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.

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.

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.

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.

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.

Multiplex analysis of cytokines/chemokines as biomarkers that differentiate healthy contacts from tuberculosis patients in high endemic settings.

Differentiation of latent tuberculosis infection (LTBI) from active disease is one of the crucial elements in the control of tuberculosis. Earlier in Indian population which is tuberculosis endemic, we identified that 10 Mycobacterium tuberculosis secreted protein fractions, induced IFN-γ response only in healthy contacts of TB patients (HCs) and not in tuberculosis patients (TB). These fractions were termed as "Contact Specific Fractions" ("CS" fractions) and found useful for differentiating HC from TB. Proteomic analysis revealed that "CS" fractions have 16 different proteins, of which three were novel T cell antigens. Using these "CS" fractions as stimulants, earlier IFN-γ, TNF-α and IL-4 cytokine responses were studied. In the present study, in order to identify the other useful cytokine biomarkers that were differentially expressed between HC and TB, Cytokine/chemokine response to "CS" fractions were analyzed using multiplex cytokine assay system. This preliminary investigation in our tuberculosis endemic population showed six cytokine (G-CSF, IL-6, IL-7, IL-8, IL-9, and PDGF) and one receptor antagonist (IL-1Ra) that were differentially expressed between HC and TB, for the first time. Especially IL-6 and PDGF were more promising biomarkers. IL-6 measurement identified seven as HC out of 10 HC analyzed. The measurement of PDGF identified eight as TB out of 10TB tested. Studies are underway to further validate these biomarkers for the differentiation of LTBI from active tuberculosis.

Immunoproteomic identification of human T cell antigens of Mycobacterium tuberculosis that differentiate healthy contacts from tuberculosis patients.

Identification of Mycobacterium tuberculosis antigens inducing cellular immune responses is required to improve the diagnosis of and vaccine development against tuberculosis. To identify the antigens of M. tuberculosis that differentiated between tuberculosis (TB) patients and healthy contacts based on T cell reactivity, the culture filtrate of in vitro grown M. tuberculosis was fractionated by two-dimensional liquid phase electrophoresis and tested for the ability to stimulate T cells in a whole blood assay. This approach separated the culture filtrate into 350 fractions with sufficient protein quantity (at least 200 microg of protein) for mass spectrometry and immunological analyses. High levels of interferon-gamma (IFN-gamma) secretion were induced by 105 fractions in healthy contacts compared with TB patients (p < 0.05). Most interesting was the identification of 10 fractions that specifically induced strong IFN-gamma production in the healthy contact population but not in TB patients. Other immunological measurements showed 42 fractions that induced significant lymphocyte proliferative responses in the healthy contact group compared with the TB patients. The tumor necrosis factor-alpha response for most of the fractions did not significantly differ in the tested groups, and the interleukin-4 response was below the detectable range for all fractions and both study groups. Proteomic characterization of the 105 fractions that induced a significant IFN-gamma response in the healthy contacts compared with the TB patients led to the identification of 59 proteins of which 24 represented potentially novel T cell antigens. Likewise, the protein identification in the 10 healthy "contact-specific fractions" revealed 16 proteins that are key candidates as vaccine or diagnostic targets.

IP-10 response to RD1 antigens might be a useful biomarker for monitoring tuberculosis therapy.

BACKGROUND: There is an urgent need of prognosis markers for tuberculosis (TB) to improve treatment strategies. The results of several studies show that the Interferon (IFN)-γ-specific response to the TB antigens of the QuantiFERON TB Gold (QFT-IT antigens) decreases after successful TB therapy. The objective of this study was to evaluate whether there are factors other than IFN-γ [such as IFN-γ inducible protein (IP)-10 which has also been associated with TB] in response to QFT-IT antigens that can be used as biomarkers for monitoring TB treatment. METHODS: In this exploratory study we assessed the changes in IP-10 secretion in response to QFT-IT antigens and RD1 peptides selected by computational analysis in 17 patients with active TB at the time of diagnosis and after 6 months of treatment. The IFN-γ response to QFT-IT antigens and RD1 selected peptides was evaluated as a control. A non-parametric Wilcoxon signed-rank test for paired comparisons was used to compare the continuous variables at the time of diagnosis and at therapy completion. A Chi-square test was used to compare proportions. RESULTS: We did not observe significant IP-10 changes in whole blood from either NIL or QFT-IT antigen tubes, after 1-day stimulation, between baseline and therapy completion (p = 0.08 and p = 0.7 respectively). Conversely, the level of IP-10 release to RD1 selected peptides was significantly different (p = 0.006). Similar results were obtained when we detected the IFN-γ in response to the QFT-IT antigens (p = 0.06) and RD1 selected peptides (p = 0.0003). The proportion of the IP-10 responders to the QFT-IT antigens did not significantly change between baseline and therapy completion (p = 0.6), whereas it significantly changed in response to RD1 selected peptides (p = 0.002). The proportion of IFN-γ responders between baseline and therapy completion was not significant for QFT-IT antigens (p = 0.2), whereas it was significant for the RD1 selected peptides (p = 0.002), confirming previous observations. CONCLUSIONS: Our preliminary study provides an interesting hypothesis: IP-10 response to RD1 selected peptides (similar to IFN-γ) might be a useful biomarker for monitoring therapy efficacy in patients with active TB. However, further studies in larger cohorts are needed to confirm the consistency of these study results.

