Firdaus Shahid

Cytokine profiles using whole-blood assays can discriminate between tuberculosis patients and healthy endemic controls in a BCG-vaccinated population

Whole-blood assays (WB) provide a simple tool for assessing immune cytokine profiles which may be useful laboratory predictors of early disease, aiding the evaluation of new tuberculosis (TB) vaccines and offering insights into disease pathogenesis. Although BCG does not provide protection against pulmonary disease in TB endemic areas, it does modulate immune responses to mycobacterial antigens. It is important, therefore, to evaluate any new tool in an endemic setting in both BCG vaccinees and patients with tuberculosis. We have assessed the optimal conditions in terms of dose and kinetics of those cytokines which are released early (TNF-alpha, IL6 and TGF-beta, IL10) or (interferon [IFN]-gamma and IL5) in WB cultures stimulated with mitogens and mycobacterial antigens. Responses were studied in parallel in untreated TB patients and endemic control groups. Optimal responses to LPS (predominantly monocyte-derived) occurred on days 1-2, whereas for PHA (predominantly T-cell-derived), they were on days 3-5. Secreted Mycobacterium tuberculosis culture filtrate proteins (CFP) provided a stronger stimulus for monocyte-derived cytokines compared to PPD, but both antigens were comparable for induction of T-cell cytokines. Using unpaired Students t-tests, pulmonary tuberculosis patients (P.TB; n=11), in response to CFP, showed higher monocyte-derived IL6 (p=0.023) and IL10 (p=0.042) compared to endemic controls (EC; n=13), and significantly suppressed T-cell-derived IFN-gamma (p=0.028) and IL5 (p=0.012) secretion but increased IL10 (p=0.047) on day 5, indicating that CFP is a strong stimulus for IL10 secretion in pulmonary TB patients. Extrapulmonary TB patients (E.TB; n=6) showed no elevation of early monocyte-derived cytokines to either PPD or CFP, but showed a marked suppression of the T-cell-derived cytokines IFN-gamma (PPD, p=0.015; CFP, p=0.05) and IL5 (PPD, p=0.05; CFP, p=0.015). Cytokine analysis in WB cultures is, therefore, able to discriminate between active tuberculosis infection and nondiseased healthy controls.

Longitudinal tracking of cytokines after acute exposure to tuberculosis: association of distinct cytokine patterns with protection and disease development

ABSTRACT Household contacts (HCs) of patients with tuberculosis (TB) are at higher risk of infection as well as the development of active disease. Longitudinal tracking of antigen-specific cytokines after acute exposure may significantly advance our understanding of the dynamic changes in cytokine patterns associated with disease establishment. To achieve this objective, we carried out a prospective cohort study with healthy HCs after exposure to TB. The patterns of cytokines (gamma interferon [IFN-γ] and interleukin 10 [IL-10]) in response to mycobacterial antigens (culture filtrate [CF] proteins) and nonspecific mitogens (phytohemagglutinin [PHA] and lipopolysaccharide [LPS]) were assessed at 0, 6, 12, and 24 months after exposure. Seven of 109 (6.4%) HCs developed active disease. Six of the seven individuals were females, and active disease developed between 12 and 15 months after exposure in 5/20 families. The most significant findings were the exponential increases (∼1,000-fold) in both the CF protein- and the PHA- or LPS-induced IFN-γ/IL-10 ratio in healthy HCs (n = 26), which peaked at 12 months, compared to the levels in HCs who developed disease (n = 7), in whom relatively flat responses were observed during the 24-month period. Linear trends for 0 to 12 and 0 to 24 months for the CF protein-induced IFN-γ/IL-10 ratio showed significant differences between the two groups, as determined by the use of the Mantel extension test for χ2 analysis (odds ratio = 0.45; 95% confidence interval = 0.295 to 0.685; P = 0.0002). Our results strongly suggest that the magnitude of the IFN-γ/IL-10 ratio at 12 months after exposure may be a critical determinant in the resolution of infection. These studies provide new insights into the cytokine responses associated with disease establishment or the resolution of infection after natural exposure to TB and have implications for TB control programs as well vaccine efficacy studies.

