PPE protein (Rv3873) from DNA segment RD1 of Mycobacterium 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.     

T-cell responses to the Mycobacterium tuberculosis-specific antigen ESAT-6 in Brazilian tuberculosis patients

The Mycobacterium tuberculosis-specific ESAT-6 antigen induces highly potent T-cell responses and production of gamma interferon (IFN-gamma), which play a critical role in protective cell-mediated immunity against tuberculosis (TB). In the present study, IFN-gamma secretion by peripheral blood mononuclear cells (PBMCs) in response to M. tuberculosis ESAT-6 in Brazilian TB patients was investigated in relation to clinical disease types, such as pleurisy and cavitary pulmonary TB. Leprosy patients, patients with pulmonary diseases other than TB, and healthy donors were assayed as control groups. Sixty percent of the TB patients indeed recognized M. tuberculosis ESAT-6, as did 50% of the leprosy patients and 60% of the non-TB controls. Nevertheless, the levels of IFN-gamma in response to the antigen ESAT, but not to antigen 85B (Ag85B) and purified protein derivative (PPD), were significantly lower in controls than in patients with treated TB or pleural or cavitary TB. Moreover, according to Mycobacterium bovis BCG vaccination status, only 59% of the vaccinated TB patients responded to ESAT in vitro, whereas 100% of them responded to PPD. Both CD4 and CD8 T cells were able to release IFN-gamma in response to ESAT. The present data demonstrate the specificity of ESAT-6 of M. tuberculosis and its ability to discriminate TB patients from controls, including leprosy patients. However, to obtain specificity, it is necessary to include quantitative IFN-gamma production in response to the antigen as well, and this might limit the use of ESAT-6-based immunodiagnosis of M. tuberculosis infection in an area of TB endemicity.     

PPE protein (Rv3425) from DNA segment RD11 of Mycobacterium tuberculosis: a potential B-cell antigen used for serological diagnosis to distinguish vaccinated controls from tuberculosis patients

Proteins encoded by a 9.5-kb DNA segment, termed the region of difference (RD), of Mycobacterium tuberculosis have been demonstrated to be important in bacterial virulence, vaccine development and the design of diagnostic reagents. This study evaluated the immunogenic properties of Rv3425, a member of the PPE family of proteins, encoded by an open reading frame found in RD11 of M. tuberculosis, in comparison with two other well-known antigens, the early secreted antigen target 6 (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10). RT-PCR demonstrated that Rv3425 mRNA is expressed in liquid culture by M. tuberculosis H37Rv. When tested in a conventional ELISA in the form of a His-tagged recombinant protein, Rv3425 revealed a statistically significant antigenic distinction between healthy bacille Calmette-Guérin (BCG)-vaccinated controls and tuberculosis (TB) patients (p <0.0001). The anti-IgG response to recombinant Rv3425 was almost equal to that for CFP-10, and was higher than that for ESAT-6. The results highlight the immunosensitive and immunospecific nature of Rv3425, which shows promise for use in the serodiagnosis of TB.     

Development of new vaccines and diagnostic reagents against tuberculosis

Tuberculosis (TB) is a major infectious disease problem with one-third of the world population infected, 8 million people developing the active disease and 2 million dying of TB each year. The attenuated Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only available vaccine against TB. However, the trials conducted in different parts of the world have shown that this vaccine doe not provide consistent protection against TB. The purified protein derivative (PPD) of Mycobacterium tuberculosis is the commonly used reagent for the diagnosis of TB. However, PPD lacks specificity because of the presence of antigens crossreactive with M. bovis BCG and other mycobacteria. The studies to identify M. tuberculosis antigens and epitopes as candidates for new protective vaccines and specific diagnostic reagents against TB have led to the identification and characterization of several major antigens of M. tuberculosis including heat shock proteins (hsp) and secreted antigens present in the culture filtrate (CF) of M. tuberculosis. Some of these antigens have shown promise as new candidate vaccines (hsp60, Ag85 and ESAT-6, etc.) and specific diagnostic reagents (ESAT-6 and CFP10, etc.) for TB. Moreover, in the mouse model of TB, vaccination with DNA-hsp60 has immunotheraputic effects and helps in eradication of persisters. In addition, identification of proper adjuvant and delivery systems has shown the promise to overcome the problem of poor immunogenicity associated with subunit and peptide based vaccines. More recently, the comparison of the genome sequence of M. tuberculosis with M. bovis BCG and other mycobacteria has led to the identification of M. tuberculosis-specific genomic regions. Evaluation of these regions for encoding proteins with immunological reactivity can lead to the identification of additional antigens of M. tuberculosis useful as new vaccines and reagents for specific diagnosis of TB.     

