Bacillus Calmette-Guérin (BCG) vaccine has failed to control the global tuberculosis (TB) epidemic, and there is a lack of safe and effective mucosal vaccines capable of potent protection against pulmonary TB. A recombinant replication-deficient adenoviral-based vaccine expressing an immunogenic Mycobacterium tuberculosis Ag Ag85A (AdAg85A) was engineered and evaluated for its potential to be used as a respiratory mucosal TB vaccine in a murine model of pulmonary TB. A single intranasal, but not i.m., immunization with AdAg85A provided potent protection against airway Mycobacterium tuberculosis challenge at an improved level over that by cutaneous BCG vaccination. Systemic priming with an Ag85A DNA vaccine and mucosal boosting with AdAg85A conferred a further enhanced immune protection which was remarkably better than BCG vaccination. Such superior protection triggered by AdAg85 mucosal immunization was correlated with much greater retention of Ag-specific T cells, particularly CD4 T cells, in the lung and was shown to be mediated by both CD4 and CD8 T cells. Thus, adenoviral TB vaccine represents a promising novel vaccine platform capable of potent mucosal immune protection against TB. Our study also lends strong evidence that respiratory mucosal vaccination is critically advantageous over systemic routes of vaccination against TB.

TNF-α has long been regarded as a proimmune cytokine involved in antimicrobial type 1 immunity. However, the precise role of TNF-α in antimicrobial type 1 immunity remains poorly understood. We found that TNF-α–deficient (TNF^–/–) mice quickly succumbed to respiratory failure following lung infection with replication-competent mycobacteria, because of apoptosis and necrosis of granuloma and lung structure. Tissue destruction was a result of an uncontrolled type 1 immune syndrome characterized by expansion of activated CD4 and CD8 T cells, increased frequency of antigen-specific T cells, and overproduction of IFN-γ and IL-12. Depletion of CD4 and CD8 T cells decreased IFN-γ levels, prevented granuloma and tissue necrosis, and prolonged the survival of TNF^–/– hosts. Early reconstitution of TNF-α by gene transfer reduced the frequency of antigen-specific T cells and improved survival. TNF-α controlled type 1 immune activation at least in part by suppressing T cell proliferation, and this suppression involved both TNF receptor p55 and TNF receptor p75. Heightened type 1 immune activation also occurred in TNF^–/– mice treated with dead mycobacteria, live replication-deficient mycobacteria, or mycobacterial cell wall components. Our study thus identifies TNF-α as a type 1 immunoregulatory cytokine whose primary role, different from those of other type 1 cytokines, is to keep an otherwise detrimental type 1 immune response in check.

T-helper 1 (Th1) cells are believed to be the major producer of the type 1 cytokine interferon-γ (IFN-γ) in cell-mediated immunity against intracellular infection. We have investigated the ability of macrophages to release type 1 cytokines and their regulatory mechanisms using both in vivo and in vitro models of pulmonary mycobacterial infection. During pulmonary infection by live Mycobacterium bovis bacilli Calmette-Guérin (BCG) in wild-type mice, lung macrophages released interleukin-12 (IL-12), IFN-γ, and tumor necrosis factor-α (TNF-α), and expressed surface activation markers. However, macrophages in infected IL-12^–/– mice released TNF-α but not IFN-γ and lacked surface activation makers. In freshly isolated lung macrophages from naive IL-2^–/– mice, mycobacteria alone released TNF-α but not IFN-γ, whereas exogenously added IL-12 alone released a minimum of IFN-γ. However, these macrophages released large quantities of IFN-γ upon stimulation with both mycobacteria and IL-12. In contrast, mycobacteria and exogenous IFN-γ released only a minimum of endogenous IFN-γ. Endogenous IL-18 (IFN-γ–inducing factor) played little role in IFN-γ responses by macrophages stimulated by mycobacteria and IL-12. Our data reveal that macrophages are a significant source of type 1 cytokines during mycobacterial infection and that both IL-12 and intracellular pathogens are required for the release of IFN-γ but not TNF-α. These findings suggest that macrophages regulate cell-mediated immunity by releasing not only IL-12 and TNF-α but also IFN-γ and that full activation of IFN-γ response in macrophages is tightly regulated.

Whether the intranasal (i.n.) route ofMycobacterium bovisBCG vaccination provides better protection against pulmonary tuberculosis than subcutaneous (s.c.) vaccination remains an incompletely solved issue. In the present study, we compared both immune responses and protection elicited by single BCG vaccinations via the i.n. or s.c. route in BALB/c mice. While both i.n. and s.c. vaccination triggered comparable levels of primary immune activation in the spleen and draining lymph nodes, i.n. vaccination led to a greater antigen-specific gamma interferon recall response in splenocytes than s.c. vaccination upon secondary respiratory mycobacterial challenge, accompanied by an increased frequency of antigen-specific lymphocytes. There was also a quicker cellular response in the lungs of i.n. vaccinated mice upon mycobacterial challenge. Mice vaccinated i.n. were found to be much better protected, particularly in the lung, than s.c. vaccinated counterparts against pulmonary tuberculosis at both 3 and 6 months postvaccination. These results suggest that the i.n. route of vaccination improves the protective effect of the current BCG vaccine.

The mechanisms underlying better immune protection by mucosal vaccination have remained poorly understood. In our current study we have investigated the mechanisms by which respiratory virus-mediated mucosal vaccination provides remarkably better immune protection against pulmonary tuberculosis than parenteral vaccination. A recombinant adenovirus-based tuberculosis (TB) vaccine expressing Mycobacterium tuberculosis Ag85A (AdAg85A) was administered either intranasally (i.n.) or i.m. to mice, and Ag-specific CD4 and CD8 T cell responses, including frequency, IFN-γ production, and CTL, were examined in the spleen, lung interstitium, and airway lumen. Although i.m. immunization with AdAg85A led to activation of T cells, particularly CD8 T cells, in the spleen and, to a lesser extent, in the lung interstitium, it failed to elicit any T cell response in the airway lumen. In contrast, although i.n. immunization failed to effectively activate T cells in the spleen, it uniquely elicited higher numbers of Ag-specific CD4 and CD8 T cells in the airway lumen that were capable of IFN-γ production and cytolytic activities, as assessed by an intratracheal in vivo CTL assay. These airway luminal T cells of i.n. immunized mice or splenic T cells of i.m. immunized mice, upon transfer locally to the lungs of naive SCID mice, conferred immune protection against M. tuberculosis challenge. Our study has demonstrated that the airway luminal T cell population plays an important role in immune protection against pulmonary TB, thus providing mechanistic insights into the superior immune protection conferred by respiratory mucosal TB vaccination.