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,
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Department of Pathobiology, University of Washington, Seattle, WA 98195; and
Infectious Disease Research Institute and
CORIXA Corporation, Seattle, WA 98104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production, was further characterized. The
N-terminal amino acid sequence of this polypeptide was determined and
used to design oligonucleotides for screening a recombinant M.
tuberculosis genomic DNA library. The gene (Mtb
8.4) corresponding to the identified polypeptide was cloned,
sequenced, and expressed in Escherichia coli. The
predicted m.w. of the recombinant protein without its signal peptide
was 8.4 kDa. By Southern analysis, the DNA encoding this mycobacterial
protein was found in the M. tuberculosis substrains
H37Rv, H37Ra, Erdman, and "C" strain, as well as in certain other
mycobacterial species, including Mycobacterium avium and
Mycobacterium bovis BCG (bacillus Calmette-Guérin,
Pasteur). The Mtb 8.4 gene appears to be absent from the
environmental mycobacterial species examined thus far, including
Mycobacterium smegmatis, Mycobacterium
gordonae, Mycobacterium chelonae,
Mycobacterium fortuitum, and Mycobacterium
scrofulaceum. Recombinant Mtb 8.4 Ag induced significant
proliferation as well as production of IFN-, IL-10, and TNF-
, but
not IL-5, from human PBMC isolated from PPD-positive healthy donors.
Mtb 8.4 did not stimulate PBMC from PPD-negative donors. Furthermore,
immunogenicity studies in mice indicate that Mtb 8.4 elicits a Th1
cytokine profile, which is considered important for protective immunity
to tuberculosis. Collectively, these results demonstrate that Mtb 8.4
is an immunodominant T cell Ag of M.
tuberculosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The only currently available vaccine against TB is the live attenuated bacillus Calmette-Guérin (BCG), derived from Mycobacterium bovis. Based on the results of many clinical trials in developing countries, the efficacy of BCG in eliciting protective immunity has been reported to vary from 0 to 80% (3, 4, 5). In addition to this variability, BCG, being a live attenuated vaccine, can also potentially cause serious disease in immunocompromised persons. There is clearly a need for a more safe and effective vaccine.
The development of a vaccine based on defined Ags of M. tuberculosis is an area of great interest. Past studies in animals have shown that effective protection against virulent challenge with M. tuberculosis occurs following immunization with living attenuated mycobacteria (BCG, R1Rv, or Mycobacterium microti) (6, 7, 8). When the same mycobacteria are heat killed and injected in the presence or absence of adjuvant, however, a protective response against a virulent challenge is not observed (9). Previous studies have also reported increased resistance against challenge with M. tuberculosis in several animal disease models after vaccination with different mycobacterial subfractions or with culture filtrate proteins (10, 11, 12, 13, 14, 15, 16, 17, 18).
As is the case for many intracellular pathogens, cell-mediated immunity
plays an important role in host protection against TB. Recent studies
have reported that acquired resistance in animals is mediated by
specific populations of sensitized T lymphocytes (19). In particular,
CD4+ Th1 and CD8+ lymphocytes, which produce
IFN-, have been shown to be important for protection of mice
(20, 21, 22). Similarly, humans who have a mutation in the gene for
IFN-R are more susceptible to mycobacterial infections, providing
further evidence of the importance of IFN- in the response to
mycobacterial infection (23, 24). Moreover, there has been some
evidence that proteins actively secreted by M. tuberculosis
during its growth may induce cell-mediated immune responses and
protective immunity (13, 18, 25, 26) by causing expansion of specific
IFN--producing T lymphocytes that are capable of recognizing and
exerting antimicrobial effects against macrophages containing
intracellular mycobacteria.
