Cross-reaction between mammalian cell entry (Mce) proteins of Mycobacterium tuberculosis

In addition to the previously cloned Mce1A and Mce1E genes of the Mce1 operon of Mycobacterium tuberculosis (Ahmad et al. Scand J Immunol 1999;50:510-8), Mce1B, Mce1D and Mce1F were cloned and expressed as glutathione-S-transferase (GST) fusion proteins in recombinant Escherichia coli. Polyclonal antibodies against a predicted B-cell epitope of each of the Mce1 proteins of M. tuberculosis were produced by immunizing rabbits with synthetic peptides coupled to keyhole limpet haemocyanin. These antibodies reacted specifically with the corresponding fusion protein, except for GST-Mce1F. A mouse monoclonal antibody, TB1-5 76C, raised against a synthetic 60-mer peptide corresponding to the residues 106-165 in the N-terminal part of Mce1A, reacted strongly with GST-Mce1A. The antibody cross-reacted with GST-Mce1F, but not with the other recombinant GST-Mce1 fusion proteins or free GST. Bioinformatic analysis revealed only slight homology between Mce1A and Mce1F, along the length of the polypeptide chains. Higher homology was found between the residues 106-165 of Mce1A and the residues 347-406, further into the mature Mce1F polypeptide chain. There was a striking, localized homology, indicating that the epitope reacting with the monoclonal antibody TB1-5 76C may be narrowed to the KRRITPKD region, the residues 131-138 in Mce1A corresponding to the residues 372-379 in Mce1F. This was confirmed in enzyme-linked immunosorbent assay, showing binding of TB1-5 76C to a 17-mer synthetic peptide containing the KRRITPKD sequence.     

Identification of a Gliadin T-Cell Epitope in Coeliac Disease: General Importance of Gliadin Deamidation for Intestinal T-Cell Recognition

Coeliac disease probably results from a T-cell response to wheat gliadin and is associated to HLA-DQ2. No gliadin epitopes recognized by intestinal T cells have yet been identified, limiting our understanding of the pathogenesis. Gut-lesion-derived DQ2-restricted T cells from coeliac disease patients were used to identify an epitope within a purified γ-type gliadin. The structure of the epitope was characterized by mass spectrometry and verified by synthesis. The epitope (QPQQSFPEQQ) results from deamidation of a distinct glutamine in the native structure. This deamidation is important for binding to DQ2 and T-cell recognition. Other gut-derived T cells fail to recognize the epitope, although deamidation of unfractionated gliadin enhances the response of all gut-derived DQ2-restricted T cells isolated from several patients. Several DQ2-restricted T-cell epitopes exist, but for all of them deamidation of glutamine residues appears to be critical for creation of active epitopes. Native gliadin has few negatively charged residues but is very rich in glutamine. After deamidation gliadin becomes a rich source of DQ2 epitopes thus providing a link between DQ2, gliadin and coeliac disease. The necessity for modification may have general immunological relevance.     

Differential T-Cell Recognition of Native and Recombinant Mycobacterium tuberculosis GroES

Mycobacterium tuberculosis GroES was purified from culture filtrate, and its identity was confirmed by immunoblot analysis and N-terminal sequencing. Comparing the immunological recognition of native and recombinant GroES, we found that whereas native GroES elicited a strong proliferative response and release of gamma interferon-gamma by peripheral blood mononuclear cells from healthy tuberculin reactors, the recombinant protein failed to do so. The same difference in immunological recognition was observed in a mouse model of TB infection. Both the native and recombinant preparations were recognized by mice immunized with the recombinant protein. Biochemical characterization including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional electrophoresis, and mass spectrometry analysis of both proteins demonstrated no differences between the native and recombinant forms of GroES except for the eight additional N-terminal amino acids derived from the fusion partner in recombinant GroES. The recombinant fusion protein, still tagged with the maltose binding protein, was recognized by T cells isolated from TB-infected mice if mixed with culture filtrate before affinity purification on an amylose column. The maltose binding protein treated in the same manner as a control preparation was not recognized. Based on the data presented, we suggest that the association of biologically active molecules from culture filtrate with the chaperone GroES may be responsible for the observed T-cell recognition of the native preparation.     

