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Journal of Clinical Microbiology, September 2000, p. 3285-3290, Vol. 38, No. 9
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Molecular and Immunological Characterization of
Mycobacterium tuberculosis CFP-10, an Immunodiagnostic
Antigen Missing in Mycobacterium bovis BCG
Davin C.
Dillon,1,*
Mark R.
Alderson,1
Craig H.
Day,1
Teresa
Bement,1
Antonio
Campos-Neto,2
Yasir A. W.
Skeiky,1
Thomas
Vedvick,1
Roberto
Badaro,3
Steven G.
Reed,1,2 and
Raymond
Houghton1
Corixa Corporation1
and Infectious Disease Research
Institute,2 Seattle, Washington 98104, and
Federal University of Bahia, Salvador, Brazil3
Received 10 April 2000/Returned for modification 29 May
2000/Accepted 12 July 2000
 |
ABSTRACT |
In order to identify antigens that may be used in the serodiagnosis
of active tuberculosis (TB), we screened a Mycobacterium tuberculosis genomic expression library with a pool of sera from patients diagnosed with active pulmonary TB. The sera used lacked reactivity with a recombinant form of the M. tuberculosis
38-kDa antigen (r38kDa), and the goal was to identify antigens that
might complement r38kDa in a serodiagnostic assay. Utilizing this
strategy, we identified a gene, previously designated lhp,
which encodes a 100-amino-acid protein referred to as culture filtrate
protein 10 (CFP-10). The lhp gene is located directly
upstream of esat-6, within a region missing in M. bovis BCG. Immunoblot analysis demonstrated that CFP-10 is
present in M. tuberculosis CFP, indicating that it is
likely a secreted or shed antigen. Purified recombinant CFP-10
(rCFP-10) was shown to be capable of detecting specific antibody in a
percentage of TB patients that lack reactivity with r38kDa, most
notably in smear-negative cases, where sensitivity was increased from
21% for r38kDa alone to 40% with the inclusion of rCFP-10. In
smear-positive patient sera, sensitivity was increased from 49% for
r38kDa alone to 58% with the inclusion of rCFP-10. In addition,
rCFP-10 was shown to be a potent T-cell antigen, eliciting
proliferative responses and gamma interferon production from peripheral
blood mononuclear cells in 70% of purified protein derivative-positive
individuals without evident disease. The responses to this antigen
argue for the inclusion of rCFP-10 in a polyvalent serodiagnostic test
for detection of active TB infection. rCFP-10 could also contribute to
the development of a recombinant T-cell diagnostic test capable of
detecting exposure to M. tuberculosis.
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INTRODUCTION |
Mycobacterium
tuberculosis, the causative agent of pulmonary tuberculosis (TB),
has infected approximately one-third of the world population and is
projected to result in 3.5 million annual deaths by the end of the year
2000 (18). The bacterium is spread primarily through
aerosolized infectious particles generated from coughing and sneezing
by individuals with TB. Due to the primary route of transmission, early
diagnosis of TB is essential in limiting the spread of M. tuberculosis within a human population. An optimal diagnostic test
for TB would be able to detect early disease with high sensitivity and
specificity, would yield results with rapidity, would be inexpensive,
and would cause little or no patient discomfort. Currently available
methods for the diagnosis of TB, including microscopic examination of
sputum smears (24), culturing of sputum samples
(8), PCR-based detection systems (8), detection of lipoarabinomannan in sera (19), chest X-ray, and the
Mantoux test, all fail to satisfy at least one of the above
requirements. An attractive methodology that continues to be explored
is the detection of M. tuberculosis-specific antibody in
patient sera, since a serodiagnostic test could potentially satisfy all
of the requirements for an optimal diagnostic test for TB.
Although numerous M. tuberculosis antigens capable of
generating specific antibody titers in TB patients have been identified (16, 24), no single antigen appears to be ideal for
serodiagnostic use. One of the best diagnostic markers isolated thus
far, the 38-kDa antigen (3, 6), has shown some potential for
use in a single-antigen diagnostic test. Studies using the antigen in
an enzyme-linked immunosorbent assay (ELISA) format detected up to 85%
of smear-positive cases (5, 10, 13). However, as a single
antigen, the 38-kDa antigen still lacks sufficient sensitivity to
create an optimal serodiagnostic test, especially for smear-negative
individuals, where sensitivity is considerably lower (13).