We have characterized M. tuberculosis culture filtrate
proteins that are immunodominant T cell Ags, capable of eliciting
proliferation and IFN- production from human PBMC of
PPD+ healthy individuals. T cell responses against such Ags
may be protective in the specific host immune response, thus providing
necessary candidates for the development of an antimycobacterial
subunit vaccine. This report describes the purification and biologic
activity of a protein obtained from the culture filtrate of M.
tuberculosis strain H37Rv, as well as cloning, DNA sequence,
expression, and preliminary immunologic characterization of this novel
immunoreactive T cell Ag of M. tuberculosis, Mtb 8.4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. tuberculosis strains H37Rv and Erdman were gifts from Dr. Sean Skerritt, Seattle Veterans Affairs Hospital, Seattle, WA; "C" strain was a gift from Dr. Lee Riley, University of California, Berkeley CA; M. bovis BCG and Mycobacterium leprae (Pasteur) were obtained from Genesis, Auckland, New Zealand. Other species of mycobacteria were obtained from the American Type Culture Collection (ATCC, Manassas, VA): M. tuberculosis H37Ra (ATCC 25177), Mycobacterium vaccae (ATCC 15483), Mycobacterium avium avium (ATCC 35718), Mycobacterium chelonae (ATCC 14472), Mycobacterium fortuitum (ATCC 6841), Mycobacterium gordonae (ATCC 14470), Mycobacterium scrofulaceum (ATCC 19981), and Mycobacterium smegmatis (ATCC 19420).
Purification of secreted polypeptides
Culture supernatants were prepared from 2- to 3-wk log phase cultures of M. tuberculosis H37Rv grown in a synthetic medium (1.5% glucose, 1% basal medium Eagle’s amino acid solution (Life Technologies, Grand Island, NY), 1% MEM nonessential amino acid solution (Life Technologies), 1% basal medium Eagle’s vitamin solution (Life Technologies), 0.05g/L ferric ammonium citrate, 4g/L K2HPO4, 2g/L citric acid, 1.2g/L MgCl2, 0.6g/L K2SO4, 2g/L NH4Cl, 0.72g/L NaOH) at 37°C. Culture supernatant was harvested and filtered through a 0.22-µm cellulose acetate membrane (Corning Glassworks, Corning, NY). The filtrate was concentrated 50x with an Amicon Centriprep-3 concentrator (Beverly, MA) and submitted for quantitation of bacterial endotoxin using a Limulus amebocyte lysate assay (LAL; BioWhittaker, Walkersville, MD). The protein concentration of the culture filtrate (CF) was determined using a commercial bicinchoninic acid assay (BCA; Pierce, Rockford, IL).
Fractionation of the CF was performed using a microbore Vydac C18 column (Vydac, Hesperia, CA) and a standard linear gradient of 0.5%/min. increase from 20 to 50% buffer B (80% acetonitrile/0.05% trifluoroacetic acid (TFA)). Aliquots of different fractions were submitted for immunologic assay and for N-terminal protein sequencing.
Amino-terminal sequencing
Amino acid identifications were performed on polypeptide(s) in positive fractions. Individual fractions were dried onto Biobrene-treated glass fiber filters (Perkin-Elmer/Applied Biosystems, Foster City, CA). The filters were loaded onto a Perkin-Elmer/Applied Biosystems Procise 492 protein sequencer, and the polypeptides were sequenced from the amino terminal using traditional Edman chemistry. The amino acid sequences obtained were compared with known amino acid sequences in the GenBank using the DNAStar system.
Preparation of M. tuberculosis genomic DNA library
Genomic DNA from M. tuberculosis Erdman was sheared
and blunt ended with Klenow, and EcoRI adapters were ligated
to these ends before ligation into bacteriophage 
ZAP II arms
(Stratagene, La Jolla, CA). Phage were packaged using Gigapack II
packaging extracts according to the manufacturer’s instructions
(Stratagene).
