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ABSTRACTBy measuring phosphate uptake by Mycobacterium tuberculosisstrains with the pstS1and pstS2genes genetically inactivated, we showed that these pstSgenes encode high-affinity phosphate binding proteins. In a mouse infection model, both mutants were attenuated in virulence, suggesting that M. tuberculosisencounters limiting phosphate concentrations during its intracellular life span.

ABSTRACTBuruli ulcer, caused by Mycobacterium ulcerans, is characterized by deep and necrotizing skin lesions, mostly on the arms and legs. Together with tuberculosis and leprosy, this mycobacterial disease has become a major health problem in tropical and subtropical regions, particularly in central and western Africa. No specific vaccine is available for Buruli ulcer. There is, however, evidence in the literature that suggests a cross-reactive protective role of the tuberculosis vaccine M. bovisBCG. To identify potential mechanisms for this cross-protection, we identified and characterized the M. ulceranshomologue of the important protective mycobacterial antigen 85 (Ag85A) from BCG. The homologue is well conserved in M. ulcerans, showing 84.1% amino acid sequence identity and 91% conserved residues compared to the sequence from BCG. This antigen was sufficiently conserved to allow cross-reactive protection, as demonstrated by the ability of M. ulcerans- infected mice to exhibit strong cellular immune responses to both BCG and its purified Ag85 complex. To further address the mechanism of cross-reactive protection, we demonstrate here that prior vaccination with either BCG or plasmid DNA encoding BCG Ag85A is capable of significantly reducing the bacterial load in the footpads ofM. ulcerans- infected mice, as determined by Ziehl-Neelsen staining and by actual counting of CFU on 7H11 Middlebrook agar. Together, the results reported here support the potential of a cross-protective Ag85-based future vaccine against tuberculosis, Buruli ulcer, and leprosy.

ABSTRACTIn this study, the hsp60 and hsp70 heat shock protein antigens ofMycobacterium tuberculosiswere tested as potential vaccine candidates, using purified recombinant protein antigens or antigens encoded in the form of a DNA plasmid vaccine. Guinea pigs vaccinated with a mixture of the two proteins showed no evidence of resistance to low-dose aerosol challenge infection and quickly developed severe lung damage characterized by necrotizing bronchointerstitial pneumonia and bronchiolitis. As a result, we turned instead to a DNA vaccination approach using a plasmid encoding the hsp60 antigen of M. tuberculosis. Although immunogenic in mice, vaccination with plasmid DNA encoding hsp60 was not protective in that model or in the guinea pig model and again gave rise to similar severe lung damage. This study seriously questions the safety of vaccines against tuberculosis that target highly conserved heat shock proteins.

ABSTRACTUsing spleen cells from mice vaccinated with liveMycobacterium bovisBCG, we previously generated three monoclonal antibodies reactive against a 22-kDa protein present in mycobacterial culture filtrate (CF) (K. Huygen et al., Infect. Immun. 61:2687–2693, 1993). These monoclonal antibodies were used to screen an M. bovisBCG genomic library made in phage λgt11. The gene encoding a 233-amino-acid (aa) protein, including a putative 26-aa signal sequence, was isolated, and sequence analysis indicated that the protein was 98% identical with the M. tuberculosisLppx protein and that it contained a sequence 94% identical with the M. leprae38-mer polypeptide 13B3 recognized by T cells from killed M. leprae-immunized subjects. Flow cytometry and cell fractionation demonstrated that the 22-kDa CF protein is also highly expressed in the bacterial cell wall and membrane compartment but not in the cytosol. C57BL/6, C3H, and BALB/c mice were vaccinated with plasmid DNA encoding the 22-kDa protein and analyzed for immune response and protection against intravenous M. tuberculosischallenge. Whereas DNA vaccination induced elevated antibody responses in C57BL/6 and particularly in C3H mice, Th1-type cytokine response, as measured by interleukin-2 and gamma interferon secretion, was only modest, and no protection against intravenous M. tuberculosischallenge was observed in any of the three mouse strains tested. Therefore, the 22-kDa antigen seems to have little potential for a DNA vaccine against tuberculosis, but it may be a good candidate for a mycobacterial antigen detection test.

