Biophysical and biochemical characterization of Rv3405c, a tetracycline repressor protein from Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis disease, is one among the deadliest pathogens in the world. Due to long treatment regimen, HIV co-infection, persistence of bacilli in latent form and development of XDR and TDR strains of Mtb, tuberculosis has posed serious concerns for managing the disease, and calls for discovery of new drugs and drug targets. Using a computational pipeline involving analysis of the structural models of the Mtb proteome and an analysis of the ATPome, followed by a series of filters to identify druggable proteins, solubility and length of the protein, several candidate proteins were shortlisted. From this, Rv3405c, a tetR family of DNA binding protein involved in antibiotic resistance, was identified as one of the good drug targets. Rv3405c binds to the upstream non-coding region of Rv3406 and causes repression of Rv3406 activity there by affecting the downstream processes involved in antibiotic resistance was further characterized. The Rv3405c gene was cloned; the gene product was over-expressed in E. coli and purified by Ni NTA chromatography. DNA binding studies by EMSA showed that the recombinant Rv3405c protein binds to the DNA sequence corresponding to the promoter region of Rv3406 and upon addition of tetracycline, the DNA binding activity was lost. ß-galactosidase reporter assay in E. coli using both wild type and a DNA binding defective mutant protein indeed proved that Rv3405c acts as a repressor.

Rv2204c, Rv0753c and Rv0009 antigens specific T cell responses in latent and active TB - a flow cytometry-based analysis.

High global prevalence of latent TB infection (LTBI) is a key challenge in distinguishing patients with active pulmonary TB (PTB) from those with LTBI. The functional profile of CD4+ and CD8+ T cell cytokines produced as a response to Mycobacterium tuberculosis antigens vary during the course of tuberculosis (TB) infection. We evaluated antigen-specific CD4+ and CD8+ T cell cytokine response after overnight in vitro stimulation of peripheral blood with mycobacterial antigens ESAT-6, CFP-10, Rv2204c, Rv0753c and Rv0009 by flow cytometry. A significantly higher frequency of antigen-specific CD4+ or CD8+ IFN-γ+ T cells were found in LTBI than in PTB. Among all the antigens used, Rv2204c-specific CD8+ IFN-γ+ displayed the positivity of 72% and 24% in LTBI and PTB respectively. In contrast to IFN-γ, the frequencies of CD4+ or CD8+ secreting TNF-α+ cells were significantly high in PTB compared to LTBI. CD8+TNF-α+ analysis showed 60% positivity in PTB and 13.6% positivity in LTBI against Rv0753c antigen stimulation. We also predicted Rv2204c specific CD8+ T cells secreting IL-10 or IL-4 showed maximum differentiation between LTBI and PTB. In conclusion, altered expression of Rv2204c-specific CD4+IFN-γ+ and CD8+IL-4+ T cells in LTBI and PTB might be a useful biomarker to differentially diagnose LTBI and active TB.

Altered expression of antigen-specific memory and regulatory T-cell subsets differentiate latent and active tuberculosis.

Although one-third of the world population is infected with Mycobacterium tuberculosis, only 5-10% of the infected individuals will develop active tuberculosis (TB) disease and the rest will remain infected with no symptoms, known as latent TB infection (LTBI). Identifying biomarkers that differentiate latent and active TB disease enables effective TB control, as early detection, treatment of active TB and preventive treatment of individuals with LTBI are crucial steps involved in TB control. Here, we have evaluated the frequency of antigen-specific memory and regulatory T (Treg) cells in 15 healthy household contacts (HHC) and 15 pulmonary TB patients (PTB) to identify biomarkers for differential diagnosis of LTBI and active TB. Among all the antigens tested in the present study, early secretory antigenic target-6 (ESAT-6) -specific CD4+ and CD8+ central memory (Tcm) cells showed 93% positivity in HHC and 20% positivity in PTB. The novel test antigens Rv0753c and Rv0009 both displayed 80% and 20% positivity in HHC and PTB, respectively. In contrast to Tcm cells, effector memory T (Tem) cells showed a higher response in PTB than HHC; both ESAT-6 and Rv0009 showed similar positivity of 80% in PTB and 33% in HHC. PTB patients have a higher proportion of circulating antigen-reactive Treg cells (CD4+  CD25+  FoxP3+ ) than LTBI. Rv2204c-specific Treg cells showed maximum positivity of 73% in PTB and 20% in HHC. Collectively, our data conclude that ESAT-6-specific Tcm cells and Rv2204c-specific Treg cells might be useful biomarkers to discriminate LTBI from active TB.