Identification of New Cytotoxic T-Cell Epitopes on the 38-Kilodalton Lipoglycoprotein of Mycobacterium tuberculosis by Using Lipopeptides

Induction of cytotoxic T lymphocytes (CTLs) by vaccination has been shown to protect against bacterial, viral, and tumoral challenge. The aim of this study was to identify CTL epitopes on the 38-kDa lipoglycoprotein from Mycobacterium tuberculosis. The identification of these CTL epitopes was based on synthesizing peptides designed from the 38-kDa lipoglycoprotein, with known major histocompatibility complex class I (MHC-I) binding motifs (H-2D^b), and studying their ability to up-regulate and stabilize MHC-I molecules on the mouse lymphoma cell line RMA-S. To improve the capacity of the identified peptides to induce CTL responses in mice, palmitic acid with a cysteine-serine-serine spacer amino acid sequence was attached to the amino terminus of the peptide. Two of five peptides with H-2D^b binding motifs and their corresponding lipopeptides up-regulated and stabilized the H-2D^b molecules on RMA-S cells. Both lipopeptides, in combination with incomplete Freund’s adjuvant, induced CTL responses in C57BL/6 (H-2^b) mice. Moreover, the lipopeptide induced stronger CTL responses than the peptide. The capacity of the various lipopeptides to induce CTL displayed a good relationship with the ability of the (lipo)peptide to up-regulate and to stabilize H-2D^b molecules. The capacity of the peptides and lipopeptides to up-regulate and stabilize MHC-I expression can therefore be used to predict their potential to function as a CTL epitope. The newly identified CTL epitopes and their lipid derivatives provide us with important information for future M. tuberculosis vaccine design.Mycobacterium tuberculosis is a facultatively intracellular bacterium that causes tuberculosis (TB). Between 1985 and 1991, TB increased 33% in Switzerland, 30% in Denmark, 20% in Norway, and 18% in both Ireland and the United States. Presently, TB affects 1.7 billion people worldwide. With 55 million cases of active disease, there are 8 million new cases and around 3 million deaths per year (2, 14). This situation, mainly in the developing countries, is directly related to a “weakness” of the immune system caused by human immunodeficiency virus infection, cancer, and the application of immunosuppressive drugs in chemotherapy (6). The situation is further complicated by the emergence of multi-drug-resistant strains (2, 6).Mycobacterium bovis BCG is the currently used vaccine against tuberculosis, but its efficacy varies widely, from 0 to 90% (15). BCG mainly activates CD4⁺ T cells, but it fails to activate CD8⁺ T cells (14). Cytotoxicity mediated by CD4⁺ cells, however, is probably not sufficient to eradicate the mycobacterium, which has an excellent capacity to survive intracellularly. Several studies have demonstrated the importance of CD8⁺ cytotoxic T lymphocytes (CTLs) against M. tuberculosis infection in mouse (CD8 knockout) models (11) and humans (12, 19, 22). However, only a few studies have described the mycobacterial antigens and the epitopes recognized by CD8⁺ CTLs (4, 31, 32, 38). Induction of cellular immunity, covering both CD4⁺ and CD8⁺ T-cell activation, may therefore be required for providing protection against M. tuberculosis (13).For the identification of CTL epitopes on viral, bacterial, and tumoral proteins, different strategies can be followed. One approach used is immunization with the whole protein to generate CTL responses or CTL clones and the subsequent mapping of the epitope by overlapping synthetic peptides (23). An alternative approach is to select 8- to 11-amino-acid (aa)-long peptides based on known major histocompatibility complex class I (MHC-I) binding motifs. These peptides are then studied for their ability to up-regulate and stabilize MHC-I expression (44). The advantage of this approach is that it limits the number of peptides to be synthesized and to be tested in animal experiments.The 38-kDa lipoglycoprotein, one of the better-studied antigens of M. tuberculosis, is actively secreted but is also partly attached to the surface of mycobacteria by a lipid tail (43). The protein induces strong antibody and T-cell responses and provides partial protection against M. tuberculosis infection in mice when it is administered either entrapped in biodegradable microparticles or in the form of a DNA vaccine (37, 46). Epitopes on the 38-kDa lipoglycoprotein have been recognized by CD4⁺ T cells and studied extensively with both mice (34, 36) and humans (35, 42). Recently, a number of epitopes recognized by CD8⁺ T cells (CTL epitopes), with an H-2^b restriction, have also been described (38, 46, 47).This report describes the identification of CTL epitopes with an H-2D^b restriction by synthesizing peptides with MHC-I binding motifs and studying their ability to up-regulate and stabilize MHC-I molecules. Positive peptides in these assays do not necessarily induce cytotoxic T-cell responses, even when they are injected with incomplete Freund’s adjuvant (IFA) (3, 9, 10, 38). The immunogenicity of these peptides, however, can be strongly improved by attachment of lipid tails, preferably with a spacer amino acid sequence between peptide and lipid (5, 18, 20, 26, 33). Previously, it has been demonstrated that the cysteine-serine-serine (CSS) spacer amino acid sequence was required for the efficient induction of CTLs against malaria epitopes (33). Therefore, palmitic acid with a CSS spacer amino acid sequence was linked to the amino terminus of the peptide, and mice were immunized with these lipopeptides in combination with IFA. Two new H-2D^b CTL epitopes were identified on the 38-kDa mycobacterial lipoglycoprotein, which increases our knowledge for future M. tuberculosis vaccine design. The relationship between the ability to up-regulate and stabilize MHC-I molecules and the capability to induce CTLs by peptides and lipopeptides will be discussed.     

