Antigens in culture supernatant of Mycobacterium tuberculosis: epitopes defined by monoclonal and human antibodies.

Antigens of Mycobacterium tuberculosis found in the supernatant of heat-treated cultures were characterized in order to explore whether antigens from this source could be used for the development of a serological test. Culture supernatants and sonicates of 12, 25 and 39 d cultures were analysed by SDS-PAGE. In culture supernatant, major protein bands of 65, 24, and 12 kDa were visible after Coomassie brilliant blue staining. Using murine monoclonal antibodies in Western blots, a pattern of protein bands distinct from that of the corresponding M. tuberculosis sonicates was found in all the culture supernatants. Gel permeation chromatography, in the presence of SDS, was used to separate the major protein bands in the culture supernatant. In ELISA, sera from 20 of 26 patients with tuberculosis reacted with fractions containing mainly 24 kDa or 12 kDa proteins, whereas none of the control sera reacted. In Western blots, each patient serum had its own characteristic banding pattern with culture supernatant, but all the sera from tuberculosis patients and control subjects reacted with protein bands of 65, 61, 58, 30 and 24 kDa. The 12 kDa protein was recognized only by sera from patients with tuberculosis in both Western blots and ELISA. This suggests that different kinds of epitopes on proteins of M. tuberculosis are detected by human antibodies in Western blots and ELISA. We assume that epitopes recognized in Western blots by patients with tuberculosis and control subjects are ubiquitous and are also present on normal commensal bacteria. Epitopes recognized by only some patients with tuberculosis in Western blots may be linear and M. tuberculosis specific. Epitopes recognized by tuberculosis patients but by none of the control subjects in ELISA may be conformation related and M. tuberculosis specific. The major protein bands found in supernatants of heat-treated cultures, 24 and 12 kDa, possess epitopes that may be M. tuberculosis specific and are potentially valuable for the development of a serological test.     

Identification of early secretory antigen target-6 epitopes for the immunodiagnosis of active tuberculosis

The early secretory antigenic target (ESAT)-6 purified protein and peptides from Mycobacterium tuberculosis were evaluated as antigens for the immunodiagnosis of tuberculosis (TB). Because the control of TB requires improved diagnostic procedures, efforts have increased to identify Mycobacterium tuberculosis-specific epitopes for the immunodiagnosis of active TB and to discriminate between active and latent states of infection. Two multiepitopic peptides from ESAT-6 protein were selected by computational analysis. Patients with active TB (7 HIV(+) and 20 HIV(-)) and control patients (17 HIV(+) and 28 HIV(-)) were enrolled. Enzyme-linked immunospot assay analysis for interferon-g expression by peripheral blood mononuclear cells was quantified after stimulation with selected ESAT-6 peptides, purified protein derivative, or the intact ESAT-6 protein. During active TB, 20 of 27 patients responded in vitro to ESAT-6 peptides and 23 of 27 patients to purified protein derivative. None of the controls without active TB, including individuals with latent TB infection, recognized ESAT-6 peptides. By contrast, latently infected individuals did respond in vitro to both intact ESAT-6 protein and purified protein derivative. Thus, high T-cell response frequencies to ESAT-6 peptides are present only during active TB and can be used to discriminate between active and latent forms of infection.     

Human T-cell responses to 25 novel antigens encoded by genes of the dormancy regulon of Mycobacterium tuberculosis

The dormancy (DosR) regulon of Mycobacterium tuberculosis is expressed in vitro during hypoxia and low-dose nitric oxide stimulation. Tubercle bacilli are thought to encounter these conditions in humans during latent infection. In this study, immune responses were evaluated to 25 most strongly induced DosR-regulon-encoded proteins, referred to as latency antigens. Proliferation assays were performed using M. tuberculosis-specific T-cell lines and peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients, tuberculin skin test positive (TST+) individuals and uninfected controls. All 25 latency antigens were able to induce production of interferon-gamma (IFN-gamma) by T-cell lines. Eighteen latency antigens were also recognized by PBMC of M. tuberculosis-infected individuals, which indicates expression of the DosR-regulon during natural infection. Differential analysis showed that TST+ individuals recognized more latency antigens and with a stronger cumulative IFN-gamma response than TB patients, while the opposite profile was found for culture filtrate protein-10. In particular Rv1733c, Rv2029c, Rv2627c and Rv2628 induced strong IFN-gamma responses in TST+ individuals, with 61%, 61%, 52% and 35% responders, respectively. In conclusion, several new M. tuberculosis antigens were identified within the DosR-regulon. Particularly strong IFN-gamma responses to latency antigens were observed in latently infected individuals, suggesting that immune responses against these antigens may contribute to controlling latent M. tuberculosis infection.     

