Hindawi Publishing Corporation
Clinical and Developmental Immunology
Volume 2011, Article ID 351573, 10 pages
doi:10.1155/2011/351573
Research Article
Human T Cell and Antibody-Mediated Responses to
the Mycobacterium tuberculosis Recombinant 85A, 85B, and
ESAT-6 Antigens
Gilson C. Macedo,1, 2 Adriana Bozzi,1 Helena Rachel Weinreich,3 Andre Bafica,4
Henrique C. Teixeira,2 and Sergio C. Oliveira1
1 Laboratory
of Immunology of Infectious Diseases, Department of Biochemistry and Immunology, Institute of Biological Sciences,
Federal University of Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
2 Department of Parasitology, Microbiology and Immunology, Biological Sciences Institute, Federal University of Juiz de Fora,
36036-900 Juiz de Fora, MG, Brazil
3 Oswaldo Cruz Health Center, Belo Horizonte-Minas Gerais, 30180-080 Belo Horizonte, MG, Brazil
4 Department of Microbiology, Immunology and Parasitology, Federal University of Santa Catarina,
88040-900 Florianopolis, MG, Brazil
Correspondence should be addressed to Sergio C. Oliveira, scozeus@icb.ufmg.br
Received 14 September 2010; Revised 1 November 2010; Accepted 5 November 2010
Academic Editor: James Triccas
Copyright © 2011 Gilson C. Macedo et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Tuberculosis remains a major health problem throughout the world causing large number of deaths. Effective disease control and
eradication programs require the identification of major antigens recognized by the protective responses against M. tuberculosis.
In this study, we have investigated humoral and cellular immune responses to M. tuberculosis-specific Ag85A, Ag85B, and ESAT-6
antigens in Brazilian patients with pulmonary (P, n = 13) or extrapulmonary (EP, n = 12) tuberculosis, patients undergoing
chemotherapy (PT, n = 23), and noninfected healthy individuals (NI, n = 7). Compared to NI, we observed increased levels
of IgG1 responses to Ag85B and ESAT-6 in P and PT groups. Regarding cellular immunity, Ag85A and ESAT-6 were able to
discriminate P, PT, and EP patients from healthy individuals by IFN-γ production and P and PT groups from EP individuals
by production of TNF-α. In summary, these findings demonstrate the ability of Ag85A, Ag85B, and ESAT-6 to differentiate TB
patients from controls by IgG1, IFN-γ and TNF-α production.
1. Introduction
Tuberculosis (TB) remains the largest single infectious
cause of death globally. It is estimated that 30% of the
word population is infected with Mycobacterium tuberculosis
resulting in approximately 2-3 million deaths each year [1].
Further, the AIDS epidemic and the appearance of multidrug
resistant strains of M. tuberculosis have contributed to
the reemergence of TB in developing countries; however,
this disease continues to be a devastating entity in the
developing world [2]. At present, the only registered vaccine
against tuberculosis, Mycobacterium bovis Bacillus CalmetteGuérin (BCG), was introduced in 1921 and has been widely
used; however, its effectiveness remains controversial because
their protection levels are extremely variable in different
population [3–5]. Furthermore, vaccination with M. bovis
BCG is contraindicated in immunocompromised subjects,
including acquired immunodeficiency syndrome patients,
who are usually at a very high risk of developing TB [6]. In
addition, the diagnostic value of the presently used skin test
reagent, purified protein derivative (PPD) of Mycobacterium
tuberculosis, is low, owing to cross-reactivity with environmental mycobacteria and vaccine strains of M. bovis BCG [7,
8]. Thus, the effective control and eradication of TB is dependent upon the availability of effective vaccines and reagents
for specific diagnosis. For this purpose, the identification
2
of major antigens recognized by the protective immune
response against M. tuberculosis remains a critical step.
Among M. tuberculosis antigens studied, the 30/32 KDa
antigen 85 (Ag85) complex has been the focus of intense
research over the past several years and comprises three
closely related proteins, 85A (32 KDa), 85B (30 KDa), and
85C (32.5 KDa) that possess enzymatic mycolyl-transferase
activity [9–11]. The Ag85 complex induces protective immunity against TB in guinea pigs [12], and strong proliferation
and IFN-γ production in peripheral blood mononuclear
cells (PBMC) from healthy tuberculin reactors [13]. Regarding, ESAT-6, the early secreted antigenic target is a lowmolecular-weight protein essentially present in pathogenic
mycobacteria including members of the mycobacterium
complex (M. tuberculosis, M bovis, and M. africanum) and
M. leprae [14]. Analysis of T-cell responses to M. tuberculosis
ESAT-6 showed an elevated range of recognition from many
tuberculosis patients [15]. Consequently, the possible use of
ESAT-6 as a marker of M. tuberculosis infection has been
proposed. Moreover, other studies have demonstrated the
ability of this protein to discriminate tuberculosis patients
from health donors in a high endemic area [16]. Additionally,
ESAT-6 is able to differentiate tuberculosis patients from
both BCG-vaccinated individuals and M. avium infected
patients [17].
The main goal of this study was to evaluate the cellular
and humoral immune responses to the recombinant proteins Ag85A, Ag85B, and ESAT-6 in Brazilian pulmonary
and extra-pulomary tuberculosis patients and individuals
undergoing chemotherapy. The recombinant proteins were
produced in E. coli and purified by affinity chromatography. Cellular proliferation and cytokine production were
evaluated in peripheral blood mononuclear cells (PBMC)
and specific antibody isotypes to Ag85A, Ag85B and ESAT6 were measured in serum of TB patients and controls. In
this study, we have shown the ability of Ag85B and ESAT-6 to
differentiate TB patients from controls by IgG1 production.
Additionally, the results here demonstrated that Ag85A and
ESAT-6 were able to discriminate P, PT, and EP patients
from healthy individuals by IFN-γ production and P and PT
groups from EP individuals by production of TNF-α.
