Mycobacterium bovis BCG-specific Th17 cells confer partial protection against Mycobacterium tuberculosis infection in the absence of gamma interferon.

Protective immunity against tuberculosis (TB) requires the integrated response of a network of lymphocytes. Both gamma interferon (IFN-γ)- and interleukin 17 (IL-17)-secreting CD4(+) T cells have been identified in subjects with latent TB infection and during experimental Mycobacterium tuberculosis infection, but the contribution of Th17 cells to protective immunity is unclear. To examine their protective effects in vivo, we transferred mycobacterium-specific IL-17- and IFN-γ-secreting CD4(+) T cells isolated from M. tuberculosis BCG-immunized IL-12p40(-/-) and IFN-γ(-/-) or wild-type mice, respectively, into M. tuberculosis-infected IL-12p40(-/-) or RAG(-/-) mice. In the absence of IL-12 and IL-23, neither IL-17-secreting (Th17) nor IFN-γ-secreting (Th1) BCG-specific T cells expanded or provided protection against M. tuberculosis. In RAG(-/-) recipients with an intact IL-12/IL-23 axis, both Th17 and Th1 cells were activated and induced significant protection against M. tuberculosis. The reduction in the bacterial load following transfer of IFN-γ(-/-) Th17 cells was associated with significant prolongation of survival compared to recipients of naïve IFN-γ(-/-) T cells. This effect was at the cost of an increased inflammatory infiltrate characterized by an excess of neutrophils. Therefore, Th17 cells can provide IFN-γ-independent protection against M. tuberculosis, and this effect may contribute to the early control of M. tuberculosis infection.

Modulation of pulmonary DC function by vaccine-encoded GM-CSF enhances protective immunity against Mycobacterium tuberculosis infection.

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.

Antigen load governs the differential priming of CD8 T cells in response to the bacille Calmette Guerin vaccine or Mycobacterium tuberculosis infection.

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.

Heterologous expression of genes in mycobacteria.

Elucidating the function of mycobacterial proteins, determining their contribution to virulence, and developing new vaccine candidates has been facilitated by systems permitting the heterologous expression of genes in mycobacteria. Mycobacterium bovis bacille Calmette Guérin (BCG) and Mycobacterium smegmatis have commonly been employed as host systems for the heterologous expression of mycobacterial genes as well as genes from other bacteria, viruses, and mammalian cells. Vectors that permit strong, constitutive expression of genes have been developed, and more recently systems that allow tightly regulated induction of gene expression have become available. In this chapter, we describe two complementary techniques relevant to the field of gene expression in mycobacteria. We first outline the methodology used for the expression and specific detection of recombinant products expressed in BCG. The expression vectors described use an epitope tag fused to the C-terminal end of the foreign protein, ablating the need for additional reagents to detect the recombinant product. Second, we describe the inducible expression of genes in recombinant M. smegmatis and the subsequent purification of gene products using affinity chromatography.

Improved protection against disseminated tuberculosis by Mycobacterium bovis bacillus Calmette-Guerin secreting murine GM-CSF is associated with expansion and activation of APCs.

Modulating the host-immune response by the use of recombinant vaccines is a potential strategy to improve protection against microbial pathogens. In this study, we sought to determine whether secretion of murine GM-CSF by the bacillus Calmette-Guérin (BCG) vaccine influenced protective immunity against Mycobacterium tuberculosis. BCG-derived GM-CSF stimulated the in vitro generation of functional APCs from murine bone marrow precursors, as demonstrated by the infection-induced secretion of IL-12 by differentiated APCs, and the ability of these cells to present Ag to mycobacterium-specific T cells. Mice vaccinated with BCG secreting [corrected] murine GM-CSF (BCG:GM-CSF) showed increased numbers of CD11c+MHCII+ and CD11c-CD11b+F480+ cells compared with those vaccinated with control BCG, and this effect was most apparent in the draining lymph nodes at 7 and 14 days postvaccination. Vaccination with BCG:GM-CSF also resulted in enhanced expression of costimulatory molecules on migratory dendritic cells in the draining lymph nodes. The increased APC number was associated with an increase in the frequency of anti-mycobacterial IFN-gamma-secreting T cells generated after BCG:GM-CSF vaccination compared with vaccination with control BCG, and this effect was sustained up to 17 wk in the spleens of immunized mice. Vaccination with BCG:GM-CSF resulted in an approximately 10-fold increase in protection against disseminated M. tuberculosis infection compared with control BCG. This study demonstrates the potential of BCG secreting [corrected] immunostimulatory molecules as vaccines to protect against tuberculosis and suggests BCG:GM-CSF merits further appraisal as a candidate to control M. tuberculosis infection in humans.

Effects of DNA- and Mycobacterium bovis BCG-based delivery of the Flt3 ligand on protective immunity to Mycobacterium tuberculosis.

The control of intracellular pathogens such as Mycobacterium tuberculosis is dependent on the activation and maintenance of pathogen-reactive T cells. Dendritic cells (DCs) are the major antigen-presenting cells initiating antimycobacterial T-cell responses in vivo. To investigate if immunization strategies that aim to optimize DC function can improve protective immunity against virulent mycobacterial infection, we exploited the ability of the hematopoietic growth factor Fms-like tyrosine kinase 3 ligand (Flt3L) to expand the number of DCs in vivo. A DNA fusion of the genes encoding murine Flt3L and M. tuberculosis antigen 85B stimulated enhanced gamma interferon (IFN-gamma) release by T cells and provided better protection against virulent M. tuberculosis than DNA encoding the single components. Vaccination of mice with a recombinant Mycobacterium bovis BCG strain secreting Flt3L (BCG:Flt3L) led to early expansion of DCs compared to immunization with BCG alone, and this effect was associated with increased stimulation of BCG-reactive IFN-gamma-secreting T cells. BCG and BCG:Flt3L provided similar protective efficacies against low-dose aerosol M. tuberculosis; however, immunization of immunodeficient mice revealed that BCG:Flt3L was markedly less virulent than conventional BCG. These results demonstrate the potential of in vivo targeting of DCs to improve antimycobacterial vaccine efficacy.

