Early Secretory Antigenic Target Research Articles

ESAT-6-ELISpot and interferon γ in the diagnosis of pleural tuberculosis

BackgroundAppropriate diagnostic methods for tuberculous pleural effusion are vital. The IFN-γ tests using specific Mycobacterium Tuberculos is antigens in samples from the site of infection may be promising in diagnosis of tuberculosis. Objective we examined the ability of ELISpot test using circulating peripheral blood mononuclear cells (PBMC) and compartmentalized pleural fluid mononuclear cells (PFMC) for diagnosis of active TB infection in patients with tuberculous pleural effusion. Methods PBMC and PFMC-based ELISpot test for IFN-γ test using specific M. tuberculosis antigen: Early Secretory Antigen Target-6 protein (ESAT-6) was used for diagnosis of active TB infection. Thirty-five patients with clinically suspected tuberculous pleural effusion were enrolled over a 12-month period. Results 11 patients out of 35 were positive by culture and PCR (31.4%). Incubation of PBMC with ESAT-6 for 8h showed sensitivity and specificity of 82% and 92%, respectively, for the PBMC–ELISpot as compared to PFMC–ELISpot that was 54% and 96% respectively. With 24h incubation of ESAT-6 there was around 2.5 fold increase in the median number of spot forming cells (SFCs) in PFMC from 30 to 74, whereas there was minimal increase of median number of SFCs in PBMC from 55 to 60. Conclusion ESAT-6 – ELISpot using PBMC and PFMC is useful as a tool for diagnosis of TB effusion. PFMC needs longer period of incubation for processing of ESAT-6 than PBMC. Moreover, IFN-γ in pleural effusion (PE) is another useful way for diagnosis of TB pleurisy which is sensitive, simple and cheap.

Rapid detection of Mycobacterium tuberculosis biomarkers in a sandwich immunoassay format using a waveguide-based optical biosensor

Early diagnosis of active tuberculosis (TB) remains an elusive challenge, especially in individuals with disseminated TB and HIV co-infection. Recent studies have shown a promise for the direct detection of pathogen-specific biomarkers such as lipoarabinomannan (LAM) for the diagnosis of TB in HIV-positive individuals. Currently, traditional immunoassay platforms that suffer from poor sensitivity and high non-specific interactions are used for the detection of such biomarkers. In this manuscript, we demonstrate the development of sandwich immunoassays for the direct detection of three TB-specific biomarkers, namely LAM, early secretory antigenic target 6 (ESAT6) and antigen 85 complex (Ag85), using a waveguide-based optical biosensor platform. Combining detection within the evanescent field of a planar optical waveguide with functional surfaces that reduce non-specific interactions allows for the ultra-sensitive and quantitative detection of biomarkers (an order of magnitude enhanced sensitivity, as compared to plate-based ELISA) in complex patient samples (urine, serum) within a short time. We also demonstrate the detection of LAM in urine from a small sample of subjects being treated for TB using this approach with excellent sensitivity and 100% corroboration with disease status. These results suggest that pathogen-specific biomarkers can be applied for the rapid and effective diagnosis of disease. It is likely that detection of a combination of biomarkers offers greater reliability of diagnosis, rather than detection of any single pathogen biomarker. NCT00341601.

Corticosteroid-modulated Immune Activation in the Tuberculosis Immune Reconstitution Inflammatory Syndrome