2. Materials and Methods
2.1. Study Population. Patients with active pulmonary TB
(P, n = 13) or active extra-pulmonary TB (EP, n = 12),
and pulmonary TB patients with 1–3 months of anti-TB
chemotherapy (PT, n = 23), diagnosed at the outpatient unit
of the Oswaldo Cruz Health Center, Belo Horizonte, Minas
Gerais, Brazil, were enrolled in this study. All TB patients had
sputum-positive bacilloscopy or culture-confirmed disease.
The EP-TB group comprised six pleural TB, five miliary TB
and one intestinal TB as shown in Table 1. Seven healthy
non-BCG vaccinated individuals (all PPD-) without prior
history of mycobacterial infection were included as control
group. All enrolled patients tested negative by ELISA for
HIV. None of the individuals had evidence of acute infections
(other than TB) at the time of sample collection. Twenty ml
of blood was taken from each patient.
Clinical and Developmental Immunology
2.2. Ethics Committee. All patients gave permission for blood
sampling after written consent, and the Ethics Committee
of the Santa Casa Hospital at Belo Horizonte, Minas Gerais,
Brazil approved the research protocol.
2.3. Mycobacterial Antigens. The recombinant Ag85A, Ag85B
e ESAT-6 was produced using the pMAL-c2 expression system. Briefly, the pMAL-85A, pMAL-85B, or pMAL-ESAT-6
construct was used to transform Escherichia coli DH5α strain
as previously described [18]. Bacterial cells were induced
using 0.42 mM IPTG (isopropyl-β-D-thiogalactoside) and
recombinant proteins fused to the maltose binding protein
(MBP) were produced. Three hours after gene expression,
the cells were harvested and lysed using thermal shock,
sonication, and lysozyme treatment. The fusion protein
recovered in the supernatant was then purified by affinity
chromatography using an amylose resin (New England
BioLabs). Residual endotoxin levels were removed from
recombinant proteins by using Triton X-114 and measured
to be <50 EU/mg recombinant protein by the LAL assay
(Limulus amoebocyte lysate). Purified protein derivative
(PPD-RT50) was obtained from Statens Serum Institute,
Copenhagen, Denmark.
2.4. T Cell Proliferation Assays. Heparinized venous blood
was obtained from all patients and controls, and peripheral
blood mononuclear cells (PBMCs) were isolated by FicollHypaque (Ficoll 6,42% (SIGMA) and Hypaque 50% (Sanofi
Synthelab)) density centrifugation. Cells were washed in
RPMI 1640 fresh medium and cultured (2, 5 × 105 cells/well)
in flat-bottom 96-well plates (Nunc Brand Products) in
200 µL of RPMI 1640 medium supplemented with 10%
AB + heat-inactivated human serum and 1% antibiotic/antimycotic (Gibco-BRL) and incubated at 37◦ C in a
humidified 5% CO2 incubator with recombinant antigens
or medium alone (control). The recombinant antigens were
titered to determine the optimal protein concentration for
proliferation assays. Ag85A, Ag85B, and PPD was used at
25 µg/ml, ESAT-6 at 50 µg/ml, and phytohaemagglutinin
(PHA) was used at 10 µg/ml. The concentration of the
recombinant antigens tested here was higher compared to the
used by other authors because we produced the mycobacterial antigens fused to MBP that has itself a molecular
mass of approximately 42.6 kDa. All antigens were plated
in triplicate. After 3 days (mitogen) or 5 days (antigens)
of incubation, [3 H] thymidine (0.5 µCi/well) was added to
the cultures. Eighteen hours later, the supernatants were
collected, and the cells were harvested. The incorporated
radioactivity in the cells was evaluated by liquid scintillation
spectroscopy as a measure of cellular proliferation. Mean
counts per minute for triplicate cultures and stimulation
index (SI) were obtained for each patient. The SI was the
ratio of mean counts per minute in the presence of antigen
to means counts per minute in medium alone.
2.5. Cytokine Measurement. Concentration of human IFN-γ,
TNF-α, IL-10, and IL-4 in cell culture supernatants of
proliferation assays was determined by enzyme-linked
immunosorbent assay (ELISA) using kits Duoset from R&D
Clinical and Developmental Immunology
3
Table 1: Clinical characteristics of TB patients and controls in this study.
Groups
Non-infected (NI)
TB patients under treatment (PT)∗
Pulmonary TB untreated (P)
Extra-pulmonary TB (EP)
(i) Pleural
(ii) Miliary
(iii) Intestinal
No. of subjects
7
25
13
12
6
5
1
TST
−
+
+
+
Males/females
05/02
14/11
09/04
08/04
Age (mean ± SD)
40.1 ± 9.5
39.8 ± 14.6
41.0 ± 15.7
41.1 ± 16.5
Age range
28–55
22–76
19–69
21–76
∗
The treatment consisted of Rifampicin (10–20 mg/kg/day), Isoniazide (10–20 mg/kg/day), and Piraminazide (30–50 mg/kg/day). PT patients were
undergoing 1–3 months of chemotherapy. TST tuberculin skin test.
Systems (Minneapolis, MN, USA) according to manufacturer’s directions.
version 5.0 (GraphPad software incorporated). Statistical
differences were considered significant at P < .05.
2.6. Detection of Antibody Responses. Detection of antibody
against recombinant Ag85A, Ag85B, and ESAT-6 antigens
in TB patients and healthy individual sera was performed
by a modified ELISA [19]. Briefly, ninety-six-well flatbottom microtiter plates (Nunc, Roskilde, Denmark) were
coated overnight at 4◦ C with 100 µL of each recombinant
antigen separately at a concentration of 5 µg/ml in 0.1 M
carbonate bicarbonate buffer (pH 9.6) per well. The plates
were then blocked with 10% bovine fetal serum in PBS
(pH 7.4) for 2 h at room temperature. Subsequently, the
plates were washed three times with PBS plus 0.05%
Tween-20 (PBS-T20). Serum samples diluted 1 : 100 in PBST20 (100 µL/well) were added in duplicate, and the plates
were incubated for 1 h at room temperature. Peroxidaselabeled anti-IgG (Sigma Chemical Co., St. Louis, MO),
anti-IgM (Sigma), and anti-IgA (Sigma) were added at
dilutions of 1 : 2,000, 1 : 2,000, and 1 : 10,000 (100 µL/well),
respectively. After 1 h at 37◦ C, the plates were washed, and
OPD (orthophenyl-diaminobenzidine) plus 0.05% hydrogen
peroxide in phosphate citrate buffer (pH 5) was added
(100 µL/well). This mixture was then incubated for 30 min
at room temperature, and the reaction was stopped by
addition of 5% H2 SO4 (50 µL/well). Absorbance was read at
492 nm using a microplate reader (BioRad, Hercules, CA).
