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Infection and Immunity, October 2000, p. 5731-5734, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Protective Antigen-Mediated Antibody Response
against a Heterologous Protein Produced In Vivo by Bacillus
anthracis
Fabien
Brossier,*
Martine
Weber-Levy,
Michèle
Mock, and
Jean-Claude
Sirard
Unité Toxines et Pathogénie
Bactériennes, Institut Pasteur, 75724 Paris Cedex 15, France
Received 17 April 2000/Returned for modification 10 June
2000/Accepted 22 July 2000
 |
ABSTRACT |
Bacillus anthracis secretes a lethal toxin composed of
two proteins, the lethal factor (LF) and the protective antigen (PA), which interact within the host or in vitro at the surfaces of eukaryotic cells. Immunization with attenuated B. anthracis
strains induces an antibody response against PA and LF. The LF-specific response is potentiated by the binding of LF to PA. In this study, we
investigated the capacity of PA to increase the antibody response against a foreign antigen. We constructed a chimeric gene encoding the
PA-binding part of LF (LF254) fused to the C fragment of tetanus toxin
(ToxC). The construct was introduced by allelic exchange into the locus
encoding LF. Two recombinant B. anthracis strains secreting
the hybrid protein LF254-ToxC were generated, one in a PA-producing
background and the other in a PA-deficient background. Mice were
immunized with spores of the strains, and the humoral response and
protection against tetanus toxin were assessed. The B. anthracis strain producing both PA and LF254-ToxC induced
significantly higher antibody titers and provided better protection
against a lethal challenge with tetanus toxin than did its PA-deficient counterpart. Thus, PA is able to potentiate protective immunity against
a heterologous antigen, demonstrating the potential of B. anthracis recombinant strains for use as live vaccine vehicles.
 |
INTRODUCTION |
Bacillus anthracis, a
spore-forming bacterium, is the etiologic agent of anthrax. The
bacterium secretes a lethal toxin composed of two proteins, the
protective antigen (PA, 83 kDa) and the lethal factor (LF, 85 kDa)
(4, 16). The pag and lef genes,
carried by the virulence plasmid pXO1 (185 kbp), encode PA and LF,
respectively (5, 20, 31). PA is the binding moiety of the
toxin, and LF is the intracellular enzyme that damages the cells. A
mode of action of the lethal toxin has been proposed from in vitro and
in vivo experiments (6, 17). PA binds to a ubiquitous receptor on the surface of mammalian cells and is cleaved by furin-like proteases (9, 14, 26). This processing results in the
release of a 20-kDa amino-terminal fragment, and the cell-associated
63-kDa fragment interacts with LF. The PA63-LF complex is then
internalized by receptor-mediated endocytosis, and at acidic pHs, LF is
translocated into the cytosol. LF displays zinc metalloprotease
activity specific for mitogen-activated protein kinase kinases 1 and 2 (8, 15, 30). The amino-terminal part of LF (LF254) binds to
PA63 (25). Fusion proteins consisting of LF254 and
heterologous antigens have been shown to be successfully delivered to
cells via PA (1, 2, 3).
The B. anthracis Sterne strain, which is attenuated, is
currently used as the live veterinary vaccine against anthrax. The immunity induced by this live vaccine is associated with stimulation of
a humoral response to the toxin components. Studies performed with
toxin-deficient B. anthracis strains (24) have
shown that the antibody response specific for LF is dependent on the
presence of PA. The level of LF-specific antibodies is much higher in
animals immunized with bacteria producing both PA and LF than in
animals receiving bacteria producing LF only. The molecular mechanisms underlying this adjuvant effect of PA have been further investigated. Strains carrying site-directed mutations in the functional domains of
toxin genes have been constructed (6). Analysis of their immunogenic properties in mice clearly indicated that potentiation of
the LF-specific antibody response requires only the binding of LF to
PA. Neither the biological activity of LF nor binding of the PA63-LF
complex to the cell receptor is involved in this phenomenon.