Specific delayed-type hypersensitivity responses to ESAT-6 identify tuberculosis-infected cattle

Human and bovine tuberculosis have long been detected by skin testing with purified protein derivative (PPD), a complex mix of partly denatured mycobacterial antigens with suboptimal specificity. In the present study, skin tests based on ESAT-6, a recombinantly produced antigen highly specific for tuberculosis infection, were investigated. Although ESAT-6 was strongly recognized in vitro and induced high levels of gamma interferon, initial investigations demonstrated that higher doses of ESAT-6 than of PPD were needed to induce substantial delayed-type hypersensitivity reactions. Also, the kinetics of the skin test response differed for the two reagents; PPD showed maximal response at 72 h, but the response to ESAT-6 often peaked later at 96 h. Tests based on an optimized strategy (400 micro g of ESAT-6 measured between 72 and 96 h), in cattle infected with Mycobacterium bovis (n = 22) and animals sensitized by exposure to environmental mycobacteria showed ESAT-6 to have a promising diagnostic potential (sensitivity, 82%; specificity, 100%; optimal cutoff, 3 mm), compared with PPD (sensitivity, 86%; specificity, 90%; optimal cutoff, 4 mm). Larger investigations are required to refine cutoff points for any new diagnostic test, but the present results indicate great potential for skin tests based on specific antigens for accurate in vivo diagnosis of tuberculosis.     

Serodiagnosis Efficacy and Immunogenicity of the Fusion Protein of Mycobacterium tuberculosis Composed of the 10-Kilodalton Culture Filtrate Protein, ESAT-6, and the Extracellular Domain Fragment of PPE68

In order to identify immunodominant antigens of Mycobacterium tuberculosis that may be used in the serodiagnosis of active tuberculosis (TB), we designed an M. tuberculosis fusion protein consisting of CFP-10 (10-kDa culture filtrate protein), ESAT-6 (6-kDa early secreted antigenic target), and the extracellular domain fragment of PPE68 (PPE68′). Then, the coding sequences of the three proteins were inserted into a prokaryotic expression vector, pET-32a(+). To enhance the immunological response, the proteins were linked together. The fusion proteins with a 6×His tag were successfully overexpressed in Escherichia coli BL21 and purified. The purified proteins were applied for detection of the total IgG titer by using an enzyme-linked immunosorbent assay (ELISA) with human sera from well-characterized TB cases and the control cases, and results were compared to those with purified protein derivative tuberculin (PPD). The ELISA results showed that among 140 cases of confirmed active TB and 70 control cases, CFP-10–ESAT-6–PPE68′ had a sensitivity of 73.3% and specificity of 94.3%, compared to a sensitivity of 66.7% and specificity of 74.3% for PPD and a sensitivity of 65% and specificity of 91.4% for CFP-10–ESAT-6. In addition, the fusion protein CFP-10–ESAT-6–PPE68′ stimulated a higher level of antigen-specific gamma interferon (IFN-γ) release for active-TB patients than PPD and CFP-10–ESAT-6. After immunization of C57BL/6 mice, the findings indicated that the total IgG titers and the concentrations of IFN-γ in mice immunized by CFP-10–ESAT-6–PPE68′ were high and induced strong, long-term humoral immunity compared to results with PPD and CFP-10–ESAT-6. Thus, our study indicates that the fusion protein CFP-10–ESAT-6–PPE68′ may be useful as an immunodominant antigen for the serodiagnosis of active TB.     

