Differential cellular recognition pattern to M. tuberculosis targets defined by IFN-γ and IL-17 production in blood from TB + patients from Honduras as compared to health care workers: TB and immune responses in patients from Honduras

  • Nancy Alvarez-Corrales1, 2,

    Affiliated with

    • Raija K Ahmed3,

      Affiliated with

      • Carol A Rodriguez1,

        Affiliated with

        • Kithiganahalli N Balaji4,

          Affiliated with

          • Rebeca Rivera1,

            Affiliated with

            • Ramakrishna Sompallae5,

              Affiliated with

              • Nalini K Vudattu6,

                Affiliated with

                • Sven E Hoffner3,

                  Affiliated with

                  • Alimuddin Zumla7,

                    Affiliated with

                    • Lelany Pineda-Garcia1Email author and

                      Affiliated with

                      • Markus Maeurer2, 8, 9Email author

                        Affiliated with

                        BMC Infectious Diseases201313:125

                        DOI: 10.1186/1471-2334-13-125

                        Received: 13 June 2012

                        Accepted: 25 February 2013

                        Published: 6 March 2013

                        Abstract

                        Background

                        A better understanding of the quality of cellular immune responses directed against molecularly defined targets will guide the development of TB diagnostics and identification of molecularly defined, clinically relevant M.tb vaccine candidates.

                        Methods

                        Recombinant proteins (n = 8) and peptide pools (n = 14) from M. tuberculosis (M.tb) targets were used to compare cellular immune responses defined by IFN-γ and IL-17 production using a Whole Blood Assay (WBA) in a cohort of 148 individuals, i.e. patients with TB + (n = 38), TB- individuals with other pulmonary diseases (n = 81) and individuals exposed to TB without evidence of clinical TB (health care workers, n = 29).

                        Results

                        M.tb antigens Rv2958c (glycosyltransferase), Rv2962c (mycolyltransferase), Rv1886c (Ag85B), Rv3804c (Ag85A), and the PPE family member Rv3347c were frequently recognized, defined by IFN-γ production, in blood from healthy individuals exposed to M.tb (health care workers). A different recognition pattern was found for IL-17 production in blood from M.tb exposed individuals responding to TB10.4 (Rv0288), Ag85B (Rv1886c) and the PPE family members Rv0978c and Rv1917c.

                        Conclusions

                        The pattern of immune target recognition is different in regard to IFN-γ and IL-17 production to defined molecular M.tb targets in PBMCs from individuals frequently exposed to M.tb. The data represent the first mapping of cellular immune responses against M.tb targets in TB patients from Honduras.

                        Keywords

                        T-cells M. tuberculosis TB Antigen-recognition Biomarkers

                        Background

                        9.4 million individuals are newly diagnosed with TB and two billion people are latently infected with M.tb worldwide; twenty two ‘High Burden Countries’ (HBC) account for 80% of TB cases. Brazil, the only HBC in America, accounts for 35% of TB cases within the region [13]. Honduras ranks number eight on the list of countries with a high TB burden in Latin America and second in Central America [3, 4]. 2901 TB cases were identified in Honduras during 2010, with an estimated incidence rate of 51/100,000 individuals [5]. The majority of patients with TB reside in three regions, i.e. Region Metropolitana de Cortes, the Region Departamental de San Pedro Sula and the Region Metropolitana de Tegucigalpa. Several factors, i.e. poor nutrition, HIV-M.tb co-infection, chronic (non-infectious) diseases, overcrowding, drug and alcohol abuse, affect the quality and magnitude of immune responses and subsequently the clinical course of TB [6].

                        Bacteriological diagnosis for pulmonary (and extra-pulmonary) TB in Honduras relies on smear microscopy-acid fast staining (AF-S), sputum culture on Löwenstein Jensen solid media and drug susceptibility testing (DST). TB diagnosis is supported by clinical findings (e.g. weight loss, coughing), individual patient history, epidemiology and X-rays. The tuberculin skin test (TST) is less frequently performed. The interferon gamma release assay (IGRA) is used for case finding in non-endemic countries as well as a corroborative test in specific populations such as children, patients with extra-pulmonary TB or immune-compromised individuals [79], IGRAs are not used to differentiate between active and latent TB. Therefore, there is still an unmet need for novel diagnostic tests to reliably diagnose extra-pulmonary TB, to differentiate between latent vs active TB or to indicate ‘immune protection’ and effective immune-surveillance in patients with latent TB. The testing of IFN-γ as well as IL-17 in anti-M.tb immune responses is biologically and clinically relevant. Both cytokines are involved in the recruitment of neutrophils, granuloma formation and in anti-M.tb directed immune responses [10]; diminished Th1 and Th17 responses appear to be associated with higher rates of extrapulmonary TB [11]; vice versa, expression of SOCS3 is associated with increased IL-17 production along with T-cell exhaustion (in peripheral blood cells from patients with TB [12].

                        Not only the nature of the immune responses, defined by cytokine production, yet also the nature of the M.tb encoded targets may determine the strength and magnitude of the anti-M.tb response. Cellular immune recognition of M.tb antigens, defined by cytokine production, may reflect preferential expression of M.tb proteins during the active and dormant phase of the infection [1316]. The aim of this study was to compare M.tb specific cellular immune responses in blood from individuals with active pulmonary (symptomatic) TB and individuals who have been frequently exposed to M.tb in response to antigens preferentially expressed by active and dormant M.tb.

                        Methods

                        Study site and subjects

                        The Honduran population receives (after birth) BCG vaccination since 1977. Different BCG vaccine strains have been used, the current BCG is provided by the Serum Institute, India, through WHO/UNICEF/GAVI. Honduras has a considerable TB incidence and limited resources in health care structures, including X-ray facilities. The definition of a “clean” and well defined (non-TB+) control group has therefore been challenging. The TST was not implemented in this study, since it is not routinely performed due to high variability in cut off interpretations and subsequent variant clinical decision making. 148 subjects were enrolled in the study between August 2008 and May 2010 in Tegucigalpa, Honduras at Instituto Nacional Cardiopulmonar (INCP) and at the Health Center El Manchen. The median age of the participants was 49 years, 62% of the study participants were female. Each subject was recruited after informed consent; HIV-testing and counseling was offered to each participant. A rapid HIV-1/HIV-2 antibody test (Abbott Laboratories) was used to screen for HIV infection. Clinical data were collected from hospital records and through structured interviews. Heparinized blood and sputum samples were obtained from each donor after written informed consent, morning (instant) sputum samples were collected after the interview and inclusion in the study. For hospitalized patients, sampling was performed in the morning as part of the diagnostic workup (3 days morning samples). The population was divided into three groups based on their clinical status and bacteriological results: Group1: TB + (n = 38) (M.tb culture and AFS positive, pulmonary TB) prior to initiation of DOTS; Group 2: TB- (n = 81) respiratory symptomatic patients (asthma, non-TB pneumonia, chronic-obstructive pulmonary disease, lung cancer, pharyngitis). Both outpatients and inpatients (in order to rule out TB, M.tb culture and AFS negative) were included in the Group 2 patients. Group 3: TB- (n = 29) health care workers from the TB units, exposed to M.tb (M.tb culture and AFS negative, no clinical signs of TB or any respiratory symptoms). LTBI was not discriminated between groups 2 and 3; however, the IGRA test was performed in both groups. All subjects tested HIV-negative. The study protocol was approved by the Institutional and National Ethical Committee, Instituto Nacional Cardiopulmonar and Comite de Etica en Investigación Biomédica (No. IRB 00003070).

