Early Secretory Antigenic Target Research Articles

Increased expression of mRNA encoding interleukin (IL)-4 and its splice variant IL-4delta2 in cells from contacts of Mycobacterium tuberculosis, in the absence of in vitro stimulation.

Expression of interleukin (IL)-4 is increased in tuberculosis and thought to be detrimental. We show here that in healthy contacts there is increased expression of its naturally occurring antagonist, IL-4delta2 (IL-4delta2). We identified contacts by showing that their peripheral blood mononuclear cells (PBMC) released interferon (IFN)-gamma in response to the Mycobacterium tuberculosis-specific antigen 6 kDa early secretory antigenic target (ESAT-6). Fresh unstimulated PBMC from these contacts contained higher levels of mRNA encoding IL-4delta2 (P=0.002) than did cells from ESAT-6 negative donors (noncontacts). These data indicate that contact with M. tuberculosis induces unusual, previously unrecognized, immunological events. We tentatively hypothesize that progression to active disease might depend upon the underlying ratio of IL-4 to IL-4delta2.

Confirmation of Mycobacterium tuberculosis infection by flow cytometry after ex vivo incubation of peripheral blood T cells with an ESAT-6-derived peptide pool.

The presence of a T-cell response to the early secretory antigenic target-6 (ESAT-6) indicates previous infection with or exposure to Mycobacterium tuberculosis. Measuring this response is useful for identifying individuals infected with M. tuberculosis. It was also reported that the frequencies of ESAT-6-specific T cells correlate with disease state. Established procedures measure secreted T-cell cytokines following whole blood stimulation with recombinant ESAT-6 protein or use Elispot as a read-out. A single ESAT-6- spanning pool of overlapping peptides (15 amino acids length with 11 overlaps) was used for overnight stimulation of peripheral blood mononuclear cells (PBMCs) from 15 patients infected with Mycobacterium tuberculosis and 11 healthy controls. T-cell responses were rated positive if interferon-gamma (IFN-gamma)-producing T cells were identified above background level, using 4-color cytokine flow cytometry. Thirteen of 15 (87%) patients, but none of the healthy controls, had a positive CD4 T-cell response to the ESAT-6 spanning peptide pool. The frequencies of IFN-gamma-producing cells varied between 1 and 167 per 10,000 CD4 T cells. The test performed as well as the tests described in the literature. Cytokine flow cytometry following PBMC stimulation with an ESAT-6 spanning peptide pool is a useful laboratory test for ESAT-6-specific T cells combining precise counting and multi-parameter phenotyping.

Enhanced protection against tuberculosis by vaccination with recombinant Mycobacterium microti vaccine that induces T cell immunity against region of difference 1 antigens.

Mycobacterium microti, the vole bacillus, which was used as a live vaccine against tuberculosis until the 1970s, confers the same protection in humans as does Mycobacterium bovis bacille Calmette-Guerin (BCG). However, because the efficacy of the BCG vaccine varies considerably, we have tried to develop a better vaccine by reintroducing into M. microti the complete region of difference 1 (RD1), which is required for secretion of the potent T cell antigens early secreted antigen target (ESAT)-6 and culture filtrate protein (CFP)-10. The resultant recombinant strain, M. microti OV254::RD1-2F9, induced specific ESAT-6 and CFP-10 immune responses in mice with CD8(+) T lymphocytes that had strong expression of the CD44(hi) activation marker. This vaccine also displayed better efficacy against disseminated disease in the mouse and the guinea pig models of tuberculosis than was seen in animals vaccinated with M. microti alone or with BCG. The M. microti OV254::RD1-2F9 vaccine was less virulent and persistent in mice and than was BCG::RD1-2F9 may represent a safer alternative to BCG::RD1-2F9.

Healthy individuals that control a latent infection with Mycobacterium tuberculosis express high levels of Th1 cytokines and the IL-4 antagonist IL-4delta2.