The successful in vivo delivery of heterologous antigens by B. anthracis has been reported. Strains producing listeriolysin O, a
hemolysin from Listeria monocytogenes, or the Ib component of iota toxin from Clostridium perfringens, which is under
the control of the pag promoter, elicit specific immune
responses and protection in mouse models (27, 28).
We investigated the ability of PA to enhance the humoral response
against a heterologous antigen fused to LF254 when delivered in vivo by
B. anthracis. We used the C fragment of tetanus toxin (ToxC). Tetanus toxin (150 kDa) is composed of a light chain (50 kDa)
carrying the catalytic site and a heavy chain (100 kDa) containing domains involved in cell binding and translocation of the toxin into
the cytoplasm (13, 21). ToxC (50 kDa) is part of the heavy
chain and corresponds to the cell-binding part of tetanus toxin
(12, 22). ToxC has been produced in various live vectors including Salmonella enterica serovar Typhi, Vibrio
cholerae, and Lactoccocus lactis (7, 11,
32). Analysis of the antibody response against ToxC secreted by
the recombinant bacteria suggested that this protein is only weakly
immunogenic. Recently, ToxC has been successfully anchored to the
surface of B. anthracis vegetative cells (19).
However, no antibody response against ToxC was observed after a single
injection of bacilli, even in the presence of adjuvant, and several
injections were required for both antibody response and protection.
Protection against tetanus is known to be antibody mediated
(10). This fact and the weak immunogenicity of ToxC in
heterologous systems make this antigen particularly suitable as a model
for analysis of the ability of PA to exert its adjuvant effect on
foreign antigens. In this study, recombinant B. anthracis strains that produced LF254-ToxC protein in a PA-producing or PA-deficient background were constructed. The capacity of the recombinant strains to stimulate a humoral response and protective immunity against tetanus toxin were analyzed.
 |
MATERIALS AND METHODS |
Bacterial strains and media.
Escherichia coli and
B. anthracis strains were cultured in Luria-Bertani and
brain heart infusion (BHI) (Difco, Detroit, Mich.) media, respectively.
Ampicillin (100 µg/ml), spectinomycin (60 µg/ml), and erythromycin
(5 µg/ml and 180 µg/ml for B. anthracis and E. coli, respectively) were added as appropriate. The B. anthracis Sterne (7702) strain and the RPL strains have been
described previously (6).
Construction of the hybrid gene lef-toxC.
A
MluI restriction site was created after the codon encoding
leucine 254 in the lef gene in the recombinant plasmid
pUC1835, using the Quickchange site-directed mutagenesis kit
(Stratagene, La Jolla, Calif.), giving rise to pLE254 (1, 5,
23). The DNA fragment encoding ToxC was amplified by PCR using
the primers toxc1
(5'-CGACGCGTTCAACACCAATTCCATTTTCTTATTC-3'),
containing an MluI restriction site (underlined), and
toxc2
(5'-CATGCCATGGTCATGAACATATCAATCTGTTTAATC-3'), containing an NcoI restriction site. Total plasmid DNA
from Clostridium tetani CN655 (kindly provided by M. R. Popoff) was used as the template. pLE254 and the 1,420-bp PCR fragment
were cleaved with MluI and NcoI and ligated, and
the resulting plasmid, pTET2541, was used to transform E. coli. Finally, the SacI-SphI fragment of
pTET2541 was ligated into the gram-positive bacterium suicide vector
pAT113 (29), giving rise to pBF254.
Mating procedure.