We were interested in determining if there are additional M. tuberculosis antigens that would complement a recombinant 38-kDa antigen (r38kDa) for serodiagnostic testing to detect active TB. Previous work of our own and by others utilizing expression screening with TB patient sera on recombinant M. tuberculosis
libraries demonstrated the effectiveness of this approach in recovering antigens recognized by patient sera (2, 9; M. J. Lodes, D. C. Dillon, R. L. Houghton, R. Raodoh, C. Day,
D. R. Benson, L. D. Reynolds, P. D. McNeill, and S. G. Reed, unpublished data). In order to isolate antigens that might
complement the r38kDa antigen, we used expression screening of a
genomic M. tuberculosis library with a pool of TB patient
sera determined previously to lack reactivity with the r38kDa antigen.
This approach is supported by a previous study that found that the
antibody response to M. tuberculosis antigens in different
infected individuals is heterogeneous (16). Additional
support is found in the preferential recognition of an 81-kDa M. tuberculosis antigen in human immunodeficiency virus
(HIV)-infected TB patients (11, 15).
Utilizing this approach, we have isolated a gene we initially referred
to as mtb11, which encodes a 100-amino-acid protein, Mtb11
(1). The same gene has recently been designated
lhp (L45 homologous protein), and the encoded protein has
been designated culture filtrate protein 10 (CFP-10) (5) and
MTSA-10 (M. tuberculosis-specific antigen 10) by another
group (7). Herein, we demonstrate that this protein is
recognized by a subset of TB patient sera and also by peripheral blood
mononuclear cells (PBMC) from a subset of healthy, purified protein
derivative-positive (PPD+) individuals. These results
suggest potential roles for this antigen both in complementing the
r38kDa antigen in the serodiagnosis of active TB and as part of a
polyvalent recombinant T-cell diagnostic test to detect M. tuberculosis exposure.
| |
MATERIALS AND METHODS |
Bacterial strains.
M. tuberculosis strains H37Rv,
H37Ra, and Erdman were gifts from the Seattle VA Hospital; the "C"
strain was a gift from Lee Riley, University of California, Berkeley;
and M. bovis BCG and M. leprae (Pasteur) were
obtained from Genesis Corp., Auckland, New Zealand. The following other
species of mycobacteria were obtained from the American Type Culture
Collection (ATCC; Manassas, Va.): M. vaccae (ATCC 15483),
M. avium subsp. avium (ATCC 35718), M. chelonae (ATCC 14472), M. fortuitum (ATCC 6841),
M. gordonae (ATCC 14470), M. scrofulaceum (ATCC
19981), and M. smegmatis (ATCC 19420). The M. tuberculosis H37Rv CFPs and membrane fraction were provided by
John Belisle, Colorado State University, Fort Collins.
Study population.
Serum samples were obtained from both male
and female individuals (>18 years of age) who had pulmonary TB alone
prior to treatment (culture and/or acid-fast bacillus smear positive
and negative). These were obtained from the Federal University of
Bahia, Salvador, Brazil. To evaluate the specificity of the recombinant
CFP-10 (rCFP-10) antigen, we obtained sera from individuals who were PPD+ (>10 mm) (culture, clinically, and radiographically
negative for TB) and from PPD
individuals (King County TB
Clinic, Seattle, Wash.). Additional healthy blood donors (United
States) were obtained from Boston Biomedica, West Bridgewater, Mass.).
PBMC were obtained from either the blood or apheresis product of
healthy PPD+ or PPD individuals by density
centrifugation over Ficoll. None of the PPD+ donors
included in the PBMC assay had a history of BCG immunization. All of
the sera and cells used came from individuals who were HIV negative.
Isolation of M. tuberculosis clones.