                        Antigens used for T-cell stimulation assays are listed in Table 1. Pools of 15-mer long peptides, overlapping by 7 amino acid residues (covering the entire protein), were synthesized by JPT Peptide Technologies, Berlin, Germany. Synthetic peptides and recombinant protein (purity > 85%) were used at final concentration of 1 μg/ml and 5 μg/ml respectively. The antigens Rv3804c, Rv1886c, Rv0288 and Rv0959 were kindly provided by the AERAS Global TB Foundation (AERAS, Rockville, USA). Recombinant proteins Rv3875 and Rv3874 were purchased from Statens Serum Institute (SSI, Copenhagen, Denmark). The recombinant PPE-proteins Rv0754, Rv0978c and Rv1917c were produced by Professor K. N. Balaji, Bangalore, India [55, 6264]. A mixture of Staphylococcal Enterotoxin A and B, (SEA/SEB; 10 ng/mlSigma Aldrich, USA) was used as the positive control for T-cell reactivity.
                        Table 1

                        Summary of M.tb test targets

                        Peptides

                        Gene locus

                        RefSeq

                        M.tb Antigens

                        aa

                        Comment

                        Rv0447c

                        NP854118 (Pool 1)

                        Probable cyclopropane fatty acyl phospholipid synthase.

                        427

                        Methyl transferase activity. Cyclopropane fatty acyl phospholipid synthase activity. Lipid biosynthetic process [17, 18]

                        Rv2940c

                        YP_976584 (Pool 2)

                        Mycocerosic acid synthase

                        2111

                        Lipid biosynthetic process. Oxido-reduction and transferase activity, Cofactor binding. Location at the cell wall. [17, 1923]

                        Rv3347c

                        YP_177963 (Pool 3)

                        PPE family protein

                        3157

                        Function unknown. Gly-Ala-Asn rich protein, interacts with the host system by inhibition of antigen processing. [18, 2326]

                        Rv2453c

                        CAA16030 (Pool 4)

                        Probable molybdopterin-guanine dinucleotide biosynthesis Protein A

                        201

                        Molybdenum cofactor biosynthesis. Molecular function as GTP binding. Located at the cytoplasm membrane. [18, 23, 24, 27]

                        Rv1886c

                        CAB10044 (Pool 5)

                        Antigen 85B

                        325

                        Fibronectin binding protein. Acyltransferase activity. Secreted protein, also located at cell wall, plasma membrane. [18, 2831]

                        Rv1690

                        CAB10947 (Pool 6)

                        Probable lipoprotein

                        127

                        Putative uncharacterized protein. Protein binding, cellular component plasma membrane.[18, 24, 32]

                        Rv3019c

                        CAA16104 (Pool 7)

                        ESAT-6 like protein

                        96

                        Belongs to ESAT-6 (esx) family, Protein-protein interaction [18, 24]

                        Rv2957

                        CAB05419 (Pool 8)

                        PGL/p-HBAD biosynthesis glycosyltrans-ferase MT3031

                        256

                        Glycosyl transferase activity, transferring hexosyl groups. Glycolipid biosynthetic function. Identified as a drug target. [18, 24, 30, 33, 34]

                        Rv1085c

                        CAA17201 (Pool 9)

                        UPF0073 membrane protein

                        242

                        Belongs to the UPF0073 (HIy-III) family. Cytolysis. Sub cellular location in cell membrane [18, 24]

                        Rv0066c

                        CAA16247 (Pool 10)

                        Isocitrate dehydrogenase, NADP-dependent- icd2.

                        745

                        Oxidoreductase function. NAD or NADH binding. Isocitrate dehidrogenase (NADP+) activity. Magnesium ion binding. Protein homodimerization. [18, 2224, 35, 36]

                        Rv2958c

                        CAB05418 (Pool 11)

                        PGL/p-HBAD biosynthesis glycosyltrans-ferase

                        428

                        Glycolipid biosynthetic process, pathogenesis, Glycosyl transferase activity. Immune evasion or - tolerance [18, 22, 24, 33, 37]

                        Rv2962c

                        CAB05415 (Pool 12)

                        PGL/p-HBAD biosynthesis rhamnosyl-transferase

                        449

                        Glycolipid biosynthetic process, pathogenesis. Glycosyl transferase activity. Evasion or tolerance concerning to the host immune response. [18, 22, 24, 30, 33, 37]

                        Rv3804c/1886

                        CAA17868/CAB10044

                        Ag85A/Ag85B Fibronecting binding protein peptide pool

                        338/325

                        Belongs to the Ag85 family, contains Esterase D. Possesses mycolyl transferase activity. [28, 29, 3841]

                        Proteins

                            

                        Rv3804c

                        CAA17868

                        Ag85A. Secreted antigen 85A. Mycolyl transferase 85A. fbpA. Ag85 complex

                        338

                        Responsible for high affinity of mycobacteria to fibronectin. Possesses mycolyl transferase for biogenesis of trehalose dimycolate. [22, 23, 29, 30, 3840, 4244]

                        Rv1886c

                        CAB10044

                        Ag85B. Secreted antigen 85B. Mycolyl transferase 85B. fbpB. Ag85 complex

                        325

                        Fibronectin binding protein. Acyltransferase activity. Response to antibiotic. Secreted protein also located at cell wall, extracellular region. [2830, 36, 38, 40, 41, 45]

                        Rv3875

                        CAA56099

                        ESAT-6. 6 KDa early secretory antigenic target. esxA

                        95

                        Function unknown. Elicit high level of IFNgamma during the first phase of protective immune response. Secreted protein, cell wall and cytoplasm. [4654]

                        Rv3874

                        CAA17966

                        CFP-10. 10 KDa culture filtrate antigen

                        100

                        ESAT-6 like protein esxB. Forms a tight 1:1 complex with EsxA. Protein binding. Host cell surface binding. Pathogenesis. Protein secretion. [22, 4749, 51, 53, 54]