Molecular cloning of rMtb 8.4 Ag
Degenerate oligonucleotides were synthesized (Life Technologies)
based on the deduced N-terminal amino acid sequence and used as probes
to screen the M. tuberculosis Erdman library. Two micrograms
of purified oligonucleotide probes were labeled with 32P by
incubation at 37°C for 30 min and inactivated by incubation at
65°C. Unincorporated nucleotides were removed using a nucleotide
removal kit (Qiagen, Chatsworth, CA). Nitrocellulose filters
(Schleicher and Schuell, Keene, NH) were placed over phage plaques
(20,000 plaque-forming units/plate x 4 plates), were then blocked
with prehybridization solution for 2 h at 60°C and probed with
denatured 32P-labeled degenerate oligonucleotides for
20 h at 48°C. The filters were washed twice at room temperature
for 20 min in 6x SSC and once at 48°C in 6x SSC/0.1% SDS. Positive
plaques were purified, and excision of the pBSK(-) phagemid was
performed using the manufacturer’s protocol (Stratagene).
DNA sequencing
Sequence analysis was done by the Taq dye terminator technique with an ABI 373-A Stretch DNA sequencer (Applied Biosystems, Perkin-Elmer). The sequence of the 1.4-kb insert of pBSK(-) was determined using M13 forward and reverse primers (Stratagene), and a number of internal synthetic oligonucleotides derived from initial sequence data which were used to join nonoverlapping fragments. Similarities to any previously reported DNA or protein sequences were determined with the BLAST and Swiss-Prot databases, respectively.
Subcloning and expression of Mtb 8.4
The coding portion of Mtb 8.4 was PCR amplified using two oligonucleotides designed to make a PCR product that would begin 3' to the possible secretory sequence and would continue to the stop codon. The 5' primer included sequence coding for six histidine residues for ease of purification with Ni-NTA resin (Qiagen), as well as an NdeI restriction enzyme site for subcloning. The 5' primer sequence (Mtb 8.4 5' His) was 5'-CAATTACATATGCATCACCATCACCATCACGATCCCGTGGACGCGGTC. The 3' primer included coding for a stop codon and an EcoRI restriction enzyme site. The 3' primer sequence (Mtb 8.4 3' End) was 5'-CAAGAATTCTTAATAGTTGTTGCAGGA.
The 1.4-kb DNA fragment encoding the Mtb 8.4 gene was purified from an agarose gel after EcoRI digestion and used as template for subcloning. Standard PCR reactions were conducted in a Peltier thermal cycler (DNA Engine PTC-200; MJ Research, Watertown, MA). The reaction amplifications were performed for 30 cycles at 94°C for 1 min, 60°C for 30 s, and 72°C for 1.5 min. The resulting PCR fragment was ligated with T4 DNA ligase in NdeI/EcoRI-digested pET 17b plasmid vector (Novagen, Madison, WI) and was transformed into Escherichia coli XL-1 Blue and BL-21 (DE3) pLysE (Novagen) by the standard procedures for DNA manipulation and protein expression, respectively (27).
To obtain E. coli BL21 lysates, single colonies were inoculated into 2x yeast tryptone (YT) broth and grown to an OD of 0.5 at 560 nm. isopropyl-ß-D-galactopyranoside was then added, and growth was continued for an additional 3 h. The bacteria were harvested by centrifugation and lysed in the presence of protease inhibitors and lysozyme, using a sonicator. The induced target protein was identified by SDS-PAGE in the insoluble inclusion body. The purification of rMtb 8.4 was performed by metal chelate column chromatography using Ni-NTA resin according to the manufacturer’s recommendations (Qiagen).
Southern analysis
Genomic DNA from several mycobacterial species was prepared using the detergent cetyltrimethylammonium bromide (CTAB, Sigma, St. Louis, MO). DNA was extracted using phenol:chloroform and precipitated. One microgram of DNA was then digested using PstI or SalI, electrophoresed in a 1.5% agarose gel, and transferred onto a nylon membrane (Schleicher and Schuell). The Mtb 8.4 insert DNA was 32P radiolabeled by the random hexamer priming method (28) and used to probe the mycobacterial DNAs. After hybridization for 18 h at 65°C, the blots were washed at 60°C, twice for 15 min with 2x SSC, once for 30 min with 2x SSC/0.2%SDS, and once for 10 min with 0.2x SSC. After drying at room temperature, blots were mounted for autoradiography at -80°C for 24 h.
