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Clin Vaccine Immunol. Feb 2014; 21(2): 133–142.
PMCID: PMC3910933

Identification of Immunological Biomarkers Which May Differentiate Latent Tuberculosis from Exposure to Environmental Nontuberculous Mycobacteria in Children

C. J. Papasian, Editor

Abstract

A positive gamma interferon (IFN-γ) response to Mycobacterium tuberculosis early secretory antigenic target-6 (ESAT-6)/culture filtrate protein-10 (CFP-10) has been taken to indicate latent tuberculosis (TB) infection, but it may also be due to exposure to environmental nontuberculous mycobacteria in which ESAT-6 homologues are present. We assessed the immune responses to M. tuberculosis ESAT-6 and cross-reactive responses to ESAT-6 homologues of Mycobacterium avium and Mycobacterium kansasii. Archived culture supernatant samples from children at 3 years post-BCG vaccination were tested for cytokine/chemokine responses to M. tuberculosis antigens. Furthermore, the IFN-γ responses to M. tuberculosis antigens were followed up for 40 children at 8 years post-BCG vaccination, and 15 TB patients were recruited as a control group for the M. tuberculosis ESAT-6 response in Malawi. IFN-γ enzyme-linked immunosorbent assays (ELISAs) on supernatants from diluted whole-blood assays, IFN-γ enzyme-linked immunosorbent spot (ELISpot) assays, QuantiFERON TB Gold-In Tube tests, and multiplex bead assays were performed. More than 45% of the responders to M. tuberculosis ESAT-6 showed IFN-γ responses to M. avium and M. kansasii ESAT-6. In response to M. tuberculosis ESAT-6/CFP-10, interleukin 5 (IL-5), IL-9, IL-13, and IL-17 differentiated the stronger IFN-γ responders to M. tuberculosis ESAT-6 from those who preferentially responded to M. kansasii and M. avium ESAT-6. A cytokine/chemokine signature of IL-5, IL-9, IL-13, and IL-17 was identified as a putative immunological biosignature to differentiate latent TB infection from exposure to M. avium and M. kansasii in Malawian children, indicating that this signature might be particularly informative in areas where both TB and exposure to environmental nontuberculous mycobacteria are endemic.

INTRODUCTION

Around half a million children worldwide from ages 0 to 14 years became ill with tuberculosis (TB) in 2011, resulting in approximately 64,000 deaths (1). Furthermore, 10 million children became orphans due to parental deaths from TB in 2009 (1). Despite the fact that children are at a higher risk of developing TB disease once infected and are more susceptible to death, pediatric TB often goes undiagnosed in children from birth to 15 years of age (2). This is because access to health services and diagnostics is often severely limited, the clinical signs and symptoms of TB in children are nonspecific, and current diagnostic tests lack sensitivity (2). This highlights the need to develop a more accurate test for TB infection than the tuberculin skin test, which lacks specificity and sensitivity due to cross-reactivity induced by BCG vaccination or exposure to environmental nontuberculous mycobacteria (NTM) (3, 4). A gamma interferon (IFN-γ) release assay based on detection of the specific IFN-γ release from antigen (Ag)-specific activated T cells that are incubated ex vivo with peptides from Mycobacterium tuberculosis antigens, such as early secretory antigenic target-6 (ESAT-6) and antigen TB7.7, has been considered, as these putative M. tuberculosis-specific antigens are genetically deleted from all Mycobacterium bovis BCG strains (5). However, ESAT-6 homologues or ESAT-6-like proteins are present in Mycobacterium leprae as well as some environmental NTM which exist in water and soil, such as Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium szulgai, and Mycobacterium avium (5,9). Thus, it has been suggested that an IFN-γ response to ESAT-6 and the 10-kDa culture filtrate protein-10 (CFP-10) on its own was not sufficient to detect M. tuberculosis infection in areas where both M. tuberculosis and environmental NTM or other pathogenic mycobacteria are endemic (10). To develop a more specific immunodiagnostic test for the detection of latent TB infection (LTBI), studies were designed to identify additional biomarkers and alternative tests to differentiate host immune responses to the M. tuberculosis ESAT-6 and CFP-10 proteins from those against their homologues in environmental NTM, particularly in regions where both TB and environmental NTM are endemic.

Since there is a high frequency of TB in the households of index TB cases in Malawi (11), children are vulnerable and are at high risk of becoming infected by adults with TB. IFN-γ responses to mycobacterial antigens have been extensively studied in cohort studies in Malawian infants in the Karonga Prevention Study (KPS) in Chilumba, Malawi (12, 13). The immune responses of the infants were followed up between 2002 and 2006 at 3 months, 12 months, and 3 years post-BCG vaccination, and 13.6% (13/98) of the infants tested at 3 years postvaccination responded to M. tuberculosis ESAT-6/CFP-10 (14). Such a result might suggest that the 13 infants who showed positive IFN-γ responses to M. tuberculosis ESAT-6/CFP-10 were infected with M. tuberculosis. However, none showed symptoms of clinical disease; an alternative explanation might be that the response shown was due to cross-reactivity with ESAT-6 homologues from other NTM which are endemic in the area, as M. leprae infection is now uncommon in Malawi. The major slow-growing NTM found in the sputa of TB patients in northern Malawi have been identified as species from the M. avium-intracellulare complex as most common, and Mycobacterium gordonae, Mycobacterium terrae, M. kansasii, and Mycobacterium malmoense were also isolated (15).

