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Background: A challenge in tuberculosis (TB) research is to develop a new immunological test that can help distinguish, among subjects responsive to QuantiFERON TB Gold In tube (QFT-IT), those who are able to control Mtb replication (remote LTBI, recent infection and past TB) from those who cannot (active TB disease). IFN-γ response to the Heparin-binding-hemagglutinin (HBHA) of Mtb has been associated with LTBI, but the cumbersome procedures of purifying the methylated and immunological active form of the protein from Mtb or M. bovis Bacillus Calmette et Guerin (BCG) have prevented its implementation in a diagnostic test. Therefore, the aim of the present study was to evaluate the IFN-γ response to methylated HBHA of Mtb produced in M. smegmatis (rHBHAms) in individuals at different stages of TB who scored positive to QFT-IT., Methodology/principal Findings: 87 individuals at different stages of TB who scored positive to QFT-IT were selected. IFN-γ response to in vitro whole blood stimulation with rHBHAms was evaluated by short-term and long-term tests and detected by ELISA or flow cytometry. We demonstrated that the IFN-γ response to rHBHAms is mediated by CD4(+) T-cells with an effector-memory phenotype. This response, evaluated by short-term-tests, is significantly lower in active TB than in remote LTBI (p = 0.0010) and past TB (p = 0.0152). These results were confirmed by long-term tests. The qualitative data confirmed that IFN-γ responses higher than the cut-off point identified by ROC analysis are associated with the status of non-active disease., Conclusions: In this study we show that the T-cell response to a recombinant and methylated HBHA of Mtb produced in M. smegmatis is useful to discriminate between active and non-active TB disease among those responsive to QFT-IT in a whole blood system. Further studies are needed to improve the accuracy of the assay.

Rationale: Due to the invasive nature of the procedures involved, most studies of Mycobacterium tuberculosis (Mtb)-specific immunity in humans have focused on the periphery rather than the site of active infection, the lung. Recently, antigens associated with Mtb-latency and -dormancy have been described using peripheral blood (PB) cells; however their response in the lung is unknown. The objective of this report was to evaluate, in patients prospectively enrolled with suspected active tuberculosis (TB), whether the latency antigen Rv2628 induces local-specific immune response in bronchoalveolar lavage (BAL) cells compared to PB cells., Material/methods: Among the 41 subjects enrolled, 20 resulted with active TB. Among the 21 without active disease, 9 were defined as subjects with latent TB-infection (LTBI) [Quantiferon TB Gold In-tube positive]. Cytokine responses to Rv2628 were evaluated by enzyme linked immunospot (ELISPOT) assay and flow cytometric (FACS) analysis. RD1-secreted antigen stimulation was used as control., Results: There was a significantly higher frequency of Rv2628- and RD1-specific CD4+ T-cells in the BAL of active TB patients than in PB. However the trend of the response to Rv2628 in subjects with LTBI was higher than in active TB in both PB and BAL, although this difference was not significant. In active TB, Rv2628 and RD1 induced a cytokine-response profile mainly consisting of interferon (IFN)-γ-single-positive over double-IFN-γ/interleukin (IL)-2 T-cells in both PB and BAL. Finally, BAL-specific CD4+ T-cells were mostly effector memory (EM), while peripheral T-cell phenotypes were distributed among naïve, central memory and terminally differentiated effector memory T-cells., Conclusions: In this observational study, we show that there is a high frequency of specific T-cells for Mtb-latency and RD1-secreted antigens (mostly IFN-γ-single-positive specific T-cells with an EM phenotype) in the BAL of active TB patients. These data may be important for better understanding the pathogenesis of TB in the lung.

