Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 101(8): 857-861, December 2006
857
Cell proliferation and interferon-γγ response to recombinant
MBP-3, NarL, MT-10.3, and 16kDa Mycobacterium tuberculosis
antigens in Brazilian tuberculosis patients
Ricardo Candido Oliveira Tavares, Jorge Salgado, Valéria Barbosa Moreira*,
Mônica Antonia S Ferreira, Fernanda Carvalho Queoz Mello**, Janaína AW Leung**,
Mahavir \Singh***, Leila de Souza Fonseca**, Maria Helena Feres Saad/+
Departamento de Micobacterioses, Instituto Oswaldo Cruz- Fiocruz, Av. Brasil 4365, 21045-360 Rio de Janeiro, RJ, Brasil *Hospital
Municipal Souza Aguiar, Rio de Janeiro, RJ, Brasil **Hospital Universitario, Instituto Microbiologia, UFRJ, Rio de Janeiro, RJ, Brasil
***Lionex Immuno Diagnostic and Therapeutic, Braunchweig, Germany
Human pulmonary tuberculosis (TB) is a worldwide public health problem. In resistant individuals, control
of the infection mainly requires development of a Th1 cell immune response with production of cytokines, of
which interferon-γ (IFN-γ)plays an important role. Several antigens from Mycobacterium tuberculosis complex
has been described for use in vaccine development or for diagnostic purposes, however little evaluation has
been done in endemic area for TB. The proliferative and IFN- γ human T cell immune responses, to four
recombinant proteins (MBP-3, NarL, MT-10.3, 16 kDa) and PPD, of 38 Brazilian TB patients (6 untreated and
32 treated) and 67 controls (38 positive and 29 negative tuberculin skin test - TST) were compared. The highest
reactivity mean rate was obtained with PPD followed by 16 kDa in TB patients. While most of the patients (87%)
and controls (> 64%) respond to the PPD, 16kDa was more specifically recognized (> 21%) although less
sensitive (54%). When TB patients were divided according to treatment status, opposite to PPD, higher average
level of IFN-γ was induced by 16kDa in untreated (505 pg/ml) compared to treated TB patients and TST+ (269.8
pg/ml x 221.6pg/ml, respectively), although the difference was not significant. These data show that in contrast
with the other recombinant proteins, the stimulatory potency of 16kDa to induce proliferative and INF-γ response
was more effective and is more recognized by active TB untreated patients, eliciting in control individuals a
more selective immune response than PPD.
Key words: tuberculosis - 16 kDa - MT10.3 - interferon-γ - immune response
Mycobacterium tuberculosis is an extremely successful pathogen infecting a third of the world’s
population, the cases being concentrated in developing
countries (WHO 2002, Guzmán et al. 2003). Koch
bacillus, which is transmitted by respiratory route from
person to person, invades macrophage, multiply and can
remain latent within the lung granuloma for years.
Disease control is based on early identification of
infected individuals followed by appropriate treatment.
Disease progression prevention through inducement of
protective immunity may be achieved through
vaccination; however the current available Bacillus
Calmette-Guérin (BCG) efficacy is controversial
(Colditz et al. 1994, Fine 1995).
Several studies have identified secreted proteins,
which are considered important targets immune response
Financial support: Brazil × Germany Science and Technology
Ministry Program grant number 52.0009/02-09, Projeto Milênio
(CNPq), Faperj grant E-26/171.546/2002, John Hopkins University grant number 1U19AI45432-01 from the National Institutes
of Health
+Corresponding author: saad@ioc.fiocruz.br
Received 19 May 2006
Accepted 19 October 2006
recognition. However, little is known about the reactivity
to these antigens in patients from Rio de Janeiro where
are described the highest rates of tuberculosis (TB)
incidence in Brazil (Hijjar 2005). The 16 kDa molecule
is a polypeptide belonging to the α-cristallin family of
low molecular weight, heat shock proteins, of which the
coding gene (Rv2031c) has been found exclusively in
the M. tuberculosis complex (Yuan et al. 1998). It has
been reported as dominant protein produced in the static
growth phase or under oxygen deprivation and required
for bacterial replication inside macrophages (Yuan et al.