Dihydrolipoamide dehydrogenase-Lpd (Rv0462)-specific T cell recall responses are higher in healthy household contacts of TB: a novel immunodominant antigen from M. tuberculosis.

The partial effectiveness against pulmonary tuberculosis (PTB), displayed by the existing tuberculosis (TB) vaccine, bacillus Calmette-Guérin (BCG), highlights the need for novel vaccines to replace or improve BCG. In TB immunology, antigen-specific cellular immune response is frequently considered indispensable. Latency-associated antigens are intriguing as targets for TB vaccine development. The mycobacterial protein, dihydrolipoamide dehydrogenase (Lpd; Rv0462), the third enzyme of the pyruvate dehydrogenase (PDH) complex, facilitates Mycobacterium tuberculosis to resist host reactive nitrogen intermediates. Multicolor flow cytometry analysis of whole-blood cultures showed higher Lpd-specific Th1 recall response (IFN-γ, TNF-α, and IL-2; P = 0.0006) and memory CD4+ and CD8+ T cells (CCR7+ CD45RA- and CCR7- CD45RA-) in healthy household contacts (HHC) of TB (P < 0.0001), which is comparable with or higher than the standard antigens, ESAT-6 and CFP-10. The frequency of Lpd-specific multifunctional T cells was higher in HHC compared with PTB patients. However, there is no significant statistical correlation. Regulatory T cell (Treg) analysis of HHCs and active TB patients demonstrated very low Lpd-specific CD4+ Tregs relative to ESAT-6 and CFP-10. Our study demonstrates that the Lpd antigen induces a strong cellular immune response in healthy mycobacteria-infected individuals. In consideration of this population having demonstrated immunologic protection against active TB disease development, our data are encouraging about the possible use of Lpd as a target for further TB subunit vaccine development.

Proteomics Analysis of Three Different Strains of Mycobacterium tuberculosis under In vitro Hypoxia and Evaluation of Hypoxia Associated Antigen's Specific Memory T Cells in Healthy Household Contacts.

In vitro mimicking conditions are thought to reflect the environment experienced by Mycobacterium tuberculosis inside the host granuloma. The majority of in vitro dormancy experimental models use laboratory-adapted strains H37Rv or Erdman instead of prevalent clinical strains involved during disease outbreaks. Thus, we included the most prevalent clinical strains (S7 and S10) of M. tuberculosis from south India in addition to H37Rv for our in vitro oxygen depletion (hypoxia) experimental model. Cytosolic proteins were prepared from hypoxic cultures, resolved by two-dimensional electrophoresis and protein spots were characterized by mass spectrometry. In total, 49 spots were characterized as over-expressed or newly emergent between the three strains. Two antigens (ESAT-6, Lpd) out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these two genes were overexpressed, purified and used for in vitro stimulation of whole blood collected from healthy household contacts (HHC) and active pulmonary tuberculosis patients (PTB). Multicolor flow cytometry analysis showed high levels of antigen specific CD4(+) central memory T cells in the circulation of HHC compared to PTB (p < 0.005 for ESAT-6 and p < 0.0005 for Lpd). This shows proteins that are predicted to be up regulated during in vitro hypoxia in most prevalent clinical strains would indicate possible potential immunogens. In vitro hypoxia experiments with most prevalent clinical strains would also elucidate the probable true representative antigens involved in adaptive mechanisms.

Dynamic IgG antibody response to immunodominant antigens of M. tuberculosis for active TB diagnosis in high endemic settings.