The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop disease and identifying what constitutes "protective immunity" is one of the holy grails of M. tuberculosis immunology. It is known that IFN-gamma is essential for protection, but it is also apparent that IFN-gamma levels alone do not explain the immunity/susceptibility dichotomy. The controversy regarding correlates of immunity persists because identifying infected but healthy individuals (those who are immune) has been problematic. We have therefore used recognition of the M. tuberculosis virulence factor early secretory antigenic target 6 to identify healthy, but infected individuals from tuberculosis (TB)-endemic and nonendemic regions (Ethiopia and Denmark) and have compared signals for cytokines expressed directly ex vivo with the pattern found in TB patients. We find that TB patients are characterized by decreased levels of Th1 cytokines and increased levels of IL-10 compared with the healthy infected and noninfected community controls. Interestingly, the healthy infected subjects exhibited a selective increase of message for the IL-4 antagonist, IL-4delta2, compared with both TB patients or noninfected individuals. These data suggest that long-term control of M. tuberculosis infection is associated not just with elevated Th1 responses but also with inhibition of the Th2 response.

Tuberculin skin testing underestimates a high prevalence of latent tuberculosis infection in hemodialysis patients

Identification of latent Mycobacterium tuberculosis infection in hemodialysis patients is hampered by reduced sensitivity of the established tuberculin skin test. We investigated whether in vitro quantitation of purified protein derivative (PPD)-specific T cells using a rapid 6-hour assay may represent an alternative approach for detecting latent infection. One hundred and twenty-seven hemodialysis patients and 218 control patients (blood donors, health care workers, and control patients) were analyzed. Specific T cells toward PPD and early secretory antigenic target-6 (ESAT-6), a protein expressed in Mycobacterium tuberculosis but absent from M. bovis bacillus Calmette-Guerin (BCG) vaccine strains, were flow cytometrically quantified from whole blood, and results were compared with skin testing. Compared to blood donors, a high proportion of both health care workers (48.6%) and hemodialysis patients (53.5%) had PPD-specific Th1-type CD4 T-cell reactivity with similar median frequencies of PPD-specific T cells (0.17%; 0.06-3.75% vs. 0.26%; 0.06-4.12%, respectively). In contrast, skin test reactivity was significantly reduced in hemodialysis patients. Whereas 85.7% of control patients with PPD reactivity in vitro were skin test-positive, the respective percentage among hemodialysis patients was 51.4% (P= 0.007). Among individuals with PPD reactivity in vitro, approximately 50% had T cells specific for ESAT-6. Unlike the skin test, measurement of PPD reactivity by in vitro quantitation of PPD-specific T cells was unaffected by uremia-associated immunosuppression. This whole-blood assay may thus be a valuable alternative to skin testing, and detection of ESAT-6-specific T cells could moreover allow distinction of latent M. tuberculosis infection from BCG-induced reactivity to PPD. The assay is well suited for clinical use and may facilitate targeting of preventative therapy in high-risk individuals.

Evaluation of T-cell responses to novel RD1- and RD2-encoded Mycobacterium tuberculosis gene products for specific detection of human tuberculosis infection.

