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Previous Article | Next Article 
Infect Immun, April 1998, p. 1648-1653, Vol. 66, No. 4
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Recombinant Live Attenuated Strain of Vibrio
cholerae Induces Immunity against Tetanus Toxin and
Bordetella pertussis Tracheal Colonization
Factor
Inês
Chen,1,2
Theresa M.
Finn,3
Liu
Yanqing,4
Qi
Guoming,4
Rino
Rappuoli,1,* and
Mariagrazia
Pizza1
IRIS, Chiron Vaccines Immunobiological
Research Institute in Siena, 53100 Siena,
Italy1;
Departamento de
Microbiologia, ICB II, Universidade de São Paulo, São
Paulo, SP 05508-900, Brazil2;
Laboratory
of Pertussis, Center for Biologics Evaluation and Research, Food
and Drug Administration, Bethesda, Maryland
20892-00293; and
Chinese Academy of
Preventive Medicine, Beijing, China4
Received 2 September 1997/Returned for modification 25 October
1997/Accepted 7 December 1997
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ABSTRACT |
An attenuated strain of Vibrio cholerae was used as a
carrier for the expression of heterologous antigens such as fragment C
from tetanus toxin (TetC) and tracheal colonization factor from Bordetella pertussis (Tcf). In vitro, high levels of
protein were obtained when the Escherichia coli nirB
promoter was used and the bacteria were grown with low aeration.
Intranasal immunization of mice with IEM101 expressing TetC elicited
serum vibriocidal activity and induced antibodies against tetanus toxin
which were protective against lethal challenge with 10 times the 50%
lethal dose of tetanus toxin. Bacterial viability was essential for the induction of anti-TetC antibodies. Intranasal administration
of IEM101 expressing Tcf induced a significant reduction in
bacterial colonization of the tracheas of mice challenged with
wild-type B. pertussis. These data are in
agreement with the putative role of Tcf in Bordetella
tracheal colonization. In conclusion, we have demonstrated that
V. cholerae may be used as a live vector to deliver
heterologous antigens in vivo and that protection to both systemic
and local challenge may be achieved.
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INTRODUCTION |
Vibrio cholerae is the
causative agent of cholera, a human diarrheal disease with high rates
of morbidity and mortality in developing countries. Infection occurs
after ingestion of contaminated water or food; the bacteria reach the
small intestine, where they penetrate the mucous layer, adhere to
epithelial cells, multiply, and produce cholera toxin. This powerful
toxin induces secretion of chloride ions by enterocytes, leading to a
severe watery diarrhea (3).
Naturally acquired infection with V. cholerae
efficiently stimulates immunity, leading to long-term protection
against the disease (24). In the last years, live attenuated
oral vaccines against V. cholerae have been developed
(9, 25, 30, 35). A naturally attenuated strain of
V. cholerae O1 El Tor, Ogawa, isolated in China
(IEM101) has been also described (26). IEM101 has been
tested for safety and immunogenicity in a human clinical study, which
showed that the strain is safe and able to colonize the intestinal
mucosa and to induce a strong immune response, eliciting high levels of
vibriocidal and antilipopolysaccharide antibodies in the sera of human
volunteers. Due to these characteristics and to the noninvasive nature
of V. cholerae infection, attenuated strains of
V. cholerae may be good candidates for the delivery of
foreign antigens to the host.
Previous work on heterologous expression in V. cholerae
has used antigens from other enteropathogens, such as Shigella
sonnei lipopolysaccharide, Escherichia coli Shiga toxin
B subunit, and enterohemorrhagic E. coli EaeA (1, 4, 6,
13, 36). In this study, we have investigated whether the
attenuated strain IEM101 is able to express antigens from nonenteric
pathogens and whether these recombinant strains induce protective
immunity to systemic and mucosal challenge.
The choice of an animal model to evaluate immune responses induced by
recombinant V. cholerae strains is a critical step, since natural infection with V. cholerae does not occur
in animals. Various animal models have been used in cholera research
(33). Infant mice or rabbits are susceptible to infection
for a short time after birth; however, given the immature immune
system of neonatal animals, they do not constitute a good model for
immunogenicity studies. Colonization by V. cholerae has been accomplished in adult rabbits, with
tincture of opium used to induce paralysis of intestinal motility, but
this model presents some limitations in terms of animal handling. It
has been recently described that germfree mice are readily
colonized by V. cholerae after oral inoculation
(5); however, these animals are usually more expensive than
nongermfree ones.