A Postgenomic Approach to Identification of Mycobacterium leprae-Specific Peptides as T-Cell Reagents

ABSTRACT To identify Mycobacterium leprae-specific human T-cell epitopes, which could be used to distinguish exposure to M. leprae from exposure to Mycobacterium tuberculosis or to environmental mycobacteria or from immune responses followingMycobacterium bovis BCG vaccination, 15-mer synthetic peptides were synthesized based on data from the M. lepraegenome, each peptide containing three or more predicted HLA-DR binding motifs. Eighty-one peptides from 33 genes were tested for their ability to induce T-cell responses, using peripheral blood mononuclear cells (PBMC) from tuberculoid leprosy patients (n = 59) and healthy leprosy contacts (n = 53) from Brazil, Ethiopia, Nepal, and Pakistan and 20 United Kingdom blood bank donors. Gamma interferon (IFN-γ) secretion proved more sensitive for detection of PBMC responses to peptides than did lymphocyte proliferation. Many of the peptides giving the strongest responses in leprosy donors compared to subjects from the United Kingdom, where leprosy is not endemic, have identical, or almost identical, sequences in M. leprae and M. tuberculosis and would not be suitable as diagnostic tools. Most of the peptides recognized by United Kingdom donors showed promiscuous recognition by subjects expressing differing HLA-DR types. The majority of the novel T-cell epitopes identified came from proteins not previously recognized as immune targets, many of which are cytosolic enzymes. Fifteen of the tested peptides had ≥5 of 15 amino acid mismatches between the equivalent M. leprae and M. tuberculosissequences; of these, eight gave specificities of ≥90% (percentage of United Kingdom donors who were nonresponders for IFN-γ secretion), with sensitivities (percentage of responders) ranging from 19 to 47% for tuberculoid leprosy patients and 21 to 64% for healthy leprosy contacts. A pool of such peptides, formulated as a skin test reagent, could be used to monitor exposure to leprosy or as an aid to early diagnosis.

Th1/Th2 Cytometric Bead Array can discriminate cytokine secretion from endogenously activated cells in pulmonary disease, recent and remote infection in tuberculosis

Differential T cell trafficking through the blood compartment towards infected foci may be occurring in different stages of tuberculosis disease and infection. The aim of the present study was to identify cytokine signatures in the blood compartment in tuberculosis patients with pulmonary disease (PTB=19), recently exposed household contacts (HC=27) and nonexposed community controls (EC=37). Diluted (1:10) whole blood was cultured for 2 days and cytokine secretion was assessed using Cytometric Bead Array (Th1/Th2 kit II; BD Biosciences) which included IL-2, TNF-α, IFN-γ (Type1/T1), IL-4, IL-6 and IL-10 (Type2/T2). All T1/T2 cytokines were elevated in PTB (AUROC>0.9) while HC showed selective elevation of IL-6 (AUROC>0.7) compared to EC. Principal component analysis (PCA) extracted two groupings with Eigen values >1; IL-6 separated into the second component for PTB, HC and EC. After rotation, IFN-γ was correlated with the first component for PTB and EC and the second component for HC indicating an absence of T1/T2 dichotomy. Therefore endogenous cytokine signatures may indicate differential T cell trafficking in different stages of tuberculosis infection and disease.

