level of cytokine IL-2 in the cell culture supernatant of B/R strain group was higher than that of BCG strain group at week 9,12 and 15 after immunization（P<0.05）.At week 12 and 15,the level of cytokine IL-2 in the cell culture supernatant of the inactivated B/R strain group was also higher than that of the BCG strain group（P<0.05）,the level of IFN-γin cell culture supernatant of B/R strain group was higher than that of BCG strain group（P<0.05）.At the week 12,the level of cytokine IFN-γin the cell culture supernatant of the inactivated B/R strain group was higher than that of the BCG strain group（P<0.05）.At week 9,12 and 15 after immunization,the spleen lymphocytes of each experimental group were stimulated by PPD.The number of T_CM and T_EM cells in CD4⁺and CD8⁺T cell typing in each experimental group was significantly higher than that in PBS group（P<0.05）.There was no significant difference between the inactivated B/R strain group and the B/R strain group.At week 15,the number of CD8⁺T_CM and T_EM cells in the inactivated B/R strain group and the B/R strain group was significantly higher than that in the BCG strain group（P<0.05）.（6）At the week 8 after the mice were immunized with each vaccine strain,high concentration BCG strain was injected into the tail vein of the mice,and the mice were infected.The lungs tissue of the mice in each group were taken for pathological examination.The results showed that the pathological changes of the lungs tissue of the mice in each experimental group were significantly lighter than those in the PBS group.The inactivated B/R strain group was basically the same as the B/R strain group and the BCG strain group.The number of colonies in the lungs and spleen tissues of mice in each experimental group was significantly lower than that in the PBS group（P<0.05）,and there was no significant difference in the number of colonies in the lungs and spleen tissues of mice in each experimental group.Conclusion:（1）The immune protective effect of inactivated B/R strain on mice is comparable to B/R strain.（2）The ability of inactivated B/R strain to induce immune memory in mice is comparable to B/R strain.（3）Heat inactivation has no effect on immune protection and immune memory induced by B/R strain.（4）Compared with BCG strain,the inactivated B/R strain may have more advantages in immune protection and induction of immune memory in mice at the late stage of inoculation.