The tuberculin skin test for diagnosing Mycobacterium tuberculosis infection suffers from antigenic cross-reactivity of purified protein derivative with BCG, resulting in poor specificity in BCG-vaccinated populations. Comparative genomics has identified several genetic regions in M. tuberculosis and M. bovis that are deleted in M. bovis BCG. Proteins encoded in these regions will form the basis of new specific T-cell-based blood tests that do not cross-react with BCG, but only two, early secretory antigen target 6 and culture filtrate protein 10, have been studied in detail in humans. We investigated four novel gene products, encoded by RD2 (Rv1989c) and RD1 (Rv3873, Rv3878, and Rv3879c), that are absent from most or all of the vaccine strains of BCG, respectively. Sixty-seven overlapping peptides were tested in ex vivo gamma interferon enzyme-linked immunospot assays in 49 patients with culture-confirmed tuberculosis and 38 healthy BCG-vaccinated donors. Forty-five percent (95% confidence interval [CI], 31 to 57%) and 53% (95% CI, 39 to 67%) of the tuberculosis patients responded to Rv3879c and Rv3873, respectively, identifying these proteins as major M. tuberculosis T-cell antigens in humans, while 35 and 25% of the patients responded to Rv3878 and Rv1989c, respectively. Of the 38 BCG-vaccinated donors, 1 (2.6%) responded to peptides from Rv3878 and Rv3879c, 3 (7.9%) responded to Rv3873, and none responded to Rv1989c. Exclusion of cross-reactive peptides encoded in conserved motifs of Rv3873, a PPE family member, increased its specificity to 97.4%. The high specificity of Rv3879c peptides and nonconserved Rv3873 sequences, together with their moderate sensitivity in tuberculosis patients, identifies these peptides as candidates for inclusion in new T-cell-based tests for M. tuberculosis infection.

Use of a T cell-based assay for monitoring efficacy of antituberculosis therapy.

Monitoring the efficacy of antituberculosis therapy is crucial both for the individual patient and for better control of the spread of tuberculosis. We studied 18 patients with microbiologically confirmed tuberculosis, both at the time of diagnosis and 3 months after they started therapy, using an in vitro assay that detects T cell-mediated interferon- gamma response to selected peptides of Mycobacterium tuberculosis-specific early secretory antigenic target 6 (ESAT-6) protein. All patients had positive results at diagnosis; however, 3 months later, the response to ESAT-6 peptides was still detectable only in the 5 patients with microbiological isolation and/or absence of clinical improvement after treatment. On the basis of these data, we conclude that our assay is a useful tool in monitoring the efficacy of antituberculosis therapy.

PPE Protein (Rv3873) from DNA Segment RD1 ofMycobacterium tuberculosis: Strong Recognition of Both Specific T-Cell Epitopes and Epitopes Conserved within the PPE Family

Proteins encoded by DNA segment RD1 of Mycobacterium tuberculosis have recently been demonstrated to play important roles in bacterial virulence, vaccine development, and diagnostic reagent design. Previously, we characterized two immunodominant T-cell antigens, the early secreted antigen target (ESAT-6) and the 10-kDa culture filtrate protein (CFP10), which are encoded by the esx-lhp operon in this region. In the present study we characterized a third putative open reading frame in this region, rv3873, which encodes a PPE protein. We found that the rv3873 gene is expressed in M. tuberculosis H37Rv and that the native protein, Rv3873, is predominantly associated with the mycobacterial cell or wall. When tested as a His-tagged recombinant protein, Rv3873 stimulated high levels of gamma interferon secretion in peripheral blood mononuclear cells isolated from tuberculosis (TB) patients, as well as from healthy tuberculin purified protein derivative-positive donors. In contrast to other RD1-encoded antigens, Rv3873 was also found to be recognized by a significant proportion of Mycobacterium bovis BCG-vaccinated donors. Epitope mapping performed with overlapping peptides revealed a broad pattern of T-cell recognition comprising both TB-specific epitopes and epitopes also recognized by BCG-vaccinated donors. The immunodominant epitope (residues 118 to 135) for both TB patients and BCG-vaccinated individuals was found to be highly conserved among a large number of PPE family members.

The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue.

Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette-Guérin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting DeltaRD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis DeltaRD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the DeltaRD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We conclude that the primary attenuating mechanism of bacillus Calmette-Guérin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness.

Production of a fusion protein consisting of the enterotoxigenic Escherichia coli heat-labile toxin B subunit and a tuberculosis antigen in Arabidopsis thaliana.