Proteins released from Mycobacterium tuberculosis during growth.

Proteins secreted from Mycobacterium tuberculosis during growth are believed to be important for protective immunity against tuberculosis. We have investigated the growth of M. tuberculosis in an enriched liquid medium. The release of isocitrate dehydrogenase from the bacilli served as a marker of autolysis and was observed during the late logarithmic growth phase. The release of proteins during the culture period was investigated by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three major groups of proteins, which differed markedly with respect to profile of release and location in intact bacilli, were defined. A short-term filtrate devoid of autolytic products was defined and found to be composed of 33 major components. Five proteins were identified by monoclonal antibodies. Pronounced superoxide dismutase activity was detected in the filtrate. The enzyme was purified and identified as a dominating component of short-term filtrate.     

The PE-PPE Family of Mycobacterium tuberculosis: Proteins in Disguise

Mycobacterium tuberculosis has thrived in parallel with humans for millennia, and despite our efforts, M. tuberculosis continues to plague us, currently infecting a third of the world’s population. The success of M. tuberculosis has recently been attributed, in part, to the PE-PPE family; a unique collection of 168 proteins fundamentally involved in the pathogenesis of M. tuberculosis. The PE-PPE family proteins have been at the forefront of intense research efforts since their discovery in 1998 and whilst our knowledge and understanding has significantly advanced over the last two decades, many important questions remain to be elucidated.This review consolidates and examines the vast body of existing literature regarding the PE-PPE family proteins, with respect to the latest developments in elucidating their evolution, structure, subcellular localisation, function, and immunogenicity. This review also highlights significant inconsistencies and contradictions within the field. Additionally, possible explanations for these knowledge gaps are explored. Lastly, this review poses many important questions, which need to be addressed to complete our understanding of the PE-PPE family, as well as highlighting the challenges associated with studying this enigmatic family of proteins.Further research into the PE-PPE family, together with technological advancements in genomics and proteomics, will undoubtedly improve our understanding of the pathogenesis of M. tuberculosis, as well as identify key targets/candidates for the development of novel drugs, diagnostics, and vaccines.     

Human T-Cell Epitopes on the Mycobacterium tuberculosis Secreted Protein MPT64

Mycobacterium tuberculosis secretes a number of proteins into the extracellular envirornment, some of which are restricted to the M. tuberculosis complex. These proteins are targets for T- and B-cell immune responses in tuberculosis (TB) patients and their contacts. The authors have mapped the immunogenic regions of the MPT64 protein of M. tuberculosis using peripheral blood mononuclear cells (PBMC) from TB patients and a set of overlapping peptides encompassing the complete sequence of the protein. T-cell epitopes which induced proliferation or interferon-gamma (IFN-γ) release were distributed over the full length of the protein. A C-terminal region of the protein, however, contains sequences recognized in the context of multiple HLA-DR phenotypes by more than 80% of the subjects tested. The nature of the T-cell response was further investigated by generating MPT64-specific T-cell lines. These lines also identified the T-cell epitopes in the C-terminal region of the protein. On stimulation with antigen the lines secreted IFN-γ, but not interleukin 4 (IL-4). A minority of TB patients (6/32) mounted an antibody response to MPT64. Sera from half (3/6) of these identified two linear antibody binding sites. These results confirm the significance of this protein in the immune response to tuberculosis infection.     