Production of rabbit polyclonal serum against the rMtb 8.4 Ag
The rMtb 8.4 Ag (150 µg) was emulsified in a mixture of 100 µg of muramyl dipeptide and 1 ml of IFA (Life Technologies) as adjuvants and injected intramuscularly at multiple sites into a New Zealand rabbit (R&R Rabbitry, Stanwood, WA). A s.c. booster injection of 100 µg of rAg with 1 ml of IFA was given 6 wk later, and 25 µg in PBS was given 3 wk later. The rabbit was sacrificed 1 wk following the last boost, and serum was collected and stored at -20°C.
PAGE and immunoblotting
Samples of crude E. coli protein extracts were separated by SDS-15% PAGE before being stained with Coomassie brilliant blue or transferred onto nitrocellulose sheets. The samples blotted on nitrocellulose were probed with polyclonal rabbit serum against the purified rMtb 8.4 protein or against M. tuberculosis H37Rv CF proteins. The detecting agent was protein A-conjugated 125I.
Immunologic evaluation of HPLC fractionated peaks and rMtb 8.4
The fractionated polypeptides from reverse phase HPLC and the
rMtb 8.4 Ag were screened for their ability to induce T cell
proliferation and IFN- production in PBMC preparations from
PPD+ healthy individuals. The donor cells were obtained
from ethnically diverse individuals (Caucasian, Middle Eastern, Asian,
and Hispanic) who all tested with PPD indurations of >20 mm. PBMC were
prepared from heparinized blood by Ficoll density gradient
(d = 1.077) centrifugation or apheresis. PBMC were
adjusted to 2 x 105 cells per well in 96-well
flat-bottom plates (Costar, Cambridge, MA) using RPMI 1640
containing 10% human serum. HPLC fractions were added in triplicate at
dilutions of 1:40 and 1:120, while rMtb 8.4 and CF were titrated at
concentrations of 20 µg/ml to 0.001 µg/ml. PHA and tetanus
toxoid were used as positive controls. Plates were cultured for 5 to 6
days at 37°C in 5% CO2 and then 50 µl of the culture
supernatants were removed for determination of cytokine levels. The
cultures were each pulsed with 1 µCi of [3H]thymidine
for 18 h, harvested, and incorporation of
[3H]thymidine was counted in a gas scintillation
counter.
Cytokine assays
The production of cytokines was quantified by sandwich ELISA.
Briefly, ELISA plates (Corning) were coated for 4 h at room
temperature with 50 µl/well cytokine capture mAb (1 µg/ml for
IFN-, IL-5, and IL-10; 2 µg/ml for TNF-; PharMingen, San Diego,
CA) in 0.1 M NaHCO3/Na2CO3 buffer
(pH 9.6). After blocking overnight at 4°C (5% (W/V) nonfat dried
milk for IFN- and 1% BSA for IL-5, IL-10, and TNF-, samples or
standards were added for 2 h at room temperature. Plates were
washed with PBS, 0.05% Tween (PBS-Tween) and then incubated for 2
h at room temperature with 100 µl/well of second Ab (rabbit
anti-human polyclonal Ab; Immunex, Seattle, WA) diluted
1:3000 in PBS-10% normal goat serum for IFN-; biotinylated
detecting Ab in PBS-Tween, 0.1% BSA at 0.5 µg/ml for IL-5 and IL-10;
and at 1 µg/ml for TNF- (PharMingen). After washing, plates were
incubated with goat anti-rabbit horseradish peroxidase in
PBS-Tween, 5% nonfat dried milk (IFN-) or with
streptavidin-peroxidase in PBS-Tween, 0.1% BSA (IL-5, IL-10, and
TNF-). Plates were developed using TMB substrate
(3,3',5,5'-tetramethylbenzidine, Kirkegaard and Perry, Gaithersburg,
MD). OD was determined at 450 nm using 570 nm as a reference
wavelength. Cytokine concentration was evaluated using the respective
standard curves.