M.
tuberculosis H37Ra genomic DNA was isolated and sheared by
sonication to a size range of 1 to 4 kb. M. tuberculosis
H37Rv genomic DNA was partially digested with Sau3A1.
Libraries were constructed in Lambda ZapII (Stratagene, La Jolla,
Calif.) using EcoRI adapters. Expression screening was
performed using a pool of eight patient sera preadsorbed with
Escherichia coli (20). These eight serum samples
were demonstrated to lack reactivity with the r38kDa antigen by ELISA.
Additionally, seven of the eight samples came from smear-negative TB patients.
Cloning of the full-length mtb11 (lhp) gene was
accomplished by isolating the 5' portion of the RaCl-1 insert, random
labeling it with [32P]dCTP, and screening approximately
75,000 plaques as previously described (20) using the H37Rv
genomic library.
Expression of recombinant M. tuberculosis
antigens.
The 1.4-kb insert within the original RaCl-1 clone was
subcloned by restriction into a pET17b vector, modified by the addition of residues encoding a six-histidine tag following the ATG start codon
within the NdeI site, followed by the inclusion of a portion of the pBSK polylinker (region from SmaI to
XhoI). The resulting clone was referred to as pET
lhp, and
the resulting recombinant protein was named rCFP-10 and contained
the amino acid sequence MHHHHHHPGCRNSARE, followed by the 95 C-terminal
residues of CFP-10 (Fig. 1).
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FIG. 1.
Amino acid sequences of native CFP-10, rCFP-10, and
rCFP-10. Residues that are identical in all three proteins are
represented by dots.
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The full-length
lhp gene contained within the RvCl-1 clone
was engineered for expression by PCR utilizing a 5' primer which
contained an
NdeI site (which included the ATG start codon
of
lhp) and a 3' primer which contained residues encoding
the C-terminal
residues of the
lhp gene, a six-histidine
tag, a termination codon,
and a
HindIII site. The
amplified product was digested with
NdeI
and
HindIII and ligated into pET17b. This clone was referred
to
as pETlhp, and the recombinant protein encoded was rCFP-10. All
DNA
manipulations were confirmed by DNA sequencing to eliminate
the
possibility of the introduction of mutations by restriction,
ligation,
and
PCR.
Expression and purification of r
CFP-10 and rCFP-10 were performed as
follows. Pellets from induced
E. coli BL-21(pLysE)
containing
the pET
lhp and pETlhp constructs were lysed, and the
recombinant
proteins were recovered in the soluble fraction. The
initial purification
of r
CFP-10 was performed by binding the sample
with Ni-nitrilotriacetic
acid (Ni-NTA) resin under nondenaturing
conditions (20 mM Tris-HCl
[pH 8], 100 mM NaCl), washing with 20 mM
Tris-HCl [pH 8]-100 mM
NaCl-10 mM imidazole, and elution in a
column with increasing
concentrations of imidazole (10 to 100 mM).
Fractions containing
protein were combined and dialyzed against 10 mM
Tris-HCl (pH
7.4). This initial purification method resulted in the
copurification
of approximately equal amounts of recombinant ESAT-6
(rESAT-6)
protein. These recombinant TB proteins were separated on a
Vydac
C
18 (218TP5115) column (1 by 150 mm) by reverse-phase
high-pressure
liquid chromatography (Perkin-Elmer/Applied Biosystems,
Foster
City, Calif.). The proteins were separated on a linear gradient
of 0 to 100% buffer B in 50 min. The two major peaks eluted at
39 and
47 min on the chromatogram. The peak fractions were loaded
directly
into the Procise 494 protein Sequencer (Perkin-Elmer/Applied
Biosystems). The amino-terminal protein sequence identified the
two
peaks as r
CFP-10 and rESAT-6, respectively. The
esat-6
gene
is located just downstream of
lhp and is present in the
pET
lhp
clone, which contains a 1.4-kb insert. This allowed the
coexpression
of r
CFP-10 and rESAT-6 in the induced
E. coli and subsequent
copurification of the proteins on Ni-NTA
resin, presumably due
to an interaction between these two
proteins.