BACKGROUND: Even though various techniques have been developed for rapid diagnosis of tuberculosis (TB), still there is an immense need for a simple, cost effective, highly sensitive and specific test. Hence, one of the possibilities is identification of Mycobacterium tuberculosis specific antibodies in infected serum by using specific antigens. METHODS: We tested 10 recombinant M. tuberculosis antigens to evaluate IgG levels among Healthy control subjects (HCS), Healthy household contacts (HHC) and pulmonary TB patients (PTB) by ELISA. RESULTS: The median IgG levels specific to all the antigens are higher in PTB than HHC and HCS. Amongst single antigens, 38-kDa antigen has showed maximum sensitivity of 50% than any other antigens at 95.5% specificity. Among the two antigen combination, 38-kDa+Rv1860 has showed maximum sensitivity of 66.6% with specificity of 92.2%. The same antigen combination (38-kDa and Rv1860) predominantly identifies smear negative and culture positive TB patients with 68% sensitivity and 92.2% specificity. Most of the antigens have exhibited higher antibody titre in cavitary TB than non cavitary. With regard to latent TB infection (LTBI) identification, Rv1860 has exhibited maximum sensitivity of 53.3% with 95% specificity. CONCLUSIONS: IgG response to combination of recombinant mycobacterial antigens (38-kDa, Rv1860, Rv2204c and Rv0753c) presents good specificity with acceptable level of sensitivity for TB diagnosis.

T cell recall response of two hypothetical proteins (Rv2251 and Rv2721c) from Mycobacterium tuberculosis in healthy household contacts of TB - Possible subunit vaccine candidates.

The demonstrated variable efficacy of the only licensed TB vaccine Mycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) encourages the need for new vaccine candidates against TB. Antigen specific cellular immune response is often considered imperative during Mycobacterium tuberculosis (M. tuberculosis) infection and antigens that are strongly associated with the latent phase of infection are drawing increasing attention for anti-TB vaccine development. Here, we investigated the phenotypic and functional profiles of two novel mycobacterial antigens Rv2251 and Rv2721c during T cell recall response via multi-color flow cytometry. Healthy household contacts of TB (latent/HHC) and active pulmonary TB (PTB) patients were recruited to investigate the difference in antigen specific T cell recall response. These two antigens induced expansion of CD45RA- CCR7+ central memory subtypes and CD45RA- CCR7- effector memory cells in latent population which suggests their possible association with HHC. Rv2251 and Rv2721c antigen specific IFN-γ, TNF-α and IL-2 response was also significantly high in HHC when compared to the PTB (p < 0.005, p < 0.05 and p < 0.05 respectively). The frequency of multifunctional T cells also was high in HHC compared to the PTB with statistical significance only for the antigen Rv2251. Often, the dominant Th1 immune response in HHC is correlated with the protection against the active TB disease. Collectively, we report the first insights into Rv2251 and Rv2721c antigen specific immune response in human donors of TB and provide the immunologic rationale for selecting them for vaccine development against TB.

Variable transcriptional adaptation between the laboratory (H37Rv) and clinical strains (S7 and S10) of Mycobacterium tuberculosis under hypoxia.

Tuberculosis continues to be a major public health problem in many parts of the world, despite intensified efforts taken to control the disease. The remarkable success of M. tuberculosis as a pathogen is largely due to its ability to persist within the host for long periods. To develop the effective intervention strategies, understanding the biology of persistence is highly required. Accumulating evidences showed oxygen deprivation (hypoxia) as a potential stimulus for triggering the transition of M. tuberculosis to a non-replicating persistent state analogous to latency in vivo. To date, in vitro hypoxia experimental models used the laboratory adapted isolate H37Rv and very little is known about the behavior of clinical isolates that are involved during disease outbreaks. Hence, we compared the transcription profiles of H37Rv and two south Indian clinical isolates (S7 and S10) under hypoxia to find differences in gene expression pattern. The main objective of this current work is to find "differentially regulated genes" (genes that are down regulated in H37Rv but upregulated in both the clinical isolates) under hypoxia. Microarray results showed, a total of 502 genes were down regulated in H37Rv under hypoxia and 10 out of 502 genes were upregulated in both the clinical isolates. Thus, giving less importance to down regulated genes based on H37Rv model strain might exclude the true representative gene candidates in clinical isolates. Our study suggests the use of most prevalent clinical isolates for in vitro experimental model to minimize the variation in understanding the adaptation mechanisms of the strains.