Immunogenicity studies
BALB/c and C57BL/6 mice were immunized in the footpads with 15
to 30 µg of the rMtb 8.4 Ag formulated in IFA as adjuvant. Mice were
also immunized with saline or IFA alone as control. Draining lymph
nodes were removed 9 days later, and cells were plated at 2.5 x
105/well for proliferation assays and at 2.5 x
106/well for cytokine assays. The lymph node cells were
cultured in the presence of anti-IL-4R (Immunex) at 3 µg/ml. The
anti-IL-4R Ab was added to the cultures because it presumably
blocks the uptake of secreted IL-4 by activated T cells, thereby
increasing the accuracy of quantitating IL-4 without affecting
proliferation or IFN- production. The lymph node cells were
restimulated in vitro with rMtb 8.4 at 1, 5, and 25 µg/ml. Plates
were cultured for 3 days at 37°C in 5% CO2. Supernatants
were then taken for cytokine ELISA, or plates were pulsed with 1 µCi
of [3H]thymidine for 18 h, harvested, and
[3H] thymidine incorporation was counted in a gas
scintillation counter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CF of M. tuberculosis has previously been reported
to contain Ags that elicit specific immune responses or protection in
animals infected with M. tuberculosis (12, 13, 14, 15, 16, 17). To study the
PBMC responses to such proteins, CF from M. tuberculosis
that tested negative for endotoxin by Limulus amebocyte
lysate assay was separated into 16 fractions on a microbore
column using a slow gradient (Fig. 1
) and
evaluated immunologically using PBMC from PPD+ individuals
(Fig. 2). As shown, fractions 8 through
16 at a 1:40 dilution were able to stimulate proliferation of PBMC from
both of the PPD+ individuals to a strong degree (Fig. 2).
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Cloning of the Mtb 8.4 gene
Several overlapping degenerate oligonucleotides were designed
based on the amino-terminal sequence data of the native Mtb 8.4
polypeptide. One of the oligonucleotides,
TGYAAYTAYGGICARGTIGTIGCSGCSCTSA (where Y = C + T; I =
deoxyinosine; R = A + G; S = C + G), representing the amino
acids Cys-Asn-Tyr-Gly-Gln-Val-Val-Ala-Ala-Leu, was used to clone a
recombinant gene with an insert size of 
1.4 kb. The
nucleotide sequence of Mtb 8.4 and the predicted amino acid
sequence coding for the Mtb 8.4 protein are shown in Figure 3. An open reading frame of 330
nucleotides coded for a protein of 110 amino acids with a consensus
signal peptide of 28 amino acids (residues 969-1052) and a predicted
molecular mass of 10.8 kDa. The DNA sequence contained an open reading
frame starting with an ATG codon at nucleotide 969 and ending with a
termination codon (TTA) at nucleotide 1299–1301. The N-terminal
sequence of the purified culture filtrate polypeptide and the deduced
amino acid sequence of the cloned DNA corresponding to Mtb 8.4without its hydrophobic secretory region were identical. Given the
known amino terminal sequence of the purified culture filtrate Ag and
the characteristics of the signal peptide sequence, it is feasible that
the signal peptidase recognition sequence (Ala-X-Ala) (31) is located
in front of the N-terminal region of the mature form of the protein at
nucleotide 1052. The structural gene encoding the mature Mtb 8.4
protein, Mtb 8.4, derived from M. tuberculosis
Erdman is thus found at nucleotide residues 1053–1298. A potential
ribosome-binding site (GGAAGG) is located at nucleotide residue
953–958. Putative Pribnow boxes (-35 and -10 sequences), similar to
the E. coli promoter-like consensus sequences, are located
at nucleotide residues 919 and 945, respectively.