HIV-tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an immunopathological reaction to mycobacterial antigens induced by antiretroviral therapy. Prednisone reduces morbidity in TB-IRIS, but the mechanisms are unclear. To determine the effect of prednisone on the inflammatory response in TB-IRIS (antigen-specific effector T cells, cytokines, and chemokines). Blood was taken from participants in a randomized placebo-controlled trial of prednisone for TB-IRIS, at 0, 2, and 4 weeks. Participants received prednisone at a dosage of 1.5 mg/kg/day for 2 weeks followed by 0.75 mg/kg/day for 2 weeks, or placebo at identical dosages. Analyses included IFN-γ enzyme-linked immunospot (ELISPOT), reverse transcription-polymerase chain reaction on peripheral blood mononuclear cells after restimulation with heat-killed Mycobacterium tuberculosis, Luminex multiplex cytokine analysis of corresponding tissue culture supernatants, and Luminex multiplex cytokine analysis of serum. Fifty-eight participants with TB-IRIS (31 receiving prednisone, 27 receiving placebo) were included. In serum, significant decreases in IL-6, IL-10, IL-12 p40, tumor necrosis factor-α, IFN-γ, and IFN-γ-induced protein-10 concentrations during prednisone, but not placebo, treatment were observed. No differences in ELISPOT responses comparing prednisone and placebo groups were shown in response to ESAT-6 (early secreted antigen target-6), Acr1, Acr2, 38-kD antigen, or heat-killed H37Rv M. tuberculosis. Purified protein derivative ELISPOT responses increased over 4 weeks in the prednisone group and decreased in the placebo group (P = 0.007). The beneficial effects of prednisone in TB-IRIS appear to be mediated via suppression of predominantly proinflammatory cytokine responses of innate immune origin, not via a reduction of the numbers of antigen-specific T cells in peripheral blood.

Peripheral T Cell Cytokine Responses for Diagnosis of Active Tuberculosis

BackgroundA test for diagnosis of active Tuberculosis (TB) from peripheral blood could tremendously improve clinical management of patients.MethodsOf 178 prospectively enrolled patients with possible TB, 60 patients were diagnosed with pulmonary and 27 patients with extrapulmonary TB. The frequencies of Mycobacterium tuberculosis (MTB) specific CD4+ T cells and CD8+ T cells producing cytokines were assessed using overnight stimulation with purified protein derivate (PPD) or early secretory antigenic target (ESAT)-6, respectively.ResultsAmong patients with active TB, an increased type 1 cytokine profile consisting of mainly CD4+ T cell derived interferon (IFN)-γ was detectable. Despite contributing to the cytokine profile as a whole, the independent diagnostic performance of one cytokine producing T cells as well as polyfunctional T cells was poor. IFN-γ/Interleukin(IL)-2 cytokine ratios discriminated best between active TB and other diseases.ConclusionT cells producing one cytokine and polyfunctional T cells have a limited role in diagnosis of active TB. The significant shift from a “memory type” to an “effector type” cytokine profile may be useful for further development of a rapid immune-diagnostic tool for active TB.

CBA/J mice generate protective immunity to soluble Ag85 but fail to respond efficiently to Ag85 during natural Mycobacterium tuberculosis infection

In CBA/J mice, susceptibility to Mycobacterium tuberculosis (M.tb) is associated with low interferon-gamma (IFN-γ) responses to antigens (Antigen 85 (Ag85) and early secreted antigenic target-6 (ESAT-6)) that have been defined as immunodominant. Here, we asked whether the failure of CBA/J mice to recognize Ag85 is a consequence of M.tb infection or whether CBA/J mice have a general defect in generating specific T-cell responses to this protein antigen. We compared CBA/J mice during primary M.tb infection, Ag85 vaccination followed by M.tb challenge, or M.tb memory immune mice for their capacity to generate Ag85-specific IFN-γ responses and to control M.tb infection. CBA/J mice did not respond efficiently to Ag85 in the context of natural infection or re-infection. In contrast, CBA/J mice could generate Ag85-specific IFN-γ responses and protective immunity when this antigen was delivered as a soluble protein. Our data indicate that although M.tb infection of CBA/J mice does not drive an Ag85 response, these mice can fully and protectively respond to Ag85 if it is delivered as a vaccine. The data from this experimental model suggest that the Ag85-containing vaccines in clinical trials should protect M.tb susceptible humans.