Later purifications of r
CFP-10 and rCFP-10 were performed as
described above, except that they were performed under denaturing
conditions, with the inclusion of 8 M urea during binding, washing,
and
elution on Ni-NTA resin. In all cases, the purity of the recombinant
protein was assessed by sodium dodecyl sulfate (SDS)-polyacrylamide
gel
electrophoresis (PAGE), followed by Coomassie blue staining,
and
N-terminal sequencing using traditional Edman chemistry with
a Procise
494 protein Sequencer (Perkin-Elmer/Applied Biosystems).
The endotoxin
level was determined to be less than 100 endotoxin
units/mg by
Limulus amebocyte lysate assay (BioWhittaker, Walkersville,
Md.).
Expression and purification of r38kDa were performed as described
previously (
11).
Molecular analysis of M. tuberculosis clones.
DNA was prepared by following the manufacturers' (Qiagen, Chatsworth,
Calif., and Promega, Madison, Wis.) protocols. DNA sequencing was
performed by automated sequencer (model 373; Perkin-Elmer/Applied Biosystems). DNA sequences and deduced amino acid sequences were used
in database searches (GenBank DNA and protein databases).
Genomic DNAs from mycobacterial strains were digested with
PstI, separated by agarose gel electrophoresis, and blotted
on
Nytran (Schleicher & Schuell, Keene, N.H.). The
lhp gene
was labeled
with [
32P]dCTP by random oligonucleotide
primers (Boehringer Mannheim,
Indianapolis, Ind.) and used as a probe.
Hybridization was performed
at 65°C in 0.2 M Na
H
2PO
4-3.6 M NaCl-0.2 M EDTA overnight and
washed to a stringency of 0.075 M NaCl-0.0075 M sodium citrate
(pH
7.0)-0.5% SDS at the temperature of
hybridization.
Immunoblot analysis.
Antiserum to rCFP-10 was raised
using adult New Zealand White rabbits (R & R Rabbitry, Stanwood, Wash.)
as described previously (9).
M. tuberculosis H37Rv lysate, CFP, and purified rCFP-10 were
subjected to SDS-12% PAGE and transferred to nitrocellulose.
Filters
were blocked with phosphate-buffered saline (PBS; pH 7.4)
containing
5% nonfat milk at 4°C overnight, washed three times
in PBS-0.1%
Tween 20 (PBS-T), and incubated for 1 h in rabbit
serum (diluted
1:250 in PBS-T) on a rocker at room temperature.
Filters were washed
three times with PBS-T, and bound antibody
was detected with
10
5 cpm of
125I-labeled protein A per ml,
followed by
autoradiography.
Serological analysis of rCFP-10.
ELISAs were performed with
96-well microtiter plates (Corning Costar, Cambridge, Mass.) which were
coated with CFP-10 and 38-kDa antigen (200 ng/well) and incubated
overnight at 4°C. ELISAs were performed as described previously
(11). The cutoff for the assays was the mean of the negative
population plus 3 standard deviations (SD) of the mean.
Proliferation and cytokine production assays.
PBMC were
cultured in 96-well round-bottom plates (Corning Costar) at 2 × 105 cells/well in a volume of 200 µl. Antigens were
tested in triplicate at 2 to 10 µg/ml. The culture medium consisted
of RPMI medium with 10% pooled human serum and gentamicin at 50 µg/ml. After 5 days of culture at 37°C in 5% CO2, 50 µl of culture supernatant was carefully aspirated for determination
of gamma interferon (IFN-
) levels and the plates were pulsed with 1 µCi of tritiated thymidine per well. After culture for a further
18 h, cells were harvested and tritium uptake was determined using
a gas scintillation counter. IFN- levels in culture supernatants
were determined by ELISA as previously described (21).
Proliferation results were considered positive if the stimulation index
(SI) compared to that of a no-antigen control was 5 or greater. This
cutoff was selected based on previous work involving approximately 20 purified recombinant M. tuberculosis antigens. It was found
that PPD donor PBMC responses to these antigens rarely
exceeded an SI of 5 (data not shown). Additionally, when the mean SI
plus 3 SD was calculated for this group of samples (healthy,
PPD PBMC) for these various antigens (including rCFP-10),
a value of approximately 5 was obtained (data not shown).
| |
RESULTS |
Isolation of lhp.