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To determine the distribution of Mtb 8.4 within species
belonging to the M. tuberculosis complex, M.
avium and M. bovis BCG, as well as in
"environmental" mycobacterial species, the 279-bp
NdeI/EcoRI Mtb 8.4 fragment from
pET/Mtb 8.4 was used as a probe in a Southern hybridization
analysis of digested total genomic DNA from various mycobacterial
strains (Fig. 4). This experiment
indicated that the Mtb 8.4 gene is present as a single copy
in the mycobacterial genome. The probe hybridized to SalI
fragments of approximately 4.4 kb in the M. tuberculosis
substrains H37Rv, H37Ra, Erdman, and the "C" strain, as well as in
M. bovis BCG (Pasteur). Hybridization to a fragment of 8
kb was observed in M. avium avium, but the probe did not
hybridize to any SalI fragments from M.
leprae, M. smegmatis,
M. vaccae, M. gordonae, M.
chelonae, M. fortuitum, or M. scrofulaceum.
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Recombinant Mtb 8.4 lacking its hydrophobic, putative signal
peptide sequence was expressed in E. coli with six
consecutive His residues at the amino-terminal portion immediately
following the initiator Met residue (N-terminal HIS-TAG) of the pET
plasmid vector (pET-17b) and a T7 RNA polymerase expression system
(Novagen). Crude protein extracts of Mtb 8.4 and purified rMtb 8.4 were
subjected to SDS-PAGE and stained with Coomassie brilliant blue (Fig. 5). To further characterize native and
recombinant Mtb 8.4, a Mtb 8.4 antiserum was raised in a rabbit and
used as a probe in an immunoblot assay. Figure 6A shows that although the
rabbit anti-Mtb 8.4 serum reacted with a single band in the CF, it
did not detect the Mtb 8.4 molecule in the M. tuberculosis
lysate H37Rv. This finding suggests that the Mtb 8.4 protein may be
rapidly processed and exported from the bacilli after synthesis. The
absence of general protease degradation of the lysate during
preparation was substantiated by probing another blot with a rabbit
polyclonal antiserum raised against the M. tuberculosis
protein Ag 85B. This antiserum recognized recombinant 85B, native 85B
in CF, and a single band in the M. tuberculosis H37Rv lysate
(data not shown). A rabbit antiserum raised against M.
tuberculosis H37Rv CF proteins was also used in Western blot
analysis. This antiserum reacted with numerous proteins in the M.
tuberculosis H37Rv lysate and the CF, as well as with rMtb 8.4,
thus confirming the presence of the native Mtb 8.4 in the culture
supernatant during growth (Fig. 6B). Preimmune rabbit serum
had no reactivity with rMtb 8.4 (data not shown).
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To determine the immunologic properties of rMtb 8.4, PBMC isolated
from PPD+ healthy individuals were stimulated with a range
of concentrations of purified rMtb 8.4 (0.0002–20 µg/ml). Individual
donors were found to elicit different responses, but a 10 µg/ml
concentration was found to be optimum for a number of donors (data not
shown). The data in Figure 7A
demonstrate that PBMC from 7 of the 10 donors with prior exposure to
M. tuberculosis proliferated in response to rMtb 8.4, with
stimulation indices (SI) > 5. The mean proliferative response of the
PPD+ healthy PBMC to rMtb 8.4 was SI = 25.6. The
differences between the mean responses of PPD+ healthy
donors to the medium control and to the rMtb 8.4 Ag were statistically
significant (p < 0.05). All of the PPD
+ healthy donors responded to CF with an SI of >5, and
90% of these donors stimulated the PBMC with an SI of >10.
None of the 10 healthy PPD- donors responded to rMtb 8.4
(10 µg/ml) with an SI of >5 (Fig. 7A). The mean levels of
proliferative responses to rMtb 8.4 were significantly different in the
PPD- and PPD+ healthy donors
(p < 0.05).
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, as well as for TNF-, IL-5, and IL-10 production.
IFN- production by PBMC from PPD+ healthy individuals in
response to rMtb 8.4 varied between individuals (< 5–870 pg/ml), with
8 of the 10 PPD+ donor PBMC eliciting IFN- levels
> 10 pg/ml. The difference in mean levels of IFN- production in the
reactivity of PPD+ healthy donors to the medium control and
to the rMtb 8.4 was statistically significant by the paired Student’s
t test (p < 0.05). In contrast, this
cytokine was undetectable in the culture supernatants of cells from
PPD- individuals. A large variation in IFN- production
(11–1028 pg/ml) was also observed in the PPD+ PBMC
stimulated with CF (Fig. 7
B).