In vitro QuantiFERON-TB gold antigen specific interleukin-1beta to diagnose TB among HIV-positive subjects.

BACKGROUND: The recently introduced IFN-γ release assay (IGRA) has been reported to improve the diagnosis of TB. However, IGRA has suboptimal sensitivity to diagnose TB among HIV co-infected subjects. Apart from IFN-γ, the pro inflammatory cytokines such as Interleukin-1beta (IL-1ß), Tumor necrosis factor-alpha (TNF-α), IL-2, IL-6, IL-8 and IL-12 are also play a major role in mycobacterial infections. This study aimed to analyze these cytokines for detecting active TB among HIV sero positive subjects. MATERIALS AND METHODS: We had prospectively enrolled 53 HIV positive subjects and 55 HIV-TB co-infected patients from India. IGRA was performed by using QuantiFERON TB-Gold In tube (QFT-GIT) method. TB antigen specific IL-1ß, TNF-α, IL-2, IL-6, IL-8 and IL-12 levels were evaluated by ELISA in plasma harvested from QFT-GIT tubes. RESULTS AND CONCLUSION: The TB antigen specific IL-1ß levels were significantly elevated in HIV-TB co-infected patients compared to HIV positive subjects (p = 0.0004). The specificity of both IL-1ß (50.94%) and QFT-GIT (52.83%) remained similar in HIV positive subjects (p = 0.24). However, IL-1ß had shown higher sensitivity (72.73%) than QFT-GIT (54.55%) to diagnose TB among HIV co-infected patients. Moreover, in culture test positive HIV-TB patients, antigen specific IL-1ß exhibited sensitivity of 84.21%; whereas QFT-GIT exhibited only 57.89% sensitivity. Unlike IFN-γ (the read out marker of QFT-GIT), antigen specific IL-1ß levels were not influenced by low CD4 counts. The other cytokine levels were not significantly differ between the 2 groups.From this study we concluded that TB antigen specific IL-1ß may be an additional biomarker for active TB diagnosis among HIV positive subjects.

PpiA antigen specific immune response is a potential biomarker for latent tuberculosis infection.

One third of the world's population is estimated to harbour latent tuberculosis infection (LTBI). Around 10% of them have the life time risk of developing active tuberculosis (PTB). Currently there is no gold standard test for identifying LTBI. Therefore identification of specific markers for LTBI will help as to develop a test specific for LTBI. Earlier, in our immunoproteomic analysis, we found that peptidyl-prolyl cis-trans isomerase A (PpiA) protein-containing fractions induced significantly higher interferon-gamma (IFN-γ) response in LTBI than in PTB. Immunological characterisation of recombinant PpiA protein was carried out in the current study. We have studied 10 cytokines and 2 chemokine responses against PpiA and standard antigens such as early secretory antigenic target-6 (ESAT-6) and culture filtrate antigen-10 (CFP-10). In healthy household contacts (HHC), all the tested antigens induced significantly higher levels of IFN-γ and Interlukin-8 (IL-8) compared with those in PTB. PpiA-specific IL-12p40 response was significantly increased in HHC compared with that in PTB. PpiA antigen-specific IFN-γ and IL-12p40 both showed 86% positivity in HHC, whereas in PTB, they showed 20% and 38% positivity, respectively. In terms of IFN-γ/TNF-α ratio, PpiA displayed 86% (30/35) positivity in HHC and 18% (7/39) positivity in PTB. In summary we found that PpiA-specific IFN-γ and IFN-γ/TNF-α ratio response were specific biomarkers for LTBI identification.

Evaluation of cytokine and chemokine response elicited by Rv2204c and Rv0753c to detect latent tuberculosis infection.