A Family of Cross-Reacting Proteins Secreted by Mycobacterium tuberculosis

Cross-reactions between five proteins actively secreted by Mycobacterium tuberculosis were studied by crossed immunoelectrophoresis, SDS-PAGE with immunoblotting, and ELISA using polyclonal rabbit antisera and mouse monoclonal antibodies to the purified proteins. The monoclonal antibody HBT4 was demonstrated to react with the MPT51 protein. The 85A, 85B and 85C constituents of the M. tuberculosis and Mycobacterium bovis BCG antigen 85 complex cross-react extensively, each of the components containing component-specific as well as cross-reacting epitopes. These components also cross-reacted with MPT51 and MPT64. N-terminal sequence studies revealed striking homology at the amino acid level between 85A, 85B, 85C and MPT51. MPT64 showed less homology. In addition, striking homology was demonstrated between two different stretches within the 85B sequence and indicated between three stretches within the MPT64 molecule. Thus, a family of at least four secreted proteins with common structural features has been demonstrated in mycobacteria. MPT64 may also belong to this family.     

Immunodominant B-cell epitope in the Mce1A mammalian cell entry protein of Mycobacterium tuberculosis cross-reacting with glutathione S-transferase

The TB1-5 76C monoclonal antibody raised against a synthetic 60-mer peptide in the N-terminal part of the Mce1A mammalian cell entry protein of Mycobacterium tuberculosis has previously been shown to react with a linear epitope in the KRRITPKD region, residues 131-138 in Mce1A, and to cross-react with Mce1F. Six additional monoclonal antibodies raised against the same peptide were also shown to cross-react with Mce1F. Four of them reacted with a linear epitope in the same area, indicating that this area is immunodominant but showed distinct differrences in fine specificity. Two monoclonal antibodies did not react with synthetic peptides from this region on the solid phase in enzyme-linked immunosorbent assay, indicating greater influence of conformation on reactivity. None of the monoclonal antibodies reacted with 14-mer synthetic peptides from the corresponding area in Mce2A, Mce3A, Mce4A, M. avium, M. smegmatis or M. leprae. The reaction pattern of the monoclonal antibodies was analysed in relation to our model of the Mce1A molecule (AK Das et al. Biochem Biophys Res Commun 2003;302:442-7).The epitope is located on the surface of Mce1A, at the distal beta-strand-loop region in the beta-domain supporting its potential role in promoting uptake of M. tuberculosis in host cells. Monoclonal antibody TB1-5 19C cross-reacted with glutathione S-transferase of Schistosoma japonicum containing a PKE triplet. Monoclonal antibody TB1-5 76C gave a major band at about 44 kDa in Western blotting of M. tuberculosis sonicate, whereas polyclonal rabbit anti-Mce1A peptide antibodies reacting with the extended TTPKNPTKRRITPKDVI area of Mce1A showed a distinct band above the 160 kDa molecular mass standard.     

New Alternative Vaccine Component Against Mycobacterium Tuberculosis– Heat Shock Protein 16.3 or its T-Cell Epitope

