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International audience; Tuberculosis remains a leading cause of mortality among infectious diseases worldwide, prompting the need to discover new drugs to fight this disease. We report here the design, synthesis, and antimycobacterial activity of isatin-mono/bis-isoniazid hybrids. Most of the compounds exhibited very high activity against Mycobacterium tuberculosis, with MICs in the range of 0.195 to 0.39 μg/ml, and exerted a more potent bactericidal effect than the standard antitubercular drug isoniazid (INH). Importantly, these compounds were found to be well tolerated at high doses (>200 μg/ml) on Vero kidney cells, leading to high selectivity indices. Two of the most promising hybrids were evaluated for activity in THP-1 macrophages infected with M. tuberculosis, among which compound 11e was found to be slightly more effective than INH. Overexpression of InhA along with cross-resistance determination of the most potent compounds, selection of resistant mutants, and biochemical analysis, allowed us to decipher their mode of action. These compounds effectively inhibited mycolic acid biosynthesis and required KatG to exert their biological effects. Collectively, this suggests that the synthesized isatin-INH hybrids are promising antitubercular molecules for further evaluation in preclinical settings.

A strain of Mycobacterium bovis BCG that secretes high levels of functional murine monocyte chemotactic protein 3 (BCGMCP-3) was developed. Mice vaccinated with BCGMCP-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 BCGMCP-3 was equivalent to that with control BCG; however, immunodeficient mice infected with BCGMCP-3 survived significantly longer than mice infected with the control BCG strain. Therefore, BCGMCP-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

Protection against intracellular pathogens such asMycobacterium tuberculosisrequires 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 againstM. tuberculosisinfection. 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. tuberculosisinfection of DCs was more potent at inducing IL-12 and IL-23 secretion than infection with the vaccine strainMycobacterium bovisbacille Calmette-Guérin (BCG), and no significant upregulation of IL-27 was observed. Coimmunization of C57BL/6 mice with DNA expressingM. tuberculosisantigen 85B (Ag85B; DNA85B) and plasmids expressing IL-23 or IL-12 stimulated stronger Ag85B-specific T-cell proliferative and IFN-γ 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 aerosolM. tuberculosischallenge. Therefore, both p2AIL-23 and p2AIL-12 are valuable as cytokine adjuvants for increasing the protective antituberculosis immunity induced by DNA vaccines.

Although the essential role of tumor necrosis factor (TNF) in resistance toListeria monocytogenesinfection is well established, the roles of the related cytokines lymphotoxin alpha (LTα) and lymphotoxin beta (LTβ) are unknown. Using C57BL/6 mice in which the genes for these cytokines were disrupted, we examined the contributions of TNF, LTα, and LTβ in the host response toListeria. To overcome the lack of peripheral lymph nodes in LTα−/−and LTβ−/−mice, bone marrow chimeras were constructed. TNF−/−and LTα−/−chimeras that lacked both secreted LTα3and membrane-bound LTα1β2and LTα2β1were highly susceptible and succumbed 4.5 and 6 days, respectively, after a low-dose infection (200 CFU). LTβ−/−chimeras, which lacked only membrane-bound LT, controlled the infection in a manner comparable to wild-type (WT) chimeras. TheListeria-specific proliferative and gamma interferon T-cell responses were equivalent in all five groups of infected mice (LTα−/−and LTβ−/−chimeras, WT chimeras, and TNF−/−and WT mice). TNF−/−mice and LTα−/−chimeras, however, failed to generate the discrete foci of lymphocytes and macrophages that are essential for bacterial elimination. Rather, aberrant necrotic lesions comprised predominantly of neutrophils with relatively few lymphocytes and macrophages were observed in the livers and spleens of TNF−/−and LTα−/−chimeras. Therefore, in addition to TNF, soluble LTα3plays a separate essential role in control of listerial infection through control of leukocyte accumulation and organization in infected organs.

We have previously shown that young adults living in a rural area of northern Malawi showed greater gamma interferon (IFN-γ) responses to purified protein derivatives (PPD) prepared from environmental mycobacteria than to PPD from Mycobacterium tuberculosis . In order to define the mycobacterial species to which individuals living in a rural African population have been exposed and sensitized, we tested T-cell recognition of recombinant and purified antigens from M. tuberculosis (38 kDa, MPT64, and ESAT-6), M. bovis (MPB70), M. bovis BCG (Ag85), and M. leprae (65 kDa, 35 kDa, and 18 kDa) in >600 non- M. bovis BCG-vaccinated young adults in the Karonga District of northern Malawi. IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA) in day 6 supernatants of diluted whole-blood cultures. The recombinant M. leprae 35-kDa and 18-kDa and purified native M. bovis BCG Ag85 antigens induced the highest percentages of responders, though both leprosy and bovine tuberculosis are now rare in this population. The M. tuberculosis antigens ESAT-6 and MPT64 and the M. bovis antigen MPB70 induced the lowest percentages of responders. One of the subjects subsequently developed extrapulmonary tuberculosis; this individual had a 15-mm-diameter reaction to the Mantoux test and responded to M. tuberculosis PPD, Ag85, MPT64, and ESAT-6 but not to any of the leprosy antigens. We conclude that in this rural African population, exposure to M. tuberculosis or M. bovis is much less frequent than exposure to environmental mycobacteria such as M. avium , which have antigens homologous to the M. leprae 35-kDa and 18-kDa antigens. M. tuberculosis ESAT-6 showed the strongest association with the size of the Mantoux skin test induration, suggesting that among the three M. tuberculosis antigens tested it provided the best indication of exposure to, or infection with, M. tuberculosis .