Transgenic plants are potentially safe and inexpensive vehicles to produce and mucosally deliver protective antigens. However, the application of this technology is limited by the poor response of the immune system to non-particulate, subunit vaccines. Co-delivery of therapeutic proteins with carrier proteins could increase the effectiveness of the antigen. This paper reports the ability of transgenic Arabidopsis thaliana plants to produce a fusion protein consisting of the B subunit of the Escherichia coli heat-labile enterotoxin and a 6 kDa tuberculosis antigen, the early secretory antigenic target ESAT-6. Both components of the fusion protein were detected using GM1-ganglioside-dependent enzyme-linked immunosorbant assay. This suggested the fusion protein retained both its native antigenicity and the ability to form pentamers.

Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis.

The live tuberculosis vaccines Mycobacterium bovis BCG (bacille Calmette-Guérin) and Mycobacterium microti both lack the potent, secreted T-cell antigens ESAT-6 (6-kDa early secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein). This is a result of independent deletions in the region of deletion-1 (RD1) locus, which is intact in virulent members of the Mycobacterium tuberculosis complex. To increase their immunogenicity and protective capacity, we complemented both vaccines with different constructs containing the esxA and esxB genes, which encode ESAT-6 and CFP-10 respectively, as well as a variable number of flanking genes. Only reintroduction of the complete locus, comprising at least 11 genes, led to full secretion of the antigens and resulted in specific ESAT-6-dependent immune responses; this suggests that the flanking genes encode a secretory apparatus. Mice and guinea pigs vaccinated with the recombinant strain BCG::RD1-2F9 were better protected against challenge with M. tuberculosis, showing less severe pathology and reduced dissemination of the pathogen, as compared with control animals immunized with BCG alone.

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.

Expression of the mceA, esat-6 and hspX genes in Mycobacterium tuberculosis and their responses to aerobic conditions and to restricted oxygen supply.

The expression of six of the mammalian cell-entry (mce1a-mce1f) genes of the mce1 operon of Mycobacterium tuberculosis has been described previously. In this study, data are presented for the expression of other mammalian cell-entry homologues (mce-2a, mce-3a and mce-4a) at the RNA level, as determined by RT-PCR. The stress responses of these genes and of other immunologically important antigens are also characterized with respect to the introduction of oxygen depletion. Analysis of the expression of the mceA genes in relation to oxygen depletion revealed that they were expressed differentially. The RT-PCR results showed that mce-1a, mce-2a, hspX (encoding the alpha-crystallin antigen Acr) and esat-6 (encoding the early secretory antigenic target-6) were expressed throughout the cultivation period, whereas the expression of mce-3a and mce-4a was downregulated in the later stages of cultivation. This study gives new insights into the expression profiles of the different mce operons and the hspX and esat-6 genes in an in vitro model of dormant-like bacilli. Identification of the genes that are differentially expressed under aerobic conditions and under oxygen-limited conditions contributes to our understanding of the bacilli involved in latent tuberculosis.

Long-lived immune response to early secretory antigenic target 6 in individuals who had recovered from tuberculosis.

We sought to understand the persistence and relevance of the long-lived immune response to early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis in humans. ESAT-6 is recognized by memory cells involved in protection of animals against tuberculosis (TB). Recent reports also showed that ESAT-6 response can be recovered in patients with TB and in those soon after anti-TB therapy. We chose 18 individuals who had recovered from pulmonary TB (some in remission for >5 years), and 14 bacille Calmette-Guérin-vaccinated healthy individuals for this study. The results showed that peripheral blood mononuclear cells of 10 (55.6%) of 18 patients with TB remission responded to ESAT-6 with stimulation indices >3.0, whereas none of the healthy controls responded. Functional analysis showed that 13 (72.2%) of 18 patients with TB remission produced significant amounts of IFN-gamma in response to ESAT-6, whereas only 1 (7.1%) of the 14 healthy control subjects did so. It appears that responses to ESAT-6 can persist in individuals who had recovered from pulmonary TB.

Enumeration of T cells specific for RD1-encoded antigens suggests a high prevalence of latent Mycobacterium tuberculosis infection in healthy urban Indians.