A pool of sera from eight individuals
with active or recently treated pulmonary TB was used in expression
screening of an M. tuberculosis H37Ra genomic library. These
eight sera had previously been shown to lack reactivity to purified
M. tuberculosis r38kDa by ELISA (data not shown). Screening
of 40,000 plaques led to the isolation of a single reactive clone,
termed RaCl-1, containing an insert of approximately 1.4 kb. Partial
DNA sequence analysis was performed on the RaCl-1 clone, which
predicted an encoded 95-amino-acid open reading frame in frame with the
N-terminal 4-kDa 
-galactosidase, creating a fusion protein of
approximately 14 kDa. The RaCl-1 clone was re-engineered to remove most
of the 5' lacZ sequence and add a six-histidine tag for use
in purification of the recombinant protein (see Materials and Methods).
The re-engineered clone was designated pETlhp, and the protein was
designated rCFP-10 (Fig. 1).
The 5' portion of the RaCl-1 insert was used as a probe to recover the
full-length gene from an
M. tuberculosis H37Rv genomic
library. Six clones were recovered, and subsequent DNA sequencing
determined that one (RvCl-1) contained the full-length gene which
included an additional 5 N-terminal amino acids not present in
the
original fusion, resulting in a predicted protein of 100 amino
acids
(Fig.
1) with a mass of 10,794 Da. No differences at the
DNA level
between the RaCl-1 and RvCl-1 clones were identified
within the coding
region of this gene (data not
shown).
We initially referred to this gene as
mtb11 and the encoded
protein as Mtb11, based on the predicted mass of the protein
(
1).
Subsequent DNA database searches with this sequence
revealed 100%
identity with an
M. bovis sequence which was
previously demonstrated
to be within a region missing in
M. bovis BCG (
17). In addition,
identity to the
M. tuberculosis lhp gene, located just upstream
of
esat-6,
was found. A search performed with the predicted amino
acid sequence
showed identity to the putative open reading frame
encoded by the
lhp gene (accession no. CAA17966), referred to
in the
database as CFP-10. The
lhp gene 3' end is located just
34 bp upstream of the
esat-6 gene, and both genes are oriented
in the same direction. Additionally, evidence has been presented
that
the two genes are part of an
M. tuberculosis operon and that
both proteins are secreted (
4,
23). The protein database
searches did not reveal significant homology with any proteins
with
known function but did show some relatedness (40% identity)
to a
predicted 100-amino-acid
M. leprae protein (accession no.
CAA75210) and low-level relatedness to ESAT-6 and TB10.4
(
22).
Hydropathy analysis (
14) of the CFP-10
amino acid sequence did
not indicate the presence of any extended
hydrophobic regions
that might serve as transmembrane domains (data not
shown).
Genomic DNAs from a number of mycobacterial species were analyzed by
Southern blotting using the
lhp gene as a probe. The
results
(Fig.
2) indicate that the gene is
present in single copy
and is conserved in two
M. tuberculosis clinical strains but not
well conserved in any of the
other mycobacterial species tested,
including
M. bovis BCG.
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FIG. 2.
Southern blot analysis of the lhp gene.
Genomic DNAs (2.5 µg) from mycobacterial strains were digested with
PstI, separated by agarose gel electrophoresis, and blotted
onto Nytran. The lhp gene was labeled with
[32P]dCTP by random oligonucleotide primers and used as a
probe. Molecular size markers (M) in kilobases are shown.
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The re-engineered clone, pET
lhp, comprised the entire 1.4-kb insert
originally recovered, which included the entire
esat-6 gene
region located immediately downstream of the
lhp gene (see
Materials and Methods). An unanticipated consequence of the expression
and purification of r
CFP-10 using Ni-NTA resin (Qiagen) under
nondenaturing conditions was the coexpression and copurification
of
recombinant ESAT-6 in amounts roughly equivalent to those of
r
CFP-10. Under the purification conditions used, the coisolation
of
rESAT-6 had to occur through interaction of the rESAT-6 protein
with
either r
CFP-10 or directly with the Ni-NTA resin. Since
the
esat-6 gene in this construct was not engineered to contain
a six-histidine tag, the interaction of rESAT-6 with r
CFP-10
is the
more likely explanation. This finding is consistent with
earlier work
indicating that these genes are coordinately expressed
(
4)
but also indicates that the two proteins may interact
directly.