Efficient Ex vivo stimulation of Mycobacterium tuberculosis-specific T cells by genetically detoxified Bordetella pertussis adenylate cyclase antigen toxoids

Mycobacterium tuberculosis is a significant threat to global health. Mycobacterium bovis BCG vaccine provides only partial protection, and the skin test reagent used to aid diagnosis of both active and latent tuberculosis, purified protein derivative (PPD), lacks specificity and sensitivity. The use of genetically detoxified Bordetella pertussis adenylate cyclase toxin (CyaA) as a delivery system for two immunodominant proteins of M. tuberculosis that are of greater specificity than PPD, early-secreted antigenic target 6-kDa protein (ESAT-6) and culture filtrate protein 10 (CFP-10), was therefore investigated. CyaA toxoids incorporating these antigens were able to restimulate T cells from more than 91% tuberculosis patients and healthy sensitized donors. Delivery of antigen by CyaA decreased by 10-fold the amount of ESAT-6 and CFP-10 required to restimulate T cells, and in low responders, the overall frequency of gamma interferon-producing cells detected by enzyme-linked immunospot assay was increased (P < 0.01 for both antigens). Delivery of ESAT-6 and CFP-10 by CyaA enabled the detection of both CD4(+) and CD8(+) T cells: these responses could be blocked by inhibition of major histocompatibility complex class II or class I, respectively. Covalent linkage of antigen to the CyaA vector was required for enhancement to occur, as a mixture of mock CyaA toxoid plus recombinant ESAT-6 did not lead to enhancement. In a simplified whole-blood model to detect tuberculosis infection, the frequency of positive responses to CFP-10 was increased by CyaA delivery, a potentially important attribute that could facilitate the identification of latent infection.     

Identification, diagnostic potential, and natural expression of immunodominant seroreactive peptides encoded by five Mycobacterium tuberculosis-specific genomic regions

Comparative genomic studies have identified several Mycobacterium tuberculosis-specific genomic regions of difference (RDs) which are absent in the vaccine strains of Mycobacterium bovis BCG and which may be useful in the specific diagnosis of tuberculosis (TB). In this study, a total of 775 synthetic peptides covering the sequences of 39 open reading frame (ORF) proteins encoded by genes predicted in five RDs of M. tuberculosis, i.e., RD1, RD4, RD5, RD6, and RD7, were tested by enzyme-linked immunosorbent assays for antibody reactivity with sera from HIV-negative pulmonary TB patients (n = 100) and M. bovis BCG-vaccinated healthy subjects (n = 100). The results identified three immunodominant peptides reactive with TB sera, i.e., amino acids (aa) 346 to 370 of RD1ORF Rv3876, aa 241 to 265 of RD6ORF Rv1508c, and aa 325 to 336 of RD6ORF Rv1516c. These peptides had significantly stronger antibody reactivity with sera from TB patients than with sera from healthy subjects (P < 0.05) and significantly higher rates of positivity with TB sera (positives = 66 to 93%) than sera from healthy subjects (positives = 10 to 28%). Antipeptide antibodies were raised in rabbits after immunization with pools of 11 peptides corresponding to each protein. Probing of culture filtrates and whole-cell lysates of M. tuberculosis with antipeptide antibodies suggested the natural expression of Rv1516c in whole-cell lysates of M. tuberculosis. The results suggest the potential of the identified immunodominant RD peptides in the serodiagnosis of TB.     

Immunological crossreactivity of the Mycobacterium leprae CFP-10 with its homologue in Mycobacterium tuberculosis

Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) (Rv3874) is considered a promising antigen for the immunodiagnosis of tuberculosis (TB) together with early secreted antigens of M. tuberculosis (ESAT-6). Both ESAT-6 and CFP-10 are encoded by the RD1 region that is deleted from all tested M. bovis bacille Calmette-Guérin (BCG) strains but present in M. leprae, M. tuberculosis, M. bovis, M. kansasii, M. africanum and M. marinum. In this study, the homologue of CFP-10 in M. leprae (ML0050) is identified and characterized. Interferon-gamma production in response to this homologue by T cells from leprosy patients, TB patients and unexposed controls shows that CFP-10 of M. leprae is a potent antigen that crossreacts with CFP-10 of M. tuberculosis at the T-cell level. This crossreactivity has implications for the use of CFP-10 of these mycobacterial species as diagnostic tool in areas endemic for both the diseases.     