The intranasal route has been shown to be highly efficient for the
induction of immune responses, at the systemic and mucosal levels, with
a variety of antigens delivered, either with mucosal adjuvants
(10, 11, 37) or microcapsules (21). This
route has also been used to deliver live Salmonella
typhimurium (19), Bordetella pertussis
(32), Mycobacterium bovis BCG (22),
and even Salmonella typhi, which also lacks a practical
small-animal model (2, 16).
In this study we have used intranasal immunization as an alternative
mucosal route. Fragment C (TetC) from tetanus toxin (TT) and tracheal
colonization factor (Tcf) from B. pertussis were used as
model antigens. TetC is the nontoxic 50-kDa C-terminal portion of TT
(18); it is immunogenic and able to protect against challenge with the toxin when either administered as purified immunogen
(12) or delivered by attenuated Salmonella
(8). Tcf is a virulence-associated factor secreted by
B. pertussis, and tcfA mutants are impaired in
their ability to colonize the mouse trachea after aerosol infection
(15). Tcf is produced as a cell-associated precursor form,
with an apparent molecular mass of 90 kDa, which is processed to
release the 60-kDa form.
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MATERIALS AND METHODS |
Strains, media, and growth conditions.
E. coli DH5
was used for cloning purposes. V. cholerae IEM101 was
grown in Luria broth or Luria broth with ampicillin (100 µg/ml) when
required. B. pertussis BP18323 Smr was grown in
Stainer-Scholte (SS) modified medium (34) or on Bordet-Gengou (BG) agar (Difco) with 20% defibrinated sheep blood and
streptomycin (40 µg/ml). Transformation of IEM101 was performed by
electroporation as previously described (17). Growth of
bacterial cultures under low-aeration conditions was achieved by
inoculation of 2 ml from an overnight culture into a completely filled,
tight-capped 50-ml Falcon tube and subsequent incubation at 37°C for
3.5 to 4 h. To recover IEM101 from organ homogenates, TCBS agar
(Difco) was used.
Plasmids and constructions.
Plasmid pTETnir15 (obtained from
G. Dougan, Imperial College, London, United Kingdom) contains the
coding sequence for TetC under the control of the E. coli
nirB modified promoter, which is induced under anaerobic
conditions (29).
tcfA was amplified from the B. pertussis BP18323
Smr chromosome by using Taq DNA polymerase
(Boehringer Mannheim). The forward primer was oligonucleotide Tcf1
(5'-ACTAGTGATCATATGCACAATTTACGGAAATA-3'), which contains the
initial codon of tcfA, comprised within the NdeI
site, and an upstream BclI site; the reverse primer was
oligonucleotide Tcf2 (5'-GTCTAGAATTCTACCAGGCGTAGCGATACC-3'),
containing the stop codon of the tcfA gene and an
EcoRI site downstream. The amplification product was
digested with BclI and EcoRI and cloned into
pBlueScript/KS+ (Stratagene) between EcoRI and
BamHI sites, giving origin to pBS-tcf. The cloned fragment
was completely sequenced, and one mutation, leading to a conservative
amino acid substitution (Met to Thr) in position 494, probably due to
Taq DNA polymerase inaccuracy, was found.
tcfA was placed under the control of the cholera toxin gene
promoter (pctx) as follows: a 190-bp sequence located
immediately upstream to the ctx structural region was
amplified from plasmid pJM17, which contains the virulence cassette
region from V. cholerae classical strain 569B
(31). Oligonucleotides Pct1
(5'-TAGATCTAGATACCTTTGCAGCGCAAGG-3') and Pct2
(5'-TATCTTTACCATATGATGCTCCC-3') were used as forward and
reverse primers, respectively; Pct1 contains an XbaI site at
its 5' extremity, whereas Pct2 corresponds to the reverse sequence of
the ctx gene, with the initiation codon (reverse) within an NdeI site. The amplified fragment was digested with
XbaI and NdeI and cloned into pBS-tcf digested
with the same enzymes, generating pCT-tcf.
