A novel recombinant BCG (BCG-DHTM), that was deficient in urease, expressed with gene encoding the fusion of BCG-derived HSP70 and M. tuberculosis-derived major membrane protein (MMP)-Ⅱ, was constructed for use as a vaccine against tuberculosis. BCG-DHTM efficiently activated dendritic cells (DC) to induce cytokine production, including IL-12, TNFα and IL-1β and phenotypic changes. The DC infected BCG-DHTM was more potent in activation of naïve T cells of CD4 and CD8 subsets than those infected vector control BCG. The activation of naïve T cells by BCG-DHTM was closely associated with phagomal maturation, and that of naïve CD8+ T cells by BCG-DHTM was induced by the activation of cytosolic cross-presentation pathway. Further, BCG-DHTM seemed to activate naïve CD4+ T cells and naïve CD8+ T cells by antigen-specific fashion. The primary infection of BCG-DHTM in C57BL/6 mice for 12 weeks efficiently produced T cells responsive to in vitro secondary stimulation with MMP-Ⅱ HSP70 and H37Rv-derived cytosolic protein and inhibited with multiplication of subsequently challenged M. tuberculosis in lungs at least partially. The effect of BCG-DHTM as a vaccine for tuberculosis is not fully convincing and need the improvement, however, our strategy in the development of new recombinant BCG for tuberculosis seems to provide useful tool.
The effectiveness of a vaccine against tuberculosis and leprosy is mainly judged by its capability to induce memory CD8+ cytotoxic T cells (CTL). It has been reported that ‘help’ from CD4+ T cells is required to induce memory CTL. However, how CD4+ T cells instruct or support memory CTL during priming phase has not been resolved in detail. Therefore, we examined the helper function of CD4+ T cells in CTL differentiation. Peptide-25 is the major T cell epitope of Ag85B of Mycobacterium tuberculosis. We found that this peptide induced the expression of T-bet and TATA box binding protein-associated factor that can induce the chromatin remodeling of ifn-γ gene, and as a result induced Th1 differentiation even in the absence of IFN-γ and IL-12. Next, we established an in vitro CTL differentiation system using Peptide-25, Peptide-25 specific CD4+ T cells, OVA specific CD8+ T cells and splenic DC. By using this system, we found that CD4+ T cells activated DC even in the absence of IFN-γ and CD40 ligand association, and the activated DC induced the functional differentiation of CTL. To identify the regulatory factors for DC activation, we analyzed the gene expression profile of helper CD4+ T cells and identified 27 genes. Taken together, these results suggest that the inducing factors for Th1 differentiation are not indispensable to induce the functional differentiation of CTL.
Instead of rapid multiplication, pathogenic mycobacteria, such as Mycobacterium tuberculosis are likely to have acquired slow but long life. Host immunity affords desirable non-competitive environment for M. tuberculosis in human lungs, where this pathogen slowly grows or arrests growing, which avoids rapid loss of living places. Mycobacterial DNA-binding protein 1 (MDP1), a unique histone-like protein associating mycobacterial GC-rich DNA, has pivotal role in realizing such slow life and pathogenesis including drug tolerance to isoniazid.