Latent TB infection (LTBI) is one of the major contributing factors for the high incidence of TB in India that in turn significantly contributes to the pool of active TB. Hence, identification and treatment of LTBI is of utmost importance. Currently, no specific diagnostic test is available for LTBI. Earlier, in our immunoproteomic analysis, we identified Rv2204c and Rv0753c protein-containing fractions induced significantly higher interferon-gamma (IFN-γ) response in LTBI than in active TB. In this study, we evaluated cytokine and chemokine response against M. tuberculosis antigens for improving LTBI identification. Two M. tb proteins Rv2204c and Rv0753c were cloned, over expressed in E. coli and purified by affinity chromatography. Antigen-specific immune response was evaluated in 39 pulmonary TB patients (PTB) and 35 healthy house-hold contacts (HHC). After whole blood culture for 6 days, the secretion of cytokines and chemokines were quantified in culture supernatants using Enzyme Linked Immune Sorbent Assay (ELISA). Antigen specific cytokines such as interferon gamma (IFN-γ), interleukin-6 (IL-6), IL-8, IL-12p40 and chemokines like monocyte chemotactic proteins MCP-1, MCP-2 were significantly higher in HHC than PTB. In contrast to other cytokines, tumor necrosis factor-alpha (TNF)-α response was significantly increased in PTB compared with HHC. Both Rv2204c and Rv0753c antigen specific IFN-γ response showed 86% positivity in HHC; whereas in PTB, these antigens showed 18% and 21% positivity respectively. Rv2204c antigen-specific IFN-γ/TNF-α response displayed maximum positivity of 91% in HHC and minimum positivity of 10% (4/39) in PTB. Rv2204c and Rv0753c specific IFN-γ and IFN-γ/TNF-α responses showed the most promising accuracy in identifying LTBI.

The influence of reduced oxygen availability on gene expression in laboratory (H37Rv) and clinical strains (S7 and S10) of Mycobacterium tuberculosis.

Mycobacterium tuberculosis has the ability to persist within the host in a dormant stage. One important condition believed to contribute to dormancy is reduced access to oxygen known as hypoxia. However, the response of M. tuberculosis to such hypoxia condition is not fully characterized. Virtually all dormant models against tuberculosis tested in animals used laboratory strain H37Rv or Erdman strain. But major outbreaks of tuberculosis (TB) occur with the strains that have widely different genotypes and phenotypes compared to H37Rv. In this study, we used a custom oligonucleotide microarray to determine the overall transcriptional response of laboratory strain (H37Rv) and most prevalent clinical strains (S7 and S10) of M. tuberculosis from South India to hypoxia. Analysis of microarray results revealed that a total of 1161 genes were differentially regulated (≥1.5 fold change) in H37Rv, among them 659 genes upregulated and 502 genes down regulated. Microarray data of clinical isolates showed that a total of 790 genes were differentially regulated in S7 among which 453 genes were upregulated and 337 down regulated. Interestingly, numerous genes were also differentially regulated in S10 (total 2805 genes) of which 1463 genes upregulated and 1342 genes down regulated during reduced oxygen condition (Wayne's model). One hundred and thirty-four genes were found common and upregulated among all three strains (H37Rv, S7, and S10) and can be targeted for drug/vaccine development against TB.

IFN-γ/TNF-α ratio in response to immuno proteomically identified human T-cell antigens of Mycobacterium tuberculosis - The most suitable surrogate biomarker for latent TB infection.

The enormous reservoir of latent TB infection (LTBI) poses a major hurdle for global TB control. The existing Tuberculin skin test (TST) and IFN-γ release assays (IGRAs) are found to be suboptimal for LTBI diagnosis. Previously we had taken an immunoproteomic approach and identified 10 protein fractions (contains 16 proteins), which are solely recognized by LTBI. In a cohort of 40 pulmonary TB patients (PTB) and 35 healthy household contacts (HHC), IFN-γ and TNF-α response were measured against 16 antigens by using 1:10 diluted whole blood assay. Among all the antigens, IFN-γ response to Rv2626c has shown positivity of 88.57% in HHC and 7.5% in PTB group. IFN-γ response to combination of Rv2626c + Rv3716c has demonstrated 100% positivity in HHC and 17.5% positivity in PTB respectively. Compared to individual cytokines (i.e. IFN-γ and TNF-α), ratio of IFN-γ/TNF-α has shown promising results for diagnosis of LTBI. IFN-γ/TNF-α ratio against Rv3716c and TrxC has exhibited a positivity of 94.29% in HHC and 5% in PTB group. Accession of Rv2626c and Rv3716c may improve the diagnostic performance of existing QFT-GIT. Independent of QFT-GIT assay, ratio of IFN-γ/TNF-α in response to either Rv3716c or TrxC may acts as suitable surrogate biomarker for LTBI.