                        Rv0754

                        CAE55319

                        PE_PGR11. PE-PGRS family protein

                        584

                        Member of M.tb PE family. PGRS subfamily gly-rich proteins. Unknown function. Protein existence predicted. [18, 21]

                        Rv0978c

                        CAE55343

                        PE_PGR17. PE-PGRS family protein

                        331

                        Member of M.tb PE family. PGRS subfamily gly-rich proteins. Unknown function. Protein existence predicted. [18, 55, 56]

                        Rv1917c

                        CAE55440

                        PPE34. PPE family protein

                        1459

                        Member of M.tb PPE family. Glycin rich proteins. Unknown function. [18, 57]

                        Rv0288

                        CAA17363

                        TB10.4. Low molecular weight protein. (M.tb)

                        96

                        Belongs to theESAT-6 (esx) family. Molecular function protein binding. Involved in virulence. Immunogenic. [18, 24, 5861]

                        Whole blood assay

                        The whole blood assay (WBA) was used to determine IFN-γ and IL-17 production in response to M.tb antigens. Venous whole blood was obtained using heparinized blood collection tubes and diluted 1:2.5 in RPMI 1640-medium supplemented with 1% Hepes, 0.5% Penicillin/ (100 IU/ml) and streptomycin (10 mg/ml), (Gibco Invitrogen). 100 μl of diluted blood was added into 96-well round bottom plates (Nunc, Roskilde, Denmark) in duplicate wells pre-coated with the specific antigen diluted in 100 μl medium. Cultures were incubated at 37°C, 5% CO2. After 7 days, 75 μl of cell culture supernatant was removed from each duplicate well, pooled, and stored at −80°C until IFN-γ and IL-17 were determined by ELISA.

                        IFN-γ and IL-17A determination

                        IFN-γ and IL-17A (IL-17) in cell culture supernatants was measured by ELISA (Eli-pair DIACLONE, Biosite, Stockholm, Sweden). The assays were performed according to the manufacturer’s instruction. In brief, Nunc-Immuno™ Maxisorp 96-well plates (Nunc, GTF, Stockholm, Sweden) were coated with the specific capture antibody overnight at 4°C. The plates were then washed with PBS containing 0.05% tween-20 and then blocked with PBS containing 5% bovine serum albumin (BSA, Karolinska Hospital, Stockholm, Sweden). Supernatants collected from WBA (150 μl) were thawed and diluted (1:1.47 in IFN-γ [75 μl] and 1:1.91 for IL-17 [55 μl] with PBS containing 1% BSA. Values were multiplied by their corresponding dilution factor, background from un-stimulated medium control were subtracted from each antigen response both for IFN-γ and IL-17; the cytokine concentration was expressed in pg/ml. The detection range for IFN-γ was 400–7 pg/ml and for IL-17 100–3.1 pg/ml. An additional standard IFN-γ recombination protein (purchased from R&D, Minneapolis, MN, USA) was used as an internal control to gauge for differences between ELISA assay performances.

                        Interferon gamma release assay (IGRA)

                        Quantiferon TB-Gold in tube (QFT-GIT) (Cellestis, Copenhagen, Denmark) [65], was performed. Briefly, 1 ml of venous blood was collected directly into three tubes containing TB-specific antigens, mitogen and nil control. Tubes were incubated at 37°C, 5% CO2 for 16 to 24 hours before centrifugation at 3000 g for 15 min. The plasma was collected and stored at +4°C until IFN-γ detection within two weeks. ELISA was performed as described by the manufacturer, in brief, 50 μl of conjugate was added as well as 50 μl of test sample; diluted standards were added to designated wells followed by 2 hours incubation. After washing, 100 μl of substrate solution were added to wells and incubated for 30 minutes, followed by 50 μl of enzyme stopping solution. OD values were calculated using the Software A-QTF-2.5-02 2.5, a free online source provided by Cellestis validating the assay by internal quality controls and setting the cut off for positive results at >0.35 International units (UI)/ml.

                        Sputum based acid-fast staining (AF-S) and Löwenstein Jensen (LJ) culture

                        Acid fast staining (Ziehl-Neelsen technique) was performed to visualize the acid-fast bacilli in sputum samples. Samples were further cultured in duplicate at 37°C and periodically revised up to 8 weeks (Löwenstein Jensen selective media produced at the Research lab). In 14/148 individuals, no cultures could be initiated due to inadequate sputum procurement; the contamination rate of cultures was 2/132. Positive cultures were confirmed using standard biochemical tests, i.e. niacin, reduction of nitrates and catalase activity [66]. Quality control of Löwenstein-Jensen media was routinely performed using M.tb control strains at the microbiology department of the Hospital.

                        Statistical analysis

                        Data were explored using dotplot and barplot analysis. A t-test was used to examine differences in IFN-γ and IL-17 production between groups. An adjustment for multiplicity was applied on the resulting p-values from the tests, only those antigens that showed significant difference at 5% level were discussed. We used the R software pairwise t-test function to compare the treatment group means with pooled Standard Deviation. The ANOVA test was performed in order to confirm significant differences between groups as well as a pairwise t-test and the Holm adjustment method for multiple corrections.

                        Results

                        Increased IFN-γ production to M.tb antigens in blood from health care workers exposed to M.tuberculosis as compared to TB patients

                        IFN-γ production in response to M.tb target antigens (overview see Table 1) was analyzed in blood from the study participants (n = 148). We identified a significant difference in IFN-γ production between the groups (see Figure 1, Table 2 and Additional file 1: Table S1a-c) i.e. a different magnitudes of IFN-γ production in response to (peptide cocktails) Rv2958c, Rv2962c, Rv3347c, Rv3804c, and Rv1886c (protein) between groups 1 (TB+) and 3 (health care workers); the antigens Rv2958c and Rv2962c were differentially recognized between group 2 (non-TB pulmonary diseases) and 3 (health care workers).
                        http://static-content.springer.com/image/art%3A10.1186%2F1471-2334-13-125/MediaObjects/12879_2012_2317_Fig1_HTML.jpg
                        Figure 1

                        IFN-γ boxplots of immune reactivity in blood stimulated with antigens/peptides that showed significant differences between groups (group 1, TB + patients, group 2, TB- other pulmonary diseases, group 3, frequent M.tb exposure, yet clinically healthy). Antigens were identified after testing a broad panel of M.tb antigens (see Table 1). Only statistically significant different IFN-γ responses are shown. Strong recognition of Ag85B (Rv1886c), PGL/p-HBAD biosynthesis glycosyltransferase (Rv2958c), PGL/p-HBAD biosynthesis rhamnosyl-transferase (Rv2962c) and PPE55 (Rv3347c). Recombinant proteins Ag85A (Rv3804) and Ag85B (Rv1886c) were differentially recognized among TB + and TB- groups defined by IFN-γ release. The thick lines (inside the boxplots) represent median values.