Mycobacterium tuberculosisinfects humans through the lung, and immunity to this chronic infection is mediated primarily by CD4+T lymphocytes. Recently we have demonstrated that the recruitment of lymphocytes to the lung during primary aerosolM. tuberculosisinfection in mice occurs predominantly through the interaction of α4β1integrin on CD4+T cells and vascular cell adhesion molecule-1 on the pulmonary endothelium. To investigate the effect of route of immunization withMycobacterium bovisBCG on the pattern of T-cell recruitment to the lung, we have analyzed the differences in expression of integrins on activated memory CD4+T cells infiltrating the lung following primary BCG immunization by aerosol, intravenous, and subcutaneous routes and after subsequent aerosol challenge withM. tuberculosis. There were marked differences in the patterns of recruitment of activated CD4+T cells to the lung following primary immunization by the three routes. Expansion of CD44hiCD62LlowCD4+T cells in the lung occurred following aerosol and intravenous BCG immunizations, and the lymphocyte recruitment was proportional to the pulmonary bacterial load. The majority of infiltrating CD4+T cells expressed α4β1integrin. On subsequent exposure to aerosol BCG rapid expansion of gamma interferon-secreting α4β1+CD4+T cells occurred to the same extent in all immunized mice, regardless of the route of immunization. Similar expansion of α4β1+CD4+memory T cells occurred followingM. tuberculosischallenge. The three routes of BCG immunization resulted in the same level of protection against aerosolM. tuberculosisor BCG challenge in both the lungs and spleen. Therefore, recruitment of effector T lymphocytes and protective efficacy against pulmonary mycobacterial infection are independent of the route of prior BCG immunization.

The resolution of pulmonary tuberculosis (TB) critically depends on the development of the Th1 type of immune responses, as exemplified by the exacerbation of TB in IL-12-deficient mice. Therefore, vaccination strategies optimizing IL-12 production by antigen-presenting cells (APC) in response to mycobacteria may have enhanced protective efficacy. Since dendritic cells (DC) are the critical APC for activation of CD4+and CD8+T cells, we examined whether stimulation ofMycobacterium bovisbacillus Calmette Guérin (BCG)-infected DC via CD40 increased their ability to generate Th1-oriented cellular immune responses. Incubation of DC with an agonistic anti-CD40 antibody activated CD40 signaling in DC, as shown by increased expression of major histocompatibility complex class II and costimulatory molecules, mRNA production for proinflammatory cytokines and interleukin 12 (IL-12) p40. This activation pattern was maintained when DC were stimulated with anti-CD40 antibody and infected with BCG. Importantly, CD40-stimulated BCG-infected DC displayed increased capacity to release bioactive IL-12 and to activate gamma interferon (IFN-γ) producing T cells in vitro. Moreover, when C57BL/6 mice were immunized with these DC and challenged with aerosolMycobacterium tuberculosis, increased levels of mRNA for IL-12 p40, IL-18, and IFN-γ were present in the draining mediastinal lymph nodes. However, the mycobacterial burden in the lungs was not reduced compared to that in mice immunized with BCG-infected non-CD40-stimulated DC. Therefore, although the manipulation of DC via CD40 is effective for enhancing immune responses to mycobacteria in vivo, additional strategies are required to increase protection against virulentM. tuberculosisinfection.