Isolation of a 19-kDa mycobacterium,bovis-specific antigen,different from MPB70/80, by chromatofocusing

Two antigens, 19-kDa each, were purified from Mycobacterium bovis culture filtrate protein extract by chromatofocusing. Antigen I had a 4.5 pI, and its amino terminal (DPVDAVINTTCNYGQVVAALNATDP) showed a 100% homology with the hypothetical protein Rv1174c. Antigen II had a pI of 6.0 pI and its amino terminal (GDLVGPGCAEYAAANPTGPASVQGM) showed a 100% homology with M. bovis MPB70/80. Antigen I is a hetero-dimer formed by a glycosylated, 10.5-kDa, monomer and a non-glycosylated 8-kDa monomer with identical amino terminal sequences. Both antigens were recognized by the sera of PPD+ animals, but antigen I did not crossreact with sera of human PPD+ individuals. Antigen I was a weak inducer of lymphocyte proliferation and IFN-gamma production. Our results show that M. bovis expresses a 19 kDa glycoprotein, homologue to the product of M. tuberculosis gen Rv1174c, which may prove useful for bovine TB diagnostic assays.     

Restoration of mycobacterial antigen-induced proliferation and interferon-gamma responses in peripheral blood mononuclear cells of tuberculosis patients upon effective chemotherapy

Peripheral blood mononuclear cells (PBMC) were obtained from culture-proven tuberculosis (TB) patients before and after 2 and 6 months of chemotherapy with a multi-drug regimen. PBMC were tested for cellular responses in antigen-induced proliferation and interferon-gamma (IFN-gamma) assays in response to complex mycobacterial antigens (whole cell Mycobacterium bovis BCG and M. tuberculosis, cell walls and short-term culture filtrate [ST-CF] of M. tuberculosis), fractionated ST-CF antigens (fractions F1-F10) and ESAT-6. The responses in TB patients before anti-TB treatment were low (median stimulation index (SI)=1-7, median delta IFN-gamma=0-12 U ml(-1), and percent responders=13-67%) to all the antigenic preparations. Following the administration of anti-TB chemotherapy for 2 months, there were significant (P<0.05) improvements in the cellular responses (median SI=9-76, median delta IFN-gamma=3-70 U ml(-1), and percent responders=33-100%) to most of the antigenic preparations tested. However, concanavalin A-induced proliferation responses of PBMC from the same patients before and after 2 months of chemotherapy were high and comparable (median SI=101 and 114, respectively, P>0.05, 100% responders). A further increase in IFN-gamma responses (median delta IFN-gamma=14-250 U ml(-1) and percent responders=43-100%) to mycobacterial antigens was observed in patients receiving chemotherapy for 6 months. Among the ST-CF fractions, F1 and F2 containing low molecular mass proteins resulted in the highest responses, whereas ESAT-6 showed responses comparable to these fractions only in a minority of the patients. HLA-DR typing of these patients showed heterogeneity in the expression of molecules encoded by HLA-DRB genes. These results show that effective chemotherapy restores cellular responses of TB patients to a large number of M. tuberculosis antigens, which could be useful in monitoring the efficacy of anti-TB treatment.     

Immunoproteomic Identification of Human T Cell Antigens of Mycobacterium tuberculosis That Differentiate Healthy Contacts from Tuberculosis Patients