                        Table 2

                        Statistically different cytokine production in groups 1(TB patients), 2 (patients with other pulmonary diseases) and 3 (negative TB cases but highly exposed)

                        Antigen

                        Groups

                        P-value

                        SEA/B

                        Group1 vs Group2

                        0.001

                        SEA/B

                        Group1 vs Group3

                        < 0.001

                        Rv1886c

                        Group1 vs Group3

                        0.015

                        Rv2958c

                        Group1 vs Group3

                        0.020

                        Rv2958c

                        Group2 vs Group3

                        0.020

                        Rv2962c

                        Group1 vs Group3

                        0.021

                        Rv2962c

                        Group2 vs Group3

                        0.021

                        Rv3347c

                        Group1 vs Group3

                        0.034

                        Rv3804c

                        Group1 vs Group3

                        0.031

                        Rv1886c

                        Group1 vs Group2

                        0.016

                        Rv1886c

                        Group1 vs Group3

                        0.002

                        Immune cells from individuals in group 2 (TB-, other respiratory diseases) and group 3 (exposed to TB, no clinical TB uninfected) produced significantly higher levels of IFN-γ in response to stimulation with Rv1886c (Ag85B) Rv2958c (glycosyl-transferase) and Rv2962c (pHBAD biosynthesis rhamnosyltransferase). The recombinant protein antigens Rv3804c (Ag85A) and Rv3347c (PPE family member) induced the strongest IFN-γ production in blood from group 3 (M.tb exposed individuals) as compared to TB + individuals (group 1). A head-to-head comparison between overlapping peptides for Rv1886c (Ag85B) and the recombinant protein (Table 2) yielded a similar trend (p = 0.015) for differences between group 1 and 3 (using peptides as the assay target) as well as for testing the recombinant protein (comparison between groups 1 and 3, p = 0.002).

                        A different M.tb target recognition pattern defined by IL-17 production

                        A more recent study revealed differences between IFN-γ and IL-17 production in response to M.tb antigens [67]. We selected therefore 14 M.tb antigens for further analysis and tested IL-17 production in response to the antigens Rv0447, Rv1886c, Rv3019c, Rv2957, Rv2958c, Rv2962c (peptides) and the recombinant proteins Rv3804c, Rv1886c, Rv3874, Rv3875, Rv0288, Rv0754, Rv0978a and Rv1917 (see Figure 2, Table 3, Additional file 1: Table S1a-c). We were able to demonstrate significantly different IL-17 production in response to Rv1886c (Ag85B) in individuals from group 2 (non TB, other respiratory disease) and 3 (health care workers, exposed to TB) as compared to individuals in group 1 (pulmonary TB). Immune cells from individuals in group 2 and group 3 produced more IL-17 in response to Rv0978c (PPE family member), Rv0288 (TB10.4) and Rv1917c (PPE family member) as compared to blood from individuals with TB (group 1, patients with pulmonary TB). The highest IL-17 production was identified in blood from exposed individuals directed against the antigens listed above.
                        http://static-content.springer.com/image/art%3A10.1186%2F1471-2334-13-125/MediaObjects/12879_2012_2317_Fig2_HTML.jpg
                        Figure 2

                        IL-17 production shows a different pattern of cellular immune recognition as compared to IFN-γ. IL-17 production in blood was statistically different in response to the antigens Ag85B (Rv1886c), TB10.4 (Rv0288), PPE - PGR11 (Rv0978c) and PPE34 (Rv1917c). The thick lines (inside the boxplots) represent median values. Identical patient cohorts as in Figure 1.

                        Table 3

                        Distribution of IFN-γ levels for the antigens associated with statistically different cytokine production between groups at 5% level

                        Antigen

                        Groups

                        P-value

                        Rv1886c

                        Group1 vs Group2

                        0.050

                        Rv1886c

                        Group1 vs Group3

                        0.029

                        Rv0288

                        Group1 vs Group3

                        0.030

                        Rv0288

                        Group2 vs Group3

                        0.027

                        Rv0978c

                        Group1 vs Group3

                        0.003

                        Rv0978c

                        Group2 vs Group3

                        0.002

                        Rv1917c

                        Group1 vs Group3

                        0.005

                        Rv1917c

                        Group2 vs Group3

                        0.008

                        Differences in IFN-γ between groups were examined using a t-test; adjustment for multiplicity was applied on the resulting p-values from the tests. (group 1: TB+, AFS and culture positive, 2: other pulmonary diseases, no exposure records to TB, 3: health care workers, frequently exposed to M.tb, yet clinically healthy). Only statistically relevant differences are listed.

                        Analysis between QFT-GIT and bacteriology

                        We analyzed the cytokine responses in blood from individuals stratified by the QFT-GIT (see online Additional file 1: Table S2-4 and Figures S1 and S2) based on the acid fast stain result, culture positivity and previous history of TB). Immune cells from individuals with AFS + M.tb + culture showed decreased IFN-γ responses to the positive (SEA/SEB) controls. ESAT-6 and CFP-10 immune reactivity was significantly higher in blood from individuals with AFS- and negative culture results– yet with a positive QFT-GIT for IFN-γ production (and for IL-17 production in response to Rv3874, CFP10). Significant differences concerning cytokine response patterns could be identified in blood from patients with TB (AFS+, QFT-GIT + and culture+) versus blood from health care workers in response to Rv3804c (Ag85A, IFN-γ p = 0.009 as well as for IL-17, p = 0.019) and Rv2962 (pHBAD, rhamnosyl-transferase, p = 0.042 for IL-17 production) (Additional file 1: Tables S2-4, Additional file 1: Figures S1 and S2).

                        Discussion

                        Tuberculosis is the result of a dynamic host-pathogen relationship. Anti-M.tb directed immune responses may be associated with immune-protection, yet also with immune-pathology, as shown for IL-17-driven cellular immune responses. IL-17 – associated immune responses can be protective or harmful in TB and may lead to lung tissue damage along with massive inflammation and influx of neutrophils [68]. Most immune assays aim to gauge M.tb responses measure IFN-γ, which is indispensable to contain M.tb, yet other immune effector cytokines, e.g. IL-17, may also be instrumental in immune protection. Further evidence for the beneficial role of IL-17 is provided from a pre-clinical model: a recombinant BCG vaccine expressing listeriolysin, leads to a statistically significant different IL-17 (yet not IFN-γ) production [69] in a murine M.tb challenge model.

                        We were able to show that IFN-γ mediated responses showed strong cellular recognition of Rv1886c (Ag85B), Rv3804c (Ag85A), Rv2958c, Rv2962c (enzymes associated with lipid-alteration) and Rv3347 (PPE family member) in blood from individuals who have been exposed to M.tb, yet are clinically healthy. The data are compiled for review in Table 4 concerning the detailed clinical diagnoses of individuals enrolled in the study; Table 5 summarizes the molecularly defined test antigens leading to IFN-γ and IL-17 production. Decreased IFN-γ production in blood from individuals with TB (AFS and culture positive) may in part be related to ‘anergy’, which is reflected in significantly lower IFN-γ responses to the positive control stimulus (SEA/SEB, Figure 1). This notion is further supported by a more detailed examination of patient subpopulations (see online Additional file 1: Figure S1 and Table S2). Only blood from individuals with a AFS+, culture+, QFT-GIT + profile showed decreased IFN-γ production, this was not found to be true for individuals with presumably latent TB (based on exposure history, QFT-GIT+, yet culture - AFS-).
                        Table 4

                        Description of the general population, clinical characteristics, microbiology and immunological test results

                        Group

                        QTF-GIT

                        AF-S

                        Culture

                        X-ray

                        Gender

                        Comments

                        TB + HIV- n = 38

                        Pos: 23

                        Pos: 26

                        MTB: 23

                        altered: 23

                        Female: 17

                        Pulmonary TB (26), Pleural TB (3) extra-pulmonary TB (9)

                        Neg: 10

                        Neg:12

                        Neg: 11

                        non altered: 5

                        Male: 21

                        Ind: 5

                        No data: 0

                        Cont: 1 No data: 3

                        no data: 10

                         

                        TB-HIV- n = 81

                        Pos: 24

                        Pos: 0

                        Neg: 79

                        altered: 33

                        Female: 56

                        Allergy (1) asthma (17), EPOC (20), (non-TB) pneumonia (8), lung cancer (4), (non-TB) pleural effusion (1), bronchitis (5), other (25) otitis, rhinitis, influenza, diabetes, heart disease.

                        Neg: 54

                        Neg: 81

                        Cont: 1

                        non altered: 7

                        Male: 25

                        Ind: 3

                        No data: 0

                        No data: 1

                        no data: 41

                         

                        TB-HIV- n = 29

                        Pos: 11

                        Pos: 0

                        Neg: 19

                        altered: 10

                        Female: 19

                        Nurses and TB/HIV health care workers (19), previous TB with successful treatment regimen in the past (at least 2 years) but no current TB (10)

                        Neg: 17

                        Neg: 19

                        No data: 10

                        non altered: 2

                        Male: 10

                         

                        Ind: 1

                        No data: 10

                         

                        no data: 17

                          

                        Ind: indeterminate; Neg: negative, Pos: positive.

                        Table 5

                        Compilation of the antigen-specific response analysis

                        Antigen

                        Group 1

                        Group 2

                        Group 3

                         

                        IFN-γ (%)

                        IL-17 (%)

                        IFN-γ (%)

                        IL-17 (%)

                        IFN-γ (%)

                        IL-17 (%)

                        Rv0447c

                        10/38 (26)

                        10/38 (26)

                        24/81 (30)

                        17/81 (21)

                        12/29 (41)

                        11/29 (38)

                        Rv2940c

                        3/38 (8)

                         

                        8/81 (10)

                         

                        3/29 (10)

                         

                        Rv3347c

                        5/38 (13)

                         

                        12/81 (15)

                         

                        12/29 (41)

                         

                        Rv2453c

                        4/38 (11)

                         

                        11/81 (14)

                         

                        9/29 (31)

                         

                        Rv1886c

                        14/38 (37)

                        10/38 (26)

                        37/81 (46)

                        27/81 (33)

                        15/29 (52)

                        14/29 (48)

                        Rv1690

                        6/38 (16)

                         

                        18/81 (22)

                         

                        8/29 (28)

                         

                        Rv3019c

                        8/38 (21)

                        10/38 (26)

                        29/81 (36)

                        37/81 (46)

                        9/29 (31)

                        16/29 (55)

                        Rv2957

                        7/38 (18)

                        11/38 (29)

                        30/81 (37)

                        33/81 (41)

                        11/29 (38)

                        12/29 (41)

                        Rv1085c

                        6/38 (16)

                         

                        11/81 (14)

                         

                        4/29 (14)

                         

                        Rv0066c

                        3/38 (8)

                         

                        14/81 (17)

                         

                        4/29 (14)

                         

                        Rv2958c

                        4/38 (11)

                        14/38 (37)

                        10/81 (12)

                        31/81 (38)

                        7/29 (24)

                        13/29 (45)

                        Rv2962c

                        3/38 (8)

                        10/38 (26)

                        10/81 (12)

                        32/81 (40)

                        8/29 (28)

                        15/29 (28)

                        Rv1886c

                        12/25(48)

                        12/25 (48)

                        40/65 (62)

                        47/65 (72)

                        11/17 (65)

                        15/17 (88)

                        Rv3804c

                        15/25 (60)

                        15/25 (60)

                        40/65 (62)

                        44/65 (68)

                        12/17 (71)

                        12/17 (71)

                        Rv3874

                        13/20 (65)

                        8/20 (40)

                        32/48 (67)

                        25/48 (52)

                        13/14 (93)

                        7/14 (50)

                        Rv3875

                        9/20 (45)

                        8/20 (40)

                        24/48 (50)

                        11/48 (23)

                        6/14 (43)

                        5/14 (36)

                        Rv0288 pep

                        3/11 (27)

                         

                        3/21 (14)

                         

                        0/3 (0)

                         

                        Rv3804c/Rv1886c

                        3/11 (27)

                         

                        3/21 (14)

                         

                        0/3 (0)

                         

                        Rv3875/3874

                        7/9 (78)

                         

                        6/21 (29)

                         

                        0/5 (0)

                         

                        Rv0754

                        3/5 (60)

                        1/5 (20)

                        1/17 (6)

                        5/17 (29)

                        1/3 (33)

                        3/3 (100)

                        Rv0978

                        2/5 (40)

                        0/5 (0)

                        4/17 (24)

                        5/17 (29)

                        0/3 (0)

                        2/3 (66)

                        Rv1917

                        2/5 (40)

                        1/5 (20)

                        2/17 (12)

                        7/17 (41)

                        0/3 (0)

                        3/3 (100)

                        Rv0288

                        1/5 (20)

                        1/5 (20)

                        1/17 (6)

                        6/17 (35)

                        0/3 (0)

                        2/3 (66)

                        Group 1, TB + patients, group 2, TB- other pulmonary diseases, group 3, frequent M.tb exposure, yet clinically healthy.