ABSTRACTListeria monocytogenesis a gram-positive, nonsporulating, food-borne pathogen of humans and animals that is able to invade many eukaryotic cells. Several listerial surface components have been reported to interact with eukaryotic cell receptors, but the complete mechanism by which the bacteria interact with all of these cell types remains largely unknown. In this work, we found thatL. monocytogenesbinds to human fibronectin, a 450,000-Da dimeric glycoprotein found in body fluids, on the surface of cells and in an insoluble component of the extracellular matrix. The binding of fibronectin to L. monocytogeneswas found to be saturable and dependent on proteinaceous receptors. Five fibronectin-binding proteins of 55.3, 48.6, 46.7, 42.4, and 26.8 kDa were identified. The 55.3-kDa protein was proved to be present at the bacterial cell surface. The binding of L. monocytogenesto fibronectin adds to the number of molecules to which the bacterium is able to adhere and emphasizes the complexity of host-pathogen interactions.

ABSTRACTGamma interferon (IFN-γ)-secreting CD4+T cells have long been established as an essential component of the protective immune response against Mycobacterium tuberculosis. It is now becoming evident from studies with the murine model of tuberculosis that an important role also exists for major histocompatibility complex (MHC) class I-restricted CD8+T cells. These cells are capable of acting as both IFN-γ secretors and cytotoxic T lymphocyte (CTL) effectors; however, their exact role in immunity against tuberculosis remains unclear. This study demonstrates the presence ofMycobacterium bovisBCG-reactive CD8+T cells in healthy BCG-vaccinated donors and that these CD8+T cells are potent cytokine producers as well as cytotoxic effector cells. Using FACScan analysis, we have shown that restimulation with live M. bovisBCG induced more CD8+-T-cell activation than the soluble antigen purified protein derivative and that these cells are actively producing the type 1 cytokines IFN-γ and tumor necrosis factor alpha (TNF-α). These CD8+T cells also contain the cytolytic granule perforin and are capable of acting as potent CTLs against M. bovisBCG-infected macrophages. The mycobacterial antigens 85A and B (Ag85A and Ag85B, respectively), and to a lesser extent the 19- and 38-kDa proteins, are major antigenic targets for these mycobacterium-specific CD8+T cells, while whole-M. bovisBCG activated effector cells from these BCG-vaccinated donors, as expected, failed to recognize the 6-kDa ESAT-6 protein. The use of metabolic inhibitors and blocking antibodies revealed that the CD8+T cells recognize antigen processed and presented via the classical MHC class I pathway. These data suggest that CD8+T cells may play a critical role in the human immune response to tuberculosis infection.

ABSTRACTVaccination of mice with plasmid DNA carrying the gene for the major secreted mycobacterial antigen 85A (Ag85A) fromMycobacterium tuberculosisis a powerful technique for generating robust specific Th1 helper T-cell responses, CD8+-mediated cytotoxicity, and protection against M. tuberculosischallenge (K. Huygen et al., Nat. Med. 2:893–898, 1996). We have now analyzed in more detail the antigen-specific immune CD4+- and CD8+-T-cell responses induced in BALB/c mice vaccinated with Ag85A DNA and have compared these responses to those generated by intravenous infection with M. tuberculosis. T-cell-epitope mapping, as measured by interleukin-2 and gamma interferon secretion from splenic T cells restimulated in vitro with synthetic 20-mer peptides spanning the complete mature sequence of Ag85A, demonstrated that DNA vaccination stimulated a stronger and broader T-cell response than did M. tuberculosisinfection. Moreover, elevated cytotoxic T lymphocyte (CTL) activity against Ag85A-transfected and peptide-pulsed P815 target cells could be generated exclusively by vaccination with plasmid DNA, not following M. tuberculosisinfection. By using DNA vaccination, three Ag85A CTL epitopes with predicted major histocompatibility complex class I binding motifs were defined. One of them was previously reported as a dominant, promiscuously recognized T-cell epitope in healthy humans with primary infections. These data strengthen the potential of DNA vaccination with respect to inducing antituberculous immunity in humans.