1998, Agrewala & Wilkinson 1998). MBP-3 is a 23 kDa
maltose binding protein found as residue in the purification process of the MPT-64 antigen. The MT-10.3
or TB10.3 (Rv3019c) antigen, together with TB12.9 and
TB10.4, comprise one subfamily within the esat-6 gene
family and seem to be expressed during the TB infection
(Skjot et al. 2002, Demissie et al. 2006). Two regulatory
system components are ubiquitously distributed among
bacteria and plants and are involved in the organisms’
virulence (Urao et al. 2000). Nar L is the product of the
response regulator narL gene of M. tuberculosis
regulatory system (Parish et al. 2003).
In the present study, we evaluated the production of
interferon-γ (IFN-γ) and the proliferative response of
peripheral blood mononuclear cells (PBMC), from TB
patients from Rio de Janeiro, stimulated with the
858
Immune response to mycobacterial antigens • Ricardo Candido Oliveira Tavares et al.
following M. tuberculosis recombinant proteins: 16kDa,
MBP-3, MT-10.3, and NarL.
density gradient centrifugation (Pharmacia, Uppsala,
Sweden). The cells were ressuspended in RPMI-1640
supplemented with 0.04 mM glutamine, 20% autologous
heat-inactivated serum, 100 U/ml penicilin and
streptomycin (Gibco, Paisley, UK). For cell culture, a
total volume of 200 µl/well of PBMC (1 × 106 cells/ml)
was plated in triplicate in 96-well-round bottom
microtitre plate (Costar Corp., Cambridge, MA) in the
presence or absence of each Ag, at 37°C in a humidified
5% CO2 chamber for five days. Supernatants (100 µl/
well for each triplicate) were pooled and stocked at
–20oC for further IFN-γ quantification. T-cells were
added with [3H] thymidine (1 µCi/well), incubated an
additional 18 h, and the cells harvested using FilterMAT
11731 (SKATRON Instruments, UK) and cell harvester
PHD (Cambridge Technology, UK). Incorporated
radioactivity was determined as counts per minute by βscintillation counting (Packard-1900 CA, US).
Lymphocyte proliferation was expressed as stimulation
index (SI) and the cut off was fixed at SI > 3.
MATERIALS AND METHODS
Study subject - TB diagnosis was performed at
district Hospital Souza Aguiar (HMSA), School-Center
Germano Sinval de Farias of National Public Health
School/Fundação Oswaldo Cruz, and University Hospital
Clementino Fraga Filho (HU-UFRJ), Rio de Janeiro city,
in the Southern region of Brazil. The criteria for TB
diagnosis and standard treatment were in accordance with
those of the Brazilian Ministry of Health (MS 2002).
Patients and control groups are described in Table I.
Pulmonary TB diagnosis was defined by at least one
sputum-positive smear by Ziehl-Neelsen staining, a
positive M. tuberculosis sputum culture or a compatible
clinical evolution. The control groups were composed
of biology students with positive tuberculin skin tests
(TST, ≥ 5 mm) or negative reaction (< 5 mm) and no TB
family history. None of the TB patients had clinical
evidence of anti-TB chemotherapy failure or miliary TB.
Patients under 18 years old and pregnant or breastfeeding
women were not included. All recruited TB patients were
negative by enzyme-linked immunosorbent assay
(ELISA) for human immunodeficiency virus (HIV)
(Organon Teknica, Boxtei, The Netherlands). Blood
samples were collected after written informed consent
was obtained. The study protocol was approved by the
National Research Ethical Committee.
IFN-γ detection - IFN-γ was measured with a standard
ELISA, using the Duo-Set-IFN-γ kit (R&D, US),
according to manufacturer’s recommendations. The
concentrations of IFN-γ in the samples were calculated
with the standard curve generated from recombinant IFNγ, and results were expressed in picograms. The cut of
was fixed at 100 pg/ml.
Statistical analysis - T-cell responses were evaluated
non-parametrically using Kruskal Wallis and MannWhitney tests for comparison of study groups. P ≤ 0.05
was considered statistically significant. Data were
reported as mean ± standard error (SE)
Mycobacterial antigens - M. tuberculosis recombinant in Escherichia coli 16kDa, MBP-3, MT10,3 and
NarL were kindly provided by Lionex GmbH
(Braunchweig, Germany). Purified protein derivative
(PPD RT23) was purchased from the Staten Serum
Institute (Copenhagen, Denmark). The recombinant Ags
and phytohemaglutinin (PHA) mitogen were utilized in
the in vitro assays in a final concentration of 5 µg/ml,
and PPD was employed in a final concentration of 2.5
µg/ml (optimal concentrations were established in
previous pilot experiments, data not shown).
