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SUMMARY has been demonstrated to synergize with BCG for induction of a T-helper-type 1 (Th1) immune response. Since successful treatment of superficial bladder cancer with BCG requires proper induction of Th1 immunity, we have developed a recombinant (r) BCG strain that functionally secretes mouse (m) IL-18. This rBCG-mIL-18 strain significantly increased production of the major Th1 cytokine IFN-g in splenocyte cultures, at levels comparable to that elicited by control BCG plus exogenous rIL-18. IFN-g production by splenocytes was eliminated by addition of neutralizing anti-IL-18 antibody. Endogenous IL-12 played a favourable role whereas IL-10 played an adverse role in rBCGmIL-18-induced IFN-g production. Enhanced host antimycobacterial immunity was observed in mice infected with rBCG-mIL-18 which showed less splenic enlargement and reduced bacterial load compared to control mice infected with BCG. Further, splenocytes from rBCG-mIL-18-infected mice, in response to BCG antigen, displayed increased production of IFN-g and GMCSF, decreased production of IL-10, elevated cellular proliferation and higher differentiation of IFN-g -secreting cells. rBCG-mIL-18 also enhanced BCG-induced macrophage cytotoxicity against bladder cancer MBT-2 cells in a dosedependent manner. Neutralizing all endogenous macrophage-derived cytokines tested (IL-12, IL-18 and TNF-a ) as well as IFN-g severely diminished the rBCG-mIL-18-induced macrophage cytolytic activity, indicating a critical role for these cytokines in this process. Cytokine analysis for supernatants of macrophage-BCG mixture cultures manifested higher levels of IFN-g and TNF-a in rBCG-mIL-18 cultures than in control BCG cultures. Taken together, this rBCG-mIL-18 strain augments BCG's immunostimulatory property and may serve as a better agent for bladder cancer immunotherapy and antimycobacterial immunization.

Host control of Mycobacterium tuberculosis is dependent on the activation of CD4+ T cells secreting IFN-γ and their recruitment to the site of infection. The development of more efficient vaccines against tuberculosis requires detailed understanding of the induction and maintenance of T cell immunity. Cytokines important for the development of cell-mediated immunity include IL-12 and IL-23, which share the p40 subunit and the IL-12Rβ1 signaling chain. To explore the differential effect of IL-12 and IL-23 during M. tuberculosis infection, we used plasmids expressing IL-23 (p2AIL-23) or IL-12 (p2AIL-12) alone in dendritic cells or macrophages from IL-12p40−/− mice. In the absence of the IL-12/IL-23 axis, immunization with a DNA vaccine expressing the M. tuberculosis Ag85B induced a limited Ag-specific T cell response and no control of M. tuberculosis infection. Codelivery of p2AIL-23 or p2AIL-12 with DNA85B induced strong proliferative and IFN-γ-secreting T cell responses equivalent to those observed in wild-type mice immunized with DNA85B. This response resulted in partial protection against aerosol M. tuberculosis; however, the protective effect was less than in wild-type mice owing to the requirement for IL-12 or IL-23 for the optimal expansion of IFN-γ-secreting T cells. Interestingly, bacillus Calmette-Guérin immune T cells generated in the absence of IL-12 or IL-23 were deficient in IFN-γ production, but exhibited a robust IL-17 secretion associated with a degree of protection against pulmonary infection. Therefore, exogenous IL-23 can complement IL-12 deficiency for the initial expansion of Ag-specific T cells and is not essential for the development of potentially protective IL-17-secreting T cells.

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One reason proposed for the failure of Mycobacterium bovis bacille Calmette Guérin (BCG) vaccination to adequately control the spread of tuberculosis is a limited ability of the vaccine to induce effective CD8 T cell responses. However, the relative capacity of the BCG vaccine and virulent Mycobacterium tuberculosis to induce activation of CD8 T cells, and the factors that govern the initial priming of these cells after mycobacterial infection, are poorly characterized. Using a TCR transgenic CD8 T cell transfer model, we demonstrate significant activation of Ag-specific CD8 T cells by BCG, but responses were delayed and of reduced magnitude compared with those following infection with M. tuberculosis. The degree of CD8 T cell activation was critically dependent on the level of antigenic stimulation, as modifying the infectious dose to achieve comparable numbers of BCG or M. tuberculosis in draining lymph nodes led to the same pattern of CD8 T cell responses to both strains. Factors specific to M. tuberculosis infection did not influence the priming of CD8 T cells, as codelivery of M. tuberculosis with BCG did not alter the magnitude of BCG-induced T cell activation. Following transfer to RAG-1−/− recipients, BCG and M. tuberculosis-induced CD8 T cells conferred equivalent levels of protection against M. tuberculosis infection. These findings demonstrate that BCG is able to prime functional CD8 T cells, and suggest that effective delivery of Ag to sites of T cell activation by vaccines may be a key requirement for optimal CD8 T cell responses to control mycobacterial infection.