Interleukins 15 and 12 in combination expand the selective loss of natural killer T cells in HIV infection in vitro.

The present study evaluated the frequency and receptor expression pattern of invariant natural killer T (iNKT) cells in human immunodeficiency virus (HIV)-infected individuals. Further, the effect of IL-15 + IL-12 stimulation on iNKT cells was also assessed. The study included 15 individuals each from normal healthy subjects, pulmonary tuberculosis patients, HIV-infected individuals, and patients with HIV and tuberculosis coinfection (HIV-TB). The frequency of iNKT cells and the expression of phenotype, cytotoxic and chemokine receptors were studied by flow cytometry. The number of iNKT cells was significantly depleted in HIV and HIV-TB patients, which upon IL-15 + IL-12 stimulation expanded in HIV. The constitutively expressed natural cytotoxicity receptor, NKp46 was increased in HIV and HIV-TB, which might be the host's response to HIV replication. The distinct expression patterns of chemokine and adhesion receptors suggest that iNKT subsets might traffic to different microenvironment and tissues. High expression of chemokine receptor CCR5 by most iNKT cells suggests that these cells might be more favorable targets of HIV infection. Our results show that IL-15 and IL-12 combination has the ability to expand the selective depletion of iNKT cells in vitro in HIV-infected individuals, but of limited value when coinfected with TB.

Role of QuantiFERON-TB Gold antigen-specific IL-1ß in diagnosis of active tuberculosis.

The main objective of the study was to evaluate whether in vitro QuantiFERON-TB Gold In-Tube (QFT-GIT) assay antigen-specific IL-1ß, TNF-α, IL-2, IL-6, IL-8 and IL-12 (p40) production is associated with active TB. In a cohort of 77 pulmonary TB patients (PTB), 67 healthy household contacts (HHC) and 83 healthy control subjects (HCS), the antigen-specific cytokines levels were determined in supernatants generated from QFT-GIT tubes. Antigen-specific IL-1ß levels were significantly higher in PTB than HHC and HCS. At a fixed cutoff point (1,108 pg/ml), IL-1ß showed positivity of 62.33% in PTB, 22.38% in HHC and 22.89% in HCS. Moreover, antigen-specific IL-1ß assay can differentiate PTB and HHC (believed to be latently infected) (p < 0.0001). Like IL-1ß, significantly higher levels of antigen-specific TNF-α were associated with PTB and displayed 43.63% positivity in PTB. The antigen-specific IL-2 levels were associated both with PTB (54.54%) and HHC (48.14%). Other cytokines levels did not differ among the groups. Our results suggest that antigen-specific IL-1ß can be used as a biomarker for active TB diagnosis as well as for differential diagnosis of PTB and LTBI.

Assessing humoral immune response of 4 recombinant antigens for serodiagnosis of tuberculosis.

Serodiagnostic potential of four recombinant proteins (38 kDa[Rv0934], MPT64[Rv1980c], Adk[Rv0733], and BfrB[Rv3874]) was evaluated in Healthy control subjects (HCS), Healthy household contacts (HHC), Pulmonary tuberculosis patients (PTB), and Human immuno deficiency virus & Tuberculosis co-infected patients (HIV-TB). All the antigens tested individually for the detection of serum IgG by indirect ELISA. All the four antigens have a significantly higher antibody response in PTB compared to healthy controls (P < 0.05). The sensitivity of individual antigens ranged from 20% to 52.5% for the prefixed specificity of 95%. When results of all 4 antigens were combined the sensitivity was increased to 75% and specificity was reduced 89% in HCS. In smear- and culture-positive (S+C+) PTB, four antigen combination gives maximum sensitivity (89.6%) with 89% specificity. In smear negative culture negative (S-C+) PTB, three antigen combination (38 kDa with MPT64 and BfrB) gives maximum sensitivity (69.5%) and specificity (91.6%). In HIV-TB, 4 antigen combinations give the maximum sensitivity of 51.2% with 89% specificity. Combining serology (Four antigen combination) with smear was able to increase the sensitivity from 70% to 92.5% in culture positive PTB. So, we propose that this serology test can be used as adjunct test along with smear for rapid diagnosis of PTB.