Nanoparticle-based adjuvant for enhanced protective efficacy of DNA vaccine Ag85A-ESAT-6-IL-21 against Mycobacterium tuberculosis infection

The goal of this study was to evaluate the protective efficacy of a cationic nanoparticle-based DNA vaccine expressing antigen 85A (Ag85A) and 6-kDa early secretory antigen target (ESAT-6) of Mycobacterium tuberculosis as well as cytokine interleukin-21 (IL-21) against M. tuberculosis infection. The results of this indicated that the anti–M. tuberculosis immune responses were induced in mice that had received the different DNA vaccines. More importantly, compared with using DNA vaccine Ag85A-ESAT-6-IL-21 alone, the nanoparticle-based DNA vaccine Ag85A-ESAT-6-IL-21 showed a statistically significant increase in the protective efficacy against M. tuberculosis infection in the immunized mice. We concluded that the nanoparticle-based DNA vaccine induced a strong immune response and markedly inhibited the growth of the M. tuberculosis in the mice. These findings highlighted the potential utility of Fe3O4-Glu-polyethyleneimine nanoparticles encapsulated with the DNA vaccine as a prophylactic vaccine in the M. tuberculosis–infected mouse model. From the Clinical EditorThis study emphasizes the potential utility of Fe3O4-Glu-polyethyleneimine nanoparticles encapsulated with DNA vaccine against TB as a prophylactic vaccine. The authors demonstrated a strong immune response and marked growth inhibition of mycobacterium tuberculosis in the mice.

Massive monoclonal expansion of CD4 T-cells specific for aMycobacterium tuberculosisESAT-6 peptide

T-cell responses towards tuberculin (purified protein derivative; PPD) or the Mycobacterium tuberculosis-specific antigens early secretory antigenic target (ESAT)-6 and culture filtrate protein-10 are indicative of prior contact with mycobacterial antigens. In this study, we investigated the exceptional case of a 75-yr-old patient who devoted more than one-third of his CD4 T-cells against PPD and ESAT-6. Antigen-specific T-cells were characterised using flow cytometric intracellular cytokine staining, ELISPOT assay, proliferation assays, and T-cell receptor spectratyping. T-cell frequencies were far above those found in age-matched controls (median 0.33%, range 0.05-6.32%) and remained at high levels for >2 yrs. The patient initially presented with haemoptysis, but active tuberculosis was ruled out by repeated analysis of sputum and bronchoalveolar lavage fluid. Skin testing was negative and haemoptyses did not have a M. tuberculosis-related aetiology. Phenotypical and functional properties of specific T-cells were consistent with a terminally differentiated effector-memory phenotype with capacity to produce interferon-γ, interleukin-2 and tumour necrosis factor-α. Epitope mapping showed that the CD4 T-cells were directed against a single peptide from ESAT-6 (amino acid 5-20) that was presented in context of HLA-DR. T-cell receptor Vβ-spectratyping and sequencing of specific CD4 T-cells revealed a prominent peak fraction of monoclonal origin. In conclusion, similar to monoclonal gammopathies of undetermined significance, this may represent the first T-cell counterpart with known specificity against M. tuberculosis.

Development of an ultrasensitive polymerase chain reaction–amplified immunoassay based on mycobacterial RD antigens: implications for the serodiagnosis of tuberculosis

Immuno-polymerase chain reaction (I-PCR) combines the versatility of enzyme-linked immunosorbent assay (ELISA) with the exponential amplification power of PCR. The present study was designed to detect antibodies to Mycobacterium tuberculosis complex–specific region of difference (RD) antigens, i.e., early secretory antigenic target-6, culture filtrate protein-10, culture filtrate protein-21, and mycobacterial protein from species tuberculosis-64, as well as antigens in pulmonary tuberculosis patients by I-PCR assay. We could detect ESAT-6 and other RD antigens up to 0.1 fg by I-PCR assay, thus resulting in 10 7 times higher sensitivity than that observed with ELISA. With paired sample analysis based on the detection of antibodies in serum and antigens in sputum of the same individual, the sensitivity of RD multi-antigen cocktail-based I-PCR assay was 72% in smear-negative cases and 91% in smear-positive cases of pulmonary tuberculosis with high specificity values. In extrapulmonary tuberculosis patients, higher sensitivity was observed by detecting cocktail of antigens by I-PCR assay as compared to sensitivity earlier observed in the same samples by ELISA.