To determine IgG subclasses levels, the previous protocol was
slightly modified. The serum dilution was changed to 1:80 for
IgG1 and IgG3 and 1:20 to IgG2 and IgG4. Diluted sera were
added to the plates, and they were incubated for 2 h at 37◦ C.
After washing, peroxidase-labeled antihuman antibody was
dispensed in each well at concentrations of 1 : 1,000 (IgG1,
IgG3) or 1 : 500 (IgG2, IgG4), and the plates were incubated
for 12–16 h at 4◦ C. The next steps were identical to those
described above.
3. Results
2.7. Statistical Analysis. Results are reported as means ±
standard errors. Differences between responses from TB
patient groups and control groups were analyzed with nonparametric Kruskall-Wallis test. Correlation between ESAT6 induced IFN-γ and proliferation responses from patients
WAS identified using Spearman Correlation. Statistical analysis was performed using the GraphPad Prism software
3.1. IgG1 Is the Predominant Antibody Isotype Present in Sera
of TB Patients. To investigate the presence of specific antiAg85A, -Ag85B or -ESAT-6 antibodies in sera of TB patients
with different clinical forms of the disease, ELISA were
performed. Figure 1 shows the levels of specific IgG, IgM
and IgA to mycobacterial antigens in sera of TB patients and
healthy donors. The levels of anti-PPD IgG were significantly
elevated in all tuberculosis patients compared to NI group.
Furthermore, increased levels of IgG anti-Ag85B and antiESAT-6 were detected in P and PT groups compared to NI
individuals. Interestingly, no significant titers of IgG antiAg85A were detected in studied patients. Levels of specific
IgA antibodies to all antigens were very low and did not differ
between the studied groups. In addition, only marginal antiAg85B and anti-PPD IgM levels were observed in the P and
PT groups.
Having observed elevated IgG levels to Ag85B and
ESTA-6 antigens in patient sera, we decided to determine
the IgG subclasses involved. The IgG subclass profile of TB
patients was characterized predominantly by IgG1 responses
to rAg85B and rESAT-6 on P and PT groups (Figure 2).
Additionally, statistically significant levels of IgG3 to rAg85B,
ESAT-6 and Ag85A were also detected in sera of PT and P
group, however at lower levels. These results demonstrate
the better performance of the Ag85B and ESAT-6 antigens
compared to Ag85A to determine humoral responses in
patients with active TB.
3.2. Proliferative Responses to Mycobacterial Antigens. In
order to determine T cell-reactivity to the mycobacterial antigens tested, lymphoproliferative responses were measured in
cells from tuberculosis patients and healthy individuals. As
shown in Figure 3, proliferative responses upon stimulation
with rESAT-6 were able to discriminate P, EP, and PT
tuberculosis patients from healthy individuals. Regarding
Ag85A and Ag85B, they were able to differentiate PT
and P tuberculosis patients from noninfected individuals.
Only when PPD was used as antigen, this assay was able
to discriminate PT and P patients from individuals with
4
Clinical and Developmental Immunology
85A
1.4
O.D. 492 nm
1.2
85B
ESAT-6
∗
1
∗
∗
0.8
PPD
∗
∗
∗
∗
0.6
0.4
∗ ∗
∗ ∗
0.2
∗
0
NI P PT EP
NI P PT EP
NI P PT EP
Groups
NI P PT EP
IgG
IgM
IgA
Figure 1: Levels of serum IgG (hatched box), IgM (black box) and IgA (empty box) to Ag85A, Ag85B, ESAT-6, and PPD were determined
in serum of non-infected individuals (NI), pulmonary tuberculosis patients (P), pulmonary tuberculosis patients undergoing treatment
(PT) and extra-pulmonary tuberculosis (EP). Results are presented as mean ± standard deviation. Differences between responses from TB
patient groups and control groups were analyzed with nonparametric Kruskall-Wallis test. ∗ Statistically significant differences in relation to
NI group (P < .05).
extrapulmonary TB. PBMC of all individuals showed high
cell proliferation after stimulation with PHA as a positive
control (data not shown).
3.3. Cytokine Profile in Response to Recombinant Mycobacterial Antigens. In order to evaluate the cytokines produced by
P, EP, and PT patient cells to the mycobacterial recombinant
antigens, IFN-γ, TNF-α, IL-10 and IL-4 were measured.
Figure 4 shows a significant production of IFN-γ after
stimulation with Ag85A and ESAT-6 in the PT group (1285 ±
1571 and 1355 ± 976, resp.), P group (1268 ± 1722 and
974 ± 218, resp.), and EP group (903 ± 785 and 920 ± 2477,
resp.) in comparison to the non-infected control group. This
elevated production of IFN-γ in ESAT6-stimulated PBMC of
TB patients correlated with positive response to ESAT6 in the
proliferation assays (Spearman test; PT group r = 0.5158,
P = .0118; P group r = 0.5659, P = .0438; EP group r =
0.6224, P = .0307). Moreover, in response to Ag85B, only PT
(1206 ± 2087) and P (1227 ± 799) patients produced significant levels of IFN-γ compared to healthy individuals. The PT
groups were the higher producers of IFN-γ in response to all
recombinant antigens; however no difference was observed
within this group among the different antigens used. All TB
patients produced high levels of IFN-γ to PPD, and this result
is in agreement with the complex antigenic mixture of PPD.
Regarding TNF-α production, we observed that PT and P
groups produced significant levels of this cytokine when
stimulated with Ag85A, Ag85B or ESAT-6. However, no
recombinant antigens tested were able to induce greater levels
of TNF-α in EP group compared to healthy individuals.
Furthermore, only Ag85A or ESAT6 antigens induced production of higher amounts of TNF-α by PT and P cells that
allowed discrimination between these groups from EP group.