Mycobacterium aviumis an opportunistic pathogen that primarily infects immunocompromised individuals, although the frequency ofM. aviuminfection is also increasing in the immunocompetent population. The antigen repertoire ofM. aviumvaries from that ofMycobacterium tuberculosis, with the immunodominant 35-kDa protein being present inM. aviumandMycobacterium lepraebut not in members of theM. tuberculosiscomplex. Here we show that a DNA vector encoding thisM. avium35-kDa antigen (DNA-35) induces protective immunity against virulentM. aviuminfection, and this protective effect persists over 14 weeks of infection. In C57BL/6 mice, DNA vaccines expressing the 35-kDa protein as a cytoplasmic or secreted protein, both induced strong T-cell gamma interferon (IFN-γ) and humoral immune responses. Furthermore, the antibody response was to conformational determinants, confirming that the vector-encoded protein had adopted the native conformation. DNA-35 immunization resulted in an increased activated/memory CD4+T-cell response, with an accumulation of CD4+CD44hiCD45RBloT cells and an increase in antigen-specific IFN-γ production. The protective effect of the DNA-35 vectors againstM. aviuminfection was comparable to that of vaccination withMycobacterium bovisBCG and significantly greater than that for previous treated infection withM. avium. These results illustrate the importance of the 35-kDa protein in the protective response toM. aviuminfection and indicate that DNA vaccination successfully promotes a sustained level of protection during chronicM. aviuminfection.

Although it is well established that CD4 + T cells are required for the protective immune response against tuberculosis (TB), there is some evidence that CD8 + T cells are also involved in the host response to Mycobacterium tuberculosis . There is, however, a paucity of information on the pulmonary CD8 + T-cell response during infection. We therefore have compared the changes in both CD8 + and CD4 + T cells following aerosol infection with M. tuberculosis . There was an observed delay between the peak of infection and the activated T-cell response in the lung. The kinetics of CD8 + and CD4 + T-cell responses in the lung were identical, both peaking at week 8, 4 weeks later than the peak of cellular response in draining lymph nodes. Similar changes in activation/memory phenotypes occurred on the pulmonary CD8 + and CD4 + T cells. Following in vitro restimulation, both subsets synthesized gamma interferon, a cytokine essential for controlling M. tuberculosis infection. Since lung CD8 + T cells are actively expanded during aerosol M. tuberculosis infection, it is important that both CD8 + and CD4 + T cells be targeted in the design of future TB vaccines.

The control of mycobacterial infections depends on the cytokine-mediated activation of mononuclear phagocytes to inhibit the growth of intracellular mycobacteria. Optimal activation requires the presence of T-cell-derived gamma interferon (IFN-γ) and other signals, including tumor necrosis factor (TNF). Recently, an 11-mer peptide based on amino acids 70 to 80 of the human TNF sequence, TNF (70-80) , was found to have TNF mimetic properties, which include the activation of human and mouse neutrophils to kill Plasmodia spp. Therefore, we investigated the capacity of TNF (70-80) to activate the murine macrophage cell line RAW264.7 infected with the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG). When RAW264.7 cells were pretreated with human TNF or TNF (70-80) in the presence of IFN-γ, there was a dose-dependent reduction in the replication of BCG as measured by the uptake of 3 H-labeled uracil and a concomitant release of nitric oxide as measured by the nitrite in the culture supernatants. TNF- or TNF (70-80) -induced macrophage activation was dependent on IFN-γ and was inhibited by neutralizing monoclonal antibody to human TNF and by anti-IFN-γ antisera. Both nitrite release and BCG growth inhibition were abrogated by competitive inhibitors of l -arginine, which blocked the activation of inducible nitric oxide synthase. A soluble form of the Type 1 TNF receptor blocked the activation of BCG-infected macrophages by human TNF and TNF (70-80) , demonstrating that the effect of TNF (70-80) is dependent on signaling through TNF receptor I. The mimetic effects of TNF (70-80) on macrophage activation in vitro suggest that treatment with TNF (70-80) may modulate mycobacterial infections in vivo.

The mycobacterial 70-kDa heat shock protein (Hsp70) is a dominant antigen during the human T-cell response to mycobacterial infection despite the conserved sequence with the human homolog. To determine whether this response is pathogen specific, CD4+ T-cell clones were isolated from Mycobacterium leprae Hsp70-reactive individuals. The cytokine profile of the clones was mixed, with all of the clones releasing interferon gamma and half releasing interleukin-4 on stimulation, while six demonstrated cytolytic activity. Five clones reacted with the N-terminal half of the molecule, and the epitopes identified were mycobacterium specific. Residues 241 to 260 were identified by three clones, one of which was restricted by HLA-DR7 (DR7), while a DR1-restricted clone identified residues 71 to 90 and residues 261 to 280 were recognized in the context of DR3. The remaining five T-cell clones reacted with the C-terminal half of the molecule, and the precise position of these epitopes was mapped with 12-mer peptides overlapping by 11 residues. Two of these clones identified overlapping epitopes from residues 411 to 425 and 412 to 428, the latter restricted by DR3. Further epitopes were mapped to residues 298 to 313 restricted by DRw53, residues 388 to 406 restricted by DRw52 or DQ2, and residues 471 to 486 restricted by DR1. The sequences of three epitopes, residues 411 to 425, 412 to 428, and 471 to 486, showed significant identity with the equivalent regions of the prototype human Hsp70. However, when amino acid substitutions that made the sequence more like the human sequence were introduced, the changes were tolerated poorly as measured by proliferation, cytokine production, and cytotoxic potential. Therefore, T-cell recognition of the M. leprae Hsp70 antigen occurs in the context of multiple HLA-DR phenotypes and is exquisitely species specific.