A toolbox for tuberculosis (TB) diagnosis: an Indian multi-centric study (2006-2008); evaluation of serological assays based on PGL-Tb1 and ESAT-6/CFP10 antigens for TB diagnosis.

BACKGROUND: The aim of this multi-centric prospective study in India was to assess the accuracy of a serological test as an additional tool for diagnosing active tuberculosis (ATB). In particular, an assay based on ELISA using a phenolic glycolipid (PGL-Tb1) or a fusion protein (ESAT-6/CFP10) was compared to the tuberculin skin test (TST) and the microbiological results according to HIV status. METHODS: Individuals with and without ATB and HIV infection were enrolled. Serology and TST results were analyzed per se and in combination with the microbiological data. RESULTS: Among the 778 ATB patients, 102 were HIV-infected, 316 HIV-uninfected and 360 had an HIV-unknown status. Of the 945 non-ATB subjects, 559 were at low risk (community adults) and 386 at high risk of M. tuberculosis exposure. Among those with ATB, the sensitivity of ELISA-PGL-Tb1 for ATB was higher than that of ELISA-ESAT-6/CFP10, both in HIV-infected (72.3% versus 63.7%, p = 0.29) and HIV-uninfected/HIV-unknown groups (40.5% versus 28.6%; p<0.0001), whereas the specificity was around 91% for both tests. Sensitivity for ATB increased when the results of the two ELISA were combined, reaching 75.5% in the HIV-infected and 50.9% in the group of HIV-uninfected/HIV-unknown ATB, with a significant decrease of the global specificity (83.9%). Analyzing the ELISA results with the microbiological results, we observed that the sensitivity of both serology tests was independent of the ATB patients' smear microscopy (SM) status and grade. Combining the results of SM with both ELISA, the detection of ATB patients significantly increased (p<0.0001), particularly in those with extrapulmonary TB (up to 45.1%) or HIV infection (up to 83.3%). No significant association was observed between TST and serology results. CONCLUSIONS: In this prospective multi-centric study, the combination of two rapid tests, such as SM and serology, might be useful in detecting ATB, especially in HIV-infected patients.

In silico analysis of potential human T Cell antigens from Mycobacterium tuberculosis for the development of subunit vaccines against tuberculosis.

In silico analysis was used to predict MHC class I and class II promiscuous epitopes and potential antigens, from 24 novel T cell antigens of Mycobacterium tuberculosis. Majority of the antigens (16/24) had high affinity peptides to both MHC class I and class II alleles and higher population coverage compared to well-proven T cell antigens ESAT-6, CFP-10 and Ag85B. Among these, highest population coverage were calculated for three novel T cell antigens Rv0733 (97.24%), Rv0462 (96.9%) and Rv2251 (96.3%). The prediction results were experimentally tested by in vitro stimulation of these novel T cell antigens with blood drawn from QuantiFERON-TB Gold In-Tube (QFT-IT) positive healthy household contacts of tuberculosis patients and pulmonary TB patients. Significantly higher level interferon-γ (IFN-γ) was observed, with these novel T cell antigens, in healthy household contacts compared to pulmonary TB subjects (p = 0.0001). In silico analysis also resulted in prediction of 36 promiscuous epitopes from the novel 24 T cell antigens. Population coverage for 4 out of the 36 promiscuous epitopes was >90% [67 VVLLWSPRS (Rv1324), 42 VVGVTTNPS (Rv1448c), 178 MRFLLSAKS (Rv0242c) and 842 IRLMALVEY (Rv3800c)]. Our results shows that these novel antigens and promiscuous epitopes identified from our analysis can further be investigated for their usefulness for subunit vaccine development.