Concentrations of IL-5 were undetectable above background in the
culture supernatants of rMtb 8.4-stimulated PBMCs from PPD+
healthy donors (Fig. 8C).
Concentrations of IL-10 in supernatants of 10 µg/ml rMtb
8.4-stimulated PPD+ PBMCs were high (mean, 2135 pg/ml
± 80) compared with the levels of <1000 pg/ml reported in the
literature for M. tuberculosis-stimulated PBMC from healthy
tuberculin reactors (36, 37). The elicitation of IFN-, TNF-, and
IL-10 was shown to be dependent on the concentration of rMtb 8.4 used
to stimulate the PPD+ healthy PBMC (Fig. 8, A–C). Interestingly, the overall profile of
cytokine production (IFN-, TNF-, and IL-10) elicited by rMtb 8.4
was very similar to that observed for CF (data not shown). Mtb
8.4-specific TNF-, IL-5, and IL-10 cytokine production was
undetectable in PBMC from PPD- donors (data not shown).
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Immunization of C57BL/6 mice with rMtb 8.4 formulated in IFA
demonstrated that this M. tuberculosis protein is a potent
immunogen. SI of 8.9 to 45 were measured in popliteal lymph node cells
(Fig. 9A). Additionally,
IFN- elicitation was substantial and varied between 3 and 68 ng/ml
according to the concentration of rMtb 8.4 used for in vitro
restimulation. (Fig. 9B). The Th2 cytokine, IL-4, could not
be detected in any of the supernatants taken from lymph node cells
cultured with Mtb 8.4.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The antigenic repertoire of an outbred human population is both diverse
and heterogeneous. It is possible, nevertheless, that a few Ags could
elicit protective immunity against TB, particularly if the component T
cell epitopes demonstrate indiscriminate reactivity and evoke responses
with different host MHC. One of the requirements of a candidate vaccine
Ag, therefore, is recognition by the immune system during the course of
infection by the majority of individuals of the target population. To
fulfill this requirement, we used PBMC from a panel of ethnically
diverse, healthy, PPD+ donors who have no history of TB,
and the majority of whom have not been BCG immunized, to assess our
candidate M. tuberculosis Ags. The parameters used as
indicators of T cell responses included Ag-specific proliferation and
the measurement of IFN- production to identify what are likely to be
CD4+ lymphocytes of the Th1 phenotype that are considered
to play an important role in the response against virulent M.
tuberculosis.
Our preliminary screening has identified Mtb 8.4 as an Ag that is
recognized with T cell proliferation and IFN- production by 70% of
the donors tested. Given that IFN- can be produced by both T cells
and NK cells, it was interesting to observe correlation between
proliferation and IFN- levels, suggesting that the IFN- recorded
was T cell derived. Equally important was the finding that
PPD+ healthy donors who elicited proliferation SI <
10 to rMtb 8.4 also correlated with relatively low levels (<100 pg/ml)
of IFN- production, indicating that the former test was
representative of the active T cell repertoire. The ability of rMtb 8.4
to elicit cytokine production from PPD+ donors even at
extremely low Ag concentrations is impressive and suggests that at low
expression levels some Ags are able to trigger vigorous immune
responses.
When rMtb 8.4 was titrated and used to stimulate PBMC from
PPD+ donors, the production of IFN-, IL-10, and TNF-,
but not IL-5, were each found to be Ag dependent (Fig. 8). IL-10 is
produced by both of the Th cell subpopulations in humans (38, 39). Clinical and experimental data in animals and humans suggest,
likewise, that TNF- can play both a protective and immunopathologic
role in TB (40, 41, 42, 43, 44). Nevertheless, the overall pattern of cytokine
production, indicative of a CD4+ Th1-like pattern, appears
to be induced by rMtb 8.4.
Immunogenicity studies in mice have indicated that rMtb 8.4 induces a
substantial amount of Ag-specific proliferation (Fig. 9A).