Immune profiling of leprosy and tuberculosis patients to 15-mer peptides of Mycobacterium leprae and M-tuberculosis GroES in a BCG vaccinated area: implications for development of vaccine and diagnostic reagents

Mycobacterium leprae (ML) GroES has been shown to induce strong T cell responses in tuberculoid as well as in exposed healthy contacts of leprosy patients, and therefore this antigen has been the focus of study as a potential vaccine candidate. Paradoxically, we have shown that ML GroES also induces extremely high titres of IgG1 antibody in leprosy patients across the disease spectrum, a response associated with disease progression. IgG1 antibodies in leprosy also show a negative association with interferon‐γ, a critical T cell cytokine responsible for macrophage activation and intracellular killing of mycobacteria. We therefore queried if antibody and T cell responses were being evoked by different epitopes in ML GroES proteins. To address the issue of epitope recognition in mycobacterial diseases, we have analysed 16 peptides (15‐mer peptides) spanning the entire ML and M. tuberculosis GroES protein in leprosy (n = 19) and tuberculosis (n = 9) patients and healthy endemic controls (n = 8). Our analysis demonstrates clearly that the dominant peptides evokingT cell and IgG subclass antibodies were different. The target of both T and B cell responses were cross‐reactive epitopes in all groups. Differences in disease and healthy states related to the strength (mean intensity) of the T cell and antibody response. IgG1 and IgG3 antibodies were associated with disseminated disease and IgG 2 and IgG4 with disease limitation. Such comprehensive immune profiling of antigen‐specific responses is critical to understanding the disease pathogenesis and also if these reagents are to be exploited for either diagnostic or vaccine purposes.

Biomarker Changes Associated with Tuberculin Skin Test (TST) Conversion: A Two-Year Longitudinal Follow-Up Study in Exposed Household Contacts

Background A high prevalence (50–80%) of Tuberculin Skin Test Positivity (TST+ ≥10 mm indurations) has been reported in TB endemic countries. This pool forms a huge reservoir for new incident TB cases. However, immune biomarkers associated with TST conversion are largely unknown. The objective of this study was to identify immune biomarkers associated with TST conversion after acute Mycobacterium tuberculosis (MTB) exposure. Methodology/Principal Findings A 24 month longitudinal study was carried out in a recently MTB exposed cohort of household contacts (HC = 93; 75% TST+). Control group consisted of unexposed community controls (EC = 59; 46%TST+). Cytokine secretion was assessed in whole blood cultures in response to either mycobacterial culture filtrate (CF) antigens or mitogens (PHA or LPS) using Elisa methodology. Compared to the EC group, the HC group at recruitment (Kruskal-Wallis Test) showed significantly suppressed IFN γ (p = 0.0001), raised IL-10 (p = 0.0005) and raised TNF α (p = 0.001) in response to CF irrespective of their TST status. Seventeen TST-HC, showed TST conversion when retested at 6 months. Post TST conversion (paired t tests) significant increases were observed for CF induced IFN γ (p = 0.038), IL-10 (p = 0.001) and IL-6 (p = 0.006). Cytokine responses were also compared in the exposed HC group with either recent infection [(TST converters (N = 17)] or previous infection [TST+ HC (N = 54)] at 0, 6, 12 and 24 months using ANOVA on repeated measures. Significant differences between the exposed HC groups were noted only at 6 months. CF induced IFN γ was higher in previously infected HC group (p = 0.038) while IL-10 was higher in recently infected HC group (p = 0.041). Mitogen induced cytokine secretion showed similar differences for different group. Conclusions/Significance Our results suggest that TST conversion is associated with early increases in IFN γ and IL-10 responses and precedes latency by several months post exposure.

Dynamic Changes in Biomarker Profiles Associated with Clinical and Subclinical Tuberculosis in a High Transmission Setting: A Four-Year Follow-Up Study