The B. anthracis mutant strains
were constructed by heterogramic mating, as previously described
(23, 29). The wild-type lef gene was replaced
with the fusion gene in two steps as previously described
(6). First, pBF254, which is resistant to erythromycin, was
introduced into the lef-inactivated strain, RPL, which
carries a spectinomycin resistance cassette in the lef gene
(6). We selected clones in which pBF254 had been integrated
into pXO1 by a single crossover event on agar containing both
erythromycin and spectinomycin. The resulting merodiploids were grown
in 5 ml of fresh BHI medium inoculated with 5 µl of a culture with an
optical density at 600 nm of 0.8. After seven subcultures, 20% of the
bacteria were sensitive to the marker antibiotics and harbored the
hybrid gene under the control of the lef promoter (Fig.
1A). One of the resulting clones, RPL254,
was used to construct its PA-deficient counterpart, RPA254. The suicide
plasmid pBAFH114, harboring a deleted pag gene inactivated
by a spectinomycin resistance cassette, was used for the mating
(27). The RPA254 strain was obtained by allelic exchange
between the wild-type pag gene on pXO1 from RPL254 and the
deletion-containing pag gene of pBAFH114. The two
recombinant strains were checked by PCR, and the junction between
lef and toxC was sequenced (Sequenase; Amersham,
Cleveland, Ohio).

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FIG. 1.
Schematic representation of the hybrid gene and Western
blot analysis of the recombinant strains. (A) Schematic representation
of the hybrid gene, lef-toxC, at the lef locus on
pXO1. plef corresponds to the promoter of the lef
gene. The MluI restriction site adds two codons encoding Thr
and Arg at the junction of the fusion protein. The open reading frame
is indicated by arrows. (B) In vitro production of LF254-ToxC by
recombinant B. anthracis strains. The recombinant strains,
RPL254 and RPA254, were grown in BHI medium in the presence of
bicarbonate to stimulate toxin production. Supernatants from 500 µl
of culture were analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and Western blotting with anti-LF and anti-PA
monoclonal antibodies and with rabbit sera specific for ToxC.
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|
Detection of B. anthracis toxin components.
Recombinant B. anthracis was grown in BHI medium
supplemented with 0.4% (wt/vol) bicarbonate at 37°C in tightly
closed flasks to an optical density at 600 nm of 0.7 (27).
The cultures were centrifuged (13,000 × g, 5 min), and
proteins were precipitated from the supernatants with 10%
trichloroacetic acid and subjected to electrophoresis in a 12%
polyacrylamide gel. The proteins were either stained with Coomassie
blue or transferred to a nitrocellulose membrane (Hybond-C; Amersham).
For immunoblot analysis, monoclonal antibodies specific for PA and LF
and polyclonal serum specific for ToxC were used at a dilution of
1/10,000. A secondary antibody conjugated to peroxidase was added at a
dilution of 1/20,000. Proteins were detected by adding luminol (ECL
kit; Amersham).
In vivo experiments.
Seven-week-old female OF/1 outbred mice
(Iffa Credo, l'Arbresle, France) were immunized by subcutaneous
injection with 3 × 108 to 5 × 108
spores per animal (in a volume of 500 µl). After 35 days, serum samples were obtained from the retro-orbital plexus as previously described (24). If necessary, a booster of 3 × 108 spores per mouse was given at 35 days and sera were
obtained on day 50. The titer of antibody specific for the tetanus
toxin was measured by enzyme-linked immunosorbent assay (ELISA) using tetanus anatoxin (a formaldehyde-inactivated protein) at 25 µg/ml to
coat the microplates. ELISA for PA-, LF- and extractable antigen 1 (EA1)-specific-antibody titers were performed as previously described
(24). An arbitrary A492 value of 0.5 was used to calculate the endpoint titers. However, for PA, LF, EA1,
and ToxC, the A492 value obtained with sera of
untreated animals diluted 1/100 never exceeded 0.02. For protection
experiments, mice were challenged intramuscularly, 35 days after the
first or 15 days after the second injection, with 4 ng of purified
tetanus toxin per animal (30 50% lethal doses [LD50], in
a volume of 250 µl). Mortality and morbidity (paralysis) were
monitored for 10 days after challenge.