Tuberculosis in elephants: antibody responses to defined antigens of Mycobacterium tuberculosis, potential for early diagnosis, and monitoring of treatment

Tuberculosis (TB) in elephants is a re-emerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current diagnosis relies on trunk wash culture, the only officially recognized test, which has serious limitations. Innovative and efficient diagnostic methods are urgently needed. Rapid identification of infected animals is a crucial prerequisite for more effective control of TB, as early diagnosis allows timely initiation of chemotherapy. Serology has diagnostic potential, although key antigens have not been identified and optimal immunoassay formats are not established. To characterize the humoral responses in elephant TB, we tested 143 serum samples collected from 15 elephants over time. These included 48 samples from five culture-confirmed TB cases, of which four were in Asian elephants infected with M. tuberculosis and one was in an African elephant with Mycobacterium bovis. Multiantigen print immunoassay (MAPIA) employing a panel of 12 defined antigens was used to identify serologic correlates of active disease. ESAT-6 was the immunodominant antigen recognized in elephant TB. Serum immunoglobulin G antibodies to ESAT-6 and other proteins were detected up to 3.5 years prior to culture of M. tuberculosis from trunk washes. Antibody levels to certain antigens gradually decreased in response to antitubercular therapy, suggesting the possibility of treatment monitoring. In addition to MAPIA, serum samples were evaluated with a recently developed rapid test (RT) based on lateral flow technology (ElephantTB STAT-PAK). Similarly to MAPIA, infected elephants were identified using the RT up to 4 years prior to positive culture. These findings demonstrate the potential for TB surveillance and treatment monitoring using the RT and MAPIA, respectively.     

Detection by flow cytometry of ESAT-6- and PPD-specific circulating CD4+ T lymphocytes as a diagnostic tool for tuberculosis

BACKGROUND: The identification of mycobacteria represents the gold standard in the diagnosis of tuberculosis (TB), but it is not applicable in all patients, and immunological tests, such as the tuberculin skin test (TST), are not specific and sensitive enough. METHODS: By flow cytometry, we measured the CD4 T-cell response to purified protein derivative (PPD) and early secretory antigenic target-6 (ESAT-6) protein using the intracellular cytokine staining technique on whole blood samples obtained from active TB (n = 16), latent TB (n = 17), Bacille Calmette-Guérin (BCG)-vaccinated (n = 11) and healthy (n = 10) donors. All the patients were also tested with conventional TST. RESULTS: The identification by flow cytometry of PPD-specific T lymphocytes upon antigen stimulation of whole blood enables the discrimination of active TB, latent TB and BCG-vaccinated subjects from healthy individuals, whereas the ESAT-6 response discriminated active TB from healthy and BCG-vaccinated individuals. Moreover, this test enables identification of active TB patients who were negative on TST and to distinguish between TB and non-typical mycobacteria TB infections. CONCLUSIONS: The identification by flow cytometry of antigen-specific T lymphocytes upon antigen stimulation of whole blood has a better positive predictive value than TST, and could represent a further tool in the diagnosis of TB infection.     

Mycobacterium tuberculosis Region of Difference (RD) 2 Antigen Rv1985c and RD11 Antigen Rv3425 Have the Promising Potential To Distinguish Patients with Active Tuberculosis from M. bovis BCG-Vaccinated Individuals

Antigens encoded in the region of difference (RD) of Mycobacterium tuberculosis constitute a potential source of specific immunodiagnostic antigens for distinguishing tuberculosis (TB) infection from BCG vaccination. We evaluated the diagnostic potential of specific T-cell epitopes selected from two immunodominant antigens, Rv1985c and Rv3425, from RD2 and RD11, respectively, on the basis of epitope mapping, in TB patients and BCG-vaccinated healthy individuals. Using a whole-blood gamma interferon release assay, a wide array of epitopes was recognized on both Rv1985c and Rv3425 in TB patients. Those epitopes that could specifically discriminate TB infection from BCG vaccination were carefully selected, and the most promising peptide pools from Rv1985c showed a sensitivity of 53.9% and a specificity of 95.5%. When the novel specific peptides from Rv1985c joined the diagnostic antigens in the QuantiFERON-TB Gold In-Tube (QFT-IT) assay, the sensitivity was increased from 86.4% to 96.2%, with no drop in specificity. These results indicate that the peptide pools selected from Rv1985c and Rv3425 have the potential to diagnose TB infection by a method that may be routinely used in clinical laboratories.     