To characterize native
M. tuberculosis CFP-10, rabbit
antiserum to purified r
CFP-10 was raised. This antiserum was used in
an immunoblot assay of the
M. tuberculosis lysate, CFP, and
membrane
fraction. The results (Fig.
3)
indicate the presence of a reactive
protein with an approximate mass of
11 kDa in the lysate, CFP,
and membrane fraction.
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FIG. 3.
Characterization of native CFP-10. M. tuberculosis H37Rv lysate (2.5 µg; lane 1), 2.5 µg of CFP
(lane 2), 2.5 µg of the cytoplasmic fraction (lane 3), 50 ng of
rCFP-10 (lane 4), no sample (lane 5), and 2.5 µg of the membrane
fraction (lane 6) were subjected to SDS-PAGE, transferred to
nitrocellulose, and reacted with rabbit antiserum generated against
rCFP-10. Molecular masses are shown in kilodaltons.
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Immunological responses to rCFP-10.
The full-length
lhp gene was reengineered for expression, removing all
flanking sequences and adding a C-terminal six-histidine tag (Fig. 1).
The resulting recombinant antigen, referred to as rCFP-10, was
expressed and purified (Fig. 4) and was
used to assess human antibody and T-cell responses.
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FIG. 4.
Purification of rCFP-10. Expression and purification of
rCFP-10 are shown with uninduced (lane 2) and induced (lane 3) E. coli lysates and 5 µg of purified protein (lane 4). Molecular
masses are shown in kilodaltons (lane 1).
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Sera from individuals with TB were assessed for specific antibody
reactivity with r38kDa and rCFP-10 in an ELISA format (Table
1). These included sera from both
smear-positive and smear-negative
TB patients. The control sera used
were from healthy PPD
+ and PPD
individuals.
Serological reactivity to rCFP-10 was observed in
28% (69 of 250) of
smear-positive TB patient sera and in 25% (13
of 52) of smear-negative
TB patient sera. Reactivity was generally
not observed in sera from
either healthy PPD
+ or healthy PPD
individuals, with an overall specificity of approximately 97%
(83 of
86).
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TABLE 1.
Serological reactivity of rCFP-10 and r38kDa in
smear-positive and smear-negative TB patients and healthy
PPD+ and PPD individuals
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Moreover, rCFP-10 was able to complement r38kDa in the detection of TB.
In smear-positive TB patient sera, detection increased
from 49% (122 of 250) with r38kDa alone to 58% (146 of 250) with
both r38kDa and
rCFP-10. More significant complementation was
observed in
smear-negative patient sera, where detection increased
from 21% (11 of
52) with r38kDa alone to 40% (21 of 52) detected
with both r38kDa and
rCFP-10. The additional sensitivity provided
by the addition of rCFP-10
to r38kDa did not result in significant
specificity problems, with no
additional rCFP-10-positive, PPD
+ sera and one additional
rCFP-10-positive, PPD
control serum. Thus, the
specificity of r38kDa alone was 93%
(80 of 86) and that of the
combination of r38 kDa and rCFP-10
was 92% (79 of 86). In total, there
were three sera that yielded
false-positive results with rCFP-10 (from
two PPD
+ individuals and one PPD
individual).
The latter was at the edge of the cutoff of the
assay and was of U.S.
origin. Of the remaining two (PPD
+) individuals, the one
with the highest ELISA reactivity was of
U.S. origin, had been in
contact with an active case, was a potential
converter, and was being
placed on therapy. The remaining false
positive person for CFP-10 was
from South
Vietnam.
