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Infection and Immunity, November 1999, p. 6187-6190, Vol. 67, No. 11
Department of Microbiology, College of
Medicine, Chungnam National University, Taejeon 301-131, Korea
Received 17 May 1999/Returned for modification 1 July 1999/Accepted 24 August 1999
The three proteins of the antigen 85 complex (85A, 85B, and 85C),
which are major secretory products of Mycobacterium
tuberculosis, were purified to homogeneity in large amounts by a
combination of chromatography on hydroxylapatite, DEAE-Sepharose, and
DEAE-Sephacel and gel filtration from M. tuberculosis
culture filtrate. Then we examined the immunological reactivity of the
three proteins in tuberculosis patients and healthy controls. Antibody
responses to the 85B and 85A proteins in patients were significantly
greater than responses to the 85C protein. In contrast, all three
antigens induced significant lymphoproliferation and gamma interferon
production in peripheral blood mononuclear cells from healthy
tuberculin reactors.
The 30/32-kDa antigen 85 (Ag85)
complex has been the focus of intensive research over the past several
years and comprises three closely related proteins, 85A (32 kDa), 85B
(30 kDa), and 85C (32.5 kDa) (17, 25-27). The Ag85 complex
induces protective immunity against tuberculosis (TB) in
guinea pigs (7), and strong T-cell proliferation and
gamma interferon (IFN- Sera.
Sera were collected from two groups. One test group
consisted of 42 patients with pulmonary TB who had been admitted at the National Masan Tuberculosis Hospital, Masan, Korea, and had been receiving therapy for over 2 months. A diagnosis of TB was based upon a
clinical evaluation, a sputum smear and culture, and/or a chest X-ray.
The other group consisted of 20 patients with pulmonary TB who were
outpatients at the Taejeon Sungmo Hospital, Taejeon, Korea. All of
these 20 outpatients received standard chemotherapy for 6 months. Sera
were taken serially from these patients before treatment began and at
about 2 and 6 months after the initiation of chemotherapy. The healthy
control sera were obtained from 104 students of the Chungnam National
University, Taejeon, Korea.
Purification of the 85A, 85B, and 85C proteins.
M.
tuberculosis H37Rv (ATCC 27294) was grown for 6 weeks at 37°C as
a surface pellicle on Sauton medium. The CF was sterilely filtered and
precipitated with ammonium sulfate (55% saturation), and the resulting
precipitate was dissolved and dialyzed against 1 mM sodium phosphate
buffer (PB) (pH 6.8). Protein concentrations were determined by a
protein assay kit (Pierce) with bovine serum albumin (BSA) as the standard.
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Purification and Immunoreactivity of Three
Components from the 30/32-Kilodalton Antigen 85 Complex in
Mycobacterium tuberculosis
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) production in peripheral blood mononuclear
cells (PBMC) from healthy tuberculin reactors (6, 8, 21). In
this regard, this complex is a candidate for a novel TB vaccine. There
may be differences in the T-cell-stimulating capacities and antibody
reactivity of the individual components of the Ag85 complex (14,
15, 18, 23-25). However, because these antigens are difficult to
purify in large amounts by biochemical techniques, very limited
information on differences in cellular and humoral immune responses to
each of the three components of the native Ag85 complex is available. In particular, the 85C protein is quantitatively minor and has not been
well characterized by other investigators. Therefore, we purified the
three components of the Ag85 complex from Mycobacterium tuberculosis culture filtrates (CF) by biochemical methods. Then, immunological reactivity against these purified antigens in TB patients
and healthy volunteers was evaluated by measuring specific serum
immunoglobulin G (IgG) antibody levels and lymphoproliferation and
IFN-
production of PBMC stimulated with the antigens.

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FIG. 1.
SDS-PAGE (A), immunoblotting (B), and natural PAGE (C)
analyses of the purified 85A, 85B, and 85C proteins. Lane 1, low
molecular weight marker. Lanes 2 through 5, products from different
stages of purification, as follows. The 55% ammonium sulfate fraction
(lane 2) of CF was applied to a hydroxylapatite column, and then the
column was washed with 1 mM PB (pH 6.8). All pass-through fractions
were pooled (lane 3) and then applied to a DEAE-Sepharose column. The
eluate fractions from DEAE-Sepharose with 5 mM PB (lane 5) and 10 mM PB
(lane 4) were dialyzed against 10 mM NaCl-20 mM Tris-HCl buffer (pH
7.4) and 5 mM PB (pH 6.8), respectively, and applied to a DEAE-Sephacel
column. Final purification was performed by gel filtration on Superdex
75. The antigens were separated by SDS-PAGE and natural PAGE and then
analyzed by Coomassie blue staining and immunoblotting with monoclonal
antibody HYT27. Lanes 6 to 8, purified 85B, 85A, and 85C proteins,
respectively.