In this study, we hypothesized that the positive IFN-γ responses to M. tuberculosis ESAT-6/CFP-10 observed in these 13 children at 3 years post-BCG vaccination might not reliably indicate M. tuberculosis infection but can be derived from cross-reactive responses to ESAT-6 homologues of environmental NTM and that cytokine/chemokine signatures may distinguish between the subjects who showed stronger IFN-γ responses to M. tuberculosis ESAT-6 and those who responded more strongly to ESAT-6 derived from NTM. To test these two hypotheses, we chose M. avium subsp. avium and M. kansasii, which have ESAT-6 homologues, among the species frequently found in the sputa of TB patients in northern Malawi. We measured the immune responses of the children at 8 years post-BCG vaccination in Malawi and assessed the cross-reactive responses between M. tuberculosis ESAT-6 and ESAT-6 homologues of M. avium and M. kansasii to identify how these responses related to the positive IFN-γ responses to M. tuberculosis ESAT-6/CFP-10 at 3 years post-BCG vaccination. In addition, we analyzed the cytokine/chemokine signatures in response to M. tuberculosis ESAT-6/CFP-10 and M. tuberculosis purified protein derivative (PPD) to identify potential biomarkers which can discriminate M. tuberculosis infection from the cross-reactive response to ESAT-6 homologues of M. avium and M. kansasii.

MATERIALS AND METHODS

Ethical permissions.

Authorization for the exportation of archives in Malawi was granted by the National Health Sciences Research Committee (NHSRC). Ethical permission for the previous studies to look at immune response in infants at 3 months, 12 months, and 3 years postvaccination was granted by the NHSRC (approval 01/38) and the London School of Hygiene & Tropical Medicine (LSHTM) ethics committee (approval 745A) in 2001. Ethical permission for a follow-up study to determine the immune responses of 40 children at 8 years post-BCG vaccination and 15 TB patients was granted by the LSHTM ethics committee (approval 5929) and the NHSRC in Malawi (approval 866) in 2011.

Consent forms and information sheets, including translation into local languages, were prepared for the parents/guardians of children from the previous cohort study group and TB patients. Appropriate informed written consent was obtained from adult TB patients and from the parents or guardians of the children recruited into the study. All of the study participants had the study explained to them and were given the opportunity to ask questions. Confidentiality was ensured by using unique study numbers and blood sample numbers on the samples and questionnaires. Forms with the ethics application, the research proposal, consent forms, and information sheets were reviewed by the ethics committees of the LSHTM and the NHSRC in Malawi.

Selection of the archived samples at 3 years post-BCG vaccination.

Previously collected culture supernatant samples obtained from Malawian infants at 3 years post-BCG vaccination, who participated in a vaccination cohort study, were retrieved from the archive at the laboratories of the KPS (14). Based on the previous results that showed that 13 of 98 infants tested had positive IFN-γ responses to M. tuberculosis ESAT-6/CFP-10 fusion protein in a whole blood assay (WBA) at 3 years postvaccination, archived culture supernatants from 13 IFN-γ responders to M. tuberculosis ESAT-6/CFP-10 and 11 nonresponders were retrieved and transported to the LSHTM laboratory to determine their cytokine/chemokine profiles. Samples which had been stimulated with M. tuberculosis PPD (batch RT49, lot 204; Statens Serum Institut [SSI], Copenhagen, Denmark), M. tuberculosis ESAT-6/CFP-10 (Bill and Melinda Gates Foundation Grand Challenge 6 [BMGF GC6] project, batch 040101) (16, 17), phytohemagglutinin-M (PHA-M) (Sigma-Aldrich, Poole, United Kingdom), and culture medium (RPMI 1640; Sigma-Aldrich) were analyzed further from each of the selected study participants. To test the sample quality following extended storage since 2006, 4 additional archived samples were stimulated with 19 different antigens: PHA-P, M. tuberculosis PPD, M. avium PPD, M. bovis BCG (SSI), tetanus toxoid, antigen 85A, soluble egg antigen, streptokinase streptodornase antigen, ESAT-6, TB10 (Rv0288), PHA, and Dormancy survival regulator (DosR) regulon encoded antigens such as M. tuberculosis Rv0081, Rv1737C, Rv1812C, Rv2006, Rv2625C, Rv3132C, Rv3133C, and Rv0574C (BMGF GC6 project) in addition to RPMI medium were also retrieved and transported to the LSHTM laboratory for IFN-γ enzyme-linked immunosorbent assays (ELISAs).

Recruitment of children at 8 years post-BCG vaccination and TB patients.

It was confirmed that 11 of the 13 subjects who previously showed positive IFN-γ responses to M. tuberculosis ESAT-6/CFP-10 were traceable under the demographic KPS in Malawi (55% male and 45% female), and they were recruited for the new follow-up study at 8 years postvaccination (14). As a control group, 11 nonresponders at 3 years postvaccination who were also confirmed to be traceable were recruited. In addition, because of the high possibility of individuals converting from nonresponder to responder during the 5 years after the last follow-up visit, 18 additional ESAT-6/CFP-10 nonresponders at 3 years post-BCG vaccination were randomly selected and recruited (59% male and 41% female). All of the recruited responders and nonresponders had a BCG vaccination within 1 week after birth. To act as a positive-control population for ESAT-6 responses, 15 TB patients were recruited from the Karonga District Hospital and the Chilumba Rural Hospital. As laboratory-confirmed cases of TB in children are rare in the Karonga District, at diagnosis or within the first 3 months of treatment we recruited adult TB patients between the ages of 18 and 50 years (40% male and 60% female). TB was confirmed by smear/culture of sputa, and the TB patients were not eligible if they were HIV positive, taking immunosuppressant medication, suffering from cancer or diabetes, pregnant, a prisoner, or unable to give consent (Fig. 1).