Natural-killer cell-derived cytolytic molecules in HIV-associated pulmonary tuberculosis-role of exogenous interleukins.

OBJECTIVE: The ability of NK cells to produce cytolytic molecules is impaired during HIV infection. The objective of the present study is to investigate whether impairment in production of innate cytokines in HIV-infected individuals is responsible for the defective NK cytolytic response. MATERIALS AND METHODS: The study included 30 subjects each of normal healthy subjects, pulmonary tuberculosis patients, HIV-infected individuals, and patients with HIV and TB co-infection. Intracellular staining method was adopted to enumerate the NK cells positive for cytolytic molecules. Highest stimulation of cytolytic molecules was seen with IL-15 + IL-12 combination. RESULTS: Stimulation with IL-15 + IL-12 showed an increased expression of perforin in NHS and HIV groups. Granzyme A was stimulated only in HIV, even with IL-15 + IL-12. Among the cytolytic molecules, maximal stimulation with IL-15 + IL-12 was seen for Granyme A and Granzyme B. Both the HIV and HIV-TB groups showed an increased response with IL-15 + IL-12 for granulysin. CONCLUSION: Supplementing IL-15 + IL-12 in vitro increased the number of NK cells that are expressing cytolytic molecules in HIV-infected individuals but in HIV-TB, the critical cytolytic molecule, perforin is not apparent perhaps due to the influence of TB on HIV.

Cytotoxicity responses to selected ESAT-6 and CFP-10 peptides in tuberculosis.

Cytotoxicity responses were studied for the ESAT-6 peptides Esp1, Esp6, Esp7, Esp8, and CFP-10 peptides, Cfp6, Cfp7, Cfp8, Cfp9 (synthetic 20-mer peptides) and the recombinant ESAT-6, CFP-10 proteins. Cytolytic molecules perforin, granzymes A and B, granulysin responses in healthy household contacts (HHC) and pulmonary tuberculosis patients (PTB), were studied by intracellular flow cytometry. Functional cytotoxicity was studied in both the groups for the peptides Esp6 and Cfp8 by an enzyme (lactate dehydrogenase) based assay. The results revealed that cytolytic molecule positive CD4+ and CD8+ T cells were increased in HHC in response to Esp1, Esp6, Cfp8 and Cfp9 immunogenic peptides compared to PTB. Functional cytotoxicity results showed higher cytotoxicity (not statistically significant) to be exhibited by the peptide Esp6 than Cfp8 in the HHC.

Cellular immune response to Mycobacterium tuberculosis-specific antigen culture filtrate protein-10 in south India.

The Mycobacterium tuberculosis (M. tuberculosis)-specific culture filtrate protein-10 (CFP-10) is highly recognized by M. tuberculosis infected subjects. In the present study, the proliferative response and IFN-gamma secretion was found for C-terminal peptides of the protein (Cfp6(51-70), Cfp7(61-80), Cfp8(71-90), and Cfp9(81-100)). The alleles HLA DRB1 *04 and HLA DRB1 *10 recognized the C-terminal peptides Cfp7, Cfp8, and Cfp9 in HHC. Cfp6 was predominantly recognized by the alleles HLA DRB1 *03 and HLA DRB1 *15 by PTB. The minimal nonameric epitopes from the C-terminal region were CFP-10(56-64) and CFP-10(76-84). These two peptides deserve attention for inclusion in a vaccine against tuberculosis in this region.

Immunological and proteomic analysis of preparative isoelectric focusing separated culture filtrate antigens of Mycobacterium tuberculosis.