RESULTS
The immunological reactivity to PPD and recombinant proteins 16kDa, MT10.3, MBP-3, and NarL was
evaluated by stimulating PBMC and measuring IFN-γ
production from TB patients and controls (Table II). As
expected, PPD induced a higher positive response and
IFN-γ release in the majority of the TB patients (87%)
and in TST positive individuals (92%) as well as in TST
negative group (64%), without significant difference in
Cellular proliferation assay - PBMC were isolated
from heparinized venous blood by Ficoll-Hypaque
TABLE I
Characteristics of Brazilian tuberculous patients and control groups
Characteristics
Total
Male
Female
TST +
TST –
BCG (%)
Age (x ± SE)
Patients
38
28
10
-
-
80
39 ± 3
Clinical form:
Pulmonary
Extra-pulmonary a
32
6
26
3
6
3
-
-
80
83
41 ± 3
35 ± 7
Treatment
Untreated
Treated (1 to 6 months)
Healthy
6
32
67
5
23
24
1
9
43
38
29
83
83
80
32 ± 6
40 ± 8
30 ± 2
BCG: Bacillus Calmette Guèrin vaccine scar; TST±: tuberculin skin test positive ( ≥ 5 mm) and negative (< 5 mm); SE:: standard error;
x: mean; a: ganglionary TB (3), aortic TB (1), skin TB (1), pleural TB (1).
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 101, 2006
the mean level of proliferative response and IFN-γ
production. Subsequent to PPD, a better response was
elicited by 16 kDa, which was recognized by 56% of TB
patients and TST positive individuals. This protein was
more specific in TST negative control group, been
recognized by only 21% of the individuals (p < 0.03).
Although the mean IFN-γ response to 16 kDa in TB
patients and control was significantly lower then PPD
(p < 0.05), there was a significant difference in the
average level of IFN-γ among the groups tested, which
was obtained exclusively for 16kDa (TB/TST positive ×
TST negative, p = 0.015). The MT-10.3 stimulated PBMC
from 41 % of TB patient; however the IFN-γ production
was detected in only 24%, with a mean response close
to the cut off. The lowest reactivity in all group tested
was obtained with NarL and MBP-3
The effect of chemotherapy on IFN-γ and proliferative
response was also evaluated in TB patients (Table III).
Chemotherapy increased mean responses to PPD,
however the frequency of responders was high for both
group of patients (> 83%). 16kDa was associated with
an increase mean reactivity in PBMC responses and
frequency of responders in untreated patients, followed
by MT-10-.3 that gave the same pattern but with lower
responses. None of the responses was significantly
different between groups. Sensitivity and specificity
values for all antigens were calculated, and although the
PPD had 87% sensitivity the specificity was low (36%).
On the other hand, only 16kDa afforded identification
of more than 50% of the patients with specificity of
80%. However if the calculation included the healthy
TST positive donors, the specificity decreased to 60%,
that is still higher than PPD.
DISCUSSION
TB control depends on early identification and
treatment completion. Several secreted antigens have
859
been characterized, and their utility for diagnostic testing
as well as their attributes as vaccine components have
been exploited (Bothamley 1995, Lodha & Kabra 2004,
Orme 2006). A very well established knowledge in
mycobacterial disease is that a strong T cell response is
associated with localization of and protection against the
pathogen. Herein, we investigated the human T cell
response in vitro against M. tuberculosis recombinant
proteins MPB-3, Narl, MT10.3, 16 kDa, and PPD in a
TB high endemic area. Our results demonstrated that
PBMC from patients and healthy controls did not elicit
strong levels of cell proliferation and INF-γ release, as
did PPD. However, we did not encounter significant
difference in PPD induced IFN-γ production in patients
compared to controls, confirming previous findings
(Surcel et al. 1994). As proliferation, in general, correlates with INF-γ production, we will solely discuss the
data concerning INF-γ. T cell response from few patients
and controls was obtained under MBP-3 and NarL
stimulation, reveling that these antigens are not well
recognized by host cell immunity. However, the T cells
recognized MT-10.3 more specifically since most of the
healthy TST negative individuals failed to respond.
