Sequential immunization with mycobacterial antigen Ag85B-expressing DNA and Mycobacterium bovis bacille Calmette-Guerin (BCG) was more effective than BCG immunization in protecting against Mycobacterium tuberculosis infection. Depletion of the CD8 ؉ T cells in the immunized mice impaired protection in their spleens, indicating that this improved efficacy was partially mediated by CD8 ؉ T cells.

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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...

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A strain of Mycobacterium bovis BCG that secretes high levels of functional murine monocyte chemotactic protein 3 (BCG MCP-3 ) was developed. Mice vaccinated with BCG MCP-3 displayed increased lymphocyte migration in vivo and augmented antigen-specific T-cell responses compared to mice vaccinated with BCG alone. The level of protection afforded by BCG MCP-3 was equivalent to that with control BCG; however, immunodeficient mice infected with BCG MCP-3 survived significantly longer than mice infected with the control BCG strain. Therefore, BCG MCP-3 may be a safer alternative than conventional BCG for vaccination of immunocompromised individuals.Tuberculosis (TB) remains a leading cause of worldwide mortality alongside human immunodeficiency virus and malaria. Recent figures suggest that each year there are approximately 8 million new cases and 2 million deaths (17). The current vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is only partially effective against lung TB, the contagious form of the disease, with vaccine efficacy ranging from 0 to 80% (median of 50%) (7). Consequently, although BCG is considered a safe vaccine and has been administered since 1921 to an estimated 3 billion people, it has proved insufficient to control the spread of TB (2). BCG is amenable to genetic manipulation, and overexpression of protective antigens in BCG can improve the efficacy of the vaccine in animal models of TB (9,20,23). An alternative approach is to express immunostimulatory molecules in BCG. Murine cytokines, including interleukin-2, interleukin-18, gamma interferon (IFN-␥), and granulocyte-macrophage colony-stimulating factor, can be secreted by BCG in a functional form; however, the ability of these vaccines to protect against TB has not been tested (13,16,18). The monocyte chemotactic proteins (MCP) are a subset of the CC or ␤-chemokines consisting of four members, MCP-1, -2, -3, and -4 (with MCP-5 in the mouse only), which are induced by viral and bacterial infections in many cell types (26, 31). MCP-3 is a pleiotropic chemokine that binds the receptors CCR1, CCR2, and CCR3 (15). These receptors are expressed on mononuclear phagocytes, T cells, B cells, natural killer cells, basophils, eosinophils, neutrophils, and particularly immature dendritic cells (1,3,11,26,32). MCP-3 is highly expressed by macrophages exposed to mycobacterial components or M. tuberculosis-infected mice (27, 30). Mice lacking CCR2 were acutely susceptible to M. tuberculosis infection and exhibited a significant reduction in early macrophage recruitment and a later defect in dendritic cell and T-cell migration (22). These results clearly demonstrate that chemokines, such as MCP-3, are vital in the response to M. tuberculosis infection, as they are required for the selective migration of the cells essential for the generation of a protective immune response.In this report, we demonstrate that murine MCP-3 (mMCP-3) can be expressed and secreted by BCG in an active form. Secretion of MCP-3 markedly improved the immunogenicity of BCG a...

The isolation of elements driving high-level expression of foreign genes in mycobacteria would significantly aid characterization of mycobacterial antigens and recombinant vaccine development. Mycobacterium smegmatis is a widely employed host for recombinant mycobacterial gene expression. This report describes the identification of strong promoter elements of M. smegmatis. Fluorescenceactivated cell sorting was employed to isolate DNA fragments permitting high-level expression of the Aequorea victoria green fluorescent protein within recombinant M. smegmatis. Ten postulated M. smegmatis promoters were identified which showed activity two to six times that of the strong L-lactamase promoter of Mycobacterium fortuitum. The utility of one of these promoters for the over-expression of foreign genes in mycobacteria was demonstrated by the efficient purification of the Mycobacterium leprae 35-kDa antigen from recombinant M. smegmatis.

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