Antibody responses to the three proteins. The enzyme-linked immunosorbent assay (ELISA) for antibody determination was performed as described previously (11). Polystyrene 96-well microplates were coated overnight with each protein at 1.0 µg/ml (0.1 ml/well) in 0.05 M carbonate buffer (pH 9.6). Each well was blocked with 1% BSA in phosphate-buffered saline (PBS), washed with PBS containing 0.05% Tween 20 (PBS-Tween 20), and incubated with 0.1 ml of serum diluted 1:200 in 1% BSA-PBS-Tween 20 for 1 h at 37°C. After washing, the plates were incubated with 0.1 ml of peroxidase-conjugated goat anti-human IgG (Sigma) diluted 1:3,000 in 0.1% BSA-PBS-Tween 20 for 1 h at 37°C. Color development was performed by adding O-phenylenediamine at 0.4 mg/ml in 0.05 M citrate-phosphate buffer (pH 5.0) containing 0.03% H2O2. After 30 min, the reactions were stopped with 8 N H2SO4 and the A492 was measured in an ELISA reader (Spectra Max Plus; Molecular Devices).
The levels of IgG antibodies to the three proteins were measured in sera from TB patients treated for at least 2 months and from healthy subjects (Table 1). There was a wide variation in antibody content in sera from individual patients. Antibody responses to the 85A and 85B proteins in patients were significantly greater than those to the 85C protein (P < 0.05), but no significant difference between the mean levels of IgG antibody in response to the 85A and 85B proteins was observed. Twenty patients were also tested before treatment began and at 2 and 6 months after initiation of treatment (Table 2). These patients had been treated successfully by standard chemotherapy for 6 months. The mean IgG antibody levels against all antigens increased significantly at about 2 months after the start of chemotherapy (P < 0.05), and then antibody levels declined by about 30% at 6 months after the start of chemotherapy. However, some patients showed a steady increase or plateau in antibody levels until 6 months. These results are in good agreement with those previously obtained with the P32 antigen (22) and may be due to the release of increasing amounts of antigen as mycobacteria are destroyed by antituberculous drugs (9). One investigation has indicated that exposure to isoniazid induces the expression of several secreted proteins, including the Ag85 complex, in M. tuberculosis (4).
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Proliferative responses and IFN-
production due to the three
proteins.
To evaluate the immunoreactivity of 85A, 85B, and 85C,
we measured the proliferative responses and IFN-
production levels of PBMC from five tuberculin-positive and five tuberculin-negative healthy subjects. Lymphocyte proliferation assays were performed as
described previously (10). IFN-
levels from culture
supernatants after 96 h were determined by ELISA with a commercial
kit for human cytokines (PharMingen). As shown in Fig.
2, proliferative responses and IFN-
production levels in response to all three antigens were significantly
greater in healthy tuberculin reactors than in negative subjects
(P < 0.05), although individual reactive patterns
showed considerable variation. These results are consistent with the
human studies of others (8, 20, 21). Previous studies also
indicate that immunological reactivities to mycobacterial antigens in
human subjects are highly heterogenous (6, 8, 13, 16, 19,
20).
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production
in PBMC from healthy tuberculin reactors. These results suggest that
the cross-reactive epitopes recognized by T cells are present in these
proteins. In mapping of the T-cell epitopes analyzed by different
workers (13, 20), there was only partial correlation
between the epitopes of 85A and 85B in healthy tuberculin reactors.
Others found that a few peptides of the two antigens were identical
(14). However, because highly diverse epitopes of this
complex may be processed and presented in vivo, it is possible that
there are no significant differences among the three proteins. In
addition, mean proliferative responses to 85B and 85A were relatively
higher than that to 85C in PBMC from healthy tuberculin reactors. One
recent report (14) indicated that the level of lymphocyte
proliferation in response to the 30-kDa protein allows more
discrimination between immunized and control animals than that in
response to the 32-kDa protein. Our findings and the above-mentioned
reports show that highly immunogenic or component-specific epitopes are
present in the Ag85 complex.
Recently, DNA vaccination with the genes encoding the 85A and 85B (but
not 85C) components of the Ag85 complex resulted in a strong
stimulation of a specific Th1-like response and of cytotoxic T-lymphocyte activity in animal models (15). Therefore,
further detailed comparison of cellular immune responses to 85B, 85A, and 85C will provide fundamental knowledge of the immunological parameters which correlate with protective immunity against TB in humans.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the Ministry of Health and Welfare of Korea (HMP-98-B-1-003) and in part by the Korea Research Foundation (1998-003-F00067).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, College of Medicine, Chungnam National University, 6 Munwha-Dong, Chung-Ku, Taejeon 301-131, Republic of Korea. Phone: 82-42-580-8242. Fax: 82-42-585-3686. E-mail: hjukim{at}hanbat.chungnam.ac.kr.
Editor: S. H. E. Kaufmann
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