FIG 1
Collection of archived samples and recruitment of study subjects. In a previous study cohort, 13 of 98 children at 3 years post-BCG vaccination showed positive IFN-γ responses to M. tuberculosis ESAT-6/CFP-10. In this study, 24 sets of archived ...

IFN-γ ELISA to test archive sample quality.

The production of IFN-γ was retested in archived samples from 4 different individuals to determine if the archived samples had retained their integrity following the extended storage period. The IFN-γ ELISA protocol for this test followed a previous protocol (12, 14) using a standard sigmoid curve fit.

Blood collection and PBMC isolation.

A total of 10 ml of blood was collected in a heparinized tube (170 IU of sodium heparin; BD Vacutainer, Plymouth, United Kingdom). Four hundred and fifty microliters was used for a diluted WBA, and the remaining blood was used for peripheral blood mononuclear cell (PBMC) isolation. PBMCs were prepared by density gradient centrifugation using Ficoll (Sigma-Aldrich) (18). The cells were diluted to 2.5 × 105 cells/180 μl in AIM-V growth medium (Fisher Scientific) and 180 μl of resuspended cells were added into enzyme-linked immunosorbent spot (ELISpot) plate wells which contained 20 μl of each peptide antigen or controls.

Diluted whole-blood assay and measurement of IFN-γ.

Blood was diluted in RPMI supplemented with 1% l-glutamine (1 in 5; Invitrogen, Paisley, United Kingdom), and 100 μl was added into each well with 100 μl each of M. tuberculosis PPD (RT50, lot 219; SSI) at a final concentration of 5 μg/ml, ESAT-6/CFP-10 (BMGF GC6 to 74 project, batch 040101) at a final concentration of 10 μg/ml (16, 17), PHA (lot 017k4029; Sigma-Aldrich) at a final concentration of 5 μg/ml, and RPMI 1640 supplemented with 1% l-glutamine (Sigma-Aldrich). After a 6-day incubation at 37°C, the culture supernatant was harvested, and the production of IFN-γ was measured in 50 μl of culture supernatant by an ELISA (19). A positive response in an IFN-γ ELISA was defined as >62.5 pg/ml, which is twice the limit of detection of the assay (19). The concentrations of IFN-γ above 4,000 pg/ml were set to be 4,000 pg/ml.

ESAT-6 overlapping peptides derived from M. tuberculosis, M. avium, and M. kansasii.

M. avium and M. kansasii, which contain homologous ESAT-6 sequences, were selected to examine the cross-reactivity between M. tuberculosis ESAT-6 and ESAT-6 homologues of NTM in Malawian children and TB patients. The protein sequences between M. tuberculosis ESAT-6 and ESAT-6 homologues of M. avium and M. kansasii are >90% identical to M. kansasii but only 27% identical to M. avium (see http://blast.ncbi.nlm.nih.gov/Blast.cgi#221229377, http://www.ncbi.nlm.nih.gov/protein/CAE55648.1?report=genpept [GenBank accession no. CAE55648.1], http://www.ncbi.nlm.nih.gov/protein/ZP_05218333.1?report=genpept [NCBI reference sequence ZP_05218333.1], and http://www.ncbi.nlm.nih.gov/protein/gb%7CACG70856.1 [GenBank accession no. ACG70856.1]). The positions of predominantly recognized epitopes are scattered throughout the ESAT-6 protein sequence, and the multiple T cell epitopes recognized are different depending on the population (20,22). Based on published papers and the SYFPEITHI program used to predict epitope sites (23), 14 overlapping peptides, including 15 mers with predicted epitopes for major histocompatibility complex (MHC) type II binding, were designed using the full-length ESAT-6 amino acid sequence from M. tuberculosis and M. avium subsp. avium (see Fig. S1 in the supplemental material). The ESAT-6 amino acid sequences are identical from amino acid 1 to 62 between M. tuberculosis and M. kansasii, and only two overlapping peptides, including different sequences, were synthesized for ESAT-6 homologues of M. kansasii. The overlapping peptides were put together into 5 peptide pools according to the positions of amino acids in the sequence alignment, i.e., M. tuberculosis1-57, M. tuberculosis55-95, M. avium2-59, M. avium57-97, and M. kansasii55-95 (see Fig. S1).

IFN-γ ELISpot assay.

The IFN-γ ELISpot assay was carried out as previously described (18). The final concentrations of the M. tuberculosis, M. avium, and M. kansasii peptides were 10 μg/ml each, and the concentration of M. tuberculosis PPD was 5 μg/ml. An anti-human CD3 monoclonal antibody (MAb) was used at 0.1 μg/ml (Mabtech, Nacka Strand, Sweden). The spots were counted using an ELISpot 4.0 reader (AID Diagnostika GmbH, San Diego, CA). The positive responses to each antigen were measured by an empirical rule (ER) which defines a positive response as an at least 2-fold increase of spot number in the experimental wells over the background with a minimum threshold of 5 spots per 100,000 PBMCs in the experimental wells (24). An M. tuberculosis ESAT-6-specific positive response was defined as spots appearing in M. tuberculosis ESAT-6/CFP-10 and either M. tuberculosis ESAT-61-57 or M. tuberculosis ESAT-655-95 stimulation alongside the positive response to anti-CD3 antibody.

QuantiFERON TB Gold-In Tube test.

The QuantiFERON TB Gold-In Tube (QFT-IT) test is a commercially available diagnostic assay for measuring cell-mediated immune responses to M. tuberculosis-specific antigens using ESAT-6, CFP-10, and TB7.7 (4). For the test, 1 ml of blood was collected directly into each of two QuantiFERON TB Gold tubes (a nil and an M. tuberculosis Ag tube, ESAT-6, CFP-10, and TB7.7 peptide Ags; Cellestis, Valencia, CA). The tubes containing blood were incubated upright at 37°C for 24 h, and plasma was harvested for IFN-γ ELISAs. The plasma samples were stored at −80°C until the recruitment of all study subjects was complete. The plasma samples were assayed using an IFN-γ ELISA according to the manufacturer's protocol (QuantiFERON-TB Gold, Cellestis). The data were analyzed using the QuantiFERON-TB Gold IT analysis software (Cellestis), and the results were expressed as positive, negative, and intermediate responses.

Nineteen- and forty-two-plex bead assays.

Culture supernatants from cells stimulated with M. tuberculosis ESAT-6/CFP-10, M. tuberculosis PPD, PHA-M, and RPMI 1640 were selected from the archive at 3 years post-BCG vaccination for a multiplex bead assay with 19 different cytokines and chemokines: interleukin-1α (IL-1α), IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17, IFN-γ, tumor necrosis factor alpha (TNF-α), IFN-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), MIP-1α (CCL3), IP-10 (CXCL10), MDC (CCL22), and MCP-3 (CCL7). The beads in the 42-plex kit for the samples from newly recruited subjects are a combination of a premixed 39-bead mix, including IL-1α, IL-1ra, IL-1β, IL-2, sIL-2Rα, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, IFN-α2, IFN-γ, TNF-α, TNF-β, sCD40L, MIP-1α (CCL3), MIP-1β (CCL4), Gro-α (CXCL1), IL-8 (CXCL8), IP-10 (CXCL10), MCP-1 (CCL2), MCP-3 (CCL7), MDC (CCL22), TGF-α, G-CSF, GM-CSF, IL-3, IL-7, eotaxin, FGF-2, Flt-3L, fractalkine (CX3CL1), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF), plus the addition of 3 chemokines, platelet-derived growth factor AA (PDGF-AA), PDGF-AB/BB, and RANTES (CCL5), which were added at 70 μl each from 3 individual vials after the beads were sonicated and vortexed. Beads were diluted 1 in 2, and the detailed protocol of the 42-plex bead assay followed the manufacturer's protocol (no. MPXHCYTO60KPMX42, MILLIPLEXMAP kit; Millipore, Billerica, MA) as described in a previous study (13). The range of the standard curve was from 3.2 to 10,000 pg/ml, and values below 3.2 pg/ml were set to 1.6 pg/ml. Considering the cost restraints, samples showing values above 10,000 pg/ml were not retested with dilution, but the values were set to 15,000 pg/ml (13).

Statistical analysis.

The Wilcoxon signed rank test and Spearman's rank correlation test were used to compare the IFN-γ concentrations measured in 2006 and 2010 in 4 different archived samples, including the culture supernatants stimulated with 19 antigens in each sample. The IFN-γ concentration measured by the IFN-γ ELISA was compared using the Mann-Whitney test between 40 children and 15 TB patients. Also, the Mann-Whitney test was used to compare cytokine responses measured by the multiplex bead assay between 13 IFN-γ responders and 11 nonresponders to M. tuberculosis ESAT-6. Agreement of the results obtained from different assays such as the IFN-γ ELISA, the IFN-γ ELISpot assay, and the QFT-IT test was assessed by kappa statistics.

RESULTS

IFN-γ responses to M. tuberculosis ESAT-6/CFP-10, PPD, and PHA.

IFN-γ responses to M. tuberculosis ESAT-6/CFP-10, M. tuberculosis PPD, and PHA-M were investigated by IFN-γ ELISAs after a 6-day culture of diluted whole blood isolated from 40 children at 8 years post-BCG vaccination and 15 TB patients at diagnosis or on treatment for <3 months. Among the 40 children recruited at 8 years post-BCG vaccination, 3 children showed positive IFN-γ responses to M. tuberculosis ESAT-6/CFP-10 (>62.5 pg/ml), while 36 of 40 children showed positive IFN-γ responses to M. tuberculosis PPD (>62.5 pg/ml). The median response of IFN-γ to M. tuberculosis PPD (853 pg/ml) was much greater than that to M. tuberculosis ESAT-6/CFP-10 (15 pg/ml), and all of the children recruited responded to PHA (Fig. 2A). Twelve out of the 15 TB patients responded to M. tuberculosis ESAT-6/CFP-10 (median concentration, 245 pg/ml), while only 3 of 40 children at 8 years post-BCG vaccination responded (median response, 15 pg/ml of all children) (Fig. 2A). One of the 3 IFN-γ-nonresponding TB patients (<62.5 pg/ml) was a patient at diagnosis, and two were patients on treatment. The IFN-γ responses to M. tuberculosis PPD were positive in all of the TB patients recruited, and the median IFN-γ response was 2,691 pg/ml, with IFN-γ responses ranging from 324 to 4,000 pg/ml. The median IFN-γ responses to both M. tuberculosis ESAT-6/CFP-10 and M. tuberculosis PPD were significantly greater in TB patients than in children (P < 0.001 and P < 0.01, respectively), while there was no difference in the median IFN-γ responses to PHA (P = 0.15) (Fig. 2A). Interestingly, only two subjects (103278 and 103738) at 8 years post-BCG vaccination had very marked increases in IFN-γ, which were >10 times greater (4,000 and 1,958 pg/ml, respectively) than the IFN-γ responses to M. tuberculosis ESAT-6/CFP-10 in the same children at 3 years post-BCG vaccination (Fig. 2B), while most previous nonresponders did not show positive IFN-γ responses to M. tuberculosis ESAT-6/CFP-10 and one subject (104043) showed a weak positive IFN-γ response (101 pg/ml) at 8 years post-BCG vaccination (Fig. 2C).