Subcloning tcfA downstream of the nirB promoter
from pTETnir15 was achieved in two steps. First, pTETnir15 was digested
with BglII and BamHI, liberating a fragment
containing the coding region for TetC and ca. 30 bp of the 5'
untranslated region, including the putative ribosomal binding site
sequence. In order to reconstitute this region, which could be
important for RNA stability and/or efficient translation initiation,
the fragment containing the vector sequence was ligated to
oligonucleotides PnirUTR1
(5'-GATCTTAATCATCCACAGGAGACTTTCATATGATATCTAGATGCATC-3') and
PnirUTR2
(5'-G ATCGATGCATCTAGATATCATATGAAAGTCTCCTGTGGATGATTAA- 3'),
corresponding to the 5' untranslated region, with an
NdeI site comprising the initiation codon, and an
XbaI site immediately downstream. This plasmid was called
pNIR100; it was digested with XbaI, filled in with Klenow
fragment from E. coli DNA polymerase I, digested with
NdeI, and ligated to the NdeI-EcoRV
fragment from pBS-tcf, which contains the tcfA gene,
generating pNIR-tcf.
Analysis of protein expression and localization.
Expression
of TetC was analyzed by sodium dodecyl sulfate-12.5% polyacrylamide
gel electrophoresis (SDS-12.5% PAGE) and immunoblotting of whole-cell
lysates, using mouse anti-TT antiserum at a dilution of 1:2,000
(obtained from S. Peppoloni, IRIS, Siena, Italy).
Tcf expression was probed by immunoblotting, using mouse antiserum
raised against the N-terminal portion of Tcf fused to MalE (15) at a dilution of 1:8,000. Culture supernatant (1 ml)
from B. pertussis or V. cholerae was
trichloroacetic acid precipitated and loaded on an SDS-polyacrylamide
gel. B. pertussis whole-cell lysate was prepared by
pelleting bacteria from a liquid culture with an optical density at 590 nm (OD590) of 1.0 and resuspension in 1× sample boiling
buffer in order to have approximately 5 × 109
cells/ml. B. pertussis outer membrane-associated proteins
(OMAP) were prepared by resuspending cells in 50 mM Tris-HCl (pH
8.0)-150 mM NaCl to a final concentration of 20 OD units/ml; the
suspension was incubated at 60°C for 1 h with gentle shaking;
intact cells were pelleted by centrifugation, and the supernatant,
containing the OMAP, was used for SDS-PAGE. Whole-cell lysates of
IEM101 were prepared by pelleting cells from liquid cultures and
resuspending them in sample boiling buffer to a final concentration of
approximately 1010 cells/ml; the V. cholerae outer membrane fraction was prepared from 109
cells, as previously described (14).
FACScan analysis was used to probe Tcf exposure on the bacterial cell
surface. Approximately 106 cells were pelleted and kept on
ice through the procedure. Cells were washed with phosphate-buffered
saline (PBS)-2% bovine serum albumin (BSA) and incubated for 1 h
with serial dilutions of anti-MalE-Tcf mouse antisera in 200 µl of
PBS-2% BSA; mouse preimmune serum was used as a negative control.
Bacterial cells were washed twice with PBS-2% BSA and incubated for
30 min with the appropriate dilution of R-phycoerythrin-labeled
F(ab')2 goat anti-mouse immunoglobulin G (IgG) antiserum.
Cells were subsequently washed twice with PBS-2% BSA and resuspended
in PBS, and 104 bacterial cells were analyzed for
cell-bound fluorescence with a FACScan flow cytometer (Becton
Dickinson, Mountain View, Calif.), using the Lysis II software program
from Becton Dickinson. The threshold of positivity was set for each
experiment by flow cytometric analysis of IEM101 previously incubated
with antisera to MalE-Tcf and with the R-phycoerythrin-labeled
secondary antibody.
Immunizations.
Female BALB/c mice (6 to 8 weeks old; Charles
River, Calco, Italy) each received 30 µl of a bacterial suspension in
PBS by the intranasal route, either without anesthesia or anesthetized with 0.2 ml of a mixture of 15% xylazine hydrochloride (Rompun) and
10% ketamine hydrochloride (Ketavet), on days 0, 28, 42, and 56. Animals were bled on days 27, 35, 49, and 63 and challenged at day 70. Nasal lavages were performed at day 70 by repeated flushing and
aspiration with 1 ml of PBS containing 0.1% BSA (Sigma).
Titration of antigen-specific serum antibodies.