T-cell responses to rCFP-10 were also evaluated. PBMC from 20 healthy
PPD
+ donors and 20 healthy PPD
donors were
analyzed for proliferation and production of IFN-
in response to CFP
or purified rCFP-10. Using an SI cutoff of
5 (see Materials and
Methods), purified rCFP-10 elicited proliferative
responses in 70% (14 of 20) PPD
+ donors but no response was observed in the 20 PPD
donors (Table
2). This
contrasted with CFP, which elicited proliferative
responses from the
PBMC of 95% (19 of 20) of healthy PPD
+ individuals but
also elicited responses from 45% (9 of 20) of
healthy
PPD
individuals. The difference in mean SI between the
PPD
+ group and the PPD
group was 17-fold for
rCFP-10, while only a 7-fold difference
was observed for CFP. Analysis
of the IFN-
production for these
same samples yielded responses
similar to the proliferation results
(data not shown).
Finally, for those samples in which both PBMC and serum were available,
we compared T-cell responses and antibody responses
to rCFP-10 (Table
3). The data show clearly that regardless
of
the presence and extent of the T-cell response to rCFP-10 observed
in PBMC in a subset of the healthy PPD
+ donors (eight of
nine with an SI of greater than 5), no detectable
antibody response was
present. No positive rCFP-10-specific T-cell
response or antibody
reactivity was observed for any of the PPD
individuals.
| |
DISCUSSION |
A significant difficulty in the development of a serodiagnostic
assay for detection of TB has been the identification of an antigen, or
a collection of antigens, that yields the desired sensitivity and
specificity. The M. tuberculosis 38-kDa antigen is
undoubtedly the best single serodiagnostic antigen to have been
identified thus far, but it still has sensitivity limitations, especially in smear-negative individuals (13). However, if
additional M. tuberculosis antigens are to be used to
complement the 38-kDa antigen to yield increased sensitivity in a
serodiagnostic assay, it cannot occur at the price of specificity. We
have demonstrated that both the serological and T-cell responses to
rCFP-10 antigen are highly specific. It is possible that the
specificity observed with rCFP-10 is due, at least in part, to the fact
that the gene encoding this antigen is not highly conserved in other
mycobacterial species and is missing in M. bovis BCG.
Consequently, cross-reactive responses are not likely to occur from
previous exposure to other mycobacterial species or through
immunization with M. bovis BCG. We have observed that
BCG-immunized individuals do not have increased rCFP-10-specific
antibody responses (data not shown). Although we have not tested sera
or PBMC from individuals infected with other mycobacterial species,
such as M. avium, the lack of conservation of this gene in
these mycobacteria predicts no rCFP-10-specific responses from the sera
or PBMC of these patients.
Although the TB patient group used in these serological studies had a
singular geographic origin (Brazil), additional studies that are
ongoing show similar serological responses to rCFP-10 from TB patients
from other geographical areas (data not shown). Additionally, because
the patient cohort was located in an area where TB is endemic, it is
unlikely that the disease in these patients was caused by
nontuberculous mycobacteria. As a stand-alone antigen, rCFP-10 would be
a poor serodiagnostic agent, detecting only 28% of smear-positive TB
patients and 25% of smear-negative TB patients. However, we found that
rCFP-10 does have potential as a supplemental antigen to r38kDa, most
notably in smear-negative TB patients, where sensitivity was increased
from 21 to 40% without a significant reduction in test
specificity. Although these two antigens together are clearly not
sufficient, lacking the necessary sensitivity of an optimal diagnostic
agent to detect active TB, these results do argue for the possibility
that a multiantigen serodiagnostic test for TB with high sensitivity
can be achieved while maintaining a high level of specificity. Current
evaluation of an additional M. tuberculosis antigen, MTB48,
that can further complement the combination of r38kDa and rCFP-10 in
the serodiagnosis of TB also supports this multiantigen diagnostic
approach (Lodes et al., unpublished). Additional evidence that specific
antigens are capable of assisting in the serodiagnosis of specific
subgroups of active TB has been found in a recent study demonstrating
the use of Mtb81 in the detection of active TB in patients coinfected with HIV (11). Collectively, the data indicate that a set of antigens will likely be needed to achieve a serodiagnostic test with
the desired sensitivity. However, each antigen component must
contribute to increasing the sensitivity of the serodiagnostic test and
maintaining a high degree of specificity for inclusion in a
multiantigen serodiagnostic test for active TB. The ability of rCFP-10
to satisfy both of these criteria qualifies it as a supplemental
antigen to r38kDa.