The control of leprosy will be facilitated by the identification of major Mycobacterium leprae-specific antigens which mirror the immune response to the organism across the leprosy spectrum. We have investigated the host response to a 35-kDa protein of M. leprae. Recombinant 35-kDa protein purified from Mycobacterium smegmatis resembled the native antigen in the formation of multimeric complexes and binding by monoclonal antibodies and sera from leprosy patients. These properties were not shared by two forms of 35-kDa protein purified from Escherichia coli. The M. smegmatis-derived 35-kDa protein stimulated a gamma interferon-secreting T-cell proliferative response in the majority of paucibacillary leprosy patients and healthy contacts of leprosy patients tested. Cellular responses to the protein in patients with multibacillary leprosy were weak or absent, consistent with hyporesponsiveness to M. leprae characteristic of this form of the disease. Almost all leprosy patients and contacts recognized the 35-kDa protein by either a T-cell proliferative or an immunoglobulin G antibody response, whereas few tuberculosis patients recognized the antigen. This specificity was confirmed in guinea pigs, with the 35-kDa protein eliciting strong delayed-type hypersensitivity in M. leprae-sensitized animals but not in those sensitized with Mycobacterium tuberculosis or Mycobacterium bovis BCG. Therefore, the M. leprae 35-kDa protein appears to be a major and relatively specific target of the human immune response to M. leprae and is a potential component of a diagnostic test to detect exposure to leprosy or a vaccine to combat the disease.

Among the first proteins encountered by the host immune system upon infection or vaccination with mycobacteria are those secreted by the bacillus during growth. The antigen 85 complex of Mycobacterium bovis bacillus Calmette-Gúerin (BCG) is composed of three closely related members. The mature 85B protein of M. bovis (MPB59) has a high degree of amino acid identity with the M. bovis 85A protein (76%) and the Mycobacterium tuberculosis 85B (99%) and 85A (76%) proteins. We have examined the regions of MPB59 which stimulate human T- and B-cell responses by use of a set of 28 synthetic peptides, 20 amino acids (aa) in length and overlapping by 10 aa. Initial proliferative assays with recombinant MPB59 demonstrated that peripheral blood mononuclear cells from 95% of BCG vaccinees and 52% of tuberculosis patients responded to the whole mature protein. Peripheral blood mononuclear cells from MPB59 responders, but not nonresponders, were stimulated by peptides in a dose-dependent fashion. Five peptides were reactive in more than half of the MPB59 responders. The T-cell-reactive regions were essentially identical in the M. bovis and M. tuberculosis 85B proteins. Subjects with a variety of HLA-DR phenotypes responded to a number of these peptides. The dominant T-cell-reactive regions were distinct from the peptides recognized by sera from tuberculosis patients (aa 71 to 100) and the murine monoclonal antibody HYT27 (aa 61 to 90). The region reactive with antibodies overlapped part of the MPB59 sequence recently shown to participate in the binding of MPB59 to fibronectin.

Sequential immunization with mycobacterial antigen Ag85B-expressing DNA andMycobacterium bovisbacille Calmette-Guerin (BCG) was more effective than BCG immunization in protecting againstMycobacterium tuberculosisinfection. 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.

More effective vaccines againstMycobacterium tuberculosismay contribute to the control of this major human pathogen. DNA vaccines encoding single mycobacterial proteins stimulate antimycobacterial T-cell responses and induce partial protection againstM. tuberculosisin animal models. The protective efficacy of these vaccines encoding a single antigen, however, has been less than that afforded by the current vaccine,Mycobacterium bovisbacillus Calmette-Guérin (BCG). The heterodimeric cytokine interleukin-12 (IL-12) potentiates the induction and maintenance of the type 1 helper T-cell response. We have developed a novel self-splicing vector based on the 2A protein of foot-and-mouth disease virus that permits the coordinate expression of both chains of IL-12 (p2AIL12). Coimmunization with this vector and DNA expressingM. tuberculosisantigen 85B or MPT64 enhanced the specific lymphocyte proliferative response and increased the frequency of specific gamma interferon-secreting T cells against the whole protein and a defined CD8+T-cell epitope on MPT64. Further, coimmunizing with p2AIL12 significantly increased the protective efficacy of DNA-85 in the lung against an aerosol challenge withM. tuberculosisto the level achieved with BCG. Therefore, codelivery of an IL-12-secreting plasmid may be a potent strategy for enhancing the protective efficacy of vaccines againstM. tuberculosis.