Neutralization assays.
Sera from animals immunized with the
RPL254 and RPA254 strains were pooled separately and heated for 30 min
at 56°C. For each mouse, 1 ng of tetanus toxin (eight
LD50) was incubated for 2 h at room temperature with
diluted serum (final dilutions from 1:10 to 1:40). The mixture was then
injected intramuscularly into naive mice (four animals per dilution)
(28). Neutralizing activity was scored as the highest
dilution at which all animals survived. Surviving animals were killed
10 days after challenge.
Statistics.
Statistical analyses were performed using
Student's unpaired t test for antibody titers and
2 for protection assessment.
 |
RESULTS AND DISCUSSION |
Characterization of recombinant B. anthracis strains
harboring the chimeric gene, lef-toxC.
The hybrid gene,
lef-toxC, was introduced by homologous recombination into
the lef gene on pXO1 of the Sterne strain, as described in
Materials and Methods (Fig. 1A). The resulting recombinant strain,
RPL254, was used as a recipient for the construction of its
PA-deficient counterpart. The wild-type pag gene was
replaced by allelic exchange with an inactivated copy carrying a
spectinomycin resistance cassette (6) to produce the RPA254
strain. The hybrid gene, lef-toxC, is under the control of
the in vivo inducible lef gene promoter. The recombinant
B. anthracis strains were then grown under conditions known
to induce the synthesis of PA and LF in vitro (27). The
secreted proteins were analyzed by immunoblotting using antibodies
specific for PA, LF, and ToxC (Fig. 1B). As expected, PA was found
exclusively in the supernatants of the parental Sterne strain and of
RPL254. LF-specific antibodies detected an 85-kDa band in the
supernatant of the Sterne strain, corresponding to the full-length LF
molecule. In contrast, the recombinant strains RPL254 and RPA254
secreted a 78-kDa protein which reacted with antibodies against LF and
ToxC. The size of this protein was as expected for the hybrid
LF254-ToxC molecule. The two recombinant strains also appeared to
produce similar amounts of LF254-ToxC.
Antibody response to tetanus toxin induced by B. anthracis strains.
Mice were injected subcutaneously with
one or two doses of RPL254 or RPA254 spores, and serum-specific
antibodies were analyzed by ELISA (Fig.
2). The in vivo development of the RPL254
and RPA254 strains was monitored by the antibody response raised
against EA1, one of the two S-layer proteins which are exclusively
found at the surface of vegetative forms of B. anthracis
(18). Titers of antibody against EA1 thus reflect the
germination of spores and the development of bacilli within mice. The
humoral responses obtained with the two strains were similar (1/12,800)
and close to those routinely obtained following immunization with
B. anthracis strains (6). The in vivo development
of the spores was therefore similar for the two LF254-ToxC producing
strains. The antibody response (1/10,600) against PA in animals
injected with RPL254 was similar to that usually found after the
injection of PA-producing strains. An antibody response to the tetanus
toxin was detected after the first injection for both recombinant
strains (Fig. 2). However, the antibody titers elicited by the
PA-producing strain were 8 times higher than those for the PA-deficient
strain (1/1,700 versus 1/200; P < 0.05). Booster
injections of RPL254 and RPA254 stimulated a secondary immune response,
as observed by the increase in antibody titers (1/5,000 versus 1/1,000,
respectively). These data clearly indicate that in vivo, LF254-ToxC is
efficiently produced and presented to the immune system. Moreover, it
is established that LF254 is the region involved in the binding of LF
to PA63 (1, 2, 3, 25). This property allows the formation of the PA63-LF254-ToxC complex. It is likely that the higher stability of
the complex in vivo accounts for the immunostimulatory effect of PA
(6). The data also provide evidence that PA is able to potentiate a humoral response against a heterologous antigen secreted by B. anthracis. This effect was clearly demonstrated by the
detection of an antibody response to ToxC after single-spore
immunization, in contrast to results reported for other delivery
systems (7, 19, 32). The adjuvant property of PA is thus of
particular interest for the delivery in vivo of proteins that are only
weakly immunogenic.

