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Infection and Immunity, November 1999, p. 6187-6190, Vol. 67, No. 11
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
Jae-Hyun
Lim,
Jeong-Kyu
Park,
Eun-Kyeong
Jo,
Chang-Hwa
Song,
Dullei
Min,
Young-Ja
Song, and
Hwa-Jung
Kim*
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
 |
ABSTRACT |
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.
| |
TEXT |
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-
) 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.
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.
The 55% ammonium sulfate fraction of the CF was applied to a column of
hydroxylapatite (Bio-Rad) equilibrated with 1 mM PB (pH 6.8) and eluted
with 1 mM PB because the Ag85 complex was not retained on the column
(11, 26). Initially, to separate the 30-kDa (85B) and 32-kDa
(85A and 85C) proteins, the fractions excluded from the hydroxylapatite
column were applied to a column of DEAE-Sepharose CL-6B
(Sigma) equilibrated with 1 mM PB (pH 7.2). The 32-kDa (85A) and
32.5-kDa (85C) proteins were coeluted with 5 mM PB (Fig.
1A, lane 5), and the 30-kDa protein
(85B) was eluted with 10 mM PB (Fig. 1A, lane 4). The 85A and 85C
proteins were further separated by DEAE-Sephacel (Pharmacia). The 85A
protein was eluted first from DEAE-Sephacel with 20 mM Tris-HCl,
followed by the 85C protein. On the other hand, the fractions from the DEAE-Sepharose column enriched for the 85B protein were dialyzed against 5 mM PB (pH 6.8) and then also applied to a DEAE-Sephacel column to remove contaminated 32-kDa protein and other proteins. The
85B protein was eluted with 10 mM PB from DEAE-Sephacel. The analysis
of eluted fractions was performed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and natural PAGE.
SDS-PAGE was performed in a discontinuous buffer system by the method
of Laemmli (12). For natural PAGE, the same gel system was
used, except that SDS and 2-mercaptoethanol were omitted from all
buffers. Each fraction from the DEAE-Sephacel column enriched for the
85B, 85A, and 85C proteins was pooled and concentrated, separately.
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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.
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|
Finally, the three proteins were purified by gel filtration on Superdex
75 (Pharmacia). Each protein yielded a single band
on SDS-PAGE and
natural PAGE gels stained with Coomassie blue
(Fig.
1A and C, lanes 6, 7, and 8). The monoclonal antibody HYT27
(provided by the United
Nations Development Program/World Bank/World
Health Organization
Special Program for Research and Training
in Tropical Disease), which
is specific to the Ag85 complex, reacted
strongly with the three
proteins (Fig.
1B). Immunoblotting analysis
was performed as described
previously (
10). In one experiment
we purified 1.52, 1.89, and 0.55 mg of 85B, 85A, and 85C, respectively,
from 484 mg of the 55%
ammonium sulfate fraction of the
CF.
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).
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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FIG. 2.
Lymphoproliferation (A) and IFN- production (B) in
response to the 85B, 85A, and 85C proteins. (A) PBMC from five healthy
purified protein derivative (PPD)-positive [PPD(+)] subjects
(designated by the letters A through E) and five PPD-negative
[PPD( )] subjects (designated by the letters F through J) were
stimulated with each of the three antigens (1.0 µg/ml) for 5 days,
and incorporation of [3H]thymidine was measured. The
results were expressed as a stimulation index (defined as counts per
minute in antigen-stimulated cultures/counts per minute in unstimulated
cultures). Error bars represent standard deviations from the means
(open symbols [as for closed symbols, respective shapes show results
for individual antigens]). (B) Supernatants from antigen-stimulated
PBMC were collected after 96 h and assayed for IFN- production
by ELISA.
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|
The Ag85 complex proteins, 85A, 85B, and 85C, share high sequence
homology at the nucleotide and protein levels both with
each other and
with Ag85 from other mycobacterial species (
2,
5). The 85A
and 85B proteins have been purified independently
by different workers
(
3,
7,
17,
26). Because the 85A
and 85C proteins had
slightly different molecular masses, they
could not be isolated in
large amounts by gel filtration and were
clearly resolved by natural
PAGE. DEAE-Sephacel was therefore
included in the purification
procedure to ensure a good separation
of the two antigens from each
other. Interestingly, 85A and 85C
were eluted in that order from
DEAE-Sephacel with 20 mM Tris-HCl
buffer (pH 7.4) containing 10 mM
NaCl.
The relationship between the individual components of the Ag85 complex
is of the greatest interest. In fact, comparison of
the immune
responses to the 85A and 85B proteins in human and
animal models have
been analyzed by different workers (
17,
18,
23). However,
detailed immunoreactivity studies of all three
native proteins have not
been carried out. In this work, sensitivities
of 76, 50, and 12% for
85B, 85A, and 85C, respectively, were shown,
which is surprising,
considering the high level of homology between
these proteins. The 85B
protein was more valuable in the diagnosis
of TB than 85A because
antibody reactivities to 85A were much
higher than those to 85B in
healthy controls as well as TB patients.
These results are in agreement
with the findings of previous studies
using 85A and 85B (
18,
22-24). Interestingly, significantly lower
antibody levels
against the 85C protein were observed in our patients.
The differences
in antibody reactivity among the three antigens
may be due to
differences in their immunogenicity, in the composition
of the Ag85
complex (
5) and in antigen availability in vivo
or in their
capacities to form complexes with IgG and fibronectin
in circulation
(
1).
In contrast to antibody reactivity, all three components of the Ag85
complex induced significant lymphoproliferation and IFN-
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.
| |
ACKNOWLEDGMENTS |
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).
| |
FOOTNOTES |
*
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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Infection and Immunity, November 1999, p. 6187-6190, Vol. 67, No. 11
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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