FIG 2
IFN-γ responses to M. tuberculosis (M. tb) ESAT-6/CFP-10, M. tuberculosis PPD, and PHA in 40 children at 8 years post-BCG vaccination and 15 TB patients. (A) TB patients showed significantly greater IFN-γ production in response to M. tuberculosis ...

Cross-reactivity between M. tuberculosis ESAT-6 and its homologues.

To determine the level of cross-reactivity of IFN-γ responses between M. tuberculosis ESAT-6 and ESAT-6 homologues of M. avium subsp. avium and M. kansasii, 5 overlapping peptide antigen pools derived from M. tuberculosis ESAT-6 and ESAT-6 homologues of M. avium and M. kansasii were synthesized (see Fig. S1 in the supplemental material), and the quantification of T cells producing IFN-γ in response to the ESAT-6 peptide antigens was measured ex vivo using PBMCs from 40 children and 15 TB patients. Among the 40 children, only 5% (2/40; subjects 103738 and 104043) showed IFN-γ-producing cells in response to the M. tuberculosis ESAT-6/CFP-10 fusion protein and either M. tuberculosis1–57 or M. tuberculosis55–95 ESAT-6 peptides (Table 1). Two more subjects (subjects 103604 and 104041) showed positive IFN-γ-producing cells in response to M. tuberculosis55–95 ESAT-6, although they did not show positive responses to the M. tuberculosis ESAT-6/CFP-10 fusion protein, and they were not counted as responders to M. tuberculosis ESAT-6 (data not shown). In all, 5 children responded to M. avium ESAT-6 peptides and 3 children showed positive responses to M. kansasii ESAT-6 peptides (Table 1). In response to PPD, IFN-γ-producing cells were detected in 75% (30/40) of the tested children. Compared with the proportion of positivity in children, TB patients showed much greater IFN-γ-positive responses to M. tuberculosis ESAT-6 in the ELISpot assay (Table 1) and in the IFN-γ ELISA (Fig. 2). The level of cross-reactivity of IFN-γ responses between M. tuberculosis ESAT-6 and ESAT-6 homologues of M. avium and M. kansasii is shown in Table 2. The two responders to M. tuberculosis ESAT-6/CFP-10 antigens and M. tuberculosis ESAT-6 peptides also showed positive IFN-γ responses to both M. avium2–59 and M. kansasii55–95. In 11 TB patients who showed positive IFN-γ-producing cells to M. tuberculosis ESAT-6, >45% and 60% of the responders showed cross-reactivity with M. avium2–59 (5/11 patients) and M. kansasii55–95 ESAT-6 (7/11 patients), respectively. In summary, more than half of the responders who showed positive IFN-γ-producing cells in response to M. tuberculosis ESAT-6 had cross-reactive IFN-γ responses to M. avium2–59 and M. kansasii55–95 ESAT-6 peptides, with greater cross-reactivity with M. kansasii ESAT-6.

TABLE 1
Detection of positive antigen-reactive T cells producing IFN-γ in Malawian children and TB patientsa
TABLE 2
Cross-reactivity of IFN-γ responses between M. tuberculosis ESAT-6 and ESAT-6 homologues of M. avium and M. kansasiia

IFN-γ responses by QFT-IT test.

Forty children at 8 years post-BCG vaccination and 15 TB patients were tested by the QFT-IT IFN-γ ELISA. Positive IFN-γ responses were detected in 4 of 40 (10%) children. Among the 4 positive responders in the children, the results of only 1 subject (no. 103738) matched with the results of the IFN-γ ELISA after the WBA and the IFN-γ ELISpot assay, while the other 3 positive responders by QFT-IT test were not found to be positive by the IFN-γ ELISA or the ELISpot assay. On the other hand, greater agreement of results of the three tests was found in the TB patient group. Thirteen of 15 TB patients showed positive IFN-γ responses by the QFT-IT test, and 12 and 11 patients showed positive responses to M. tuberculosis ESAT-6/CFP-10 by the IFN-γ ELISA and the ELISpot assay, respectively. Among the 13 QFT-IT responders, all 5 of the TB patients at diagnosis were positive by the QFT-IT test, and 8 of 10 patients who were on treatment showed positive IFN-γ responses.

Agreement between the results from different assays.

In this study, 3 different methodologies, the IFN-γ ELISA, the IFN-γ ELISpot assay, and the QFT-IT test, were used to measure IFN-γ responses in 40 children at 8 years post-BCG vaccination and in 15 TB patients. The outcomes of the different assays were discordant, and kappa statistics was applied to quantify this. The concordance of the ELISA and the ELISpot assay was highest with 89% agreement (kappa, 0.7130, P < 0.01). The agreement between the ELISpot assay and the QFT-IT test was 86% (kappa, 0.6358, P < 0.01) and 82% between the IFN-γ ELISA and the QFT-IT test (kappa, 0.5600, P < 0.01). In the 40 children only, the concordance between the IFN-γ ELISA and the ELISpot assay was also high (kappa, 0.7872, P < 0.01), while the agreement between the QFT-IT test and both the IFN-γ ELISA and the ELISpot assay was low (kappa, 0.2188 and 0.2857, P > 0.05 and P < 0.05, respectively).

Cytokine/chemokine signatures in children at 3 years post-BCG vaccination.

In order to examine if the cytokine proteins in the archived samples still remained intact, an IFN-γ ELISA was performed, and the level of IFN-γ production was compared with previous data obtained in 2006. The levels of IFN-γ measured from the archived samples collected from 4 infants at 3 years post-BCG vaccination were similar to those in the previous data from 2006 and slightly higher in some of the supernatant aliquots than the IFN-γ detection in the past (see Fig. S2A in the supplemental material). However, no significant differences were found in the IFN-γ concentrations of 19 culture supernatant samples from each of 4 subjects (indicated by lab number) when measured in 2006 and again in 2010 (subject 38289 [P = 0.49], subject 38290 [P = 0.14], subject 38291 [P = 0.36], and subject 38633 [P = 0.50] by the Wilcoxon signed rank test) (see Fig. S2A). In addition, the Spearman correlation coefficient calculated using the IFN-γ data obtained from all 76 samples was 0.9808 (P < 0.0001), indicating a strong correlation between the IFN-γ values obtained in 2006 and 2010 (see Fig. S2B in the supplemental material). Based on the IFN-γ production in response to M. tuberculosis ESAT-6/CFP-10, 17 cytokines and chemokines were analyzed among the IFN-γ responders and nonresponders. IL-4 and IL-15 were excluded from this analysis, as they were produced at levels below the limit of detection of the assay. M. tuberculosis ESAT-6/CFP-10 stimulation differentiated 13 IFN-γ responders from 11 nonresponders using 5 cytokines and chemokines, IL-1α, IL-10, MIP-1α, IP-10, and GM-CSF, with median responses showing a difference of >5-fold in the two groups. Furthermore, IL-5, IL-9, IL-13, and IL-17 were not produced in most of those tested, irrespective of whether they were IFN-γ responders or nonresponders (23/24) (Fig. 3). In response to PHA, the median concentration of most cytokine and chemokine responses measured was high apart from those of IL-2 and IL-4, which were below levels of detection (data not shown).

FIG 3
Cytokine/chemokine responses in archived samples from children at 3 years post-BCG vaccination. The levels of cytokines and chemokines in response to M. tuberculosis ESAT-6/CFP-10 measured by multiplex bead assays were compared between positive IFN-γ ...

Comparison of cytokine/chemokine signatures between 3 and 8 years post-BCG vaccination.

To examine how the immune responses had changed over the 5 years since the vaccinees had been studied and to determine if the cytokine responses other than IFN-γ may differentiate between two strongly positive IFN-γ responders to M. tuberculosis ESAT-6/CFP-10 (subjects 103278 and 103738) and nonresponders at 8 years post-BCG vaccination, the cytokine and chemokine responses at 3 and 8 years post-BCG vaccination were compared in 11 of the previous 13 responders who showed positive IFN-γ responses to M. tuberculosis ESAT-6/CFP-10 at 3 years post-BCG vaccination. From the 11 subjects, only two subjects (103278 and 103738) showed strong positive IFN-γ responses (Fig. 2B) at 8 years post-BCG vaccination (see Fig. 4, marked in red). The production of IL-12p70, IL-1α, IL-10, IP-10, MDC, and GM-CSF in response to M. tuberculosis ESAT-6/CFP-10 was greater in the two IFN-γ responders than in the other subjects (Fig. 4). IL-17 and the Th2 type cytokines IL-5, IL-9, and IL-13 were not produced in response to M. tuberculosis ESAT-6/CFP-10 at 3 years post-BCG vaccination, and 9 of the 11 previous IFN-γ responders still showed low levels of those cytokines 5 years later (Fig. 4). However, the two IFN-γ responders at 8 years post-BCG vaccination showed increases in IL-17, IL-5, IL-9, and IL-13 in response to M. tuberculosis ESAT-6/CFP-10 (Fig. 4), and one of the two responders (103738) also showed large increases in IL-5, IL-9, and IL-13 in response to M. tuberculosis PPD since 3 years postvaccination (data not shown). No remarkable differences in the cytokine and chemokine responses to M. tuberculosis PPD and PHA were found between the two IFN-γ responders and others at 8 years post-BCG vaccination (data not shown).

FIG 4
Cytokine/chemokine responses to M. tuberculosis ESAT-6/CFP-10 at 3 and 8 years post-BCG vaccination. Among the 11 subjects who had positive IFN-γ responses to M. tuberculosis ESAT-6/CFP-10 at 3 years post-BCG vaccination, only two subjects showed ...

Cytokine/chemokine signatures between IFN-γ responders to M. tuberculosis ESAT-6 and those responding to ESAT-6 homologues of M. avium and M. kansasii.

Cytokine and chemokine signatures were compared between the subject who showed a higher frequency of IFN-γ-producing cells to M. tuberculosis ESAT-6 peptides (subject 103738) and those who responded more strongly to M. avium or M. kansasii ESAT-6 (subjects 104043 and 104041) in the ELISpot assay (Fig. 5A). In response to M. tuberculosis ESAT-6/CFP-10, subject 103738, who had stronger IFN-γ responses to M. tuberculosis ESAT-6 peptides, showed about 10-fold-greater production of IFN-γ, sIL-2Rα, IL-17, IL-5, IL-13, and sCD40L than an individual who showed positive responses to M. avium57-97 and M. kansasii55-95 (subject 104041) and the subject who showed a strong response to M. kansasii55-95 (subject 104043) in the IFN-γ ELISpot assay (Fig. 5B; see also Fig. S3 in the supplemental material). However, sIL-2Rα and sCD40L were also highly produced in some other subjects who did not respond to M. tuberculosis ESAT-6/CFP-10 (data not shown). In the M. tuberculosis ESAT-6 responder (subject 103738), TNF-α, IL-9, IL-10, IL-12p70, MDC, and GM-CSF were also highly produced, and IL-9 and IL-12p70 production was still greater in response to M. tuberculosis PPD, while the other cytokines that were exclusive to subject 103738 in response to M. tuberculosis ESAT-6/CFP-10 did not show significant differences in response to M. tuberculosis PPD (data not shown). We noticed that the level of MCP-1 in the background without stimulation with M. tuberculosis antigens was very high in two IFN-γ responders (subjects 103738 and 103278) compared with those in the others.

FIG 5
Comparison of cytokine and chemokine signatures between the subject who responded to M. tuberculosis ESAT-6 and those who responded to ESAT-6 homologues of M. avium and M. kansasii. (A) Subject 103738 had a higher number of spot-forming cells (SFCs) in ...

DISCUSSION

This study provides preliminary evidence that multiple cytokine/chemokine signatures may identify potential biomarkers for a better diagnosis of M. tuberculosis infection in children and supports the observation that IFN-γ on its own is not sufficient for diagnosing M. tuberculosis infection based upon M. tuberculosis ESAT-6/CFP-10 stimulation in this setting. At the 8-year follow up, only two children showed strong positive IFN-γ responses to M. tuberculosis ESAT-6/CFP-10 in IFN-γ ELISAs after a 6-day WBA compared to those of the original 13 responders 5 years earlier. In the ELISpot assay, >50% of the IFN-γ responders to M. tuberculosis ESAT-6 showed positive IFN-γ-producing T cells to M. avium ESAT-6 or M. kansasii ESAT-6 as well, while the magnitudes of IFN-γ responses to M. tuberculosis ESAT-6 were greater than those to ESAT-6 homologues of M. avium and M. kansasii. These data indicate that an IFN-γ response to M. tuberculosis ESAT-6 alone cannot differentiate M. tuberculosis infection from infection with NTM in this setting, as shown in a report by Arend and colleagues (7). The analysis of multiple cytokine/chemokine signatures demonstrated that the signatures of IL-17, IL-5, IL-9, and IL-13 in response to M. tuberculosis ESAT-6/CFP-10 were exclusively restricted to the two strong M. tuberculosis ESAT-6 IFN-γ responders, while the IFN-γ nonresponders and the one weakly positive responder did not produce these cytokines at 8 years post-BCG vaccination. In addition, these cytokines differentiated the IFN-γ responder to M. tuberculosis ESAT-6 from those who showed stronger responses to ESAT-6 homologues of M. avium and M. kansasii, although it was not possible to determine the statistical significance of these findings due to the small sample size. None of the 40 children recruited at 8 years post-BCG vaccination and none of the 13 IFN-γ-positive responders at 3 years post-BCG vaccination had any clinical symptoms suggestive of active TB disease, such as coughing for >2 weeks, weight loss, or hemoptysis.

All of the 13 previous IFN-γ responders to M. tuberculosis ESAT-6/CFP-10 at 3 years post-BCG vaccination showed limited production of IL-17, IL-5, IL-9, and IL-13, while the cytokine levels increased in the two IFN-γ responders to M. tuberculosis ESAT-6/CFP-10 at 8 years post-BCG vaccination. The production of IL-1α, IFN-γ, IP-10, MIP-1α, and GM-CSF, which was highly detected in previous IFN-γ responders at 3 years post-BCG vaccination, was also highly detected in all of the IFN-γ responders at 8 years post-BCG vaccination regardless of the preferential IFN-γ responses to M. tuberculosis ESAT-6 or ESAT-6 homologues of M. avium and M. kansasii in the ELISpot assay (Fig. 5). These data suggest that most of the positive IFN-γ responses observed in children at 3 years post-BCG vaccination may have been cross-reactive responses with ESAT-6 homologues of environmental NTM. However, it is also possible that the 11 nonresponders who showed positive responses at 3 years post-BCG vaccination might have been transiently infected with M. tuberculosis which had been cleared during the subsequent 5 years.

Previous and recent reports showing cytokine and chemokine production in latent and active TB disease support the findings observed in this study, i.e., greater production of IL-17, IL-5, IL-9, and IL-13 upon M. tuberculosis antigen stimulation in positive IFN-γ responders (25,30). The proportions of CD4+ T cells expressing IFN-γ, IL-17, and IL-22 were observed to be significantly greater upon mycobacterial antigen stimulation in both latent and active TB disease than in healthy controls (25). Another report demonstrated that IL-17 production was significantly increased in household contacts, while it was decreased in TB cases in response to mycobacterial antigen stimulation (26), suggesting a protective role for IL-17 in disease progression to active TB. In humans, IL-13 mRNA and IL-4 mRNA were significantly expressed in TB patients compared with those in the controls (27), while higher levels of IL-13 and sCD40L were also observed in TB patients who quickly responded to anti-TB therapy than in slow responders (28). In contrast, it was also reported that the production of IL-4 and IL-5 is associated with progression to active disease (29). The enhanced production of both IFN-γ and IL-13 in our study is consistent with the previous finding that IL-13 and IFN-γ production in response to M. tuberculosis PPD and ESAT-6/CFP-10 in WBAs was significantly greater in tuberculin skin test-positive individuals in a West African cohort (30).

The peptides of M. kansasii ESAT-6 used in this study were derived from the amino acid positions 55 through 95, which include two different amino acids than those in M. tuberculosis ESAT-6 (see http://www.ncbi.nlm.nih.gov/protein/gb%7CACG70856.1 [GenBank accession no. ACG70856.1]). The small difference in only two amino acids between M. tuberculosis and M. kansasii ESAT-6 may not indicate that the peptides would act as an epitope that is specific to M. kansasii, as we showed a high percentage of cross-reactivity between the M. tuberculosis ESAT-6 and M. kansasii ESAT-6 peptides. However, changing a single residue in a 20-mer amino acid peptide can result in a lack of MHC binding and may lead to a loss of recognition by T cells that were specific for the wild-type peptide (31). In cattle, M. bovis ESAT-6 (which is identical to M. tuberculosis ESAT-6) and M. kansasii ESAT-6 were differentially recognized by bovine T cells depending on their MHC types (8).

The IFN-γ ELISA after a 6-day WBA, the IFN-γ ELISpot assay, and the QFT-IT test showed low discordance measured by the kappa statistic (0.56 ≤ kappa ≤ 0.71, P < 0.01). Any discordances among the tests are derived from the fact that different parameters are measured in each assay. The IFN-γ ELISA and multiplex bead assay measured the magnitude of IFN-γ production following a 6-day culture of whole blood with M. tuberculosis ESAT-6/CFP-10, while the ELISpot and QFT-IT assays measured overnight responses. The ELISpot assay measures the frequency of IFN-γ-producing cells, and the QFT-IT test measures secreted cytokines; effector T cell function is measured in the ELISpot and QFT-IT assays, while the WBA measures the memory recall responses. Compared with an ELISpot assay which uses a fixed number of isolated PBMCs, the QFT-IT test uses a whole-blood sample and may have a greater variability in results depending on the lymphocyte count.

The current study was derived from a cohort study with a larger adequately powered group of infants recruited in 2002 and examined the expression of genetic markers and immune responses in 590 infants at 3 months and 552 infants at 12 months post-BCG vaccination. A group of 113 children at the 3-year follow-up time point was recruited to look at the maintenance of the immune response between 3 months and 3 years post-BCG vaccination, and the study group was adequately powered for that purpose. However, based on the proportion of positive IFN-γ responders to M. tuberculosis ESAT-6/CFP-10 at 3 years (13 among 98 tested) and 8 years (3 among 40 tested, including the initial 11 responders) post-BCG vaccination, a much larger sample size than that of the initial study with 590 children would be needed in this setting to validate these findings. Alternatively, these potential biomarkers could be validated in another setting with a higher incidence of LTBI in children than is present in Karonga, Malawi.

There have been many studies to address T cell responses to M. tuberculosis region of difference 1-encoded antigens, while no studies have been published regarding biomarkers to distinguish M. tuberculosis infection from the exposure to environmental NTM, which can affect the diagnosis of TB or LTBI. The results from this study suggested putative biomarkers (IL-5, IL-9, IL-13, and IL-17) to distinguish between LTBI and exposure to M. avium and M. kansasii (Fig. 6). These findings, although preliminary in nature due to the small number of subjects involved, contribute knowledge to the ongoing development of novel diagnostic tests with higher specificities to predict M. tuberculosis infection in children. However, taking the small number of potential LTBI cases into consideration, further studies using these candidate biomarkers should be taken forward in a larger study population or cohorts with higher incidence of childhood latent TB infection to validate the diagnostic value of the suggested cytokine signature.

FIG 6
The cytokines induced by M. tuberculosis (M. tb) ESAT-6/CFP-10 in 3 categorized groups. The diagram shows how the cytokine production following M. tuberculosis ESAT-6/CFP-10 stimulation can improve the diagnosis of latent TB infection. Among the cytokines ...

Supplementary Material

Supplemental material:

ACKNOWLEDGMENT

This study was funded by Hospitals and Homes of St Giles Leprosy Fund, the Gordon Smith Traveling Scholarship, and in part by the University of London Central Research Fund. Additional support was provided by the Bill and Melinda Gates-funded Gates Grand Challenge GC6-74 and EU FP7-funded NEWTBVAC consortia. The M. tuberculosis ESAT-6/CFP-10 fusion protein and the M. tuberculosis antigens used to stimulate archived samples were provided through the GC6-74 consortium funded by the Bill and Melinda Gates Foundation. The funders had no role in study design, data collection or analysis, the decision to publish, or preparation of the manuscript.

We thank Lyn Ambrose and staff of the KPS for their assistance in collecting samples obtained from the children 3 years after BCG vaccination in a previous cohort study, and we also thank Kees Franken and Annemieke Friggen for producing the M. tuberculosis ESAT-6/CFP-10 antigen.

Footnotes

Published ahead of print 27 November 2013

Supplemental material for this article may be found at http://dx.doi.org/10.1128/CVI.00620-13.

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