Additionally, all patients produced high levels of TNF-α to
PPD.
As for IL-10, a regulatory cytokine, we observed that
the group of TB patients who had initiated chemotherapy
(PT) produced higher amounts of IL-10 compared to P or
EP groups when Ag85A, Ag85B and ESAT-6 were tested.
These values were significant to differentiate PT from P
and EP groups. None of the tested recombinant antigens or
PPD induced detectable production of IL-4 by PBMC of all
individuals tested (data not shown).
4. Discussion
For the development of new vaccines and diagnostic
reagents, there is an urgent need for assessment of immune
responses to M. tuberculosis antigens in areas of TB endemicity. In the present study T-cell and antibody responses to
recombinant Ag85A, Ag85B or ESAT-6 were investigated
in Brazilian patients with pulmonary or extra-pulmonary
TB and patients undergoing treatment compared to noninfected individuals. Several studies have detected antibodies
in sera of patients with active TB against a variety of M. tuberculosis antigens [20–22]. Herein, patients with active disease
or undergoing >2 months of treatment presented elevated
levels of IgG anti-ESAT-6 and anti-Ag85B but not to Ag85A.
High levels of antibodies against filtered M. tuberculosis
antigens in the first two months of chemotherapy have been
associated with intense stimulation of the humoral response
by antigens released from killed bacteria combined with the
disappearance of circulating mycobacterial antigens so that
specific antibodies are no longer trapped in the immune
complexes [23]. Therefore, large amounts of IgG antibodies
against secreted Ag85B and ESAT-6 antigens appear to be
associated with viable and metabolically active bacilli. Little
attention has been given to the subclasses involved in TB
[24]. In our study, the analysis of IgG subclasses to the
mycobacterial recombinant antigens revealed the predominance of IgG1 but not IgG2 and IgG4 to ESAT-6 and Ag85B
in sera of patients of the P or PT group. However, we also
detected significant levels of IgG3 against Ag85A, Ag85B and
ESAT-6 in these groups of patients. Our results are consistent
Clinical and Developmental Immunology
5
IgG1
1.5
∗
1.5
IgG2
1
O.D. 492 nm
O.D. 492 nm
∗
∗
∗
∗
∗
0.5
0
85A
85B
ESAT-6
1
∗ ∗
∗
0.5
0
PPD
85A
85B
Antigens
(a)
IgG3
1.5
1
∗
∗
∗
0.5
PPD
(b)
O.D. 492 nm
O.D. 492 nm
1.5
ESAT-6
Antigens
∗
∗
IgG4
1
0.5
∗
∗
∗
0
0
85A
85B
ESAT-6
Antigens
PPD
PT
EP
NI
P
(c)
85A
85B
ESAT-6
Antigens
PPD
PT
EP
NI
P
(d)
Figure 2: Levels of serum IgG1 (a), IgG2 (b), IgG3 (c) and IgG4 (d) to Ag85A, Ag85B, ESAT-6, and PPD were determined in serum of
non-infected individuals (NI), pulmonary tuberculosis patients (P), pulmonary tuberculosis patients undergoing treatment (PT) and extrapulmonary tuberculosis (EP). Results are presented as mean ± standard deviation. Differences between responses from TB patient groups
and control groups were analyzed with nonparametric Kruskall-Wallis test. ∗ Statistically significant differences in relation to NI group
(P < .05).
with other studies that also observed predominance of IgG1
antibodies in the sera of patients with active TB [19, 24,
25]. Since Ag85A and Ag85B share around 77% of amino
acids identity, one could expect them to have common
immunodominant epitopes. Despite pronounced sequence
homology among these Ag85 members, D’Souza et al. [26]
have shown that different Ag85-specific immunodominant
T-cell epitopes were identified in BALB/c and C57BL/6
mouse strains. These differences in MHC-restriction during
Ag85A and Ag85B epitope mapping might be one of the
reasons why we did not observed significant levels of antiAg85A IgG in our TB patients. Similarly, Van Vooren et al.
[27] suggested that Ag85B was the most useful component
of the Ag85 complex for serodiagnosis of the active form of
TB.
Recently, commercial immunodiagnostic tests for TB
have been introduced. These tests are based on the M.
tuberculosis ESAT-6 and culture filtrate protein 10 (CFP-10)
and include a whole-blood IFN-γ ELISA (QuantiFERONTB Gold, Cellestis Ltd, Victoria, Australia) and an ELISPOT
assay (T-SPOT.TB, Oxford Immunotec, Oxfordshire, UK).
Both tests have shown promising results in the detection
of latent TB and the potential use for differential diagnoses
of active tuberculosis [28, 29]. However, the sensitivities
and specificities of these assays vary among the different
populations studied, due mostly to the different HLA
genetic backgrounds, the prevalence of TB infection, and the
coverage of M. bovis BCG vaccination [30]. Furthermore,
there is a need to develop new diagnostic tools to detect
extra-pulmonary TB and sputum negative cases. In our
study, pulmonary or extra-pulmonary TB patients and
individuals undergoing chemotherapy responded to ESAT6 as evaluated by lymphoproliferative responses or by IFNγ production determined in the supernatants of stimulated
Clinical and Developmental Immunology
Stimulation index
6
12
11
10
9
8
7
6
5
4
3
2
1
0
85A
85B
∗
∗
∗
ESAT-6
∗
∗
∗
∗
NI P PT EP
NI P PT EP
NI P PT EP
Groups
PPD
∗
∗
δ
δ
NI P PT EP
Figure 3: Lymphocyte proliferation in response to recombinant Ag85A, Ag85B and ESAT-6. Freshly isolated PBMC from non-infected
individuals (NI), pulmonary TB patients (P), pulmonary TB patients in treatment (PT) and extra-pulmonary TB (EP) were cultured in
the presence of Ag85A (25 µg/ml), 85B (25 µg/ml), PPD (25 µg/ml) or ESAT-6 (50 µg/ml) for 5 days and incorporation of [3 H] thymidine
was measured. Results are expressed as stimulation index (SI) mean of triplicate cultures. Horizontal bars indicate mean values. Differences
between responses from TB patient groups and control groups were analyzed with nonparametric Kruskall-Wallis test. (∗ ) Statistically
significant differences in relation to NI group and (δ) in relation to EP group (P < .05).