A toolbox for tuberculosis (TB) diagnosis: an Indian multicentric study (2006-2008). Evaluation of QuantiFERON-TB gold in tube for TB diagnosis.

BACKGROUND: The aim of this multicentric prospective study in India was to assess the performance of the QuantiFERON TB-Gold in tube (QFT-GIT), Tuberculin Skin Test (TST) and microbiological results as additional tools for diagnosing active tuberculosis (TB) and latent infection (LTBI) according to Human Immunodeficiency Virus (HIV) status. METHODS: Individuals with and without active TB and HIV infection were enrolled between 2006-2008. QFT-GIT and TST results were analyzed per se and in combination with microbiological data. RESULTS: Among the 276 individuals (96 active pulmonary TB and 180 no active TB) tested by QFT-GIT, 18 indeterminate results (6.5%) were found, more significantly numerous in the HIV-infected (15/92; 16.3%) than the HIV-uninfected (3/184; 1.6%)(p<0.0001). QFT-GIT sensitivity for active TB was 82.3% and 92.9% respectively after including or excluding indeterminate results. Clinical sensitivity was significantly lower in the HIV-infected (68.4%) than the HIV-uninfected (91.4%) patients (p = 0.0059). LTBI was detected in 49.3% of subjects without active TB but varied according to TB exposure. When the TST and QFT-GIT were concomitantly performed, the respective sensitivity for active TB diagnosis was 95.0% and 85.0% in the HIV-uninfected (p = 0.60), and 66.7% and 51.5% in the HIV-infected patients (p = 0.32). QFT-GIT and TST respective specificity for active TB in the HIV-uninfected was 25.0% and 57.1% (p = 0.028), and 64.8% and 83.3% in the HIV-infected (p = 0.047). In those with active TB, QFT-GIT results were not associated with microbiological parameters (smear grade, liquid culture status, time-to-positivity of culture) or clinical suspicion of active TB score (provided by the clinicians at enrollment). Combining microbiological tests with both immunological tests significantly increased sensitivity for active TB diagnosis (p = 0.0002), especially in the HIV-infected individuals (p = 0.0016). CONCLUSION: QFT-GIT and TST have similar diagnostic value for active TB diagnosis. In HIV-infected patients, combining microbiological tests with both immunological tests significantly increases the sensitivity for active TB diagnosis.

Comparison of whole blood and PBMC assays for T-cell functional analysis.

BACKGROUND: Tuberculosis remains the foremost cause of morbidity and mortality, more than any other single infectious disease in the world. Cell mediated immune response plays a crucial role in the control of tuberculosis. Therefore, measuring cell mediated immune response against the antigens is having a vital role in understanding the pathogenesis of tuberculosis, which will also help in the diagnosis of and vaccination for tuberculosis. FINDINGS: The aim of the present study was to compare and optimize the assay conditions to measure the cell mediated immune response against M. tuberculosis specific antigens. Because the conventional PBMC assays (due to requirement of large volume of blood sample) are unable to screen more number of antigens within the same blood sample. So, here we have compared 6 days culture supernatants of 1:5 and 1:10 diluted blood and PBMCs from healthy laboratory volunteers, to assess the proliferative response of T lymphocytes and secreted IFN-γ levels against purified recombinant antigen of M. tuberculosis (MPT51, Rv3803c), crude antigens of M. tuberculosis (PPD) and mitogen (PHA). CONCLUSIONS: We have observed good correlation between each assay and also the mean difference of these assays did not reach the statistical significance (p>0.05). From these results, we conclude that 1:10 diluted whole-blood cultures can be well-suited as an alternative assay to measure cytokine production and lymphocyte proliferation in comparison to the conventional PBMC assays. Moreover, 1:10 diluted blood assays require less volume of blood when compared to PBMC assays which will be useful particularly in paediatric and field studies in endemic countries, where blood volume is a limiting factor.