Background: Interferon-gamma (IFN-gamma) release assays (IGRAs) were designed to detect latent tuberculosis infection (LTBI). However, discrepancies were found between the tuberculin skin test (TST) and IGRAs results that cannot be attributed to prior Bacille Calmètte Guerin vaccinations. The aim of this study was to evaluate tools for improving LTBI diagnosis by analyzing the IFN-gamma response to RD1 proteins in prolonged (long-term response) whole blood tests in those subjects resulting negative to assays such as QuantiFERON-TB Gold In tube (QFT-IT)., Methods: The study population included 106 healthy TST+ individuals with suspected LTBI (recent contact of smear-positive TB and homeless) consecutively enrolled. As controls, 13 healthy subjects unexposed to M. tuberculosis (TST-, QFT-IT-) and 29 subjects with cured pulmonary TB were enrolled. IFN-gamma whole blood response to RD1 proteins and QFT-IT were evaluated at day 1 post-culture. A prolonged test evaluating long-term IFN-gamma response (7-day) to RD1 proteins in diluted whole blood was performed., Results: Among the enrolled TST+ subjects with suspected LTBI, 70/106 (66.0%) responded to QFT-IT and 64/106 (60.3%) to RD1 proteins at day 1. To evaluate whether a prolonged test could improve the detection of LTBI, we set up the test using cured TB patients (with a microbiologically diagnosed past pulmonary disease) who resulted QFT-IT-negative and healthy controls as comparator groups. Using this assay, a statistically significant difference was found between IFN-gamma levels in cured TB patients compared to healthy controls (p < 0.006). Based on these data, we constructed a receiver operating characteristic (ROC) curve and we calculated a cut-off. Based on the cut-off value, we found that among the 36 enrolled TST+ subjects with suspected LTBI not responding to QFT-IT, a long term response to RD1 proteins was detected in 11 subjects (30.6%)., Conclusion: These results indicate that IFN-gamma long-term response to M. tuberculosis RD1 antigens may be used to detect past infection with M. tuberculosis and may help to identify additional individuals with LTBI who resulted negative in the short-term tests. These data may provide useful information for improving immunodiagnostic tests for tuberculosis infection, especially in individuals at high risk for active TB.

Rationale: Existing data on the effect of treatment of latent tuberculosis infection (LTBI) on T-cell responses to Mycobacterium tuberculosis (MTB)-specific antigens are contradictory. Differences in technical aspects of the assays used to detect this response and populations studied might explain some of these discrepancies. In an attempt to find surrogate markers of the effect of LTBI treatment, it would be important to determine whether, among contacts of patients with contagious tuberculosis, therapy for LTBI could cause changes in MTB-specific immune responses to a variety of RD1-antigens., Methods and Results: In a longitudinal study, 44 tuberculin skin test+ recent contacts were followed over a 6-month period and divided according to previous exposure to MTB and LTBI treatment. The following tests which evaluate IFN-gamma responses to RD1 antigens were performed: QuantiFERON TB Gold, RD1 intact protein- and selected peptide-based assays. Among the 24 contacts without previous exposure that completed therapy, we showed a significant decrease of IFN-gamma response in all tests employed. The response to RD1 selected peptides was found to be more markedly decreased compared to that to other RD1 antigens. Conversely, no significant changes in the response to RD1 reagents were found in 9 treated subjects with a known previous exposure to MTB and in 11 untreated controls., Conclusion: These data suggest that the effect of INH prophylaxis on RD1-specific T-cell responses may be different based on the population of subjects enrolled (recent infection versus re-infection) and, to a minor extent, on the reagents used.

Background: Interferon (IFN)-gamma response to region of difference (RD) 1 proteins (culture filtrate 10 and early secreted antigenic target 6) or overlapping peptides is a novel diagnostic marker of tuberculosis (TB) infection. Because we have recently shown that the response to certain peptides selected from RD1 allows discrimination between active TB (A-TB) and successfully treated TB (T-TB), we analyzed here the effector memory T cell profile and RD1-specific responses under the same clinical conditions., Methods: T cell responses to RD1 antigens were analyzed in patients with either severe or mild A-TB (classified on the basis of radiological lesions) and in 2 sets of healthy control subjects--those who had been successfully treated (the T-TB control subjects) and those whose tuberculin skin test (TST) results were negative (the TST-negative control subjects). IFN-gamma -producing CD4+ effector T cells were monitored by flow-cytometric analysis and ex vivo enzyme-linked immunospot (ELISPOT) assay, whereas a "cultured" ELISPOT assay was used to determine the frequency of memory T cells., Results: In the patients with severe A-TB, both CD4-mediated effector memory and central memory responses to the selected RD1 peptides were almost absent, whereas these responses were found in the majority of the patients with mild A-TB. In contrast, recognition of the selected RD1 peptides was detected in the T-TB control subjects only by expanding the central memory T cell pool., Conclusions: These data suggest a protective role for RD1 peptide-specific CD4+ effector T cells, which undergo clonal expansion during Mycobacterium tuberculosis replication and then a contraction phase after disease resolution, culminating in the generation of CD4+ memory T cells.