PBMC. Additionally, Ag85A was also recognized by all
TB patient cells compared to non-infected individuals as
measured by IFN-γ secretion. This data demonstrates that
ESAT-6 and Ag85A are recognized by T cell from many
tuberculosis patients undergoing distinct periods of clinical
disease and is consistent with Ulrichs et al. [15] findings by
which PBMCs from tuberculosis patients, but not healthy
donors, respond to ESAT-6. Furthermore, as described previously by Antas et al. [31], the Ag85A and Ag85B proteins
were also recognized by PBMC of pulmonary tuberculosis
patients and individuals undergoing treatment measured
by elevated proliferation and IFN-γ production. Regarding
TNF-α, clinical studies have associated the use of TNFblockers with progression from latent tuberculosis infection
to disease [32]. Further, Caccamo et al. [33] have reported
high percentage of CD4+ T cells expressing IFN-γ/IL-2/TNFα in active TB patients and it seems to be associated with live
bacterial loads, as indicated by the decrease in frequency of
multifunctional T cells in TB patients after completion of
antimycobacterial therapy. In our study, we have observed
elevated levels of TNF-α to Ag85A, Ag85B, and ESAT6 in patients with pulmonary tuberculosis or undergoing
treatment but not in extra-pulmonary TB patients. However,
only Ag85A and ESAT-6 antigens were able to discriminate
PT and P patients from EP. This data might be associated
to differential production of TNF-α by CD4+, and CD8+ T
cells and the compartmentalization of immune response at
site of disease. Marei et al. [34] demonstrated a differential
expression of IFN-γ and TNF-α in CD4+ T cells and CD8+
T cells after stimulation with ESAT-6. In their study, CD4+
T cells are the main producer of TNF-α while IFN-γ was
produced by either CD4+ or CD8+ T cells. In addition, other
studies have provided evidence for compartmentalization of
Th1 cytokines at the site of disease in humans [35, 36]. We
suggest that compartmentalization of the immune response
in EP patients can lead to sequestration of TNF-α producing
cells at the disease site and it contributes to reduced
production of this cytokine in peripheral blood in response
to Ag85A, Ag85B and ESAT-6. Herein, our results suggest
that Ag85A and ESAT-6 are able to differentiate P, PT and EP
patients from healthy individuals by IFN-γ production and
from P and PT groups to EP individuals by production of
TNF-α. In a recent study, it was reported that similar levels
of cytokine and antibody responses to M. tuberculosis ESAT6/CFP-10 fusion protein were detected in PPD+ and PPD−
groups from an endemic area of Juiz de Fora, Minas Gerais,
Brazil [19]. Herein, the NI group (PPD−) used also lives in
a TB endemic area in Brazil and probably has been exposed
to multiple forms of environmental mycobacteria. However,
to confirm the diagnostic potential of these antigens further
studies using BCG vaccinated controls (PPD+) are required.
Antibodies conventionally are considered to play little
role in defence against mycobacteria, and the function
of antibodies in pathogenesis is yet to be determined.
Macrophages in which mycobacteria resides, and multiply
have high affinity receptors (Fcγ1 and Fcγ3) for IgG1 and
IgG3 antibodies, and the presence of IgG1 and IgG3 antibodies may enhance bacterial uptake and clearance of pathogen
via the Fc receptor [37]. Hussain et al. [38] reported that
opsonizing antibodies upregulate macrophage proinflammatory cytokines TNF-α and IL-6 in mycobacterial-stimulated
macrophages thus suggesting a role for this isotype in TB,
since TNF-α synergizes with IFN-γ in its tuberculostatic
activity. Since IgG responses against proteins are T cell
dependent, antigen recognition by IgG isotypes implies that
helper T cells also recognize these mycobacterial antigens.
Further, IFN-γ produced by Th1 cells induces murine
IgG2a and IgG2b and human IgG1 and IgG3 [39, 40].
Clinical and Developmental Immunology
7
85A
ESAT-6
85B
∗
5000
4500
PPD
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
IFN-γ (pg/mL)
4000
3500
3000
2500
2000
1500
1000
500
0
NI P PT EP
NI P PT EP
NI P PT EP
Groups
NI P PT EP
(a)
5000
∗
85A
85B
ESAT-6
∗
∗
∗
δ
4500
∗
∗
PPD
∗
δ
δ
δ
∗
∗
TNF-α (pg/mL)
4000
3500
3000
2500
2000
1500
1000
500
0
NI P PT EP
NI P PT EP
NI P PT EP
Groups
NI P PT EP
(b)
85A
85B
ESAT-6
∗
∗
∗
δ
4000
3500
#
#
#
δ
#
PPD
δ
δ
δ
IL-10 (pg/mL)
3000
2500
2000
1500
1000
500
0
NI P PT EP
NI P PT EP
NI P PT EP
Groups
NI P PT EP
(c)
Figure 4: Individual IFN-γ (a), TNF-α (b), and IL-10 (c) production in response to recombinant Ag85A, Ag85B, and ESAT-6. Freshly isolated
PBMC from non-infected individuals (NI), pulmonary TB patients (P), pulmonary TB patients in treatment (PT), and extra-pulmonary
TB (EP) were cultured in the presence of Ag85A (25 µg/ml), Ag85B (25 µg/ml), PPD (25 µg/ml), or ESAT6 (50 µg/ml). Supernatants were
harvested after five days and the cytokines measured by ELISA. Horizontal bars represent mean values. Differences between responses from
TB patient groups and control groups were analyzed with nonparametric Kruskall-Wallis test. ∗ Statistically significant differences in relation
to NI group, (δ) in relation to EP group, and (#) in relation to P group (P < .05).
8
Human IgG1 and IgG3 counterparts of murine IgG2a and
IgG2b share the ability to fix complement and function
as opsonins. In this study, we observed that the increased
IgG1 was coincident with augmented levels of IFN-γ and
TNF-α detected in PBMCs of patients with active TB and
individuals undergoing treatment stimulated with Ag85B
and ESAT-6. These results suggest a possible correlation of
IgG1 production with Th1 and inflammatory response in TB.