                        Of interest is the quite different cellular reactivity concerning M.tb target pattern recognitions if IFN-γ and IL-17 are analyzed. Ag85B (Rv1886c) showed a similar trend concerning T-cell recognition, both for IFN-γ and IL-17 production (see Figures 1 and 2). Yet the antigen Rv0288 (TB10.4) and two PPE family members (Rv0978c, Rv1917c) exhibited only statistical differences in IL-17 responses, yet not concerning the capacity to induce IFN-γ production. Vice versa, the PPE family member Rv3347c, the enzymes Rv2958c (glycosyl-transferrase) and Rv2962 (rhamosyl-transferase) showed only differences in IFN-γ production between the populations (groups 1, TB + and 3, health care workers). The data consolidate our previous observations in a population from Belarus, where cellular immune responses, defined by IFN-γ production, were more frequently directed against the antigen glycsoltransferase (Rv2958c) in healthy individuals frequently exposed to M.tb[70]. The gene product Rv2958c adds a second rhamnosyl unit and a fucosyl residue to form the species-specific triglycosyl appendage of PGL-tb and p-HBAD. A differential expression of Rv2958c in BCG vaccine strains has been speculated to be associated with different levels of protection from TB [71]. The data reported in the current study consolidates also the strong immune recognition of Ag85A in blood from individuals after BCG vaccination or M.tb exposure [72].

                        Several explanations may apply for the differential recognition patterns defined by IFN-γ and IL-17 production: Rv2958c and Rv2962 were tested as overlapping peptides; some peptides may be degraded and therefore not efficiently processed and presented to T-cells; the antigen processing and presentation of peptides may be different as compared to recombinant proteins. We produced in the meantime an (LPS-free) Rv2958c protein which leads as well as to IL-17 production in blood from TB + individuals (our unpublished data), suggesting that IL-17 production may require the intact protein structure and subsequent cellular processing and presentation.

                        Other cellular mechanism may be important to initiate IL-17 production, i.e. the priming and activation of antigen presenting cells which will subsequently present the recombinant target protein to antigen-specific T-cells. For instance, the PPE family members (tested in the current panel) have been shown to mature dendritic cells via TLR-2 stimulation (in a murine system) leading to a different quality of antigen-presentation and expansion of antigen-specific immune cells [62]. A similar observation has been reported for differences in BCG and rBCG, expressing listeriolysin; ‘components’ released from rBCG in the cytosol of macrophages, may lead to a different array of pathogen-associated signaling patterns leading to stimulation of antigen-presenting cells and subsequent expansion of Th17+ immune cells [69]. It could very well be that the PPE family members described in the current report lead to different IFN-γ/IL-17 production via activation of antigen-presenting cells (see Figure 2).

                        Of interest is the strong recognition, defined by IL-17 production, of TB10.4 (Rv0288c) and Ag85B (Rv1886c), two components of several TB vaccine candidates, in blood from healthy TB- exposed individuals. A similar, statistically significant trend, was found to be true for IL-17 production in response to the PPE family members Rv0978c (p = 0.003, difference between groups 1 and 3) and Rv1917c (p = 0.008, difference between groups 1 and 3). Both proteins signal via TLR2 and are able to mature dendritic cells [62]. Future studies will show whether recombinant target proteins, used in the current study, are able to induce critical feedback regulators that would preferentially expand IL-17 –producing immune cells [12]. The strong TB10.4 recognition in blood from healthy individuals appears to be in contrast to the study of Sutherland and coworkers who reported a significant difference in IFN-γ production, yet not IL-17 production, by comparing cytokine responses in blood from TB + cases (West Africa) and a TST- control cohort [67]. Several reasons may account for these differences, i.e. a different exposure history of the test population to M.tb and/or environmental mycobacteria and subsequent expansion of IL-17+ producing immune cells. Not only exposures to mycobacterial species, yet other (environmental pathogens) may contribute of shaping the immune response leading to preferential IL-17 production, i.e. natural killer, natural killer T-cells, lymphoid tissue inducer and TCRγδ + T-cells are contributing to IL-17 production (for review see [73]). Future studies may therefore need to dissect the role of the cell source of IL-17 production in response to M.tb targets and require the stratification of immune response analysis based on the distribution of immune cells subsets (T-cells, NK, NKT, TCRγδ + T-cells) in the test samples.

                        Conclusions

                        In summary, we report for the first time the cellular immune recognition pattern against M.tb in a clinically defined Honduran population characterized by differential immune recognition patterns in regard to IFN-γ and IL-17 production. The limitations of the current study are the number of study participants, the difficulty to obtain age and sex-matched control individuals, to challenge to gauge and control for multiple M.tb exposures and MOTT, as well as the challenge of defining latent TB infection in general, particularly in a resource-restrained country. Of particular interest is the antigen Rv2958, which is currently being evaluated as part of a new TB vaccine from our group in pre-clinical models. Screening of M.tb exposed, yet clinically healthy, individuals (as compared to TB + patients and non-Mtb exposed control cohorts, such as those described in the current study) may help to better identify immunological markers which help to define M.tb exposure and immune protection.

                        Declarations

                        Acknowledgment

                        This study was supported by the Swedish International Development Cooperation Agency (Sida) [Sida contribution N° 75007345], Vetenskapsrådet, Vinnova and HLF (Hjärt Lungfonden). We would like to thanks the staff of the Instituto Nacional Cardiopulmonar and the patients who participated and made this study possible.

                        Authors’ Affiliations

                        (1)
                        Escuela de Microbiología, Universidad Nacional Autónoma de Honduras (UNAH)
                        (2)
                        Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet
                        (3)
                        Swedish Institute for Communicable Disease Control (SMI)
                        (4)
                        Department of Microbiology and Cell Biology, Indian Institute of Science
                        (5)
                        Department of Pathology, University of Iowa
                        (6)
                        Department of Immunobiology, Yale University
                        (7)
                        Department of Infection, University College London Medical School, Windeyer Institute of Medical Sciences
                        (8)
                        Center for allogeneic stem cell transplantation (CAST), Karolinska University Hospital
                        (9)
                        Department of Laboratory Medicine, Division of Therapeutic Immunology, Karolinska Institutet