Protection against intracellular pathogens such asTuberculosis (TB) is a global health emergency, with an estimated nine million new cases of active disease and approximately 2 million deaths per year (11a). The development of more effective vaccines than the current vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) may improve the control of this pandemic. New approaches to the design of TB vaccines include the preparation of recombinant BCG oversecreting mycobacterial antigens (32), attenuated strains of M. tuberculosis (54), and subunit vaccines based on DNA or protein antigens (33, 55). DNA vaccines encoding M. tuberculosis proteins, such as antigen 85A (Ag85A) or Ag85B (DNA85), induce partial protection against experimental TB (34, 36). However, the degree of protection gained from DNA vaccination alone is less than that afforded by BCG vaccination. Strategies to improve antimycobacterial immunity from subunit vaccines include the development of fusion proteins containing multiple protective antigens (46) and the use of immunostimulatory molecules as adjuvants (50).The development of acquired cellular immunity is critical for the control of M. tuberculosis infection. The key cytokine required for cell-mediated immunity is gamma interferon (IFN-␥), which functions by stimulating infected macrophages to induce phagolysosomal fusion and killing of intracellular bacteria (10,20). The heterodimeric cytokines interleukin-12 (IL-12) and IL-18 are critical for the induction of Th1-like CD4 ϩ cells and are produced primarily by dendritic cells (DCs) (44,59,67). Humans and mice lacking the p40 chain of IL-12 or its receptors are highly susceptibility to M. tuberculosis infection (6, 11). Plasmids expressing either IL-12 or IL-18 have been used as adjuvants in several infectious models (42,45,50). Coadministration of plasmids expressing IL-12 or IL-18 increased the IFN-␥ T-cell response in DNA vaccination to Ag85B, but only plasmids expressing IL-12 increased protective efficacy (62).Recently, two further cytokines, IL-23 and IL-27, have been found to contribute to the development of Th1-like CD4 ϩ T-cell responses. The heterodimeric cytokine IL-23 is secreted by activated macrophages and DCs and induces clonal expansion of memory CD4 ϩ T cells (49). IL-23 is composed of a p40 subunit, shared with IL-12, and a unique p19 subunit, signaling through the receptor IL-12R␤, and a unique IL-23R chain (49). In addition to its direct action on T cells, IL-23 also induces the secretion of IL-12 and IFN-␥ by DCs in vitro (4). This suggests that IL-23 has indirect involvement in the activation of antigen-presenting cells (APCs). Studies with genedeficient mice reveal that a number of roles that were previously accredited to IL-12 may be dependent on IL-23 (12). In

The rational design of new vaccines engineered to target key components of the host immune response is crucial to aid control of important infectious diseases such as tuberculosis. In this report, we determined whether modifying the function of pulmonary APC could improve protection against infection with Mycobacterium tuberculosis. Targeted delivery to the lung of the cytokine GM‐CSF, expressed by the Mycobacterium bovis BCG vaccine strain, increased pulmonary DC numbers and secretion of the immunoregulatory cytokine IL‐12, compared with parental BCG immunization. This impact on APC number by BCG:GM‐CSF resulted in accelerated priming of antigen‐specific CD4+ T cells in the mediastinal lymph nodes and increased migration of activated CD4+ T cells into the lung. i.n. administration of BCG:GM‐CSF resulted in significantly increased protection against M. tuberculosis infection compared with mice vaccinated with BCG alone. BCG:GM‐CSF exhibited an improved safety profile, as immunodeficient RAG1−/− mice vaccinated i.n. with BCG:GM‐CSF survived significantly longer than control BCG‐vaccinated mice. These data demonstrate that manipulating immune cells in the lung by BCG‐based delivery of GM‐CSF can assist the development of protective mucosal immunity against pulmonary bacterial infection.

Tuberculosis (TB) kills 2 million individuals per year and is the greatest cause of death by a single bacterial agent (26). The worldwide incidence of TB continues to rise (26), due in part to the variable efficacy displayed by the only registered vaccine for human use, Mycobacterium bovis bacillus Calmette-Guérin (BCG) (10). This has fuelled the search for more effective anti-TB vaccines, and approaches ranging from DNA vaccines to live, attenuated strains of Mycobacterium tuberculosis have been assessed; the majority of these approaches have failed to deliver improved protective efficacy compared to BCG in animal models (5). A small number of vaccines have recently entered human trials, two of which aim to improve expansion of T cells directed against a single member of the M. tuberculosis antigen 85 (Ag85) complex (15,24). While these two approaches have shown promise in animal models and initial testing in humans, the fact that no single mycobacterial antigen is recognized by all M. tuberculosis-infected individuals (32) may limit the breadth of coverage delivered by these vaccines within the human population.One approach to improve anti-TB vaccination is to target components of the immune response required for optimal protective efficacy. We have previously demonstrated that vaccination strategies designed to augment gamma interferon (IFN-␥) production, such as codelivery of the cytokines interleukin-12 (IL-12) and IL-23 during DNA vaccination, significantly improve protection against TB (29, 39). IL-12 is critical for the induction of Th1-like CD4 ϩ cells, and humans and mice lacking the p40 chain of IL-12 or its receptors are highly susceptible to M. tuberculosis infection (2, 6, 8). M. tuberculosis infection of either murine or human dendritic cells (DCs), but not macrophages, polarizes naïve T cells to the Th1 phenotype due to production of 14). Infection of mice with BCG and subsequent analysis of DC and macrophage populations revealed that DCs exclusively produced IL-12 and presented antigen to T cells (17). After aerosol infection of mice with M. tuberculosis it was observed that DCs, and not macrophages, migrated specifically to the draining lymph nodes (DLNs) and initiated primary Th1 responses (4). These results indicate that DCs play a pivotal role in priming the adaptive immune response to counter infection with M. tuberculosis. It is possible that limited numbers of DCs at the site of antigen production is one factor hindering development of vaccines against diseases such as TB which are reliant on optimal priming of T-cell responses.The Fms-like tyrosine kinase 3 ligand (Flt3L) influences the development of multiple hematopoietic lineages, in particular DCs (23). Administration of soluble Flt3L to both mice (21) and humans (22) increases the numbers of DCs in secondary lymphoid organs and blood. We therefore reasoned that codelivery of Flt3L may be a feasible strategy to improve the efficacy of anti-TB vaccines. In this report, we demonstrate that Flt3L, delivered as plasmid DNA fused to the d...