Heat shock protein 16.3 (Hsp16.3) of Mycobacterium tuberculosis (MTB) containing T-cell and B-cell epitopes not only plays an important role in the survival of MTB against macrophages, but also has great potential to be used to develop new TB vaccines. In order to study whether Hsp16.3 can be replaced with its T-cell epitope for producing a vaccine against TB, we expressed and purified Hsp16.3 protein of MTB H37Rv strain and confirmed by immunoblotting. The immune responses and protection against the H37Rv induced by Hsp16.3 protein were compared with its T-cell epitope synthetic peptide in mice. The results showed that both Hsp16.3 and its synthetic peptide induced significantly stronger specific antibodies than the classical TB vaccine–BCG (bacillus Calmette-Guérin). Compared with BCG, the stimulation index in the splenolymphocyte proliferation of both recombinant protein and its synthetic peptide were remarkably enhanced, but the levels of IFN-γ release were lower. Dramatic reduction in the numbers of MTB colony forming units (CFU) in the spleens and lungs was observed in the mice immunized with Hsp16.3 or its synthetic peptide. The protection provided by Hsp16.3 or its synthetic peptide in the lungs was equivalent to that provided by BCG. Both Hsp16.3 and its T-cell epitope are effective components and Hsp16.3 can be replaced with its T-cell epitope while developing the vaccine against TB, without requiring the complicated procedure of expressing and purifying Hsp16.3.     

Characterization of the Delayed Type Hypersensitivity-Inducing Epitope of MPT64 from Mycobacterium tuberculosis

Mycobacterium tuberculosis secretes several proteins into the extracellular environment, some of which are restricted to the M. tuberculosis complex. One of these antigens is MPT64. Recently, the authors showed that native as well as recombinant MPT64 is able to distinguish between an M. tuberculosis infection and a BCG Danish 1331 vaccination. Improved distinction between tuberculin purified protein derivative (PPD) sensitivity conferred by an M. tuberculosis infection and that induced by a BCG vaccination or infection with environmental mycobacteria would be useful in the control of tuberculosis. In this study, the authors report the mapping and characterization of a Dth-inducing epitope by the use of synthetic peptides in guinea-pigs vaccinated with BCG Danish 1331 or Tokyo. Studies with overlapping synthetic peptides have pinpointed the biological activity to a single Dth-inducing epitope at the carboxyterminal region of MPT64 consisting of 15 residues between amino acids Gly-173 and Ala-187, the core epitope (CE15). A fine mapping using truncated versions of CE15 indicates the epitope is restricted to 13 residues between amino acids Val-174 to Glu-186. However, the optimal Dth reactivity is obtained by CE15. Different modifications of CE15 revealed that a lysine tree construction improves the skin reactivity to a maximum level approaching that of the reactivity to tuberculin PPD.     

Identification of Amino Acid Residues of the T-Cell Epitope of Mycobacterium tuberculosis α Antigen Critical for Vβ11+ Th1 Cells

Stimulation of Mycobacterium tuberculosis-primed lymph node cells from C57BL/6 mice with alpha antigen (also known as antigen 85B and MPT59) induced cell proliferation, production of interleukin 2 and gamma interferon, and expansion of Vbeta11(+) CD4(+) T cells in conjunction with antigen-presenting cells in an I-A(b)-restricted manner. Using a series of 15-amino-acid peptides that overlapped each other by 5 amino acids and spanned the mature alpha antigen, we identified the antigenic epitope for alpha antigen-specific Vbeta11(+) Th1 cells. That peptide (peptide-25), which corresponds to amino acid residues 240 to 254 of alpha antigen, contains a motif that is conserved in I-A(b) and requires processing by antigen-presenting cells. Using peptide-25-reactive Vbeta11(+) T-cell clones and substituted peptide-25 mutants, we determined which amino acid residues within peptide-25 were critical for T-cell receptor (TCR) recognition. Our results showed that the amino acid residues at positions 245, 246, 248, 250, and 251 are important for recognition of TCRVbeta11 and that residues at positions 244, 247, 249, and 252 are I-A(b) contact residues. We also observed that active immunization of C57BL/6 mice with peptide-25 can lead to decreased bacterial load in the lungs of M. tuberculosis H37Rv-infected mice. These results should provide us with a useful tool for delineating the regulation of Vbeta11(+) Th1-cell development during M. tuberculosis infection and for developing a vaccine inducing a Th1-dominant immune response.     