Identification of Mycobacterium tuberculosis antigens inducing cellular immune responses is required to improve the diagnosis of and vaccine development against tuberculosis. To identify the antigens of M. tuberculosis that differentiated between tuberculosis (TB) patients and healthy contacts based on T cell reactivity, the culture filtrate of in vitro grown M. tuberculosis was fractionated by two-dimensional liquid phase electrophoresis and tested for the ability to stimulate T cells in a whole blood assay. This approach separated the culture filtrate into 350 fractions with sufficient protein quantity (at least 200 μg of protein) for mass spectrometry and immunological analyses. High levels of interferon-γ (IFN-γ) secretion were induced by 105 fractions in healthy contacts compared with TB patients (p < 0.05). Most interesting was the identification of 10 fractions that specifically induced strong IFN-γ production in the healthy contact population but not in TB patients. Other immunological measurements showed 42 fractions that induced significant lymphocyte proliferative responses in the healthy contact group compared with the TB patients. The tumor necrosis factor-α response for most of the fractions did not significantly differ in the tested groups, and the interleukin-4 response was below the detectable range for all fractions and both study groups. Proteomic characterization of the 105 fractions that induced a significant IFN-γ response in the healthy contacts compared with the TB patients led to the identification of 59 proteins of which 24 represented potentially novel T cell antigens. Likewise, the protein identification in the 10 healthy “contact-specific fractions” revealed 16 proteins that are key candidates as vaccine or diagnostic targets.Tuberculosis (TB)1 is a major health problem throughout the world. A recent World Health Organization report shows that TB has been increasing at a rate of 1% per year, and an estimated 9.2 million new cases arise each year (1). Although TB is preventable, there has been an increase in its incidence in recent years. Re-emergence of TB is mainly due to its association with human immunodeficiency virus infection (2) and also due to the occurrence of multidrug-resistant strains of the causative agent, Mycobacterium tuberculosis (3).Vaccination of general population is cost effective and represents one of the best biological measures for disease control. The current vaccine against tuberculosis, Bacille Calmette-Guérin (BCG), has been administered to more people than any other vaccine. The side effects of BCG are tolerable, and it prevents miliary and meningeal tuberculosis in young children. In striking contrast, it affords limited and highly variable protection (0–80%) against pulmonary TB (4). Thus, BCG does not seem to be a satisfactory vaccine (5, 6) and necessitates exploration of newer strategies to improve BCG or to develop a more effective vaccine.One of the potential strategies for the development of an improved TB vaccine involves the use of the proteins secreted by M. tuberculosis during growth. There is evidence that proteins actively secreted by M. tuberculosis during growth induce cell-mediated immune responses by causing expansion of specific interferon-γ (IFN-γ)-producing T lymphocytes that are capable of recognizing and exerting antimicrobial effects against infected macrophages (7). The importance of IFN-γ pathways in host defense against M. tuberculosis was clarified by experimental studies on IFN-γ knock-out mice as well as the identification and characterization of humans with mutations in IFN-γ receptor (8, 9).Several studies have been carried out to define the secreted proteome of M. tuberculosis. The earliest study aimed at the identification of mycobacterial culture filtrate proteins, using chromatography and N-terminal sequencing to identify eight culture filtrate proteins (10). Later, many studies used two-dimensional (2D) PAGE combined with sensitive mass spectrometric methods for identification of proteins. The above mentioned approaches have identified nearly 300 culture filtrate proteins (11–13).Identification of T cell antigens in a complex mixture was first done by a T cell Western blot method (14). Later, two-dimensional separation methods were used that involved protein separation by either IEF (15) or chromatography (16) in the first dimension and preparative SDS-PAGE followed by whole gel elution (17) in the second dimension. Mouse T cell antigens of M. tuberculosis were identified using this method (15). Mycobacterial antigens that induce an immune response in healthy household contacts and treated TB patients were also mapped using this approach (16).In the present study, 2D liquid phase electrophoresis (LPE) along with an in vitro IFN-γ assay and LC-MS/MS were used to identify potential human T cell antigens. Systematic screening of the M. tuberculosis culture filtrate (CF) proteome and comparative evaluation of cellular immune responses between TB patients and healthy contacts led to the identification of 59 proteins in the most immunogenic 2D LPE fractions. Twenty-four potentially novel T cell antigens were identified, and 16 proteins were identified in 10 2D LPE fractions that differentiated healthy contacts from TB patients based on IFN-γ responses.     