Distinct Clinical Features in Nontuberculous Mycobacterial Disease with or without Latent Tuberculosis Infection

Nontuberculous mycobacteria (NTM) diseases are in the face of a progressive increase even in immune-competent subjects, and the clinical features of NTM diseases are heterogenous. The decision to institute treatment of the patients should be made after a period of follow up, because therapy is often prolonged, and frequently ineffective. The reasons why some patients develop severe NTM diseases are not clear. Here we observed the involvement of latent tuberculosis infection (LTBI) in clinical and laboratory features of NTM diseases. We evaluated various tuberculosis-related inflammatory markers including osteopontin (OPN), pentraxin-3 (PTX-3), and soluble IL-2 receptor (sIL-2R) in NTM infected patients with or without LTBI. Eight NTM and 5 tuberculosis (TB) patients, and 5 healthy subjects were enrolled. Polymerase Chain Reaction (PCR) analysis confirmed the absence of tuberculosis specific gene (RD1 region), among clinical isolates from NTM patients. Interferon-γ (IFN-γ) release assay (IGRA) using Early Secreted Antigenic Target-6 (ESAT-6) and CFP-10, the RD1-encoded protein, was employed for determining LTBI. IGRA was positive in 4/8 NTM (NTM with LTBI, 50%) and 5/5 TB patients. Only 2 of 4 NTM with LTBI were under chemotherapy among all NTM patients, and others were followed up. The plasma levels of OPN, PTX3 and sIL-2R were significantly higher in NTM patients with LTBI than in those without LTBI (P < 0.05). The two patients under therapy showed the highest OPN levels that persisted after treatment. The increased inflammatory levels in NTM patients with LTBI indicate enhanced inflammatory reaction. Extensive therapy may be necessary in such patients.

Les nouveaux tests sanguins dans le diagnostic de l’infection tuberculeuse

The diagnosis of latent tuberculosis infection was based until now on the positivity of the tuberculin skin test. This test poses considerable technical problems of implementation, reading and interpretation. Moreover, it lacks both specificity in cases of previous BCG vaccination and sensitivity in case of immunosuppression. The understanding of cellular immunity with the contribution of molecular biology have helped to develop blood tests that measure in vitro release of interferon Gamma by T cells after stimulation by highly specific antigens of Mycobacterium tuberculosis that are ESAT6 (Early Secretory Antigenic Target 6Kda) and CFP10 (culture filtrate protein 10) and recently the Ag tb 7.7. These antigens are absent in the BCG vaccine and most nontuberculous mycobacteria. Two tests are commercialised, one measures the interferon-secreting cells by ELISpot method: the Enzyme-Linked Immuno Spot. As another measure directly interferon by ELISA: Enzyme-Linked Immuno-Sorbent Assay. The current clinical data suggest that these blood tests are more specific than the tuberculin skin test because they did not cross-react with BCG. This high specificity avoids the costs and possible side effects of unnecessary treatment among persons vaccinated with a positive tuberculin test. However in cases of active tubersulosis, the ELISA has the same sensitivity as the TST, while ELISpot appears more sensitive. In patients with negative tuberculin test with a high risk to progress to tuberculosis disease, the sensitivity of these blood tests can be considered a treatment for tuberculosis. The purpose of this paper is to recall the principles of these diagnostic tests, review the evidence on their usefulness, their limitations and their indications.

