Studies On Protection And Immune Mechanism Of Recombinant Bb-ESAT-6-MPT64 Vaccine Of Mycobacterium Tuberculosis

ObjectiveTo compare the protective immunity and immune mechanism in BALB/c mice by immunization with the recombinant rBb-ESAT-6-MPT64 vaccines against subsequent challenge with H37Ra by different inoculation routines, and to measure the levels of IFN-γ,IL-12,TNF-αand IL-10. After BALB/c mice were immunized by rBb-ESAT-6-MPT64 vaccine, the levels of serum IgE,IgG and its subclasses were detected, the levels of CD4+ and CD8+ subsets of T lymphocyte did so, the levels of IFN-γ,IL-12,TNF-αand IL-10 were measured,the proliferation of splenocytes did so.The protective immunity and immune mechanism induced in BALB/c mice by the recombinant Bb-ESAT-6-MPT64 vaccine could be explained and a kind of valuable vaccine to control tuberculosis would be provided.Methods65 female BALB/c mice were randomly divided into 5 groups,13 in each group.Group A:5×106CFU rBb-ESAT-6-MPT64 vaccines were suspended in 100μl PBS and subcutaneously injected once;Group B:5×106CFU rBb-ESAT-6-MPT64 vaccines were suspended in 100μl PBS and intramuscularly injected once;Group C:5×105CFU rBb-ESAT-6-MPT64 vaccines were suspended in 10μl PBS and intranasally inoculated once;Group D:Bb was subcutaneously injected;GroupE:100μl PBS served subcutaneously as control.All the mice were intranasally challenged with 5×105CFU H37Ra of Mycobacterium tuberculosis on 8w after immunization. After 6w, all the mice respectively were killed,the lungs and livers were removed respectively and the number of CFU was detected in those organs;serum antibodies of specific IgG,IgG1,IgG3,IgG2a,IgG2b and IgE were respectively detected by ELISA;the splenocytes were gathered and cultured with or without MTB-Ag and ConA or LPS stimulatious in vitro,IFN-γ,IL-12,TNF-αand IL-10 were respectively detected by ELISA.To study the types and dynamic change of immune responses in BALB/c mice immunized with the recombinant Bb-ESAT-6-MPT64 vaccine by subcutaneous inoculation and intranasal inoculation.60 female BALB/c mice were randomly divided into 2 groups, 30 in each group.GroupⅠ:5×106CFU rBb-ESAT-6-MPT64 vaccines were suspended in 100μl PBS and subcutaneously injected once;GroupⅡ:5×105CFU rBb-ESAT-6-MPT64 vaccines were suspended in 10μl PBS and intranasally inoculated once.Serum antibodies of specific IgG,IgG1,IgG2a,IgG2b,IgG3and IgE were respectively detected by ELISA at the time of pre-immunization and at the ends of 2,4,6,8 and 10w after immunization, IFN-γ,IL-12,TNF-αand IL-10 were respectively detected by ELISA,the number of CD4+ and CD8+ subsets of T lymphocyte were detected by FCM,the proliferation of splenocytes were measured by MTT method.ResultsOn the 6th week after mice challenged with H37Ra,the number of CFU in the lungs and livers of immunization group reduced remarkably compared with that in PBS control.The levels of serum specific antibodies were detected by ELISA:for groups A~C,the levels of antibodies IgG,IgG1,IgG2a,IgG2b were remarkably higher than that in the control groups D and E; the levels of IgG,IgG1,IgG2a,IgG2b in group C were remarkably higher than that in the group A and B,but there were no differences among all groups for IgG3 and IgE ;All antibodies for group A had no difference with group B.The levels of cytokine were detected by ELISA:for groups A~C,the levels of IFN-γ,IL-12and TNF-αin supernatant of spleen cells stimulated by MTBAg and ConA or LPS were remarkably higher than that in PBS control; for groups A~C,the levels of IL-10 in supernatant of spleen cells stimulated by MTBAg and ConA were remarkably lower than that in PBS control. In BALB/c mice by immunization with the recombinant Bb-ESAT-6-MPT64 vaccine,in the subcutaneous group,the levels of IgG incresaed on 2 week after immunization and reached the highest level on 8 week.The levels of IgG1 didn't only decrease on 8 week.The levels of IgG2a