结核分枝杆菌可视化抗体检测蛋白芯片的制备

目的：利用克隆表达的7种结核分枝杆菌优势表位抗原,建立可视化抗体检测蛋白芯片,用于结核病辅助诊断。方法：将7种结核分枝杆菌优势表位抗原,即38kD、ESAT-6、CFP10、MPT64、Mtb8、Mtb8.4和Mtb16.3点于修饰的基片上,制备可检测7种结核抗体的多靶点蛋白微阵列,建立免疫金银染色检测系统;使用该芯片对48例临床结核病患者血液样品进行检测,并与“金标准”痰涂片（48例）和痰培养（其中的29例）检测结果进行比较,分析其敏感性;对30名献血员血液样品进行检测,分析其特异性。结果：可视化抗体检测蛋白芯片的敏感性分别为98.5％和96.6％,而痰涂片和痰培养检测方法的敏感性分别为35.4％和48.3％;可视化抗体检测蛋白芯片的特异性为93.3％。结论：建立的结核分枝杆菌可视化抗体检测蛋白芯片检测敏感性显著高于痰涂片和痰培养方法,可用于结核病的临床辅助诊断,提高痰涂片和痰培养假阴性的检出率。     

Immunoreactive proteins of Campylobacter concisus, an emergent intestinal pathogen

Campylobacter concisus is an emerging pathogen of the human gastrointestinal tract. Recently, a significantly higher prevalence of C.?concisus DNA and higher levels of antibodies specific to C.?concisus was detected in children with Crohn's disease when compared with controls. The aim of this study was to identify C.?concisus immunoreactive antigens. Proteins from C.?concisus were separated using two-dimensional gel electrophoresis, and sera from 10 C.?concisus-positive children with Crohn's disease were employed for immunoprobing. The patients' sera reacted with 69 spots, which corresponded to 31 proteins identified by mass spectrometry. The proteins were functionally classified as involved in chemotaxis, signal transduction, flagellar motility, surface binding and membrane protein assembly. Although the individual patients' sera reacted to different sets of proteins, common antigens that were recognized by all patients were flagellin B, ATP synthase F1 alpha subunit, and outer membrane protein 18. Cross-reactivity between proteins of the Campylobacter genus was tested using patients' sera absorbed with Campylobacter showae, Campylobacter jejuni and Campylobacter ureolyticus. Most of the C.?concisus immunoreactive proteins identified in this study showed cross-reactivity with other species except for three antigens. In conclusion, this study has identified C.?concisus proteins that are immunoreactive within patients with Crohn's disease.     

Isolation of Mycobacterium tuberculosis protein antigens ES-3 1, ES-43 and EST-6 of diagnostic interest from tubercle bacilli by affinity chromatography

Immunodiagnostically useful M. tuberculosis H37Ra protein antigens ES-31, ES-43 and EST-6 were isolated from detergent soluble sonicate (DSS) antigen using monospecific antibodies by affinity chromatography and compared with similar antigens isolated from M. tuberculosis culture filtrate for seroreactivity in tuberculosis sera by Indirect Enzyme Linked Immunosorbent Assay. Recovery of affinity purified ES-31, ES-43 and EST-6 antigen from DSS antigen was approximately 3, 3.5 and 4% respectively, compared to 10, 9 and 6.3% from culture filtrate. Affinity purified ES-31, ES-43 and EST-6 antigens from both culture filtrate as well as DSS antigen showed similar seroreactivity with overall sensitivity 85, 80 and 75% respectively and specificity of 85% at optimum concentration of 50 pg protein of each antigen. The results suggest that DSS antigen may be a promising antigen source for isolating antigens of diagnostic interest obviating the need for cumbersome, time-consuming culture techniques of mycobacteria.     

Multiplex analysis of cytokines/chemokines as biomarkers that differentiate healthy contacts from tuberculosis patients in high endemic settings

Differentiation of latent tuberculosis infection (LTBI) from active disease is one of the crucial elements in the control of tuberculosis. Earlier in Indian population which is tuberculosis endemic, we identified that 10 Mycobacterium tuberculosis secreted protein fractions, induced IFN-γ response only in healthy contacts of TB patients (HCs) and not in tuberculosis patients (TB). These fractions were termed as “Contact Specific Fractions” (“CS” fractions) and found useful for differentiating HC from TB. Proteomic analysis revealed that “CS” fractions have 16 different proteins, of which three were novel T cell antigens. Using these “CS” fractions as stimulants, earlier IFN-γ, TNF-α and IL-4 cytokine responses were studied. In the present study, in order to identify the other useful cytokine biomarkers that were differentially expressed between HC and TB, Cytokine/chemokine response to “CS” fractions were analyzed using multiplex cytokine assay system. This preliminary investigation in our tuberculosis endemic population showed six cytokine (G-CSF, IL-6, IL-7, IL-8, IL-9, and PDGF) and one receptor antagonist (IL-1Ra) that were differentially expressed between HC and TB, for the first time. Especially IL-6 and PDGF were more promising biomarkers. IL-6 measurement identified seven as HC out of 10 HC analyzed. The measurement of PDGF identified eight as TB out of 10 TB tested. Studies are underway to further validate these biomarkers for the differentiation of LTBI from active tuberculosis.     

Detection of in vitro and in vivo released antigens of diagnostic interest in Mycobacterium tuberculosis by immunoblotting

Identification of in vitro and in vivo released mycobacterial antigens are of considerable interest in diagnosis of Mycobacterium tuberculosis. Isolation of in vitro released antigen from M. tb excretory-secretory culture filtrate protein and in vivo released circulating tuberculous antigen from smear positive pulmonary tuberculosis sera by ammonium sulphate precipitation is reported. The antigens were resolved by SDS-PAGE and immunoblotting was performed using pooled serum of smear positive, smear negative pulmonary tuberculosis sera and normal sera to identify reactive tuberculous antigens. In vitro and in vivo released mycobacterial antigens showed reactivity at 100, 31, 43 and 20 kDa with smear positive and smear negative pulmonary tuberculosis patients. Further, the in vitro released antigen showed strong reactivity exclusively at 55 kDa antigen with smear positive and 24 kDa antigen with smear negative pulmonary tuberculosis sera. In vivo released antigen reacted exclusively at 170 and 16 kDa with smear positive and 19 kDa antigen with smear negative pulmonary tuberculosis patients. Antigens of 24 and 19 kDa which are reactive with sputum negative sera will be of diagnostic interest and need further study in patients with low bacillary load. The in vitro and in vivo released mycobacterial 100, 31,43 and 20 kDa antigens, reactive with patients sera are of diagnostic interest in tuberculosis.     

Immune Response to Nocardia brasiliensis Extracellular Antigens in Patients with Mycetoma

The ability of culture-filtrate proteins to induce a cellular immune response in infected mice and humans was investigated. A crude extract culture filtrate of Nocardia brasiliensis (CFA) and five semi-purified CFA fractions (P1, P2, P3, P4, P5) were used to stimulate BALB/c mice spleen-cell cultures. The animals were divided into three groups: the first group was infected with 1 × 10⁷ CFU of N. brasiliensis in the footpad, the second group was immunized with heat-killed bacteria, and the third was injected with sterile saline. IFN-γ, IL-1α, and IL-4 concentrations were determined in culture supernatants. Protein fractions eliciting IFN-γ production in mice, as well as the CFA, were used to stimulate IFN-γ production and in vitro cell proliferation assays with peripheral blood mononuclear cells of patients with actinomycetoma by N. brasiliensis, individuals with pulmonary tuberculosis, and healthy controls. In mice, CFA and three of the protein fractions (P3, P4 and P5) induced significant IFN-γ production in the infected group. In humans, only the CFA-induced IFN-γ production and cell proliferation in the group of patients with actinomycetoma. There was no stimulation in tuberculosis patients nor healthy controls. These results suggest that some culture-filtrate antigens are recognized by patients with active actinomycetoma and do not cross-react with M. tuberculosis antigens, being therefore potential candidates to develop a diagnostic test.     

Characterization of antigens recognized by new monoclonal antibodies raised against culture filtrate proteins of Mycobacterium bovis bacillus Calmette-Guérin

Effective protection against Mycobacterium tuberculosis may be achieved in experimental animals by immunization with proteins secreted by tuberculous bacilli in the extracellular milieu during growth. In this study, monoclonal antibodies were raised against Mycobacterium bovis bacillus Calmette-Guérin (BCG) culture filtrate proteins or live BCG, in an attempt to identify novel mycobacterial secretion antigens: the localization of the antigens recognized by the monoclonal antibodies within the mycobacterial cell was studied and interspecies reactivity was also investigated. The monoclonal antibodies obtained recognized proteins of molecular mass ranging from 5 to 82 kDa, with a prevailing frequency in the 30 kDa region. Three of the monoclonal antibodies recognized proteins present only in culture filtrates, one reacted with a cytoplasmic antigen, while the remaining antibodies recognized components which were mainly associated with the cell wall and the cytoplasmic membrane. The chemical nature and possible identity of the antigens was checked. Three monoclonal antibodies are likely to react with novel mycobacterial antigens of 5, 42 and 82 kDa, respectively.     

Recognition of Human Anti-DNA Autoantibodies by Secretory Antigen 85 Complex of Mycobacterium Tuberculosis H37Rv

Secreted mycobacterial protein antigens were isolated from the culture filtrate of Mycobacterium tuberculosis H₃₇R_v strain grown in Sauton's medium. These secretory proteins of Mycobacterium tuberculosis culture filtrate (MTCF) were separated by SDS-PAGE. High titre anti-DNA autoantibodies from the sera of systemic lupus erythematosus (SLE) patients showed remarkable binding and specificity towards MTCF proteins in dot blot and solid phase immunoassays. The major immunodominant secretory protein in MTCF, fractionated by Sephadex G-200 chromatography was the antigen 85 complex comprising of 30 and 31 kDa molecular weight components. Binding of SLE anti-DNA autoantibodies to the antigen 85 complex was further confirmed by Western blotting. The results suggest the possible involvement of secretory mycobacterial protein antigens in anti-DNA antibody induction in SLE.     

Immunoreactivity of antigen extracts of Aspergillus fumigatus isolated from different sources

Allergic bronchopulmonary aspergillosis (ABPA) is the result of hypersensitivity to Aspergillus antigens in patients with long-standing atopic asthma. In the present study mycelial and culture filtrate antigens from Aspergillus fumigatus cultures isolated from diverse sources were tested against sera of 10 ABPA patients and 10 control individuals by an ELISA methodology. The results indicate higher antibody reactivity against both antigens in the sera of ABPA patients, while culture filtrate antigens also gave non-specific reactivity with control sera. Mycelial extracts, in general, were useful in the diagnosis of ABPA.     

High level expression of recombinant Mycobacterium tuberculosis culture filtrate protein CFP32 in Pichia pastoris

Difficulty in obtaining large quantities of Mycobacterium tuberculosis (MTB) proteins remains a major obstacle in the development of subunit vaccines and diagnostic reagents for tuberculosis. A major reason is because Escherichia coli has not proven to be an optimal host for the expression of MTB genes. In this article, we used the yeast Pichia pastoris to express high levels of CFP32, a culture filtrate protein restricted to the MTB complex and a potential target antigen for serodiagnosis of tuberculosis in patients. Using shaker flasks, we generated a P. pastoris clone expressing CFP32 as a secreted protein fused to the myc-(His)₆ tag, at a yield of 0.5 g of purified protein per liter of culture. Recombinant CFP32 (rCFP32) produced in P. pastoris has a molecular weight of 35 kDa, which is slightly higher than that of the native protein We identified putative acylation and glycosylation sites in the CFP32 amino acid sequence that suggested post-translational modifications may contribute to the size difference. The NH₂-terminal peptide sequencing of rCFP32 showed that the signal peptide alpha factor is correctly excised. In addition, rCFP32 reacted with the sera of patients with tuberculosis. These data are the first to show that P. pastoris is a suitable host for high-yield production of good quality mycobacterium antigens, and especially culture filtrate proteins that have vaccine and diagnostic potential.     

Cytokine profiles in tuberculosis patients and healthy subjects in response to complex and single antigens of Mycobacterium tuberculosis

Peripheral blood mononuclear cells (PBMC) were obtained from tuberculosis (TB) patients and Mycobacterium bovis bacillus Calmette-Guerin vaccinated healthy subjects. PBMC were tested for secretion of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-5 (IL-5) and IL-10 in response to complex (whole cells, culture filtrate and cell walls), single secreted (Ag85B, ESAT6, MPT64, PstS and MPT70) and single cytosolic (DnaK, GroES and GroEL) antigens of Mycobacterium tuberculosis. In the absence of antigens, detectable concentrations of TNF-alpha, IFN-gamma and IL-10 were secreted by PBMC of both donor groups, but the concentrations of only IL-10 were significantly higher (P=0.015) in TB patients than in healthy subjects. In the presence of complex antigens, PBMC secreted IFN-gamma and TNF-alpha in response to all three preparations, whereas IL-10 was secreted in response to whole cells and cell walls only. In the presence of single antigens, IFN-gamma was secreted in response to Ag85B, ESAT6 and MPT64 in TB patients and ESAT6 in healthy donors. Except for GroEL and DnaK, single antigens did not induce TNF-alpha and IL-10 secretion from PBMC in either donor group. The secretion of IFN-gamma, but not IL-10, in the presence of Ag85B, ESAT6 and MPT64 supports their potential as subunit vaccine candidates against TB.     

The use of immunoenzyme analysis and immunoblotting for the serological characterization of mycobacterial antigens]

The enzyme immunoassay and immunoblotting were used for the study of the serological activity of different mycobacterial antigens and the spectrum of antibodies to them in patients with different forms of tuberculosis and healthy persons. Antibodies in patients' sera were shown to bind antigens with different molecular weight. The level and spectrum of antibodies to purified protein fraction I made it possible to differentiate between patients with various forms of tuberculosis and healthy persons.     

Inhibition of anti-tuberculosis T-lymphocyte function with tumour necrosis factor antagonists

Reactivation of latent Mycobacterium tuberculosis (Mtb) infection is a major complication of anti-tumour necrosis factor (TNF)-alpha treatment, but its mechanism is not fully understood. We evaluated the effect of the TNF antagonists infliximab (Ifx), adalimumab (Ada) and etanercept (Eta) on anti-mycobacterial immune responses in two conditions: with ex vivo studies from patients treated with TNF antagonists and with the in vitro addition of TNF antagonists to cells stimulated with mycobacterial antigens. In both cases, we analysed the response of CD4+ T lymphocytes to purified protein derivative (PPD) and to culture filtrate protein (CFP)-10, an antigen restricted to Mtb. The tests performed were lymphoproliferation and immediate production of interferon (IFN)-gamma. In the 68 patients with inflammatory diseases (rheumatoid arthritis, spondylarthropathy or Crohn's disease), including 31 patients with a previous or latent tuberculosis (TB), 14 weeks of anti-TNF-alpha treatment had no effect on the proliferation of CD4+ T lymphocytes. In contrast, the number of IFN-gamma-releasing CD4+ T lymphocytes decreased for PPD (p < 0.005) and CFP-10 (p < 0.01) in patients with previous TB and for PPD (p < 0.05) in other patients (all vaccinated with Bacille Calmette-Guérin). Treatments with Ifx and with Eta affected IFN-gamma release to a similar extent. In vitro addition of TNF antagonists to CD4+ T lymphocytes stimulated with mycobacterial antigens inhibited their proliferation and their expression of membrane-bound TNF (mTNF). These effects occurred late in cultures, suggesting a direct effect of TNF antagonists on activated mTNF+ CD4+ T lymphocytes, and Ifx and Ada were more efficient than Eta. Therefore, TNF antagonists have a dual action on anti-mycobacterial CD4+ T lymphocytes. Administered in vivo, they decrease the frequency of the subpopulation of memory CD4+ T lymphocytes rapidly releasing IFN-gamma upon challenge with mycobacterial antigens. Added in vitro, they inhibit the activation of CD4+ T lymphocytes by mycobacterial antigens. Such a dual effect may explain the increased incidence of TB in patients treated with TNF antagonists as well as possible differences between TNF antagonists for the incidence and the clinical presentation of TB reactivation.     

Expression and purification of recombinant antigens of Mycobacterium tuberculosis for application in serodiagnosis

Accurate diagnosis is essential for the treatment, prevention, and control of tuberculosis. Poor specificity of the tuberculin skin test in BCG-vaccinated populations and constraints to implementation of PCR and CMI-based diagnostic assays in developing countries warrant development of easy-to perform robust serological tests. Due to great heterogeneity in humoral response in TB patients, it will be necessary to include several antigens in any diagnostic assay to achieve useful levels of sensitivity and specificity. This needs production of recombinants, soluble versions of mycobacterial antigens in high yields. We have cloned, expressed, and purified a number of mycobacterial proteins in Escherichia coli. This paper describes the expression and purification of four promising sero-reactive proteins namely, ESAT6, CFP10, MTC28, and 14-kDa antigen of Mycobacterium tuberculosis. The protocol involves regulated and slow expression of proteins by using a T7 promoter-based expression vector for obtaining soluble protein followed by a three-step column chromatography procedure employing media with high binding capacity and flow characteristics. The yields of these proteins obtained were several folds higher than previously reported. The purified proteins were useful in detecting antibodies in sera of TB patients (smear positive, smear negative, and extra-pulmonary categories) and in combination with other immunodominant antigens will be useful in increasing the sensitivity to detect M. tuberculosis specific antibodies.