Identification of RD5-EncodedMycobacterium tuberculosisProteins As B-Cell Antigens Used for Serodiagnosis of Tuberculosis

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, all encoded proteins from DNA segment RD5 of Mycobacterium tuberculosis, that is, Rv3117–Rv3121, were recombined and evaluated by enzyme-linked immunosorbent assays for antibody reactivity with sera from HIV-negative pulmonary TB patients (n = 60) and healthy controls (n = 32). The results identified two immunodominant antigens, that is, Rv3117 and Rv3120, both of which revealed a statistically significant antigenic distinction between healthy controls and TB patients (P < 0.05). In comparison with the well-known early-secreted antigen target 6 kDa (ESAT-6) (sensitivity 21.7%, specificity 90.6%), the higher detection sensitivity and higher specificity were achieved (Rv3117: sensitivity 25%, specificity 96.9%; Rv3120: sensitivity 31.7%, specificity 96.9%). Thus, the results highlight the immunosensitive and immunospecific nature of Rv3117 and Rv3120 and indicate promise for their use in the serodiagnosis of TB.

Novel M tuberculosis antigen-specific T-cells are early markers of infection and disease progression.

Background Mycobacterium tuberculosis Region-of-Difference-1 gene products present opportunities for specific diagnosis of M. tuberculosis infection, yet immune responses to only two gene-products, Early Secretory Antigenic Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10), have been comprehensively investigated.MethodsT-cell responses to Rv3873, Rv3878 and Rv3879c were quantified by IFN-γ-enzyme-linked-immunospot (ELISpot) in 846 children with recent household tuberculosis exposure and correlated with kinetics of tuberculin skin test (TST) and ESAT-6/CFP-10-ELISpot conversion over six months and clinical outcome over two years.ResultsResponses to Rv3873, Rv3878, and Rv3879c were present in 20–25% of contacts at enrolment. Rv3873 and Rv3879c responses were associated with and preceded TST conversion (P = 0.02 and P = 0.04 respectively), identifying these antigens as early targets of cell-mediated immunity following M. tuberculosis exposure. Responses to Rv3873 were additionally associated with subsequent ESAT-6/CFP-10-ELISpot conversion (P = 0.04). Responses to Rv3873 and Rv3878 predicted progression to active disease (adjusted incidence rate ratio [95% CI] 3.06 [1.05,8.95; P = 0.04], and 3.32 [1.14,9.71; P = 0.03], respectively). Presence of a BCG-vaccination scar was associated with a 67% (P = 0.03) relative risk reduction for progression to active tuberculosis.ConclusionsThese RD1-derived antigens are early targets of cellular immunity following tuberculosis exposure and T-cells specific for these antigens predict progression to active tuberculosis suggesting diagnostic and prognostic utility.

The CFP10/ESAT6 complex of Mycobacterium tuberculosis may function as a regulator of macrophage cell death at different stages of tuberculosis infection

Tuberculosis is a human disease caused by infection with Mycobacterium tuberculosis. M. tuberculosis (Mtb) is a facultative intracellular pathogen. The alveolar macrophages provide a critical niche for the intracellular pathogen. It has been shown that virulent strains mycobacteria (Mtb-H37Rv, Mycobacterium bovis) induce less apoptosis in host macrophage than avirulent mycobacteria strains (Bacillus Calmette-Guérin, H37Ra). Comparative genomics analysis has revealed that the region of difference (RD1) of M. tuberculosis is absent from all strains of Bacillus Calmette-Guérin (BCG). On the contrary, it presents in all virulent strains of M. tuberculosis and M. bovis. The culture filtrate protein 10 (CFP10) and early secretory antigenic target protein 6 (ESAT6) are encoded by RD1 genes Rv3874 and Rv3875, respectively. Recent studies indicated that the CFP10 and ESAT6 played an important role in M. tuberculosis virulence. It has been shown that incorporation of the RD1 region into BCG to restore the expression of CFP10 and ESAT6 leads to increasing the virulence and immunogenicity of bacterium. On the contrary, deletion of the genes encoding CFP10 and ESAT6 from the virulent M. bovis strain results in attenuation of virulence. Meanwhile, several studies showed that CFP10 and ESAT6 could inhibit and/or promote the production of tumor necrosis factor α (TNF-α) from macrophages. Furthermore, TNF-α can induce apoptosis and necrosis of infected macrophages in tuberculosis. Considering above results, we hypothesize that the CFP10 and ESAT6 may be involved in the virulence of Mycobacterium through modulating macrophage cell death.

Protection against Mycobacterium tuberculosis challenge in mice by DNA vaccine Ag85A‐ESAT‐6‐IL‐21 priming and BCG boosting

Tuberculosis (TB) is one of most important chronic infectious diseases caused by Mycobacterium tuberculosis and remains a major global health problem. In the study, we developed the DNA vaccine encoding fusion protein of antigen 85 A and 6 kDa early secretory antigen target of M. tuberculosis as well as the cytokine IL-21 to investigate its immune protective efficacy against M. tuberculosis challenge in mice after the DNA vaccine priming and Bacille Calmette-Guérin (BCG) boosting. Compared with the different control groups, the intranasal DNA vaccine priming twice and BCG boosting once markedly increased the cytotoxicities of natural killer cells and splenocytes and enhanced the interferon-γ level in the splenocyte supernatant as well as sIgA level in bronchoalveolar lavage in the vaccinated mice. Importantly, this heterologous prime-boost strategy significantly decreased the bacterial load in the mouse lungs in contrast to that of intranasal or subcutaneous BCG immunization alone. These findings provide further approaches for mucosal-targeted prime-boost vaccination to fight against TB.

Evaluation of a Tuberculosis Whole-Blood Interferon-γ Chemiluminescent Immunoassay among Chinese Military Recruits

China is a country with a high prevalence of latent tuberculosis (TB) infection (LTBI) and has a policy of routine bacillus Calmette-Guérin (BCG) vaccination. The purified protein derivative (PPD) skin test cannot distinguish TB infection from BCG vaccination, and the diagnosis of LTBI lacks an accepted standard method. The primary objective of this study was to assess the potential of a highly sensitive whole-blood interferon (IFN)-γ release assay that uses recombinant culture filtrate protein (CFP)-10/early secretory antigenic target (ESAT)-6 fusion protein (rCFP-10/ESAT-6) as a stimulus for diagnosis of LTBI. Between December 2009 and March 2010, a total of 892 new recruits to the army in Beijing, China, were interviewed and routinely examined by chest radiographs. IFNγ released from whole blood in response to stimulation with rCFP-10/ESAT-6 was detected with an enzyme-linked chemiluminescent immunoassay. The recruits were also intradermally injected with PPD to assess 72-hour skin induration (the PPD skin test). Of the 892 participants, 450 (50.4%) had a positive PPD skin test and 244 (27.4%) had a positive whole-blood IFNγ release assay. Of 442 PPD-negative subjects, 88 (19.9%) had a positive whole-blood IFNγ test. Of 450 PPD-positive subjects, 156 (34.7%) had a positive whole-blood IFNγ test. Of 132 strongly PPD-positive subjects, 62 (47.0%) had a positive whole-blood IFNγ test. The agreement between the two tests was 57.2%. Of the 892 participants, 579 (64.9%) had clear vaccination scars on their arms, and of these, 382 (66.0%) had positive PPD skin responses and 162 (28.0%) were positive for the whole-blood IFNγ test. The new whole-blood IFNγ release assay might be a better indicator of LTBI than the PPD test in China.

Multiple Cytokines Are Released When Blood from Patients with Tuberculosis Is Stimulated with Mycobacterium tuberculosis Antigens

Background Mycobacterium tuberculosis (Mtb) infection may cause overt disease or remain latent. Interferon gamma release assays (IGRAs) detect Mtb infection, both latent infection and infection manifesting as overt disease, by measuring whole-blood interferon gamma (IFN-γ) responses to Mtb antigens such as early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and TB7.7. Due to a lack of adequate diagnostic standards for confirming latent Mtb infection, IGRA sensitivity for detecting Mtb infection has been estimated using patients with culture-confirmed tuberculosis (CCTB) for whom recovery of Mtb confirms the infection. In this study, cytokines in addition to IFN-γ were assessed for potential to provide robust measures of Mtb infection.MethodsCytokine responses to ESAT-6, CFP-10, TB7.7, or combinations of these Mtb antigens, for patients with CCTB were compared with responses for subjects at low risk for Mtb infection (controls). Three different multiplexed immunoassays were used to measure concentrations of 9 to 20 different cytokines. Responses were calculated by subtracting background cytokine concentrations from cytokine concentrations in plasma from blood stimulated with Mtb antigens.ResultsTwo assays demonstrated that ESAT-6, CFP-10, ESAT-6+CFP-10, and ESAT-6+CFP-10+TB7.7 stimulated the release of significantly greater amounts of IFN-γ, IL-2, IL-8, MCP-1 and MIP-1β for CCTB patients than for controls. Responses to combination antigens were, or tended to be, greater than responses to individual antigens. A third assay, using whole blood stimulation with ESAT-6+CFP-10+TB7.7, revealed significantly greater IFN-γ, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1β, and TNF-α responses among patients compared with controls. One CCTB patient with a falsely negative IFN-γ response had elevated responses with other cytokines.ConclusionsMultiple cytokines are released when whole blood from patients with CCTB is stimulated with Mtb antigens. Measurement of multiple cytokine responses may improve diagnostic sensitivity for Mtb infection compared with assessment of IFN-γ alone.

Evaluation of immunogenicity and protective efficacy against Mycobacterium tuberculosis infection elicited by recombinant Mycobacterium bovis BCG expressing human Interleukin-12p70 and Early Secretory Antigen Target-6 fusion protein

ESAT-6 protein of Mycobacterium tuberculosis is absent in Mycobacterium bovis BCG and Mycobacterium microti and has been demonstrated to stimulate strong cell-mediated immunity. IL-12 can play crucial roles in regulating IFN-γ production and Th1 effectors production. In this study, we constructed three rBCG vaccines that could express proteins of human IL-12p70 and/or ESAT-6 and evaluated their immunogenicity and protective efficacy in mice. Our experiments illustrated that the rBCG-IE (expressing a fusion protein of human IL-12p70 and ESAT-6) was capable of inducing stronger Th1 type cell-mediated immune responses than conventional BCG, or rBCG-I (expressing human IL-12p70), or rBCG-E (expressing ESAT-6). However, the results of protective experiments showed that rBCG-IE could only confer similar and even lower protective efficacy against M. tuberculosis H37Rv infection compared with BCG vaccine.

Tuberculosis-specific T-cell response after recent treatment and remote cure

To the Editors: Tuberculosis (TB) remains a major global health problem. A major factor contributing to the current TB epidemic is the resistance of Mycobaterium tuberculosis to first-line anti-TB treatment. Therefore, there is a need for an appropriate and reliable tool to evaluate treatment efficacy. Several studies using the T-SPOT®. TB assay (Oxford Immunotec Ltd, Oxford, UK) reported a decline in the number of spots after successful anti-TB treatment, particularly in the 6-kDa early secretory antigenic target (ESAT-6) panel [1–3]; however, to our knowledge, no data have been published on cured TB patients who contracted TB before isoniazid and rifampicin became available. Therefore, the naturally long-term range of ESAT-6- and culture filtrate protein (CFP)-10-specific T-cell responses in patients with TB who did not receive modern anti-TB treatment is not known. Because most patients in prior studies were born in high-burden, TB-endemic countries, the T-cell kinetics might be influenced by reinfection. Therefore, the aim of this report is to compare the ESAT-6- and CFP-10-specific T-cell responses in a low-burden country with a low risk of reinfection between patients with a history of active TB before 1955, and patients with recently treated active TB. Venous blood for T-SPOT®. TB was collected from 24 outpatient subjects with a history of active TB before 1955 (group 1), obtained by clinical history-taking, patients’ medical records or documentation of a sanatorium admission. 16 of these patients had been admitted to a sanatorium, two patients underwent surgery and six patients were treated at home with para-aminosalicylic acid therapy or bedrest. Furthermore, 27 patients with recent active TB (group 2) were prospectively enrolled between March and May 2008. Active TB was defined as fulfilling one or more of the following criteria: 1) positive culture for M. tuberculosis (n=22); 2) positive PCR for M. …