Cytokine secretion patterns (IL-4 and IFN-) in mice have also
indicated that a Th1 lineage of cells develops upon stimulation with
rMtb 8.4, even in the presence of IFA, an adjuvant that is reported to
induce both IFN- and IL-4 from a mixed Th cell profile with M.
tuberculosis Ags (Fig. 9B). A central issue in the
broad spectrum of T cell cytokine responses produced in response to
M. tuberculosis and the outcome of infection is that Th1
cells are crucial for protection early in the disease process (45, 46).
Later on, during the chronic phase of the disease, a mixed T cell
profile is observed with concomitant production of IFN-, IL-4, and
IL-5 (47, 48, 49). Mechanisms by which containment of the disease
occurs is incompletely understood, but IFN- production by Th1
CD4+ T cells is considered essential. Given that rMtb 8.4
elicits high levels of IFN- without any IL-4, rMtb 8.4 may be
considered as a component in the development of a subunit M.
tuberculosis vaccine.
The distribution of Mtb 8.4 in M. tuberculosis substrains and other mycobacterial strains was examined in this study. Southern blot experiments demonstrated the presence of Mtb 8.4 in the M. tuberculosis substrains Ra, Rv, Erdman, and "C" strain, as well as in M. avium and M. bovis BCG Pasteur. The hybridization studies showed a difference in the genomic DNA restriction enzyme pattern only within the M. avium species. The difference observed could be the result of either different localization of Mtb 8.4 on the chromosome or a chromosomal mutation of the SalI site or a product of both possibilities. Given that Southern analysis has shown that Mtb 8.4 is present in M. bovis BCG, it was important that the majority of our donors had not previously received BCG vaccination, to ensure that the T cell responses measured were in recognition of relevant Ags encountered by the host immune system during previous infection with M. tuberculosis.
Like Mtb 8.4, various Ags such as ESAT-6 (17, 37), the Ag 85 complex (15, 49), and MPT64 (30, 31, 32), have been previously identified in viable mycobacteria and recorded to elicit T cell responses in animal models or from healthy M. tuberculosis-infected individuals, but not TB patients (49). The question remains whether Mtb 8.4 is also recognized only by individuals with latent infection vs those with active disease. Such a finding would argue that the host immune system develops a protective response against Mtb 8.4 and would determine its utility as an agent for differentiating the two infection outcomes.
This study has revealed that Mtb 8.4 is an immunoreactive T cell
Ag in individuals with latent M. tuberculosis infection.
Mtb 8.4 may play an important role in determining the outcome
of infection with pathogenic mycobacteria, given that it is one of the
Ags encountered by the host immune system during M.
tuberculosis infection and given that it elicits abundant
levels of the Th1 cytokine IFN-. As resistance to TB depends on
Ag-specific T cell activation of macrophages, and as the IFN-
pathway has been shown to be crucial in the human response to
mycobacterial infection, the elicitation of high levels of IFN- by
rMtb 8.4 in M. tuberculosis-sensitized donors is
significant.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Steven G. Reed, Infectious Disease Research Institute, 1124 Columbia Street, Suite 464, Seattle, WA 98104.
3 Abbreviations used in this paper: TB, tuberculosis; BCG, bacillus Calmette-Guerin; PPD, purified protein derivative; CF, culture filtrate; TFA, trifluoroacetic acid; SI, stimulation index.
Received for publication January 5, 1998. Accepted for publication April 30, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-receptor gene and susceptibility to mycobacterial infection. N. Engl. J. Med. 335:1941.-receptor deficiency in an infant with fatal bacille Calmette-Guerin infection. N. Engl. J. Med. 335:1956.-interferon, interleukin-4, and tumor necrosis factor. Infect. Immun. 58:2675.-mediated growth inhibition of Mycobacterium tuberculosis by human alveolar macrophages. J. Immunol. 152:743.[Abstract]
in tuberculous pleuritis. J. Immunol. 142:1114.[Abstract]
is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2:561.[Medline]
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