However a small number of healthy TST positive
individuals (17%) and tuberculous patients (24%)
yielded an in vitro response. This may suggest that MT10.3 is expressed and recognized in some individuals
during active infection, which is supported by the fact
that 50% of the untreated patients produced INF-γ against
only 19% among those treated (Table III). However,
further prospective study must be undertaken in order to
confirm this data. The other possibility is that the result
merely reflects a cross reaction, as MT-10.3 has high
homology with M. bovis and M. avium proteins (Skjot
et al. 2002). This may be a misconception because more
than 80% of our sampling (including TST negative
controls, which did not recognize this Ag) was vaccinated
TABLE II
Proliferative response and interferon-γ (INF-γ) production by peripheral blood mononuclear cells from tuberculosis (TB) patients and
controls groups stimulated with recombinant proteins and PPD
Proliferation (x ± SE)
Number of positive/Total (%)
Proteins
IFN-γ (x ± SE)
Number of positive/Total (%)
TB
TST+
TST–
TB
TST+
TST–
47.98 (14.88)
33/38(87)
35.90 (7.24)
34/37(92)
15.57 (4,83)
18/28(64)
1.038,13 (184.8)
33/38 (87)
1.222.77 (147.12)
34/37 (92)
696.37 (138.5)
16/28 (64)
16kDa
7.3 (1.86) a
13/24(54)
4.29 (0.9)
15/27(56)
2.72 (0,7)
4/20(20)
321 (83.07) b
13/24 (54)
217.4 (40.0) b
15/27 (56)
71 (21.4)
4/19 (21)
MT-10.3
4.20 (1.21)
15/37(41)
4.37 (1.2)
6/35(17)
2.74 (1,01)
2/26(8)
103,78 (46.99) b
9/38 (24)
75.94 (29.34)
6/35 (17)
70.83 (50.59)
2/26 (8)
NarL
1.58 (0.35)
1/16(6)
1.18 (0,2)
3/23(13)
1.58 (0.25)
2/17(12)
105.4 (102.0)
1/16 (6)
40.3 (19.86)
3/23 (13)
53.56 (39.17)
2/15 (13)
MBP-3
1.41 (0.2)
0/17(0)
1.87 (0,51)
1/25(4)
1.52 (0.41)
3/17(18)
2.80 (2.75)
0/17 (0)
8.4 3 (4.46)
1/25 (4)
69 (34.85)
3/17 (18)
PPD
TST±: healthy control tuberculin skin test ≥ 5 mm and < 5mm, respectively; a: one-way analysis of variance (Kruskal Wallis Multiple
Comparison Test): p < 0.05, TB x TST–; b: TST+ x TST–; x: mean; SE: standard error. The cut-of f for proliferetive response was
3 IS and 100 pg/ml for IFN-γ production.
860
Immune response to mycobacterial antigens • Ricardo Candido Oliveira Tavares et al.
TABLE III
Proliferative response and interferon-γ (INF-γ) production by peripheral blood mononuclear cells from the tuberculosis patients
untreated and treated stimulated with recombinant proteins and PPD
TB patients
Proteins
PPD
16kDa
MT-10.3
NarL
MBP-3
Untreated
Mean
No. +/Total (%)
Proliferation
36.71
6/6 (100)
14.00
3/6 (50)
8.12
3/6 (50)
2.08
0/6 (0)
1.25
0/6 (0)
Treated
Mean
No. +/Total (%)
IFN-γ
514.04
5/6 (83)
505.00
4/5 (80)
59.76
13/6 (50)
8.04
0/6 (0)
0.65
0/6 (0)
Proliferation
50.16
30/31(97)
5.41
9/22 (41)
3.44
12/31 (39)
1.50
2/13 (15)
1.44
1/19 (5)
IFN-γ
1.136,40
28/32 (88)
269.80
9/19 (47)
92.95
6/32 (19)
121.62
1/14 (7)
2.97
0/15 (0)
The cut-off for proliferative response was 3 IS and 100 pg/ml for IFN-γ production.
with BCG. T cell response to MT-10.3 may merely be
more specific, although not sensitive, and the responders
among TST positive and negative donors may be related
to true latent infection. In this investigation MT-10.3
did not prove to be strongly recognized T cell antigen,
corroborating previous studies reporting that the
synthetic peptides of this Ag are less reactive than those
from its TB10.4 highly homologous protein (Alderson
et al. 2000, Skjot et al. 2002).