Mycobacterium tuberculosis (MTB) is a slow growing bacterium. Therefore, the immune responses associated with resolution of infection or development of disease post‐exposure may take several months to evolve. We have carried out a prospective longitudinal study in a high TB transmission setting to determine the evolution of biomarkers in a recently exposed household contact (HC = 77) and their respective sputum positive index cases (TB = 17). Mycobacterium‐induced cytokines [interferon‐γ (IFN‐γ), tumour necrosis factor‐α, interleukin‐6 (IL‐6) and IL‐10)] were assessed in whole blood cultures and immunoglobulin G (IgG1) antibodies in plasma. When compared with non‐exposed community controls (endemic controls = 59) the HC group at intake showed changes in biomarkers commensurate with recent exposure. The HC group showed significant increases in IFN‐γ between 0 and 6 months (paired t‐test; P = 0.001) and IL‐0 between 6 and 12 months (P = 0.001), most likely reflecting the role of these cytokines in resolution and immune recovery from infection as this HC cohort remained symptom‐free for 4 years without prophylactic treatment. When the TB group post‐treatment was compared with the HC group, the best discriminators (ANOVA; repeated measures) were IL‐10 responses at 0 (P = 0.004) and 6 months (P = 0.001) and IgG1 at 6 (P = 0.004) and 12 months (P = 0.014) with a 3–4 fold higher responses in the TB group. Therefore, within each group, biomarkers show unique profile of responses. These studies highlighted the importance of assessing multiple biomarkers in longitudinal studies for providing better understanding of protective biomarker profiles associated with resolution of clinical and subclinical infections in TB.

Leprosy patients with lepromatous disease recognize cross-reactive T cell epitopes in the Mycobacterium leprae 10-kD antigen.

T cell responses play a critical role in determining protective responses to leprosy. Patients with self‐limiting tuberculoid leprosy show high T cell reactivity, while patients with disseminated lepromatous form of the disease show absent to low levels of T cell reactivity. Since the T cell reactivity of lepromatous patients to purified protein derivative (PPD), a highly cross‐reactive antigen, is similar to that of tuberculoid patients, we queried if lepromatous patients could recognize cross‐reactive epitopes in Mycobacterium leprae antigens as well. T cell responses were analysed to a recombinant antigen 10‐kD (a heat shock cognate protein) which is available from both M. tuberculosis (MT) and M. leprae (ML) and displays 90% identity in its amino acid sequence. Lymphoproliferative responses were assessed to ML and MT 10 kD in newly diagnosed leprosy patients (lepromatous, n = 23; tuberculoid, n = 65). Lepromatous patients showed similar, but low, lymphoproliferative responses to ML and MT 10 kD, while tuberculoid patients showed much higher responses to ML 10 kD. This suggests that the tuberculoid patients may be recognizing both species‐specific and cross‐reactive epitopes in ML 10 kD, while lepromatous patients may be recognizing only cross‐reactive epitopes. This was further supported by linear regression analysis. Lepromatous patients showed a high concordance in T cell responses between ML and MT 10 kD (r = 0.658; P < 0.0006) not observed in tuberculoid patients (r = 0.203; P > 0.1). Identification of cross‐reactive T cell epitopes in M. leprae which could induce protective responses should prove valuable in designing second generation peptide‐based vaccines.

Association of plasma cytokines with radiological recovery in pulmonary tuberculosis patients.

Objective/Background: The characterization of tuberculosis (TB) patients as slow or fast responders post anti-TB treatment has always been a matter of tremendous interest as slow responders are most likely to relapse and/or develop complications. Pulmonary tissue healing as assessed with radiology is the only available tool for tissue recovery but is not predictive at intake. The objective of the current study was to assess biomarkers associated with fast and slow recovery in TB patients at recruitment. Methods: Pulmonary TB patients (N = 15) were assessed for radiological recovery serially in parallel with clinical signs and symptoms, hematological parameters, and plasma cytokines at 0months, 6months, 12months, and 24months. On the basis of differential radiological healing, patients were characterized into slow (>12 months), intermediate (<12months), and fast (<6months) responders. Results: Baseline plasma cytokines (interleukin [IL]-2, -4, -6, -10, tumor necrosis factor-α, and interferon-γ) were determined using cytometric bead array. IL-2 and -4 were able to accurately differentiate slow and fast responders into two distinct clusters using hierarchal clustering analysis. Compared with fast responders, slow responders showed significantly high IL-2 and -4 at baseline (p = .001 Mann–Whitney U test). Conclusion: In-depth analysis of cytokines and its association with radiological recovery in TB patients may be useful in monitoring TB patients postchemotherapy for both clinicians and TB control program.