Vibriocidal
activity was assayed by incubating 107 CFU of IEM101 with
20% rabbit serum as the complement source and serial dilutions of
immunized animal sera in 100 µl of PBS in 96-well tissue culture
plates (Costar), for 1 h at 37°C; 100 µl of brain heart
infusion (BHI) (Difco) was then added to each well, and plates were
further incubated for 1 h at 37°C; absorbance at 570 nm was then
measured. Titers were calculated as the dilution of the serum that gave
50% growth inhibition compared to preimmune serum diluted 1:25.
Titers of anti-TetC antibodies were determined by enzyme-linked
immunosorbent assay (ELISA). Plates were coated overnight at 4°C with
200 ng of tetanus toxoid (formaldehyde-inactivated TT) in 100 µl of
PBS/well. After three washes with PBS-0.05% Tween 20 (PBS-T), plates
were blocked with 1% BSA in PBS-T (200 µl/well) for 2 h at
37°C, washed three times with PBS-T, and incubated with serial
dilutions of sera from immunized animals in PBS-T-0.5% BSA for
1.5 h at 37°C; plates were then incubated with PBS-T-0.5% BSA
containing alkaline phosphatase-conjugated anti-mouse IgG antibody
(Sigma) for 1.5 h at 37°C. Bound antibodies were revealed by
using p-nitrophenylphosphate as a substrate (Sigma). Titers were calculated as the dilution that gave 2.5 times the absorbance (OD405) of preimmune serum diluted 1:100.
Mucosal IgA titers were measured by using biotin-conjugated goat
anti-mouse IgA (-chain specific; Sigma) followed by
streptavidin-horseradish peroxidate conjugate (Dako). Bound antibodies
were revealed by using o-phenylendiamine as a substrate.
Titers were determined as the dilution that gave 2.5 times the
absorbance (OD490) of nasal washes of nonimmunized animals.
TT challenge.
Mice were challenged subcutaneously with 10 times the 50% lethal dose (10 × LD50) of TT. Paralysis
and death were recorded for 7 days after the challenge.
Bordetella intranasal challenge.
B.
pertussis 18323 Smr was grown for 2 days on a BG plate
and then inoculated into 100 ml of SS modified medium; the culture was
grown until it reached an OD590 of 0.5. Bacteria were then diluted in PBS to a concentration of 3.3 × 108
CFU/ml; the concentration was confirmed by serial dilution and plating.
Animals received 30 µl of the bacterial suspension (corresponding to
approximately 106 CFU) intranasally, under light
anesthesia. Colonization of the trachea and lungs was monitored by
counting CFU recovered from organ homogenates.
| |
RESULTS |
Expression of TetC in IEM101.
The plasmid pTETnir15, which
contains the gene coding for TetC under the control of the modified
E. coli nirB promoter, was electroporated into V. cholerae IEM101. Total cell extracts were prepared from both
IEM101 and IEM101(pTETnir15) strains and analyzed by Western
blotting. As shown in Fig. 1,
transformants produced TetC, and expression was induced when bacteria
were grown with low aeration, indicating that the nirB
promoter was functional in IEM101.
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FIG. 1.
TetC expression by IEM101. Whole-cell lysates
(corresponding to approximately 2.5 × 108 CFU of
bacteria) were fractionated by SDS-PAGE and probed with anti-TT mouse
polyclonal antiserum. Purified TetC (100 ng) (lane a) was used as a
standard. IEM101 was grown under aerobic conditions (lane b) or
low-aeration conditions (lane c); the same conditions were used for
growing IEM101(pTETnir15) (lane d, aerobic; lane e, low
aeration).
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Expression and localization of tcfA in IEM101.
tcfA was subcloned under the control of the ctx
promoter (pCT-tcf) and nirB promoter (pNIR-tcf); the
recombinant plasmids were electroporated into IEM101. Total cell
extracts were prepared from each of the recombinant strains, and the
levels of Tcf were visualized by immunoblotting. The results are shown
in Fig. 2.
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FIG. 2.
Tcf expression by IEM101 under control of different
promoters. Whole-cell lysates (corresponding to approximately 5 × 108 CFU of IEM101 or 5 × 107 CFU of
BP18323) were fractionated by SDS-PAGE and probed with anti-Tcf
antiserum. Samples shown are BP18323 (lane a), IEM101 (lane b),
IEM101(pCT-tcf) (lane c), IEM101(pNIR-tcf) grown under aerobic
conditions (lane d), and IEM101(pNIR-tcf) grown with low aeration
(lane e).