An additional diagnostic tool would be one that could detect exposure
of individuals to M. tuberculosis. The current diagnostic agent PPD, although both sensitive and inexpensive, has specificity problems, yielding positive results in some individuals infected with
other mycobacterial strains or in BCG-immunized individuals (12). A superior diagnostic may be found in a collection of recombinant M. tuberculosis antigens that yield specific
T-cell responses, such as rCFP-10, that could be utilized in a skin
test or an in vitro T-cell diagnostic assay. In fact, rCFP-10 appears to be able to contribute more robustly as a T-cell diagnostic antigen,
with proliferative responses from PBMC seen in 70% of PPD+
donors and no positive responses observed in the PPD
donors tested. Consistent with the role of CFP-10 as a potential T-cell
diagnostic antigen, a recent study with this antigen has demonstrated
that it is capable of eliciting delayed-type hypersensitivity in
M. tuberculosis-infected guinea pigs but not in M. bovis BCG-infected or M. avium-infected guinea pigs
(7). Additionally, it has been recently demonstrated that
PBMC from TB patients generated IFN- production in response to
recombinant CFP-10 (22). These data, along with the data
presented here on responses by healthy PPD+ donors, support
the use of rCFP-10 as a component of a T-cell diagnostic antigen for
the detection of exposure to M. tuberculosis.
One unexpected result of the characterization of CFP-10 was the
discovery that it may interact with ESAT-6. Supporting this interaction
are the adjacent location of these two genes, the evidence that they
are cotranscribed (4), and the finding presented here
describing the copurification of rESAT-6 with rCFP-10 when expressed
in E. coli. Lastly, there is also some relatedness between ESAT-6 and CFP-10 at the amino acid level (22).
Collectively, these data encourage speculation that is there not only
coordinated expression but also some direct interaction between CFP-10
and ESAT-6 in M. tuberculosis. Like ESAT-6, CFP-10 can be
found in CFP from M. tuberculosis in vitro cultures.
However, the level of CFP-10 expression and protein localization in
active and dormant M. tuberculosis in a human infection is
not known at this time.
It has been well established that the generation of substantive
antibody responses to protein antigens is dependent on the existence of
T-cell epitopes within the antigen recognized by helper T lymphocytes.
Thus, specific serological responses elicited during pulmonary TB
provide a mechanism by which to identify M. tuberculosis
T-cell antigens (9). These same antigens, while generating a
detectable humoral response in susceptible individuals, may also be a
target of cellular responses in the more frequent outcome of M. tuberculosis infection, i.e., that of acquired protective immunity. Support exists for these dual responses to the M. tuberculosis 38-kDa antigen (25), and the data
presented herein support the idea that CFP-10 is one such antigen. It
is recognized by the immune systems of a substantial percentage of
individuals infected with M. tuberculosis, both those with
active disease and those that have developed protective responses. The
data demonstrate that the immunological responses to rCFP-10 by
protected and susceptible individuals are qualitatively different at
the time of measurement. The dual responses to this antigen afford it
potential utility both as a component of a serodiagnostic test for
active TB and as a component of an improved skin test antigen to detect
exposure to M. tuberculosis.
| |
ACKNOWLEDGMENTS |
We thank Steve Johnson and John Webb for the M. tuberculosis H37Ra and H37Rv genomic libraries. We thank Dan Hoppe
for DNA sequencing efforts and Karen Kinch for assistance in manuscript preparation.
We thank John Belisle for providing CFP (provided through NIAID-NIH
Tuberculosis Research Materials contract N01-AI-25147).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Corixa Corp.,
1124 Columbia St., Seattle, WA 98104. Phone: (206) 754-5701. Fax: (206) 754-5715. E-mail: dillon{at}corixa.com.
| |
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Abstract
Introduction