enhanced on 2 week after immunization and reached the highest level on 6 week.The levels of IgG2b added only on 2 week after immunization.The levels of IgG3 decreased on 2 week after immunization and reached the lowest level on 8 and 10 week.The levels of IgE reduced on 2 week after immunization and reached the lowest level on 8 and 10 week.In the intranasal group,the levels of IgG incresaed on 2 week after immunization and reached the highest level on 8 week.The levels of IgG1 didn't only decrease on 8 and 10 week.The levels of IgG2a enhanced on 2 week after immunization and reached the highest level on 6 week.The levels of IgG2b added only on 2 weeks after immunization.The levels of IgG3 decreased on 2 week after immunization and reached the lowest level on 8 and 10 week.The levels of IgE reduced on 2 week after immunization and reached the lowest level on 8 and 10 week.The dynamical observation on the changes in cytokines of splenocytes in BALB/c mice by immunization with the recombinant Bb-ESAT-6-MPT64 vaccine,the levels of IFN-γincresaed on 2 week after immunization and reached the highest level on 6 week in the subcutaneous group,while it reached the highest level on 8 week in the intranasal group.The levels of IL-12 incresaed on 2 week after immunization and reached the highest level on 4 week in the subcutaneous group,while it reached the highest level on 4,6 and 8 week in the intranasal group.The levels of TNF-αincresaed on 2 week after immunization and reached the highest level on 6 week in the subcutaneous group,while it reached the highest level on 6 and 8 week in the intranasal group.The levels of IL-10 incresaed on 2 week after immunization and reached the highest level on 6 and 8 week in the subcutaneous group,while it reached the highest level on 6 week in the intranasal group.The levels of CD4+ and CD8+ subsets of T lymphocyte were detected by FCM: the levels of CD4+ subsets incresaed on 2 week after immunization and were reached remarkably higher than both control groups in the subcutaneous group and the intranasal group.The levels of CD8+ subsets were slightly higher on 4 week than the control groups in the subcutaneous group and the intranasal group,but had no statistic value.The proliferation of splenocytes were measured by MTT method:in the subcutaneous group, the proliferation of splenocytes incresaed on 2 week after immunization and reached the highest level on 2 week in MTBAg group and ConA group.In the intranasal group, the proliferation of splenocytes incresaed on 2 week after immunization and reached the highest level on 2 week in MTBAg group,while it reached the highest level on 4 weeks in ConA group.ConclusionProtective test with different immune routine showed that the number of CFU in the lungs and livers of the immunization group reduced remarkably compared with that in PBS control,the recombinant Bb-ESAT-6-MPT64 vaccine induced mice to product the high level of IgG, IgG1,IgG2a and IgG2b ,the routine of intranasal inoculation was better. Before and after challenge, the level of IFN-γ,IL-12 and TNF-αincreased remarkably in mice induced with recombinant Bb-ESAT-6-MPT64 vaccine;after mice were immunized, the level of IL-10 increased;after mice were challenged, the level of IL-10 decreased.It suggested that the mixed response is induced in mice by recombinant Bb-ESAT-6-MPT64 vaccine.The level of IgG, IgG2a and IgG2b increased remarkably in mice induced with recombinant Bb-ESAT-6-MPT64 vaccine,the level of IgG1,IgG3and IgE decreased;it hinted that IgG, IgG2a and IgG2b could participate in the mechanism of humoral immune response.The recombinant Bb-ESAT-6-MPT64 vaccine was able to elicit strong specific CD4+Th1 cellular immune,humoral immune and weak CD8+CTL respones. The protective immunity induced by the recombinant Bb-ESAT-6-MPT64 vaccine with intranasal inoculation is better than that of subcutaneous or intramuscular inoculation.