Knowledge of the prevalence of latent Mycobacterium tuberculosis infection is crucial for effective tuberculosis control, but tuberculin skin test surveys have major limitations, including poor specificity because of the broad antigenic cross-reactivity of tuberculin. The M. tuberculosis RD1 genomic segment encodes proteins, such as early secretory antigenic target (ESAT)-6, that are absent from M. bovis bacille Calmette-Guérin (BCG) and most environmental mycobacteria. We recently identified circulating ESAT-6-specific T cells as an accurate marker of M. tuberculosis infection. Here, interferon-gamma-secreting T cells specific for peptides derived from ESAT-6 and a second RD1 gene product, CFP10, were enumerated in 100 prospectively recruited healthy adults in Bombay (Mumbai), India. Eighty percent responded to >/=1 antigen, and many donors had high frequencies of T cells that were specific for certain immunodominant peptides. In contrast, of 40 mostly BCG-vaccinated, United Kingdom-resident healthy adults, none responded to either antigen. This study suggests an 80% prevalence of latent M. tuberculosis infection in urban India.

Efficient protection against Mycobacterium tuberculosis by vaccination with a single subdominant epitope from the ESAT-6 antigen.

We have investigated the vaccine potential of two peptides derived from the 6-kDa early secretory antigenic target (ESAT)-6 antigen in the mouse model of tuberculosis. The peptides were both strongly immunogenic in B6CBAF1 (H-2b,k) mice and primed recall responses of the same intensity after immunization. However, both tuberculosis infection and immunization with ESAT-6 resulted in responses focused towards ESAT-61-20. Multiple antigen peptide constructs as well as free peptides were emulsified with dimethyl dioctadecylammonium bromide/monophosphoryl lipid A/IL-2 and tested as experimental vaccines in an i.v. and aerosol model of tuberculosis in mice. The peptide were highly immunogenic and induced cellular responses of the same magnitude. However, only vaccines based on the subdominant ESAT-651-70 epitope promoted significant levels of protective immunity and the level of protection was equivalent to that achieved with ESAT-6 and BCG. These findings demonstrate the potential of peptide-based vaccines against tuberculosis and indicate that there is not direct correlation between the hierarchy of response to naturally processed peptides and their ability to induce protective immunity against Mycobacterium tuberculosis.

Multiple epitopes from the Mycobacterium tuberculosis ESAT-6 antigen are recognized by antigen-specific human T cell lines.

A synthetic-peptide approach was used to map epitope regions of the Mycobacterium tuberculosis 6-kDa early secreted antigen target (ESAT-6) by testing human CD4(+) T cell lines for secretion of IFN-gamma in response to recombinant ESAT-6 (rESAT-6) and overlapping 20-mer peptides covering the antigen sequence. The results demonstrate that all of the ESAT-6 peptides screened were able to induce IFN-gamma secretion from one or more of the T cell lines tested. Some of the individual T cell lines showed the capacity to respond to all peptides. Human leukocyte antigen (HLA-DR) typing of the donors showed that rESAT-6 was presented to T cells in association with multiple HLA-DR molecules. The results suggest that frequent recognition of the M. tuberculosis ESAT-6 antigen by T cells from patients with tuberculosis is due to the presence of multiple epitopes scattered throughout the ESAT-6 sequence.

Human cytolytic and interferon gamma-secreting CD8+ T lymphocytes specific for Mycobacterium tuberculosis.

Protective immunity to Mycobacterium tuberculosis is poorly understood, but mounting evidence, at least in animal models, implicates major histocompatibility complex class I-restricted CD8+ T cells as an essential component. By using a highly sensitive assay for single cell interferon gamma release, we screened an array of M. tuberculosis antigen-derived peptides congruent with HLA class I allele-specific motifs. We identified CD8+ T cells specific for epitopes in the early secretory antigenic target 6 during active tuberculosis, after clinical recovery and in healthy contacts. Unrestimulated cells exhibited peptide-specific interferon gamma secretion, whereas lines or clones recognized endogenously processed antigen and showed cytolytic activity. These results provide direct evidence for the involvement of CD8+ cytotoxic T lymphocytes in host defense against M. tuberculosis in humans and support current attempts to generate protective cytotoxic T lymphocyte responses against M. tuberculosis by vaccination.