分析5种结核杆菌耐药基因突变与耐药水平的关系

目的：分析5种结核杆菌（M.tb)耐药基因突变的情况，了解基因突变和耐药水平的关系。方法：134例临床分离株均做传统梯度药敏试验和聚合酶链反应－单链构象多态性I（PCR－SSCP）试验。结果：耐PZA(pncA),SM(rpsL),REP(rpoB),INH(katG),EMB(embB)基因突变率分别为42．7％、72％、78％、69％和43．9％，其中，上述高耐株基因突变率分别为70％、87．2％、93．4％、80％、43．9％。低耐株分别为12．5％、28．5％、45．4％、18．7％，EMB在低耐区无基因突变，结论：M.tb耐药基因变与耐药水平联系密切，多数M.tb耐药基因突变易发生在高耐药区，也有少数菌基因突变易发生在低耐药区。     

Identification of secreted proteins of Mycobacterium tuberculosis by a bioinformatic approach

Proteins secreted by Mycobacterium tuberculosis are usually targets of immune responses in the infected host. Here we describe a search for secreted proteins that combined the use of bioinformatics and phoA' fusion technology. The 3,924 proteins deduced from the M. tuberculosis genome were analyzed with several computer programs. We identified 52 proteins carrying an NH(2)-terminal secretory signal peptide but lacking additional membrane-anchoring moieties. Of these 52 proteins-the TM1 subgroup-only 7 had been previously reported to be secreted proteins. Our predictions were confirmed in 9 of 10 TM1 genes that were fused to Escherichia coli phoA', a marker of subcellular localization. These findings demonstrate that the systematic computer search described in this work identified secreted proteins of M. tuberculosis with high efficiency and 90% accuracy.     

Molecular Characterization and Human T-Cell Responses to a Member of a Novel Mycobacterium tuberculosis mtb39 Gene Family

We have used expression screening of a genomic Mycobacterium tuberculosis library with tuberculosis (TB) patient sera to identify novel genes that may be used diagnostically or in the development of a TB vaccine. Using this strategy, we have cloned a novel gene, termed mtb39a, that encodes a 39-kDa protein. Molecular characterization revealed that mtb39a is a member of a family of three highly related genes that are conserved among strains of M. tuberculosis and Mycobacterium bovis BCG but not in other mycobacterial species tested. Immunoblot analysis demonstrated the presence of Mtb39A in M. tuberculosis lysate but not in culture filtrate proteins (CFP), indicating that it is not a secreted antigen. This conclusion is strengthened by the observation that a human T-cell clone specific for purified recombinant Mtb39A protein recognized autologous dendritic cells infected with TB or pulsed with purified protein derivative (PPD) but did not respond to M. tuberculosis CFP. Purified recombinant Mtb39A elicited strong T-cell proliferative and gamma interferon responses in peripheral blood mononuclear cells from 9 of 12 PPD-positive individuals tested, and overlapping peptides were used to identify a minimum of 10 distinct T-cell epitopes. Additionally, mice immunized with mtb39a DNA have shown increased protection from M. tuberculosis challenge, as indicated by a reduction of bacterial load. The human T-cell responses and initial animal studies provide support for further evaluation of this antigen as a possible component of a subunit vaccine for M.tuberculosis.     

T-Cell Recognition of Mycobacterium tuberculosis Culture Filtrate Fractions in Tuberculosis Patients and Their Household Contacts

We examined the immune responses of patients with active pulmonary tuberculosis (TB) and their healthy household contacts to short-term culture filtrate (ST-CF) of Mycobacterium tuberculosis or molecular mass fractions derived from it. Our goal was to identify fractions strongly recognized by donors and differences among the donor groups of possible relevance for vaccine development. The study population consisted of 65 human immunodeficiency virus-negative donors from the Hossana Regional Hospital, Hossana, Ethiopia. Peripheral blood leukocytes from the donors were stimulated with different antigens and immune responses were determined. Household contacts produced significantly higher levels of gamma interferon (IFN-gamma) than the TB patients in response to antigens present in ST-CF and the 10 narrow-molecular-mass fractions. A similar difference in leukocyte proliferative responses to the antigens between the two groups was also found. In general, while all fractions stimulated immune responses, the highest activity was seen with the low-molecular-mass fractions, which include well-defined TB antigens such as ESAT-6. Leukocytes from contacts of TB patients with severe disease produced higher levels of antigen-specific IFN-gamma than those from contacts of patients with minimal disease. Both groups of contacts exhibited higher cell-mediated responses than the patients themselves. The enhanced immune response of healthy contacts, especially those of patients with severe disease, to secreted mycobacterial antigens is suggestive of an early stage of infection by M. tuberculosis, which could in time result in overt disease or containment of the infection. This possibility is currently being investigated by follow-up studies of the household contacts.     