                        References

                        1. WHO: Global TB Control 2010. In Region of the Americas. 2010: 99–120 ISBN 978 92 4 156406 9; 2010. http://​www.​doh.​state.​fl.​us/​disease_​ctrl/​tb/​trends-stats/​Fact-Sheets/​US-Global/​WHO_​Report2010_​Global_​TB_​Control.​pdf.
                        2. WHO: The global plan to Stop TB 2011–2015. Transforming the fight. WHO Stop TB Partnership; 2011.
                        3. PAHO: Plan Regional de Tuberculosis 2006–2015. In National Tuberculosis Control Programs, Ministries of Public Health. Washington, DC: PAHO, 2006: 5–23 ISBN 92 75 12673 9; 2006. http://​www.​stoptb.​org/​assets/​documents/​global/​plan/​tb-reg-plan-2006-15%20​AMRO.​pdf.
                        4. PortalSida: Situación Epidemiológica Tuberculosis. Honduras: MCP Honduras; 2007:1–2.
                        5. WHO: Tuberculosis profile. Honduras: World Health Organization; 2011.
                        6. Varela Martinez C: Plan estratégico nacional para el control de la tuberculosi, PENTB 2009–2015. Resumen. Rev Med Hondur 2010, 78:1.
                        7. O’Grady J: New and improved diagnostics for detection of drug-resistant pulmonary tuberculosis. Curr Opin Pulm Med 2011,17(3):134–41.PubMedView Article
                        8. McNerney R: Screening of immigrants in the UK for latent tuberculosis. Expert Rev Respir Med 2011,5(4):483–6.PubMedView Article
                        9. Ferrara G: A decade of interferon-gamma release assays: quest for the holy grail to diagnose latent infection with mycobacterium tuberculosis? Am J Respir Crit Care Med 2011,183(12):1732.PubMed
                        10. Curtis MM, Way SS: Interleukin-17 in host defence against bacterial, mycobacterial and fungal pathogens. Immunology 2009,126(2):177–85.PubMedView Article
                        11. Kumar NP: Suppressed type 1, type 2, and type 17 cytokine responses in active tuberculosis in children. Clin Vaccine Immunol 2011,18(11):1856–64.PubMedView Article
                        12. Kleinsteuber K: SOCS3 promotes interleukin-17 expression of human T cells. Blood 2012,120(22):4374–82.PubMedView Article
                        13. Hanekom WA, Abel B, Scriba TJ: Immunological protection against tuberculosis. S Afr Med J 2007,97(10 Pt 2):973–7.PubMed
                        14. Kaufmann SH: How can immunology contribute to the control of tuberculosis? Nat Rev Immunol 2001,1(1):20–30.PubMedView Article
                        15. Flynn JL: Immunology of tuberculosis and implications in vaccine development. Tuberculosis (Edinb) 2004,84(1–2):93–101.View Article
                        16. Demissie A: Recognition of stage specific Mycobacterial antigens differentiates between acute and latent infections with Mycobacterium tuberculosis. Clinical and vaccine immunology 2005, 179–186.
                        17. Garnier T: The complete genome sequence of Mycobacterium bovis. Proc Natl Acad Sci USA 2003,100(13):7877–82.PubMedView Article
                        18. Cole ST: Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998,393(6685):537–44.PubMedView Article
                        19. Mathur M, Kolattukudy PE: Molecular cloning and sequencing of the gene for mycocerosic acid synthase, a novel fatty acid elongating multifunctional enzyme, from Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guerin. J Biol Chem 1992,267(27):19388–95.PubMed
                        20. Rainwater DL, Kolattukudy PE: Fatty acid biosynthesis in Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guerin. Purification and characterization of a novel fatty acid synthase, mycocerosic acid synthase, which elongates n-fatty acyl-CoA with methylmalonyl-CoA. J Biol Chem 1985,260(1):616–23.PubMed
                        21. Camus JC: Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv. Microbiology 2002,148(Pt 10):2967–73.PubMed
                        22. Mawuenyega KG: Mycobacterium tuberculosis functional network analysis by global subcellular protein profiling. Mol Biol Cell 2005,16(1):396–404.PubMedView Article
                        23. Gu S: Comprehensive proteomic profiling of the membrane constituents of a Mycobacterium tuberculosis strain. Mol Cell Proteomics 2003,2(12):1284–96.PubMedView Article
                        24. Fleischmann RD: Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J Bacteriol 2002,184(19):5479–90.PubMedView Article
                        25. van Pittius NC G: Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions. BMC Evol Biol 2006, 6:95.View Article
                        26. Singh KK: Immunogenicity of the Mycobacterium tuberculosis PPE55 (Rv3347c) protein during incipient and clinical tuberculosis. Infect Immun 2005,73(8):5004–14.PubMedView Article
                        27. Guse A: Biochemical and structural analysis of the molybdenum cofactor biosynthesis protein MobA. J Biol Chem 2003,278(28):25302–7.PubMedView Article
                        28. De Wit L, Palou M, Content J: Nucleotide sequence of the 85B-protein gene of Mycobacterium bovis BCG and Mycobacterium tuberculosis. DNA Seq 1994,4(4):267–70.PubMed
                        29. Harth G: Novel insights into the genetics, biochemistry, and immunocytochemistry of the 30-kilodalton major extracellular protein of Mycobacterium tuberculosis. Infect Immun 1996,64(8):3038–47.PubMed
                        30. Raman K, Yeturu K, Chandra N: targetTB: a target identification pipeline for Mycobacterium tuberculosis through an interactome, reactome and genome-scale structural analysis. BMC Syst Biol 2008, 2:109.PubMedView Article
                        31. Anderson DH: An interfacial mechanism and a class of inhibitors inferred from two crystal structures of the Mycobacterium tuberculosis 30 kDa major secretory protein (Antigen 85B), a mycolyl transferase. J Mol Biol 2001,307(2):671–81.PubMedView Article
                        32. Steyn AJ, Joseph J, Bloom BR: Interaction of the sensor module of Mycobacterium tuberculosis H37Rv KdpD with members of the Lpr family. Mol Microbiol 2003,47(4):1075–89.PubMedView Article
                        33. Perez E: Characterization of three glycosyltransferases involved in the biosynthesis of the phenolic glycolipid antigens from the Mycobacterium tuberculosis complex. J Biol Chem 2004,279(41):42574–83.PubMedView Article
                        34. Berg S: The glycosyltransferases of Mycobacterium tuberculosis - roles in the synthesis of arabinogalactan, lipoarabinomannan, and other glycoconjugates. Glycobiology 2007,17(6):35–56R.PubMedView Article
                        35. Banerjee S: Comparison of Mycobacterium tuberculosis isocitrate dehydrogenases (ICD-1 and ICD-2) reveals differences in coenzyme affinity, oligomeric state, pH tolerance and phylogenetic affiliation. BMC Biochem 2005, 6:20.PubMedView Article
                        36. Malen H: Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv. Proteomics 2007,7(10):1702–18.PubMedView Article
                        37. Miller BH, Shinnick TM: Evaluation of Mycobacterium tuberculosis genes involved in resistance to killing by human macrophages. Infect Immun 2000,68(1):387–90.PubMedView Article
                        38. Armitige LY: Disruption of the genes encoding antigen 85A and antigen 85B of Mycobacterium tuberculosis H37Rv: effect on growth in culture and in macrophages. Infect Immun 2000,68(2):767–78.PubMedView Article