Key epitopes on the ESAT-6 antigen recognized in mice during the recall of protective immunity to Mycobacterium tuberculosis.

The recall of long-lived immunity in a mouse model of tuberculosis (TB) is defined as an accelerated accumulation of reactive T cells in the target organs. We have recently identified Ag 85B and a 6-kilodalton early secretory antigenic target, designated ESAT-6, as key antigenic targets recognized by these cells. In the present study, preferential recognition of the ESAT-6 Ag during the recall of immunity was found to be shared by five of six genetically different strains of mice. Overlapping peptides spanning the sequence of ESAT-6 were used to map two T cell epitopes on this molecule. One epitope recognized in the context of H-2b,d was located in the N-terminal part of the molecule, whereas an epitope recognized in the context of H-2a,k covered amino acids 51 to 60. Shorter versions of the N-terminal epitope allowed the precise definition of a 13-amino acid core sequence recognized in the context of H-2b. The peptide covering the N-terminal epitope was immunogenic, and a T cell response with the same fine specificity as that induced during TB infection was generated by immunization with the peptide in IFA. In the C57BL/6j strain, this single epitope was recognized by an exceedingly high frequency of splenic T cells (approximately 1:1000), representing 25 to 35% of the total culture filtrate-reactive T cells recruited to the site of infection during the first phase of the recall response. These findings emphasize the relevance of this Ag in the immune response to TB and suggest that immunologic recognition in the first phase of infection is a highly restricted event dominated by a limited number of T cell clones.

Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis.

A novel immunogenic antigen, the 6-kDa early secretory antigenic target (ESAT-6), from short-term culture filtrates of Mycobacterium tuberculosis was purified by hydrophobic interaction chromatography and anion-exchange chromatography by use of fast protein liquid chromatography. The antigen focused at two different pIs of 4.0 and 4.5 during isoelectric focusing, and each of these components separated into three spots ranging from 4 to 6 kDa during two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent differences in molecular masses or pIs of these isoforms were not due to posttranslational glycosylation. The molecular weight of the purified native protein was determined by applying gel filtration and nondenaturing polyacrylamide gel electrophoresis and found to be 24 kDa. ESAT-6 is recognized by the murine monoclonal antibody HYB 76-8, which was used to screen a recombinant lambda gt11 M. tuberculosis DNA library. A phage expressing a gene product recognized by HYB 76-8 was isolated, and a 1.7-kbp fragment of the mycobacterial DNA insert was sequenced. The structural gene of ESAT-6 was identified as the sequence encoding a polypeptide of 95 amino acids. The N terminus of the deduced sequence could be aligned with the 10 amino-terminal amino acids derived from sequence analyses of the native protein. N-terminal sequence analysis showed that the purified antigen was essentially free from contaminants, and the amino acid analysis of the antigen was in good agreement with the DNA sequence-deduced amino acid composition. Thus, the heterogeneities observed in the pI and molecular weight of the purified antigen do not derive from contaminating proteins but are most likely due to heterogeneity of the antigen itself. Native and recombinant ESAT-6 are immunologically active in that both elicited a high release of gamma interferon from T cells isolated from memory-immune mice challenged with M. tuberculosis. Analyses of subcellular fractions of M. tuberculosis showed the presence of ESAT-6 in cytosol- and cell wall-containing fractions. Interspecies analyses showed the presence of ESAT-6 in filtrates from M. tuberculosis complex species. Among filtrates from mycobacteria not belonging to the M. tuberculosis complex, reactivity was observed in Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum.