Early Secretory Antigenic Target Research Articles

Polyantigenic Interferon-γ Responses Are Associated with Protection from TB among HIV-Infected Adults with Childhood BCG Immunization

BackgroundSurrogate immunologic markers for natural and vaccine-mediated protection against tuberculosis (TB) have not been identified.MethodsHIV-infected adults with childhood BCG immunization entering the placebo arm of the DarDar TB vaccine trial in Dar es Salaam, Tanzania, were assessed for interferon gamma (IFN-γ) responses to three mycobacterial antigen preparations – secreted Mycobacterium tuberculosis antigens 85 (Ag85), early secretory antigenic target 6 (ESAT-6) and polyantigenic whole cell lysate (WCL). We investigated the association between the number of detectable IFN-γ responses at baseline and the subsequent risk of HIV-associated TB.ResultsDuring a median follow-up of 3.3 years, 92 (9.4%) of 979 placebo recipients developed TB. The incidence of TB was 14% in subjects with no detectable baseline IFN-γ responses vs. 8% in subjects with response to polyantigenic WCL (P = 0.028). Concomitant responses to secreted antigens were associated with further reduction in the incidence of HIV-associated TB. Overall the percentage of subjects with 0, 1, 2 and 3 baseline IFN-γ responses to mycobacterial preparations who developed HIV-associated TB was 14%, 8%, 7% and 4%, respectively (P = 0.004). In a multivariate Cox regression model, the hazard of developing HIV-associated TB was 46% lower with each increment in the number of detectable baseline IFN-γ responses (P<0.001).ConclusionsAmong HIV-infected adults who received BCG in childhood and live in a TB-endemic country, polyantigenic IFN-γ responses are associated with decreased risk of subsequent HIV-associated TB.Trial RegistrationClinicalTrials.gov NCT0052195

The auxiliary diagnosis value of enzyme-linked immunosorbent spot assay and flow cytometry in detection of IFN-y in tuberculous pleurisy

Objective To explore the value of IFN-γ produced or secreted by CD+4 T Lymphocytes from pleural effusion mononuclear cells for the diagnosis of tuberculous pleurisy(plTB).Methods The PEMCs of 40 patients with tuberculous pleural effusion and 30 patients with malignancy pleural effusion were selected as the tuberculosis and disease control groups, then co-cultured with the early secretory antigenic target 6 (ESAT-6) and culture filtered protein 10 (CFP-10) fusion protein (E/C).The numbers of spot forming cells(SFC) secreting IFN-γ were enumerated by ELISpot and the ratios of cells producing IFN-γ were detected by flow cytometry and intracellular cytokine staining.Moreover, the two indicators were compared between tuberculosis and disease control groups to evaluate the 2 methods detecting IFN-γ in the diagnosis of plTB.Results After E/C stimulation, the numbers of SFC were 205(125-450)SFC/5×104 PEMC in tuberculosis group and 5(2-18)SFC/5×104 PEMC in disease control group by ELISpot.The difference between two groups was statistically significant (U= 20.00, P<0.01).The proportion of IFN-γ-secreting CD+4 T lymphocytes was 3.27% (1.81%-7.34%) in tuberculosis group and 0.12% (0.06%-0.46%) in control group detected by FCM. The difference between the two groups was statistically significant (U=45.00, P<0.01).The indicators of ELISpot in detection of IFN-γ which was secreted by PEMC after co-cultured with E/C were as follows: sensitivity 92.5% (37/40), specificity 80.0% (24/30), positive predictive value 0.86, negative predictive value 0.89, positive likelihood ratio 4.63, negative likelihood ratio 0.09 and accuracy 87.1%;and for FCM, they were 87.5% (35/40), 90.0% (27/30), 0.92, 0.84, 8.75 and 0.14, respectively and accuracy 88.6%.Conclusion After E/C stimulation, the assay for IFN-γ-secreting CD+4 T lymphocytes by FCM and ELISpot is highly sensitive and specific for diagnosis of plTB as an auxiliary method. Key words: Tuberculosis, pleural; Immunoenzyme techniques; Flow cytometry

A novel recombinant &amp;lt;italic&amp;gt;Mycobacterium bovis&amp;lt;/italic&amp;gt; bacillus Calmette&amp;minus;Guerin strain expressing human granulocyte macrophage colony-stimulating factor and &amp;lt;italic&amp;gt;Mycobacterium tuberculosis&amp;lt;/italic&amp;gt; early secretory antigenic target 6 complex augments Th1 immunity

Since Mycobacterium bovis bacillus Calmette-Guerin strain (BCG) fails to protect adults from pulmonary tuberculosis (TB), there is an urgent need for developing a new vaccine. In this study, we constructed a novel recombinant BCG strain (rBCG) expressing human granulocyte macrophage colony-stimulating factor (GM-CSF) and the 6 kDa early secretory antigenic target (ESAT6) of Mycobacterium tuberculosis, named rBCG:GE (expressing GMCSF-ESAT6 complex), and evaluated the immunogenicity of the construct in BALB/c mice. Our results indicated that the rBCG:GE was able to induce higher titer of antibody than the conventional BCG, the rBCG:G (expressing GM-CSF) and the rBCG:E (expressing ESAT6). Moreover, the rBCG:GE also elicited a longer-lasting and stronger Th1 cellular immune responses than the other groups, which was confirmed by the incremental proliferation of splenocytes, the increased percentages of CD4(+) and CD8(+) T cells of spleen, the elevated level of interferon-γ in splenocyte culture after tuberculin-purified protein derivative stimulation, and the increased concentration of GM-CSF in serum. The data presented here suggested the possibility that the recombinant BCG:GE might be a good vaccine candidate to TB.

Hepatitis B virus core particles displaying Mycobacterium tuberculosis antigen ESAT-6 enhance ESAT-6-specific immune responses

Early secreted antigenic target-6 (ESAT-6), an important Mycobacterium tuberculosis T-cell antigen, is an attractive candidate antigen for tuberculosis subunit vaccine development. Because ESAT-6 has a low inherent immunogenicity, we used Hepatitis B virus core (HBc) protein as an immune carrier to enhance ESAT-6 immunogenicity. The ESAT-6 gene was inserted into the major immunodominant region of the HBc molecule by fusion PCR. The recombinant protein, HBc-ESAT-6 (HE6), was expressed in Escherichia coli, and electron microscopy confirmed the formation of virus-like particles. The immunogenicity of the chimeric particles was assessed in mice. Serological assays and in vitro Th1-biased cytokine assays found that immunization with HE6 particles elicited significantly higher ESAT-6-specific antibodies and CD4 +/CD8 + T cell responses in mice compared to immunization with recombinant ESAT-6 protein. These data demonstrate the feasibility of HBc particles serving as an efficient immune carrier for ESAT-6 and suggest that HE6 has potential for use in a tuberculosis subunit vaccine.

Novel monoclonal antibodies to ESAT-6 and CFP-10 antigens for ELISA-based diagnosis of pleural tuberculosis

To elucidate the potential of monoclonal antibodies (mAbs) of culture filtrate protein 10 (CFP-10) and early secretory antigenic target 6 (ESAT-6) in tuberculosis (TB) diagnosis. We generated and characterised monoclonal and polyclonal antibodies against Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 by immunising BALB/c mice with an ESAT-6/CFP-10 fusion protein. Stable hybridoma cell lines were established and mAbs were specifically identified by immunoblotting and immunoprecipitation. The mouse mAbs were used to coat plates, and biotin-labelled polyclonal antibodies were used to detect the antigens. One hundred and seventy-three samples of sputum culture supernatants and pleural effusion aspirates have been tested. The ESAT-6 enzyme-linked immunosorbent assay (ELISA) detected the culture supernatants and pleural effusion specimens that were positive for M. tuberculosis, but failed to identify M. tuberculosis-positive specimens in the non-M. tuberculosis culture supernatants or control specimens. This yielded a sensitivity of 95.4% and a specificity of 100% for the ESAT-6-specific ELISA. The CFP-10 ELISA presented less satisfactory sensitivity and specificity, of respectively 81.6% and 92.2%. Results showed positive detection rates of ESAT-6 and CFP-10 of 86.8% (33/38) and 76.3% (29/38) for the diagnosis of tuberculous pleural effusion in patients bacteriologically negative for M. tuberculosis culture. The ESAT-6 and CFP-10 ELISAs incorporating mAbs generated in this study serve as potential tools in the laboratory diagnosis of TB.

T Cell Monitoring of Chemotherapy in Experimental Rat Tuberculosis

Mycobacterium tuberculosis is the causative agent of a pulmonary epidemic that is estimated to infect one-third of the world's population and that has an increased incidence of multidrug resistance. The evaluation of new chemical entities against M. tuberculosis is hampered by the lack of biological tools to help predict efficacy, from early drug development to clinical trials. As the rat is the animal species of choice in the pharmaceutical industry, we have developed a rat model of acute and chronic phases of M. tuberculosis infection for drug efficacy testing. In this model, we have evaluated the impact of tuberculosis drugs on T cell response using the enzyme-linked immunospot assay methodology. Infected rats treated with isoniazid (INH) or rifampin (RIF) responded to therapy, the potency of which was comparable to that seen in the mouse. Peripheral blood mononuclear cells from infected rats produced gamma interferon (IFN-γ) in response to RD-1 antigens, such as the 6-kDa early secretory antigen target (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10). A decrease in IFN-γ spot-forming cells (SFCs) was consistently observed in response to drug treatment. In both the acute- and chronic-phase models, the T cell response was more sensitive to ESAT-6 than to CFP-10. The SFC count in response to ESAT-6 appears to be an indicator of bacterial killing in the rat. Collectively, our data suggest that the ESAT-6 response could be used as a potential surrogate of drug efficacy in the rat and that such a readout could help shorten drug testing during preclinical development.

Assessment of in vivo and in vitro tuberculosis diagnostic tests in Mycobacterium caprae naturally infected caprine flocks

Caprine tuberculosis in Spain is mainly caused by Mycobacterium caprae although the progression of the disease and lesion severity is similar to that caused by Mycobacterium bovis. In this study, the sensitivity of the gamma-interferon (IFN-γ) assay using an antigen cocktail containing early secretory antigenic target-6 kDa (ESAT-6) and culture filtrate protein 10 (CFP-10) peptides for stimulation was determined and compared with those obtained in single intradermal tuberculin (SIT) and single intradermal cervical comparative tuberculin (SICCT) tests and IFN-γ assay using purified protein derivative (PPD) in three different flocks infected with M. caprae under different epidemiological conditions. Correlation between specific IFN-γ production and severity of lesions was also evaluated. Sensitivities of the diagnostic tests varied greatly in the three flocks studied, with higher values in those where higher lesion scores were observed. The results show that IFN-γ assay applied in goats using PPD or the ESAT-6/CFP-10 peptides cocktail for stimulation yielded similar sensitivity values. A significant yet weak positive correlation between specific IFN-γ production and lesion scores was detected after the stimulation with PPDs ( p = 0.004) whereas when the blood samples were stimulated with ESAT-6/CFP-10 peptides, the correlation was not significant ( p > 0.05). Therefore, specific-IFN-γ production after the stimulation with PPDs or ESAT-6/CFP-10 was not an accurate indicator of lesion severity in naturally tuberculosis infected goats with M. caprae.

Role of BAT3 in modulating the immune response (111.12)

Abstract HLA-B-associated transcript 3 (BAT3) also known as Scythe or BAG6, is a nuclear protein that has recently been implicated in the control of apoptosis and in NK-DC cell cross-talk. Here, we demonstrate that BAT3 modulates the immune response by regulating the function of macrophages and dendritic cells. BAT3 is secreted by macrophages and dendritic cells in vitro and extracellular BAT3 down-regulates the production of nitric oxide and proinflammatory cytokines in IFN-γ and LPS stimulated macrophages. Extracellular BAT3 also down-regulates the LPS-stimulated maturation of dendritic cells. The immunodominant Mycobacterium tuberculosis antigen ESAT-6 (early secreted antigenic target-6) up-regulates BAT3 expression in macrophages and dendritic cells in vitro. Intracellular BAT3 regulates ESAT-6 induced apoptosis of macrophages by interacting with the anti-apoptotic protein BCL-2 (B-cell lymphoma 2) indicating that BAT3 may play a role in pathogenesis of tuberculosis. Taken together, the data suggest that BAT3 may exert different immunomodulatory regulations depending on whether it is intra-or extracellular.

Rv3615c is a highly immunodominant RD1 (Region of Difference 1)-dependent secreted antigen specific for Mycobacterium tuberculosis infection

The 6-kDa early secretory antigenic target of Mycobacterium tuberculosis (ESAT-6) and the 10-kDa culture filtrate antigen (CFP-10), encoded in region of difference 1 (RD1) and secreted by the ESAT-6 system 1 (Esx-1) secretion system, are the most immunodominant and highly M. tuberculosis (MTB)-specific antigens. These attributes are responsible for their primary importance in tuberculosis (TB) immunodiagnosis and vaccine development. Rv3615c [Esx-1 substrate protein C (EspC)], encoded outside RD1, is similar in size and sequence homology to CFP-10 and ESAT-6, suggesting it might be a target of cellular immunity in TB. Using ex vivo enzyme-linked immunospot- and flow cytometry-based cytokine-secretion assay, we comprehensively assessed cellular immune responses to EspC in patients with active TB, latently infected persons, and uninfected bacillus Calmette-Guérin (BCG)-vaccinated controls. EspC was at least as immunodominant as ESAT-6 and CFP-10 in both active and latent TB infection. EspC contained broadly recognized CD4(+) and CD8(+) epitopes, inducing a predominantly CD4(+) T-cell response that comprised functional T-cell subsets secreting both IFN-γ and IL-2 as well as functional T-cell subsets secreting only IFN-γ. Surprisingly, T-cell responses to EspC were as highly specific (93%) for MTB infection as responses to ESAT-6 and CFP-10, with only 2 of 27 BCG-vaccinated controls responding to each antigen. Using quantitative proteomics and metabolically labeled mutant and genetically complemented MTB strains, we identified the mechanism of the specificity of anti-EspC immunity as the Esx-1 dependence of EspC secretion. The high immunodominance of EspC, equivalent to that of ESAT-6 and CFP-10, makes it a TB vaccine candidate, and its high specificity confers strong potential for T-cell-based immunodiagnosis.

ESAT‐6‐ and CFP‐10‐Specific Th1, Th22 and Th17 Cells in Tuberculous Pleurisy May Contribute to the Local Immune Response Against Mycobacterium Tuberculosis Infection

Th1 cell-mediated adaptive immune response is very important but may not be sufficient to control Mycobacterium tuberculosis (M. tuberculosis) infection. The roles of the various T cell subsets and cytokines in the inflammatory processes are not clearly elucidated. We investigated whether Th1, Th22 and Th17 cells mediated cellular immunity at the local site of M. tuberculosis infection in patients with tuberculous pleurisy (TBP). The results showed that the cytokines IFN-γ and IL-22 but not IL-17 were elevated in tubercular pleural fluid. Following stimulation with immune-dominant peptides of early secreted antigenic target-6 (ESAT-6), culture filtrate protein-10 (CFP-10) or Bacille Calmette-Guerin, pleural fluid mononuclear cells expressed high levels of cytokines IFN-γ, IL-22 and IL-17 as revealed by mRNA and protein measurements. In addition, we showed that cytokines IFN-γ, IL-22 and IL-17 were produced in M. tuberculosis-specific immune response by distinct subsets of CD4+ T cells with the phenotype of CD45RA-CD62L-CCR7+CD27+ . Our results demonstrated for the first time that ESAT-6- and CFP-10-specific Th1, Th22 and Th17 cells existed in the patients with TBP and might play an essential role against M. tuberculosis infection. The findings of this study raised the possibility of unravelling the critical targets for therapeutic intervention in chronic inflammatory diseases such as TBP.

A multistage tuberculosis vaccine that confers efficient protection before and after exposure

All tuberculosis vaccines currently in clinical trials are designed as prophylactic vaccines based on early expressed antigens. We have developed a multistage vaccination strategy in which the early antigens Ag85B and 6-kDa early secretory antigenic target (ESAT-6) are combined with the latency-associated protein Rv2660c (H56 vaccine). In CB6F1 mice we show that Rv2660c is stably expressed in late stages of infection despite an overall reduced transcription. The H56 vaccine promotes a T cell response against all protein components that is characterized by a high proportion of polyfunctional CD4(+) T cells. In three different pre-exposure mouse models, H56 confers protective immunity characterized by a more efficient containment of late-stage infection than the Ag85B-ESAT6 vaccine (H1) and BCG. In two mouse models of latent tuberculosis, we show that H56 vaccination after exposure is able to control reactivation and significantly lower the bacterial load compared to adjuvant control mice.

Cytokine and Chemokine Responses to Selected Early Secreted Antigenic Target-6 and Culture Filtrate Protein-10 Peptides in Tuberculosis

Cytokine [tumor necrosis factor-α, interleukin-2 (IL-2), IL-4] and chemokine [regulated upon activation normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein-1] responses to selected early secreted antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) peptides were studied in healthy household contacts and patients with pulmonary tuberculosis (PTB). It was observed that Th1 cytokines and chemokine RANTES positive T cells were elevated in response to the peptides Esp1, Esp6, Cfp6, and Cfp8 in healthy household contacts. IL-4 positive T cells were enhanced by Esp1 and Esp6 in PTB. Monocyte chemoattractant protein-1 positive monocytes increased in response to the peptides Esp1, Esp6, Cfp8, and Cfp9 in PTB. These peptides deserve attention for further immune studies.

Using a Label-free Proteomics Method to Identify Differentially Abundant Proteins in Closely Related Hypo- and Hypervirulent Clinical Mycobacterium tuberculosis Beijing Isolates

Although the genome of the Mycobacterium tuberculosis H37Rv laboratory strain has been available for over 10 years, it is only recently that genomic information from clinical isolates has been used to generate the hypothesis of virulence differences between different strains. In addition, the relationship between strains displaying differing virulence in an epidemiological setting and their behavior in animal models has received little attention. The potential causes for variation in virulence between strains, as determined by differential protein expression, have similarly been a neglected area of investigation. In this study, we used a label-free quantitative proteomics approach to estimate differences in protein abundance between two closely related Beijing genotypes that have been shown to be hyper- and hypovirulent on the basis of both epidemiological and mouse model studies. We were able to identify a total of 1668 proteins from both samples, and protein abundance calculations revealed that 48 proteins were over-represented in the hypovirulent isolate, whereas 53 were over-represented in the hypervirulent. Functional classification of these results shows that molecules of cell wall organization and DNA transcription regulatory proteins may have a critical influence in defining the level of virulence. The reduction in the presence of ESAT-6, other Esx-like proteins, and FbpD (MPT51) in the hypervirulent strain indicates that changes in the repertoire of highly immunogenic proteins can be a defensive process undertaken by the virulent cell. In addition, most of the previously well characterized gene targets related to virulence were found to be similarly expressed in our model. Our data support the use of proteomics as a complementary tool for genomic comparisons to understand the biology of M. tuberculosis virulence.

Interferon γ Responses to Mycobacterial Antigens Protect against Subsequent HIV‐Associated Tuberculosis

The cellular immune responses that protect against tuberculosis have not been identified. We assessed baseline interferon γ (IFN‐γ) and lymphocyte proliferation assay (LPA) responses to antigen 85 (Ag85), early secretory antigenic target 6 (ESAT‐6), and Mycobacterium tuberculosis whole cell lysate (WCL) in human immunodeficiency virus (HIV)-infected and bacille Calmette‐Guérin (BCG)-immunized adults with CD4 cell counts of >or= 200 cells/μL who received placebo in the DarDar tuberculosis vaccine trial in Tanzania. Subjects were followed prospectively to diagnose definite or probable tuberculosis. Tuberculosis was diagnosed in 92 of 979 subjects during a mean follow‐up of 3.2 years. The relative risk of tuberculosis among subjects with positive IFN‐γ responses to Ag85 was 0.51 (95% confidence interval [CI], 0.26-0.99; P = .049), to ESAT‐6 was 0.44 (95% CI, 0.23-0.85; P = .004), and to WCL was 0.67 (95% CI, 0.49-0.88; P = .002). The relative risk of tuberculosis was not significantly associated with baseline LPA responses. In a multivariate Cox regression model, subjects with IFN‐γ responses to ESAT‐6 and WCL had a lower hazard of developing tuberculosis, with a hazard ratio for ESAT‐6 of 0.35 (95% CI, 0.16–0.77; P = .009) and a hazard ratio for WCL of 0.30 (95% CI, 0.16-0.56; P < .001). Baseline IFN‐γ responses to ESAT-6 and WCL were associated with protection from subsequent tuberculosis among HIV-infected subjects with childhood BCG immunization in a region of high tuberculosis prevalence. Trial registration. ClinicalTrials.gov identifier: NCT00052195.

Specific cytokine patterns of pulmonary tuberculosis in Central Africa

Different cytokines have been suggested to be involved in the pathogenesis of pulmonary tuberculosis (TB). The frequencies of Mycobacterium tuberculosis (MTB) specific CD4(+) and CD8(+) T cells, CD4(+)CD25(+) Forkhead Box Protein (FoxP)3(+) T cells, interleukin (IL)-6, interferon (IFN)-γ, Tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β and IL-10 were assessed in HIV-negative, pulmonary tuberculosis (TB) patients (n=30) and in healthy controls (n=23) in Gabon. Peripheral blood mononuclear cells (PBMC) were stimulated with purified protein derivative (PPD) and early secretory antigenic target-6 (ESAT-6). In patients, a pronounced pro-inflammatory cytokine response with highly significant increased levels of IL-6 and TNF-α accompanied by increased TGF-β was detectable. Differences in IFN-γ responses between patients and healthy individuals were less pronounced than expected. FoxP3 expression did not differ between groups. A distinct cytokine pattern is associated with active pulmonary TB in patients from Central Africa.

Immunological Diagnosis of Tuberculosis Based on Recombinant Antigens ESAT-6 and CFP-10 in Children from an Endemic Area in Northeast Brazil

Diagnostic tests for tuberculosis (TB) using interferon gamma (IFN-γ) responses produced by T lymphocytes after stimulation by early secretory antigen target 6 (ESAT-6), culture filtrate protein 10 (CFP-10) or purified protein derivate (PPD) were carried out using ELISA (enzyme-linked immunosorbent assay) in whole blood culture supernatants from children with suspected TB disease (n=21), latent TB infection (LTBI; n=17) and negative controls (NC; n=21) from Recife, Pernambuco, Brazil. The results were analysed using the ROC (receiver operating characteristic) curves and the areas under the curve (AUC) generated varied from 0.5 to 1.0 with higher values indicating increased discriminatory ability. Comparisons of AUCs were made using non-parametric assumptions, and the differences were considered significant if P<0.05. The ROC curve showed a statistical difference (P = 0.015) between the LTBI and NC groups with an AUC of 0.731, TB disease and NC (AUC=0.780; P=0.002) and a group with TB (latent infection+disease, n=38) and NC (AUC=0.758; P = 0.001) when the antigen used was ESAT-6. No statistical difference was found between the groups when CFP-10 or PPD was used. In conclusion, the ESAT-6 test may be the most appropriate for diagnosis of childhood TB, both LTBI and TB disease, when associated with epidemiological and clinical data, especially in endemic areas such as Brazil.

Use of the T-spot.TB test for the diagnosis of latent tuberculosis infection

Background:Tuberculosis (TB) represents a major health problem both in developing and both in industrialized countries.The identification of individuals latently infected with Mycobacterium tuberculosis (Mtb) play a key role for the efficacy of TB control. These individuals with a latent tuberculosis infection (LTBI), especially those with high risk of reactivation (e.g. HIV + / AIDS-infected individuals, patients undergoing immunosuppressive therapy and children younger than 5 years) could benefit from a preventive treatment with isoniazid reducing the risk of progression from LTBI to active TB. Until recently, detection of LTBI has relied on the tuberculin skin test (TST), but despite the widespread use in clinical practice,TST does not reliably diagnose LTBI because several drawbacks, e.g. lacking in specificity, particularly in who were exposed to non-tuberculous mycobacteria (NTM) or were vaccinated with Bacille Calmette-Guerin (BCG) In addition, in young subjects,TST sensitivity is hampered by impaired T cell function leading frequently to false negative results.These several drawbacks limit the use of TST for the diagnose an LTBI in patients who may benefit from preventive chemotherapy. On the other hand, an accurate diagnosis of LTBI avoid the over-treatment of those patients with a positive TST results but not latently infected with Mtb. Recently, new tests based on the detection of interferon-gamma (IFN-γ) after stimulation with Mtb-specific antigens: Early secretory Antigenic Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10) have been proposed for the diagnosis of active TB and LTBI. Methods: During the period from January 2009 to June 2009, in our laboratory 70 patients were tested with T-SPOT.TB (Oxford Immunotech, Abingdon, United Kingdom).We enrolled transplant patients and subjects ongoing transplant, patients immigrants from high prevalence TB countries, patients screened for immunosuppressive treatment, HIV / AIDS – infected individuals.We also tested 3 patients with clinical / radiological suspicion of active TB and 3 patients with positive tuberculin skin test and with a positive direct examination for mycobacteria in the urinary sediment. Results: In 2 patients with symptoms suggestive of TB in place,T-SPOT.TB showed a higher response of (IFN-g), more than 100 spots.Among individuals ongoing renal transplant, 6 patients tested T-SPOT.TB positive and 4 subjects were T.SPOT.TB -negative. Two patients with an autoimmune disease showed an high response to Mtb-specific antigens with T-SPOT.TB test tested before to start any treatment.T-SPOT.TB test tested strongly negative in 4 paediatric patients and in one HIV-infected individuals, regardless a positive response to a internal positive response (phytohaemagglutinin (PHA), suggesting a normal immune response. Conclusions:This preliminary data suggest that the T.SPOT.TB showed high sensitivity and specificity, producing a strongly negative response to Mtb-specific antigens in subjects who had a history of previous BCG-vaccination. In addition, T-SPOT.TB test provides, unlike the TST, indication about the potential immunosuppression of tested patient with an internal positive control that can highlight the production of IFN- γ by lymphocytes resulting in the application of this test in immunocompromised patients, e.g. children and transplantated patients and others.

Improved Immunogenicity of Recombinant Mycobacterium bovis Bacillus Calmette‐Guérin Strains Expressing Fusion Protein Ag85A‐ESAT‐6 of Mycobacterium tuberculosis

Early secretory antigen target 6 (ESAT-6) is a dominant target for cell-mediated immunity in the early phase of tuberculosis (TB) in patients with TB, causing T-cell proliferation and gamma interferon (IFN-γ) production, which has been considered to be a protective antigen that can be used for future vaccine development. Ag85A is the most essential component for bacterial survival within macrophages and has been used in numerous vaccine preparations, which can induce strong cellular immune responses. In this study, we constructed a new recombinant bacilli Calmette-Guérin (BCG) strain (rBCG-AE) that could express fusion protein Ag85A-ESAT-6 of Mycobacterium tuberculosis and evaluated its immunogenicity in BALB/c mice. There was no evidence for increased virulence of this rBCG. Our experiments illustrated that the rBCG-AE was able to induce higher titer of antibody and elicit more long-lasting and stronger Th1 type cellular immune responses than the parental BCG strain, or rBCG-A (expressing Ag85A) strain, or rBCG-E (expressing ESAT-6) strain, which are characterized by the strong antibody response, the proliferation rate of splenocytes, the ratio of CD4(+) T and CD8(+) T cells stimulated by tuberculin-purified protein derivative and elevated levels of IFN-γ in antigen-stimulated splenocyte cultures. The results show that rBCG-AE is an improved TB vaccine for further study.

Potential role for IL-2 ELISpot in differentiating recent and remote infection in tuberculosis contact tracing.

Interferon (IFN)-γ release assays (IGRA) have improved tuberculosis contact tracing, but discrimination of recent from remote Mycobacterium tuberculosis contacts is not possible by IGRA alone. We present results of a tuberculosis contact investigation with a new early-secretory-antigenic-target (ESAT)-6 and culture-filtrate-protein (CFP)-10 specific interleukin (IL)-2 ELISpot in addition to ESAT-6 and CFP-10 specific IFN-γ ELISpot and tuberculin skin testing (TST). Results of the TST, IFN-γ ELISpot and IL-2 ELISpot were positive in 6/172 (3.4%), 7/167 (4.2%) and 6/196 (3.1%) of contacts, respectively. Close contact (≥100 hours) to the index case increased the risk of positive results in the IFN-γ ELISpot, TST, and IL-2 ELISpot by 40.8, 19.3, and 2.5 times, respectively. Individuals with a positive IFN-γ ELISpot/negative IL-2 ELISpot result had a median (IQR) duration of index case exposure of 568 hours (133_1000) compared to individuals with a positive IFN-γ ELISpot/positive IL-2 ELISpot result (median = 24 hours; 20_130; p-value = 0.047). Combination of a M. tuberculosis specific IFN-γ ELISpot with a M. tuberculosis specific IL-2 ELISpot significantly improved the identification of individuals with the highest risk of recent M. tuberculosis infection and is a promising method that should be explored to target tuberculosis preventive chemotherapy.

Diagnostic usefulness of enzyme-linked immunospot assay for interferon-gamma for tuberculosis in cancer patients

The interferon-γ enzyme-linked immunospot assay (ELISPOT) has been demonstrated to be useful in the diagnosis of active tuberculosis (TB). In this study we aimed to evaluate the diagnostic performance of the ELISPOT assay in cancer patients with suspected pulmonary TB. Eighty-one cancer patients with suspected pulmonary TB were prospectively enrolled from April 2007 to December 2008, to investigate the diagnostic sensitivity and specificity of the ELISPOT assay. Of the 38 patients with TB, 33 (86.8%) had positive ELISPOT results. Of the 43 patients without TB, the results of the ELISPOT assay were negative in 35 (81.3%) patients. The overall sensitivity was 86.8%, specificity 81.3%, positive predictive value 80.5% and negative predictive value 87.5%. No significant difference was noted for the diagnostic performance of the ELISPOT assay for diagnosing TB between solid cancer and haematological cancer patients. In addition, a quantitative study did not show that TB patients with solid cancers have a better response than haematological cancer patients as measured by spot-forming cells per 10(6) peripheral blood mononuclear cells after exposure to early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10). In conclusion, the ELISPOT assay could be a useful supplementary tool for the diagnosis of pulmonary TB among cancer patients, irrespective of cancer type.