Secretion of functional monocyte chemotactic protein 3 by recombinant Mycobacterium bovis BCG attenuates vaccine virulence and maintains protective efficacy against M. tuberculosis infection.

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.

Interleukin-23 restores immunity to Mycobacterium tuberculosis infection in IL-12p40-deficient mice and is not required for the development of IL-17-secreting T cell responses.

Host control of Mycobacterium tuberculosis is dependent on the activation of CD4+ T cells secreting IFN-gamma 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-12Rbeta1 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. Co-delivery of p2AIL-23 or p2AIL-12 with DNA85B induced strong proliferative and IFN-gamma-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-gamma-secreting T cells. Interestingly, bacillus Calmette-Guérin immune T cells generated in the absence of IL-12 or IL-23 were deficient in IFN-gamma 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.

Contribution of L-alanine dehydrogenase to in vivo persistence and protective efficacy of the BCG vaccine.

The tuberculosis (TB) vaccine strain Mycobacterium bovis BCG is unable to utilise alanine and this deficiency is thought to inhibit the growth of the vaccine in vivo and limit vaccine efficacy. In this report we demonstrate that L-alanine catabolism can be conferred on BCG by introduction of the gene encoding L-alanine dehydrogenase (Ald) of Mycobacterium tuberculosis. Restoration of Ald activity did not change the in vivo growth of BCG in macrophages or mice, and protection against aerosol M. tuberculosis infection was not altered by addition of ald to the BCG vaccine. These results demonstrate that the inability to utilise L-alanine is not a contributing factor to the attenuated phenotype of BCG and does not influence the protective efficacy of the vaccine against TB.

Plasmid interleukin-23 (IL-23), but not plasmid IL-27, enhances the protective efficacy of a DNA vaccine against Mycobacterium tuberculosis infection.

Protection against intracellular pathogens such as Mycobacterium tuberculosis requires the development of Th1-like T-cell responses. This in turn is dependent on the pattern of cytokine produced from dendritic cells (DCs) after infection. Three heterodimeric cytokines, interleukin-12 (IL-12), IL-23, and IL-27, as well as IL-18, contribute to the differentiation and expansion of naive CD4(+) T cells. In this study we compared the effects of plasmids expressing both chains of IL-12, IL-23, or IL-27 as adjuvants for DNA immunization against M. tuberculosis infection. The genes encoding p19 and p40 chains of IL-23 or EBI3 and p28 chains of IL-27 were cloned on either side of a self-cleaving peptide from the FMDV2A protein. The secretion of functional cytokines from transfected cells was detected with bioassays. Supernatant from p2AIL-23-transfected cells induced the release of IL-17 from activated lymphocytes, confirming the presence of bioactive IL-23. Further, supernatant from p2AIL-27-transfected cells stimulated a significant increase in the proliferation of peptide-stimulated transgenic CD4(+) T cells. In initial experiments, M. tuberculosis infection of DCs was more potent at inducing IL-12 and IL-23 secretion than infection with the vaccine strain Mycobacterium bovis bacille Calmette-Guérin (BCG), and no significant upregulation of IL-27 was observed. Coimmunization of C57BL/6 mice with DNA expressing M. tuberculosis antigen 85B (Ag85B; DNA85B) and plasmids expressing IL-23 or IL-12 stimulated stronger Ag85B-specific T-cell proliferative and IFN-gamma responses than DNA85B alone, whereas the addition of p2AIL-27 had no effect. Interestingly, DNA85B codelivered with p2AIL-12, but not p2AIL-23, reduced the immunoglobulin G antibody response. Both p2AIL-23 and p2AIL-12, but not p2AIL-27, enhanced the protective efficacy of DNA85B against aerosol M. tuberculosis challenge. Therefore, both p2AIL-23 and p2AIL-12 are valuable as cytokine adjuvants for increasing the protective antituberculosis immunity induced by DNA vaccines.

Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria.

New tools are required to study the growing number of uncharacterised genes derived from genome sequence projects that are specific to bacterial pathogens such as Mycobacterium tuberculosis. We have developed a series of vectors that permit the specific detection of recombinant proteins expressed in mycobacterial species. Gene expression in these vectors is driven by the strong hsp60 promoter of Mycobacterium bovis BCG and detection of expressed products is facilitated by C-terminal fusion of residues 409-419 of the human c-myc proto-oncogene. Using the M. tuberculosis Ag85B as a reporter of gene expression, we demonstrate that the vectors permit the specific detection of recombinant products expressed in the host species M. bovis BCG. BCG over-expressing Ag85B was a potent inducer of Ag85B-specific T cells in immunised mice, indicating that the C-terminal c-myc tag did not alter the characteristics of the recombinant protein. The versatility of the epitope-tagging vectors was demonstrated by the efficient secretion and detection of recombinant products in BCG. The vectors described in this study will facilitate the expression of foreign proteins in mycobacterial host systems.