Antigens such as 16 kDa (former 14 kDa) have been
extensively studied and reported to have pronounced
immunogenicity by the presence of specific immunoglobulin in smear positive and negative patients with
pulmonary TB. However, the specificity is compromised
by the presence of immune reactivity in sera from healthy
hospital workers and household contacts of TB patients
(Jackett et al. 1988, Bothamley et al. 1992). Improved
sensitivity and specificity has been described in extra
pulmonary and pediatric TB for 16 kDa, suggesting bacilli
Ag production in early stage infection (Umadevi et al.
2002, 2003). In this study the sensitivity and specificity
of T cell response for 16 kDa were 54% and 60 %,
respectively. Similar results were obtained with the most
reactive synthetic peptide sequence (91-110) of 16 kDa,
50% and 66.7% (Friscia et al. 1995).
INF-γ has been well established as a protective
cytokine in animal models of TB. In this study, the in
vitro production of INF-γ clearly decreased in response
to PPD in human TB untreated patients, but was
successfully restored after specific therapy. The over
time change in T cell immune response during TB
infection has already been described (Hernandez-Pando
& Rook 1994, Hernandez-Pando et al. 1996, Wilkinson
et al. 1998, Ulrichs et al. 2000). Our results for 16 kDa
and MT-10.3 responses revealed a tendency to increased
T cell response in untreated patients. Previous work, using
the same Ags to investigate the human T and B cell
immune response disclosed a higher rate of responder
in healthy TST+ donors than in patients treated < 1 month
(Ulrichs et al. 2000), but in the present study, the
frequency of responder in the untreated TB patient group
was higher (80%) than in the TST+ group (56%).
However, it has been divulged that prior to therapy,
patients phenotypic of CD4 cell clones stimulated with
16 kDa were both Th 1 (produced IFN-γ) and
predominately Th0 (produced both IFN-γ and IL-4)
(Caccamo et al. 2005). Thus may support our findings
concerning the average level of IFN-γ in untreated
patients since 16kDa may quite possibly stimulate both
T cell clones to produce IFN-γ, besides this Ag could be
preferentially produced in the early stages of infection.
Recently it has been suggested that 16 kDa can be a
good marker for latent infected individuals because T
cells from individuals living in endemic area, previously
exposed to M. tuberculosis but without any symptoms
or known recent exposure to TB, preferentially recognized this protein (Demissie et al. 2006). In our study,
opposite to PPD, to which most of TST positive and
negative healthy individuals responded, 16 kDa was
recognized by only 56 and 21% of, respectively, TST
healthy individuals. Considering that they did not show
symptoms or recent contact with TB infected person,
this may suggest a pattern of latent infection in these
patients.
Our data contribute to the better understanding of the
specificity of T cell response in an endemic area and
show that among recombinant proteins tested, 16kDa is
more recognized by active TB untreated patients and in
healthy individuals eliciting a more selective immune
response than PPD. Gene encoding 16 kDa is over
expressed during the stationary growth phase of the TB
bacilli (Yuan et al. 1996), so a complementary
prospective study in TB patients before and during the
treatment and in recent healthy household contacts and
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 101, 2006
healthy individuals with previous exposure should be
addressed to clarify the diagnostic potential use of this
protein.
ACKNOWLEDGMENTS
To Drs Francisco Eduardo da Rocha Caldeira and Antonio
Orlando Resteita and other members of Clinical Analyses Laboratory of Hospital Municipal Souza Aguiar, Rio de Janeiro, Brazil, that provided further laboratory information. To the time from
Centro de Saúde, Escola Germano Silval Farias, ENSP-Fiocruz,
Rio de Janeiro that supplied patient’s medical records information. To Mitchell Raymond Lishon, Native of Chicago, IL, US,
for revising this manuscript.
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