Isolation of the secreted proteins and studying the immune response they induce is an essential prerequisite for understanding the pathogenesis of M. tuberculosis. In this study, preparative liquid-phase isoelectric focusing was used for the separation of culture filtrate protein (CFP) of M. tuberculosis. This procedure resolved culture filtrate proteins into 20 fractions with a pI range of 2.59 to 12.9. These 20 fractions were subjected to immunological analysis in healthy laboratory volunteers from our endemic area. Eleven fractions (Fractions 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, and 19) showed increased interferon gamma (IFN-gamma) secretion and 5 fractions induced increased proliferative response, when compared to unfractionated CFP. In the 11 fractions which showed increased IFN-gamma secretion, mass spectrometric analysis identified 19 different proteins. Apart from the already reported immunodominant antigens like FbpB, CFP-10 and ESAT-6, two new T cell antigens (AcpM and PpiA) were also identified in the immunologically active fractions. Immunoinformatic analysis showed that PpiA was predicted to bind more number of class I and class II HLA alleles compared with the immunodominant ESAT-6 and CFP-10. Population coverage calculations also showed that PpiA protein (85%) had a higher population coverage compared with ESAT-6 (79%) and CFP-10 (77%). This result shows that the PpiA protein has a potential to be a novel T cell antigen.

Improved diagnosis of pulmonary tuberculosis by detection of antibodies against multiple Mycobacterium tuberculosis antigens.

Two secreted antigens (38 and 30 kDa) and 1 cytosolic antigen (16 kDa) were purified in our laboratory from Mycobacterium tuberculosis culture filtrate and cytosol using chromatographic/electrophoretic methods. One recombinant antigen (27 kDa, MPT51) expressed in Escherichia coli was also isolated. All the 4 antigens were tested individually for detection of serum IgG, IgA, and IgM (a total of 476 sera from 5 groups) by indirect enzyme-linked immunosorbent assay. Keeping the well-reported 38 kDa as the main candidate, the usefulness of the other antigens, which may add to the test positivity in cases not diagnosed by 38 kDa, was analyzed. The individual antigens ranged in their sensitivity from 57% to 67% (IgG). Addition of other antigen results, with that of 38 kDa, offered a sensitivity of 91% in smear- and culture-positive tuberculosis (TB), 78% in smear-negative culture-confirmed TB, and 97% specificity in normal healthy subjects. IgG antibody to multiple antigens (38, 30, and 16 kDa) may be a sensitive, specific, rapid, and cost-effective test to rule-in clinical suspicion of pulmonary TB.

Evaluation of the diagnostic potential of region of deletion-1-encoded antigen culture filtrate protein-10 in pulmonary tuberculosis.

The ability of region of deletion-1 (RD-1)-encoded culture filtrate protein-10 (CFP-10) to supplement the sensitivity of 38-kDa antigen was studied using enzyme-linked immunosorbent assay in pulmonary tuberculosis (TB) patients and controls. The sensitivities for individual antigens ranged from 50% to 60%, and the specificity was 100% for immunoglobulin (Ig) G alone. When IgA results were added to IgG, the sensitivity increased. On combination of the isotype results, sensitivity increased to 61.8% and 57.2% (for 38-kDa antigen and CFP-10), respectively, and specificity changed to 98.8% and 99.4% for 38-kDa and CFP-10, respectively. Combination of results of both the antigens gave 82.4% sensitivity in smear-positive and culture-positive group, and 98.1% specificity. In smear-negative and culture-positive group, the sensitivity was 66.7%. In smear-negative and culture-negative cases, a sensitivity of 65.6% was obtained. This study demonstrates that the use of RD-1-encoded CFP-10 enhances the sensitivity of 38-kDa antigen and can be a useful diagnostic marker in 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.

Clinical value of specific detection of immune complex-bound antibodies in pulmonary tuberculosis.

Two actively secreted (38 and 30 kDa) and 1 cytosolic (16 kDa) antigens were purified from Mycobacterium tuberculosis culture filtrate and cytosol, respectively, using a combination of chromatographic and electrophoretic methods. One recombinant antigen (27 kDa) overexpressed in Escherichia coli was also isolated. The diagnostic test characteristics of circulating immune complex (CIC)-bound antibodies to purified protein antigens, singly and in combination, were evaluated in patients with pulmonary tuberculosis. The individual antigens ranged in their sensitivity from 73% to 88%, while considering the IgG response. Addition of IgA results improved the sensitivity. The combination of IgG results for 38, 30, and 16 kDa offered >95% sensitivity and specificity for the smear- and culture-positive tuberculosis, as well as for the smear-negative, culture-positive group. CIC-bound antibodies promise to be a better diagnostic tool than serum antibodies.

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.

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.