Identification of a Novel 27-kDa Protein from Mycobacterium tuberculosis Culture Fluid by a Monoclonal Antibody Specific for the Mycobacterium tuberculosis Complex

Mycobacterium tuberculosis antigens inducing species-specific immune responses are likely to be particularly important for serodiagnosis or for skin testing of tuberculosis. In the present study, we describe the characterization of two novel monoclonal antibodies (MoAbs) A3h4 (IgG2a) and B5gl (IgM) that are directed to M. tuberculosis 27-kDa and 25-kDa proteins respectively. Specificity analysis by immunoblotting using 20 different species of mycobacterial sonicates revealed that MoAb A3h4 was specific for M. tuberculosis complex alone while MoAb B5gl showed a limited cross-reactivity. Direct comparison with previously characterized MoAbs revealed that these MoAbs A3h4 and B5gl defined new antigenic determinants of M. tuberculosis. By using M. tuberculosis complex-specific MoAb A3h4 we have identified a distinct 27-kDa protein in the M. tuberculosis H37Rv culture fluid. Since this MoAb did not bind to the previously characterized MPT44, MPT59, MPT45, MPT51 and MPT64 proteins as well as the 23-kDa superoxide dismutase (SOD) protein of M. tuberculosis, we conclude that MoAb A3h4 recognizes a novel protein in the M. fuberculosis H37Rv culture fluid. Studies of the subccllular distribution of these MoAb-reaetive proteins indicate that the MoAb A3h4-reactive 27-kDa protein is present not only in the culture fluid bnt also in the cytosol and the cell wall of M. tuberculosis. By contrast. B5gl-reactive protein is mainly a cytosolic protein. When these MoAbs were tested in a previously established ELISA with intact mycobacteria derived from early cultures, only MoAb A3h4 showed the positive reactivity to mycobacteria belonging to the M. tuberculosis complex. In addition, during the present comparative studies of MoAbs we have also found that the previously described MoAb F116-5, which is known to recognize the mycobacterial 23-kDa SOD protein [17], cross-reacted with the MPT44, MPT59, MPT45 and MPT51 secreted proteins but not with MPT64 and MPB70. These findings indicate that the family of four secreted proteins of M. tuberculosis share a common epitope with M. tuberculosis SOD protein.     

Identification of Mycobacterium tuberculosis ornithine carboamyltransferase in urine as a possible molecular marker of active pulmonary tuberculosis

Although the antigen detection assay has the potential to discriminate active tuberculosis from latent infection, development of such a test for the accurate diagnosis of this serious disease has only recently become a matter of interest. Here we present evidence that a Mycobacterium tuberculosis protein (ornithine carboamyltransferase, coded for by MT_1694; Rv1656 [argF]) is an interesting candidate molecule for this test development. The protein was initially discovered by mass spectroscopy in urine of patients with pulmonary tuberculosis and shown by Western blot analysis to be present in M. tuberculosis crude cell extract as well as in the culture supernatant ("secreted" protein). In addition, a recombinant ornithine carboamyltransferase (rMT1694) produced in Escherichia coli was recognized by immunoglobulin G (IgG) antibodies from patients with active tuberculosis but not by IgG from uninfected healthy subjects. Moreover, rMT1694 was strongly recognized by peripheral blood mononuclear cells from both healthy tuberculin purified protein derivative (PPD)-positive individuals and patients with pulmonary tuberculosis. More importantly, a capture enzyme-linked immunosorbent assay formatted with rabbit IgG antibodies specific to rMT1694 was able to identify the presence of this antigen in urine samples from 6 of 16 patients with pulmonary tuberculosis and in none of 16 urine samples collected from healthy PPD(+) controls. These results indicate that an improved antigen detection assay based on M. tuberculosis ornithine carboamyltransferase may represent an important new strategy for the development of a specific and accurate diagnostic test for tuberculosis.