Identification of human T-cell responses to Mycobacterium tuberculosis resuscitation-promoting factors in long-term latently infected individuals

The Mycobacterium bovis BCG vaccine is the only tuberculosis (TB) vaccine available, yet it provides limited protection against pulmonary TB in adults and fails to protect against TB reactivation. We hypothesized that immunity against Mycobacterium tuberculosis "resuscitation-promoting factors" (Rpfs), which are small bacterial proteins that promote proliferation of dormant mycobacteria, may be relevant in the human immune response to M. tuberculosis. In previous unpublished work, we found that Rpfs Rv0867c and Rv2389c induced gamma interferon (IFN-γ) production in the blood of TB patients' healthy household contacts in several different African populations. Here we examine these two dominant Rpf antigens in more detail and define the nature of the responding T-cell subsets. Multiparameter cytokine profiling showed that Rv2389c and, to a lesser extent, Rv0867c were recognized by mycobacterium-responsive healthy Dutch individuals; peptide-scanning revealed several epitopes, including a single immunodominant epitope in Rv2389c. Rv0867c and, to a lesser extent, Rv2389c Rpf-specific T-cell responses were maintained for decades in long-term M. tuberculosis nonprogressors. Prominent Rv0867c-specific double- and single-cytokine-producing CD8(+) T-cell subset responses were found, including a large population of CD8(+) effector memory and effector T-cell subsets. We conclude that M. tuberculosis Rpf antigens are important targets in the human immune response to M. tuberculosis and represent interesting TB vaccine candidate antigens.     

重组结核抗原痘苗病毒Ankara株的构建及其免疫原性研究

目的 构建5种不同类型的表达结核杆菌特异抗原的重组痘苗病毒,并研究其特异免疫原性.方法 运用同源重组技术将含结核分泌抗原Ag85A和ESAT-6的基因片段插入痘苗病毒表达质粒p18中.重组质粒导入痘苗病毒Ankara(MVA)后构建重组痘苗病毒,经筛选和Western blot鉴定,得到5个种类的带有结核抗原基因的重组病毒.用构建的5种重组病毒免疫小鼠,MTT法检测免疫后小鼠脾淋巴细胞对特异结核抗原的增殖反应;ELISA检测小鼠脾淋巴细胞培养上清液中IFN-γ的含量;结核菌素纯蛋白衍化物(PPD)皮内试验以检测重组病毒引发的针对结核抗原的特异细胞免疫应答.结果 构建的5种蘑组病毒介导的细胞表达产物经Western blot鉴定确认相对分子质量与结核抗原一致.免疫小鼠两次后,5种重组病毒免疫组脾淋巴细胞体外与Ag85A-ESAT-6融合蛋白共培养后表现出明显的增殖活性(P<0.01),培养上清液中IFN-γ的浓度均较同组细胞经生理盐水刺激明显增高(P<0.05);与空痘苗病毒或生理盐水免疫后小鼠相比,5种重组MVA免疫组脾淋巴细胞与AgB5A.ESAT-6融合蛋白共培养后同样表现出明显的增殖活性(P<0.01),与Ag85A-ESAT-6融合蛋白共培养的细胞上清液中IFN-γ的浓度均升高(P<0.01).与空痘苗病毒或生理盐水免疫后小鼠相比,5种重组MVA免疫组小鼠对PPD都表现出显著的迟发型超敏反应应答(P<0.05).结论 成功构建了5种不同类型的表达结核杆菌抗原的重组痘苗病毒疫苗,其免疫小鼠后可激发针对结核杆菌抗原的特异性细胞免疫.     

Antigens of Mycobacterium tuberculosis expressed during preclinical tuberculosis: serological immunodominance of proteins with repetitive amino acid sequences

Four antigens of Mycobacterium tuberculosis that are expressed in vivo after aerosol infection but prior to the development of clinical tuberculosis (TB) in rabbits were identified by immunoscreening of an expression library of M. tuberculosis genomic DNA with sera obtained 5 weeks postinfection. Three of the proteins identified, PirG (Rv3810), polymorphic GC-repetitive sequence (PE-PGRS; Rv3367), and proline-threonine repetitive protein (PTRP) (Rv0538), have multiple tandem repeats of unique amino acid sequences and have characteristics of surface or secreted proteins. The fourth protein, MtrA (Rv3246c), is a response regulator of a putative two-component signal transduction system, mtrA-mtrB, of M. tuberculosis. All four antigens were recognized by pooled sera from TB patients and not from healthy controls, confirming their in vivo expression during active infection in humans. Three of the antigens (PE-PGRS, PTRP, and MtrA) were also recognized by retrospective preclinical TB sera obtained, prior to the clinical manifestation of TB, from human immunodeficiency virus-TB patients, suggesting that they are potential candidates for devising diagnostic tests for active, preclinical TB.     