Interestingly, IL-10 production was elevated only in
patients under treatment in response to Ag85, Ag86, and
ESAT-6. In this study, IL-10 levels could differentiate individuals undergoing chemotherapy from pulmonary or extrapulmonary patients. Priya et al. [41] have shown that
TB patients from India at the beginning of chemotherapy
produced similar levels of IFN-γ and IL-10 to Ag85A and
the ratio of IFN/IL-10 increases after successful treatment.
Furthermore, Meyaard et al. [42] demonstrated that IL-12 is
able to induce T cells to produce IL-10 and suggest that IL-10
is a negative regulator of IL-12- induced T cell response. In
another study testing TB patients, Priya et al. [43] observed
that high levels of IL-10 detected in active TB decreased in
patients considered cured. These results and our data led us
to hypothesize that elevated production of IL-10 encountered
in PT group is probably a modulatory effect in response to
IL-12 and IFN-γ production and can be associated to the
regulation of immune response.
Th2 responses characterized by inteleukin-4 (IL-4) production have been associated with a lack of protection in TB
[44]. In our study, IL-4 levels in response to mycobacterial
recombinant antigens were not detected in all groups
analyzed. These results are in accordance with others that
show that PBMC from TB patients do not produce significant amounts of IL-4 [45–47]. These results confirm the
polarization of immune response to recombinant antigens to
Th1 profile characterized by the production of high levels of
IFN-γ and TNF-α and no IL-4.
Finally, TB causes a staggering burden of mortality
worldwide, killing an estimated 1.9 million persons annually.
Effective treatment of tuberculosis in developing countries is
hampered by the cost of antituberculosis drugs, inability to
ensure completion of therapy, and rising drug resistant rates.
Vaccination is the most cost-effective strategy to control
and eventual elimination of tuberculosis. The current BCG
vaccine provides some degree of protection against the most
severe manifestations of childhood tuberculosis. However,
protection is incomplete, and BCG vaccine does not reduce
TB rates in adults. In fact, MVA85A, a recombinant modified
vaccinia virus Ankara expressing Ag85A, is the first candidate
TB subunit vaccine to enter human trials since BCG was
first introduced over 80 years ago. More recently, Dissel
et al. [48] demonstrated that vaccination of human naı̈ve
volunteers with adjuvanted Ag85B-ESAT-6 subunit vaccine
elicited strong antigen-specific T-cell responses. Since a basic
principle for selecting novel antigen candidates for designing
a TB subunit vaccine is based on their ability to induce
a protective Th1 response [16], our study also confirmed
the value of Ag85A, Ag85B and ESAT-6 as potential vaccine
candidates based upon specific T cell responses measured by
IFN-γ and TNF-α production in all studied patients.
Clinical and Developmental Immunology
5. Conclusions
Currently, there are no accurate surrogate biomarkers of
protective immunity and diagnoses in TB but clearly host
defense against TB depends critically on Th1 responses
and IFN-γ production. In this study, we have shown
that Ag85A and ESAT-6 are antigens able to differentiate pulmonary, extra-pulmonary and tuberculosis patients
undergoing chemotherapy from healthy individuals by IFNγ production and pulmonary and under treatment patients
from extra-pulmonary TB by TNF-α. Therefore, not only
IFN-γ production but also TNF-α to Ag85A and ESAT-6
could be used as biomarkers for the clinical status of TB
patients while IL-10 could be useful monitoring TB successful treatment. Finally, the Th1 cytokine profile induced in
PBMC of TB patients by all tested antigens reinforces their
position as potential vaccine candidates.
Acknowledgments
This work was supported by grants from the Brazilian
funding agencies CNPq, CAPES (PROCAD and PNPD),
FAPEMIG, and INCT-Vacinas.
References
[1] World Health Organization (WHO), Global Tuberculosis
Control. A short update to the 2009 report, WHO, Geneva,
Switzerland, 2009, http://www.who.int/tb/publications/global
report/2009/update/tbu 9.pdf.
[2] E. L. Corbett, C. J. Watt, N. Walker et al., “The growing burden
of tuberculosis: global trends and interactions with the HIV
epidemic,” Archives of Internal Medicine, vol. 163, no. 9, pp.
1009–1021, 2003.
[3] G. A. Colditz, T. F. Brewer, C. S. Berkey et al., “Efficacy of
BCG vaccine in the prevention of tuberculosis: meta-analysis
of the published literature,” Journal of the American Medical
Association, vol. 271, no. 9, pp. 698–702, 1994.
[4] W. J. Britton and U. Palendira, “Improving vaccines against
tuberculosis,” Immunology and Cell Biology, vol. 81, no. 1, pp.
34–45, 2003.
[5] P. E. M. Fine, “Variation in protection by BCG: implications
of and for heterologous immunity,” Lancet, vol. 346, no. 8986,
pp. 1339–1345, 1995.
[6] E. A. Talbot, M. D. Perkins, S. F.M. Suva, and R. Frothingham,
“Disseminated bacille Calmette-Guerin disease after vaccination: case report and review,” Clinical Infectious Diseases, vol.
24, no. 6, pp. 1139–1146, 1997.
[7] P. Andersen, M. E. Munk, J. M. Pollock, and T. M. Doherty,
“Specific immune-based diagnosis of tuberculosis,” Lancet,
vol. 356, no. 9235, pp. 1099–1104, 2000.
[8] E. Lee and R. S. Holzman, “Evolution and current use of the
tuberculin test,” Clinical Infectious Diseases, vol. 34, no. 3, pp.
365–370, 2002.
[9] J. H. Lim, J. K. Park, E. K. Jo et al., “Purification and
immunoreactivity of three components from the 30/32- kilodalton antigen 85 complex in Mycobacterium tuberculosis,”
Infection and Immunity, vol. 67, no. 11, pp. 6187–6190, 1999.
[10] K. Huygen, J. P. Van Vooren, M. Turneer, R. Bosmans, P.