                        39. Abou-Zeid C: Characterization of fibronectin-binding antigens released by Mycobacterium tuberculosis and Mycobacterium bovis BCG. Infect Immun 1988,56(12):3046–51.PubMed
                        40. Belisle JT: Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis. Science 1997,276(5317):1420–2.PubMedView Article
                        41. Wilkinson RJ: An increase in expression of a Mycobacterium tuberculosis mycolyl transferase gene (fbpB) occurs early after infection of human monocytes. Mol Microbiol 2001,39(3):813–21.PubMedView Article
                        42. Borremans M: Cloning, sequence determination, and expression of a 32-kilodalton-protein gene of Mycobacterium tuberculosis. Infect Immun 1989,57(10):3123–30.PubMed
                        43. Ronning DR: Mycobacterium tuberculosis antigen 85A and 85C structures confirm binding orientation and conserved substrate specificity. J Biol Chem 2004,279(35):36771–7.PubMedView Article
                        44. Braunstein M: Identification of genes encoding exported Mycobacterium tuberculosis proteins using a Tn552′phoA in vitro transposition system. J Bacteriol 2000,182(10):2732–40.PubMedView Article
                        45. Garbe TR, Hibler NS, Deretic V: Isoniazid induces expression of the antigen 85 complex in Mycobacterium tuberculosis. Antimicrob Agents Chemother 1996,40(7):1754–6.PubMed
                        46. Sorensen AL: Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. Infect Immun 1995,63(5):1710–7.PubMed
                        47. Berthet FX: A Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate protein (CFP-10). Microbiology 1998,144(Pt 11):3195–203.PubMedView Article
                        48. Fortune SM: Mutually dependent secretion of proteins required for mycobacterial virulence. Proc Natl Acad Sci USA 2005,102(30):10676–81.PubMedView Article
                        49. Brodin P: Dissection of ESAT-6 system 1 of Mycobacterium tuberculosis and impact on immunogenicity and virulence. Infect Immun 2006,74(1):88–98.PubMedView Article
                        50. DiGiuseppe Champion PA: ESX-1 secreted virulence factors are recognized by multiple cytosolic AAA ATPases in pathogenic mycobacteria. Mol Microbiol 2009,73(5):950–62.PubMedView Article
                        51. Guinn KM: Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol Microbiol 2004,51(2):359–70.PubMedView Article
                        52. Renshaw PS: Conclusive evidence that the major T-cell antigens of the Mycobacterium tuberculosis complex ESAT-6 and CFP-10 form a tight, 1:1 complex and characterization of the structural properties of ESAT-6, CFP-10, and the ESAT-6*CFP-10 complex. Implications for pathogenesis and virulence. J Biol Chem 2002,277(24):21598–603.PubMedView Article
                        53. Singh A: Dissecting virulence pathways of Mycobacterium tuberculosis through protein-protein association. Proc Natl Acad Sci USA 2006,103(30):11346–51.PubMedView Article
                        54. Renshaw PS: Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6. EMBO J 2005,24(14):2491–8.PubMedView Article
                        55. Narayana Y: Differential B-cell responses are induced by Mycobacterium tuberculosis PE antigens Rv1169c, Rv0978c, and Rv1818c. Clin Vaccine Immunol 2007,14(10):1334–41.PubMedView Article
                        56. Adindla S, Guruprasad L: Sequence analysis corresponding to the PPE and PE proteins in Mycobacterium tuberculosis and other genomes. J Biosci 2003,28(2):169–79.PubMedView Article
                        57. Sampson SL: Expression, characterization and subcellular localization of the Mycobacterium tuberculosis PPE gene Rv1917c. Tuberculosis (Edinb) 2001,81(5–6):305–17.View Article
                        58. Skjot RL: Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens. Infect Immun 2000,68(1):214–20.PubMedView Article
                        59. Majlessi L: CD8 + −T-cell responses of Mycobacterium-infected mice to a newly identified major histocompatibility complex class I-restricted epitope shared by proteins of the ESAT-6 family. Infect Immun 2003,71(12):7173–7.PubMedView Article
                        60. Okkels LM, Andersen P: Protein-protein interactions of proteins from the ESAT-6 family of Mycobacterium tuberculosis. J Bacteriol 2004,186(8):2487–91.PubMedView Article
                        61. Lightbody KL: Characterisation of complex formation between members of the Mycobacterium tuberculosis complex CFP-10/ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility. FEMS Microbiol Lett 2004,238(1):255–62.PubMed
                        62. Bansal K: PE_PGRS antigens of Mycobacterium tuberculosis induce maturation and activation of human dendritic cells. J Immunol 2010,184(7):3495–504.PubMedView Article
                        63. Bansal K: Src homology 3-interacting domain of Rv1917c of Mycobacterium tuberculosis induces selective maturation of human dendritic cells by regulating PI3K-MAPK-NF-kappaB signaling and drives Th2 immune responses. J Biol Chem 2010,285(47):36511–22.PubMedView Article
                        64. Chaturvedi R: The multifunctional PE_PGRS11 protein from Mycobacterium tuberculosis plays a role in regulating resistance to oxidative stress. J Biol Chem 2010,285(40):30389–403.PubMedView Article
                        65. FDA: Products and medical procedures. Devices approvals and clearances. Food and Drug Administration;
                        66. WHO: Manual para el diagnóstico bacteriológico de la tuberculosis. Normas y guia técnica. WHO; 2008.
                        67. Sutherland JS: Production of TNF-alpha, IL-12(p40) and IL-17 can discriminate between active TB disease and latent infection in a West African cohort. PLoS One 2010,5(8):e12365.PubMedView Article
                        68. Torrado E, Cooper AM: IL-17 and Th17 cells in tuberculosis. Cytokine Growth Factor Rev 2010,21(6):455–62.PubMedView Article
                        69. Desel C: Recombinant BCG DeltaureC hly + induces superior protection over parental BCG by stimulating a balanced combination of type 1 and type 17 cytokine responses. J Infect Dis 2011,204(10):1573–84.PubMedView Article
                        70. Ahmed RK: Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus. BMC Infect Dis 2012, 12:41.PubMedView Article
                        71. Brosch R: Genome plasticity of BCG and impact on vaccine efficacy. Proc Natl Acad Sci USA 2007,104(13):5596–601.PubMedView Article
                        72. Borgstrom E: Detection of proliferative responses to ESAT-6 and CFP-10 by FASCIA assay for diagnosis of Mycobacterium tuberculosis infection. J Immunol Methods 2011,370(1–2):55–64.PubMedView Article
                        73. Korn T: IL-17 and Th17 Cells. Annu Rev Immunol 2009, 27:485–517.PubMedView Article
                        74. Pre-publication history

                          1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2334/​13/​125/​prepub

                        Copyright

                        © Alvarez-Corrales et al; licensee BioMed Central Ltd. 2013

                        This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.