Field evaluation of a novel differential diagnostic reagent for detection of Mycobacterium bovis in cattle

In the search for improved tools with which to control bovine tuberculosis, the development of enhanced immunodiagnostic reagents is a high priority. Such reagents are required to improve the performance of tuberculin-based reagents and allow the discrimination of vaccinated cattle from those infected with Mycobacterium bovis. In this study, we identified the immunodominant, frequently recognized peptides from Rv3873, Rv3879c, Rv0288, and Rv3019c, which, together with peptides comprising the current lead diagnostic antigens, ESAT-6 and CFP-10, were formulated into a peptide cocktail. In a test of naturally infected cattle, this cocktail was significantly better than tuberculin was for identifying skin test-negative animals with confirmed bovine tuberculosis. In addition, the specificity of this cocktail was not compromised by Mycobacterium bovis BCG vaccination. In summary, our results prioritize this peptide-based, fully synthetic reagent for assessment in larger trials.     

Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens

Culture filtrate from Mycobacterium tuberculosis contains protective antigens of relevance for the generation of a new antituberculosis vaccine. We have identified two previously uncharacterized M. tuberculosis proteins (TB7.3 and TB10.4) from the highly active low-mass fraction of culture filtrate. The molecules were characterized, mapped in a two-dimensional electrophoresis reference map of short-term culture filtrate, and compared with another recently identified low-mass protein, CFP10 (F. X. Berthet, P. B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel. Microbiology 144:3195-3203, 1998), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested in cultures of peripheral blood mononuclear cells from human tuberculosis (TB) patients, Mycobacterium bovis BCG-vaccinated donors, and nonvaccinated donors. The two ESAT-6 family members, TB10.4 and CFP10, were very strongly recognized and induced gamma interferon release at the same level (CFP10) as or at an even higher level (TB10.4) than ESAT-6. The non-ESAT-6 family member, TB7.3, for comparison, was recognized at a much lower level. CFP10 was found to distinguish TB patients from BCG-vaccinated donors and is, together with ESAT-6, an interesting candidate for the diagnosis of TB. The striking immunodominance of antigens within the ESAT-6 family is discussed, and hypotheses are presented to explain this targeting of the immune response during TB infection.     

Tuberculin skin testing compared with T-cell responses to Mycobacterium tuberculosis-specific and nonspecific antigens for detection of latent infection in persons with recent tuberculosis contact.

The tuberculin skin test (TST) is used for the identification of latent tuberculosis (TB) infection (LTBI) but lacks specificity in Mycobacterium bovis BCG-vaccinated individuals, who constitute an increasing proportion of TB patients and their contacts from regions where TB is endemic. In previous studies, T-cell responses to ESAT-6 and CFP-10, M. tuberculosis-specific antigens that are absent from BCG, were sensitive and specific for detection of active TB. We studied 44 close contacts of a patient with smear-positive pulmonary TB and compared the standard screening procedure for LTBI by TST or chest radiographs with T-cell responses to M. tuberculosis-specific and nonspecific antigens. Peripheral blood mononuclear cells were cocultured with ESAT-6, CFP-10, TB10.4 (each as recombinant antigen and as a mixture of overlapping synthetic peptides), M. tuberculosis sonicate, purified protein derivative (PPD), and short-term culture filtrate, using gamma interferon production as the response measure. LTBI screening was by TST in 36 participants and by chest radiographs in 8 persons. Nineteen contacts were categorized as TST negative, 12 were categorized as TST positive, and 5 had indeterminate TST results. Recombinant antigens and peptide mixtures gave similar results. Responses to TB10.4 were neither sensitive nor specific for LTBI. T-cell responses to ESAT-6 and CFP-10 were less sensitive for detection of LTBI than those to PPD (67 versus 100%) but considerably more specific (100 versus 72%). The specificity of the TST or in vitro responses to PPD will be even less when the proportion of BCG-vaccinated persons among TB contacts evaluated for LTBI increases.