Dierckx, and J. De Bruyn, “Specific lymphoproliferation,
gamma interferon production, and serum immunoglobulin G
Clinical and Developmental Immunology
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
directed against a purified 32 kDa mycobacterial protein antigen (P32) in patients with active tuberculosis,” Scandinavian
Journal of Immunology, vol. 27, no. 2, pp. 187–194, 1988.
S. Nagai, H. Gotten Wiker, M. Harboe, and M. Kinomoto,
“Isolation and partial characterization of major protein
antigens in the culture fluid of Mycobacterium tuberculosis,”
Infection and Immunity, vol. 59, no. 1, pp. 372–382, 1991.
M. A. Horwitz, B. W. E. Lee, B. J. Dillon, and G. Harth,
“Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium
tuberculosis,” Proceedings of the National Academy of Sciences
of the United States of America, vol. 92, no. 5, pp. 1530–1534,
1995.
M. Torres, T. Herrera, H. Villareal, E. A. Rich, and E.
Sada, “Cytokine profiles for peripheral blood lymphocytes
from patients with active pulmonary tuberculosis and healthy
household contacts in response to the 30-kilodalton antigen of
Mycobacterium tuberculosis,” Infection and Immunity, vol. 66,
no. 1, pp. 176–180, 1998.
M. Harboe, T. Oettinger, H. G. Wiker, I. Rosenkrands, and
P. Andersen, “Evidence for occurrence of the ESAT-6 protein
in Mycobacterium tuberculosis and virulent Mycobacterium
bovis and for its absence in Mycobacterium bovis BCG,”
Infection and Immunity, vol. 64, no. 1, pp. 16–22, 1996.
T. Ulrichs, M. E. Munk, H. Mollenkopf et al., “Differential
T cell responses to Mycobacterium tuberculosis ESAT6 in
tuberculosis patients and healthy donors,” European Journal of
Immunology, vol. 28, no. 12, pp. 3949–3958, 1998.
F. L. L. Cardoso, P. R. Z. Antas, A. S. Milagres et al., “Tcell responses to the Mycobacterium tuberculosis-specific
antigen ESAT-6 in Brazilian tuberculosis patients,” Infection
and Immunity, vol. 70, no. 12, pp. 6707–6714, 2002.
A. D. Lein, C. F. Von Reyn, P. Ravn, C. R. Horsburgh Jr., L.
N. Alexander, and P. Andersen, “Cellular immune responses
to ESAT-6 discriminate between patients with pulmonary
disease due to Mycobacterium avium complex and those
with pulmonary disease due to Mycobacterium tuberculosis,”
Clinical and Diagnostic Laboratory Immunology, vol. 6, no. 4,
pp. 606–609, 1999.
C. F. A. Brito, C. T. Fonseca, A. M. Goes, V. Azevedo,
A. J. G. Simpson, and S. C. Oliveira, “Human IgG1 and
IgG3 recognition of Schistosoma mansoni 14 kDa fatty acidbinding recombinant protein,” Parasite Immunology, vol. 22,
no. 1, pp. 41–48, 2000.
A. M.M. Mattos, C. S. de Almeida, K. L.M.C. Franken
et al., “Increased IgG1, IFN-γ, TNF-α and IL-6 responses
to Mycobacterium tuberculosis antigens in patients with
tuberculosis are lower after chemotherapy,” International
Immunology, vol. 22, no. 9, pp. 775–782, 2010.
B. L. Wang, Y. Xu, Z. M. Li, Y. M. Xu, X. H. Weng, and
H. H. Wang, “Antibody response to four secretory proteins
from Mycobacterium tuberculosis and their complex antigen
in TB patients,” International Journal of Tuberculosis and Lung
Disease, vol. 9, no. 12, pp. 1327–1334, 2005.
K. Lyashchenko, R. Colangeli, M. Houde, H. Al Jahdali,
D. Menzies, and M. L. Gennaro, “Heterogeneous antibody
responses in tuberculosis,” Infection and Immunity, vol. 66, no.
8, pp. 3936–3940, 1998.
M. S. Imaz, M. A. Comini, E. Zerbini et al., “Evaluation of the
diagnostic value of measuring IgG, IgM and IgA antibodies
to the recombinant 16-kilodalton antigen of Mycobacterium
tuberculosis in childhood tuberculosis,” International Journal
of Tuberculosis and Lung Disease, vol. 5, no. 11, pp. 1036–1043,
2001.
9
[23] M. S. Imaz and E. Zerbini, “Antibody response to culture
filtrate antigens of Mycobacterium tuberculosis during and
after treatment of tuberculosis patients,” International Journal
of Tuberculosis and Lung Disease, vol. 4, no. 6, pp. 562–569,
2000.
[24] S. Gupta, N. Shende, A. S. Bhatia, S. Kumar, and B. C.
Harinath, “IgG subclass antibody response to mycobacterial
serine protease at different stages of pulmonary tuberculosis,”
Medical Science Monitor, vol. 11, no. 12, pp. CR585–CR588,
2005.
[25] R. Hussain, G. Dawood, N. Abrar et al., “Selective increases in
antibody isotypes and immunoglobulin G subclass responses
to secreted antigens in tuberculosis patients and healthy
household contacts of the patients,” Clinical and Diagnostic
Laboratory Immunology, vol. 2, no. 6, pp. 726–732, 1995.
[26] S. D’Souza, V. Rosseels, M. Romano et al., “Mapping of
murine Th1 helper T-cell epitopes of mycolyl transferases
Ag85A, Ag85B, and Ag85C from Mycobacterium tuberculosis,” Infection and Immunity, vol. 71, no. 1, pp. 483–493, 2003.
[27] J. P. Van Vooren, A. Drowart, M. De Cock et al., “Humoral
immune response of tuberculous patients against the three
components of the Mycobacterium bovis BCG 85 complex
separated by isoelectric focusing,” Journal of Clinical Microbiology, vol. 29, no. 10, pp. 2348–2350, 1991.
[28] P. Andersen, T. M. Doherty, M. Pai, and K. Weldingh, “The
prognosis of latent tuberculosis: can disease be predicted?”
Trends in Molecular Medicine, vol. 13, no. 5, pp. 175–182, 2007.