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FIG. 2.
Serum titers of antibody against tetanus toxin in mice
immunized with recombinant B. anthracis. Animals were
immunized once or twice with RPL254 or RPA254 spores. Tetanus
toxin-specific antibodies were determined by ELISA. Circles represent
individual titers, and the black bar represents the arithmetic mean of
the group. The strains used for immunization are indicated at the
top.
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|
Protective immunity against tetanus toxin induced by recombinant
strains.
The ability of recombinant B. anthracis to
protect mice against a challenge with the tetanus toxin (30 LD50) was assessed (Fig. 3).
All control animals died within 2 days. A single immunization was
sufficient to protect 75 to 90% of mice injected with the RPL254
strain whereas, under the same conditions, only 11 to 20% of animals
injected with the RPA254 strain were protected (Fig. 3A). After the
booster, the level of protection was 100 and 80% for mice immunized
with the RPL254 and RPA254 strains, respectively (Fig. 3B).

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FIG. 3.
Efficacy of protection against lethal tetanus toxin
challenge. Mice were immunized by a single injection (A) or two
injections (B) of RPA254 ( ) or RPL254 ( ) spores. Untreated
animals were used as controls (×). Animals were challenged with 30 LD50 of tetanus toxin, and mortality was monitored over 10 days.
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The toxin-neutralizing activity of sera from mice immunized with the
recombinant strains was analyzed (Table
1). Tetanus toxin and sera were mixed and
injected intramuscularly into naive mice. A 1/20 dilution of sera from
mice immunized twice with the RPL254 strain (titer of antibody against
ToxC, 1/5,000) fully neutralized the lethal effect of tetanus toxin and
protected mice against the toxin challenge. In contrast, no protection
was observed, even with a 1/10 dilution, with sera from untreated mice
or mice immunized once with RPA254 or RPL254, or twice with RPA254.
However, mice immunized once with RPL254 or twice with RPA254 died
later than those immunized once with RPA254. Therefore, under our
experimental conditions, a tetanus toxin-specific antibody titer of
1/5,000 was required for full neutralization.
The data obtained in active and passive protection experiments clearly
indicate a tight correlation between antibody titer and protection
against tetanus toxin. These results are consistent with studies
showing that protection against tetanus is exclusively mediated by
antibodies and that ToxC itself harbors protective epitopes
(10).
In this work, the presentation of LF254-ToxC to the immune system via
PA seemed to be an efficient strategy for inducing (i) a humoral
response against ToxC without booster or adjuvant and (ii) protection
against tetanus toxin after a single injection. Recombinant strains of
B. anthracis producing foreign antigens under the control of
the pag gene promoter have already proved successful as
vaccines (27, 28). Moreover, the genetic constructs are
stable on the resident plasmid pXO1. This work thus extends the
possibility of using B. anthracis as a live vaccine vehicle.
 |
ACKNOWLEDGMENTS |
We thank M. R. Popoff for the gift of total plasmid DNA from
C. tetani CN655 and A. Fouet for critical reading of the manuscript.
F.B. was supported by the Ministère de l'Enseignement
supérieur et de la Recherche.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité
Toxines et Pathogénie Bactériennes (URA 1858, CNRS),
Institut Pasteur, 28, rue du Dr. Roux, 75724 Paris Cedex 15, France.
Phone: (33) 1.45.68.82.59. Fax: (33) 1.45.68.89.54. E-mail:
fbrossie{at}pasteur.fr.
Present address: Institut Suisse de Recherche Expérimentale
sur le Cancer, 1066 Epalinges, Switzerland.
Editor:
W. A. Petri Jr.
 |
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Infection and Immunity, October 2000, p. 5731-5734, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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