B lymphocytes that migrate to tuberculous pleural fluid via the SDF‐1/CXCR4 axis actively respond to antigens specific for Mycobacterium tuberculosis

B-cell biology has been largely uncharacterized in the field of tuberculosis (TB). In this study, we investigated the immunophenotypical and functional characteristics of B cells obtained from the pleural fluid (PF) and peripheral blood of patients with tuberculous pleuritis (TP). Our results indicated that the total numbers of B cells, CD27(+) memory B cells and plasmablasts were clearly lower in the PF than in peripheral blood. Furthermore, we found significantly higher expression of CXCR4 on B cells in the PF, and a chemotaxis assay showed that B cells in the PF were more responsive to stromal cell-derived factor-1 (SDF-1) than B cells from peripheral blood. In addition, SDF-1 levels in PF were remarkably high compared with SDF-1 levels in plasma, suggesting that the SDF-1/CXCR4 axis might facilitate the migration of circulating B cells into tuberculous pleural space. Importantly, we observed that significantly more antibodies were produced by B cells in the PF following stimulation with BCG, early secretory antigenic target (ESAT-6)/culture filtrate protein-10 (CFP-10) or ESAT-6 protein. Collectively, these data demonstrate that Mycobacterium tuberculosis-specific B cells exist at local sites of infection in TP patients and this localization might influence the immune response to M. tuberculosis.

Diagnostic value of early secretory antigenic target⁃6 and interferon⁃γ in cerebrospinal fluid for the diagnosis of tuberculous meningitis

Objective To explore the value of early secretory antigenic target-6 (ESAT-6) and interferon-γ (IFN-γ) in cerebrospinal fluid (CSF) for the diagnosis of tuberculous meningitis (TBM). Methods Eighty-one patients were examined in our hospital or outpatient clinic. Referenced to the diagnostic criteria of TBM, patients were divided into 3 groups: 21 cases were patients with TBM; 28 were patients with intracranial infection of non-TBM; and 32 controls without nervous system disease. CSF sample was collected by standard lumbar puncture and was used for detection of ESAT-6 and IFN-γ with enzyme-linked immunosorbent assay (ELISA). Results The concentration of ESAT-6 in the CSF of TBM group was 4.46 pg/ml [(2.20-10.55) pg/ml] and the IFN-γ was 34.86 ng/L [(25.62-241.71) ng/L]. The average concentrations of ESAT-6 and IFN-γ in the intracranial infection of non-TBM group and the control group were (1.18 ± 0.49) pg/ml, (12.00 ± 3.37) ng/L and (1.05 ± 0.47) pg/ml, (14.58 ± 3.46) ng/L, respectively. The levels of ESAT-6 and IFN-γ were significantly higher in the TBM group than in the intracranial infection of non-TBM group (H = 35.695, P = 0.000) and the control group (H = 31.560, P = 0.000), but were not significantly different between the intracranial infection of non-TBM group and the control group (t ＝ 1.226, P = 0.226; t = 0.060, P = 0.952, respectively). The Spearman rank correlation analysis showed positive correlation between ESAT-6 and IFN-γ detected in the TBM group (rs = 1.000, P = 0.000). Conclusion The levels of ESAT-6 and IFN-γ in the TBM group were significantly different from the intracranial infection of non-TBM group and the control group. The 2 indicators present certain correlation and can be helpful in the clinical diagnosis of TBM. DOI：10.3969/j.issn.1672-6731.2011.05.014