[29] C. B. E. Chee, S. H. Gan, K. W. KhinMar et al., “Comparison
of sensitivities of two commercial gamma interferon release
assays for pulmonary tuberculosis,” Journal of Clinical Microbiology, vol. 46, no. 6, pp. 1935–1940, 2008.
[30] D. Menzies, M. Pai, and G. Comstock, “Meta-analysis: new
tests for the diagnosis of latent tuberculosis infection: areas
of uncertainty and recommendations for research,” Annals of
Internal Medicine, vol. 146, no. 5, pp. 340–354, 2007.
[31] P. R. Z. Antas, F. L. L. Cardoso, K. C. Pereira et al., “T
cell immune responses to mycobacterial antigens in Brazilian
tuberculosis patients and controls,” Transactions of the Royal
Society of Tropical Medicine and Hygiene, vol. 99, no. 9, pp.
699–707, 2005.
[32] J. Harris and J. Keane, “How tumour necrosis factor blockers
interfere with tuberculosis immunity,” Clinical and Experimental Immunology, vol. 161, no. 1, pp. 1–9, 2010.
[33] N. Caccamo, G. Guggino, S. A. Joosten et al., “Multifunctional
CD4+ T cells correlate with active Mycobacterium tuberculosis infection,” European Journal of Immunology, vol. 40, no. 8,
pp. 2211–2220, 2010.
[34] A. Marei, A. Ghaemmaghami, P. Renshaw et al., “Superior T
cell activation by ESAT-6 as compared with the ESAT-6-CFP10 complex,” International Immunology, vol. 17, no. 11, pp.
1439–1446, 2005.
[35] S. K. Sharma, D. K. Mitra, A. Balamurugan, R. M. Pandey, and
N. K. Mehra, “Cytokine polarization in miliary and pleural
tuberculosis,” Journal of Clinical Immunology, vol. 22, no. 6,
pp. 345–352, 2002.
[36] P. F. Barnes, S. Lu, J. S. Abrams, E. Wang, M. Yamamura, and
R. L. Modlin, “Cytokine production at the site of disease in
human tuberculosis,” Infection and Immunity, vol. 61, no. 8,
pp. 3482–3489, 1993.
[37] R. Hussain, H. Shiratsuchi, J. J. Ellner, and R. S. Wallis, “PPDspecific IgG1 antibody subclass upregulate tumour necrosis
factor expression in PPD-stimulated monocytes: possible
link with disease pathogenesis in tuberculosis,” Clinical and
Experimental Immunology, vol. 119, no. 3, pp. 449–455, 2000.
10
[38] R. Hussain, H. Shiratsuchi, M. Phillips, J. Ellner, and R.
S. Wallis, “Opsonizing antibodies (IgG1) up-regulate monocyte proinflammatory cytokines tumour necrosis factoralpha (TNF-α) and IL-6 but not anti-inflammatory cytokine
IL-10 in mycobacterial antigen-stimulated monocytes—
implications for pathogenesis,” Clinical and Experimental
Immunology, vol. 123, no. 2, pp. 210–218, 2001.
[39] F. D. Finkelman, I. M. Katona, T. R. Mosmann, and R. L.
Coffman, “IFN-γ regulates the isotypes of Ig secreted during
in vivo humoral immune responses,” Journal of Immunology,
vol. 140, no. 4, pp. 1022–1027, 1988.
[40] C. M. Snapper, T. M. McIntyre, R. Mandler et al., “Induction
of IgG3 secretion by interferon γ: a model for T cellindependent class switching in response to T cell-independent
type 2 antigens,” Journal of Experimental Medicine, vol. 175,
no. 5, pp. 1367–1371, 1992.
[41] V. H. S. Priya, G. Suman Latha, S. E. Hasnain, K. J. R.
Murthy, and V. L. Valluri, “Enhanced T cell responsiveness
to Mycobacterium bovis BCG r32-kDa Ag correlates with
successful anti-tuberculosis treatment in humans,” Cytokine,
vol. 52, no. 3, pp. 190–193, 2010.
[42] L. Meyaard, E. Hovenkamp, S. A. Otto, and F. Miedema, “IL12-induced IL-10 production by human T cells as a negative
feedback for IL-12-induced immune responses,” Journal of
Immunology, vol. 156, no. 8, pp. 2776–2782, 1996.
[43] V. H. S. Priya, B. Anuradha, S. L. Gaddam, S. E. Hasnain, K. J.
R. Murthy, and V. L. Valluri, “In vitro levels of interleukin 10
(IL-10) and IL-12 in response to a recombinant 32-kilodalton
antigen of Mycobacterium bovis BCG after treatment for
tuberculosis,” Clinical and Vaccine Immunology, vol. 16, no. 1,
pp. 111–115, 2009.
[44] J. L. Flynn, “Immunology of tuberculosis and implications in
vaccine development,” Tuberculosis, vol. 84, no. 1-2, pp. 93–
101, 2004.
[45] S. Arruda, M. Chalhoub, S. Cardoso, and M. BarralNetto, “Cell-mediated immune responses and cytotoxicity to
mycobacterial antigens in patients with tuberculous pleurisy
in Brazil,” Acta Tropica, vol. 71, no. 1, pp. 1–15, 1998.
[46] F. O. Sanchez, J. I. Rodriguez, G. Agudelo, and L. F. Garcia,
“Immune responsiveness and lymphokine production in
patients with tuberculosis and healthy controls,” Infection and
Immunity, vol. 62, no. 12, pp. 5673–5678, 1994.
[47] Y. Lin, M. Zhang, F. M. Hofman, J. Gong, and P. F. Barnes,
“Absence of a prominent Th2 cytokine response in human
tuberculosis,” Infection and Immunity, vol. 64, no. 4, pp. 1351–
1356, 1996.
[48] J. T. van Dissel, S. M. Arend, C. Prins et al., “Ag85B-ESAT6 adjuvanted with IC31 promotes strong and long-lived
Mycobacterium tuberculosis specific T cell responses in naı̈ve
human volunteers,” Vaccine, vol. 28, no. 20, pp. 3571–3581,
2010.
Clinical and Developmental Immunology