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Tcf production was observed when either the ctx or
nirB promoters were used (Fig. 2, lanes c and d,
respectively). The highest level of expression was achieved when the
pnirB construct was induced by growth with low aeration
(Fig. 2, lane e).
In order to analyze Tcf localization in the bacteria, we performed
immunoblotting experiments using different cellular fractions, and the
results are reported in Fig. 3. In
B. pertussis, Tcf could be found in the culture supernatant
(Fig. 3, lane b), confirming previously reported data (15),
and in the OMAP preparation (Fig. 3, lane a), which indicates that it
is loosely associated with the outer membrane. In V. cholerae, Tcf localized in the outer membrane fraction (Fig. 3,
lane d), although small amounts could be detected in the culture
supernatant (Fig. 3, lane e). Tcf appears to be processed at a
different site in V. cholerae, since the molecular mass
of the protein appears to be less than that of the 60-kDa form detected
in Bordetella extracts and supernatant (Fig. 3, lanes a and
b, respectively).
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FIG. 3.
Tcf localization in IEM101. OMAP preparations from
B. pertussis 18323 (from approximately 2 × 108 CFU), outer membrane protein fractions from IEM101
(from approximately 109 CFU), and trichloroacetic
acid-precipitated culture supernatant (corresponding to 1 ml) were
loaded on SDS-polyacrylamide gels and probed with anti-Tcf antiserum.
Samples shown are B. pertussis 18323 OMAP (lane a),
B. pertussis 18323 culture supernatant (lane b), IEM101
outer membrane fraction (lane c), IEM101(pNIR-tcf) outer membrane
fraction (lane d), and IEM101(pNIR-tcf) culture supernatant (lane
e).
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Flow cytometry analysis was performed to verify whether Tcf was exposed
on the V. cholerae surface. The percentage of cells emitting fluorescence was 31% in the case of B. pertussis
and 14% in the case of IEM101(pNIR-tcf), suggesting that the
protein is less exposed in V. cholerae than in B. pertussis (data not shown).
Intranasal administration of V. cholerae.
The
intranasal route was chosen to deliver V. cholerae to
mice since it has been reported to be more efficient than the oral route in inducing an immune response against foreign antigens expressed
by live recombinant bacterial strains (16, 19).
Bacterial administration conditions were defined. Animals under
anesthesia could receive intranasally up to 5 × 107
CFU without mortality. A higher bacterial dose (5 × 108 CFU) could be delivered either by inoculation of
nonanesthetized animals or by using heat-inactivated bacteria. Both
treatments resulted in similar serum vibriocidal activity in both
groups, as presented in Fig. 4. The
titers in mice inoculated with live bacteria were slightly higher than
those in mice inoculated with heat-inactivated bacteria, but the
difference was not statistically significant.
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FIG. 4.
Serum vibriocidal activities. Serum vibriocidal
activities in BALB/c mice after receiving one to four doses of 5 × 108 bacteria, either heat inactivated (A) or live (B),
with six animals per group, are presented. Results are shown as
individual values of log10 vibriocidal activity titers.
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After one intranasal administration of 5 × 108 CFU
IEM101(pTETnir15) to nonanesthetized animals, bacterial persistence
in trachea, lung, small intestine, and stool was analyzed. The results,
reported in Fig. 5, show that viable
bacteria could be recovered from mouse tracheas 24 h after
inoculation, and furthermore, most of the recovered bacteria could be
cultured on ampicillin plates, showing that bacteria had not lost the
plasmid. However, only some of the animals presented bacteria in their
lungs (not shown). Bacteria could also be recovered from small
intestine and stool, possibly due to the animals' swallowing inoculum
during administration. No bacteria were detected in any organ after
72 h.
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FIG. 5.
IEM101(pTETnir15) persistence in vivo. BALB/c mice
received intranasally 108 CFU of IEM101(pTETnir15)
(dose indicated by arrow on y axis); the presence of
bacteria in the mouse trachea was assessed after 2, 24, and 72 h,
with three animals sacrificed per time point. Bacterial counts were
performed on TCBS agar plates, with (TCBS-Ap) or without (TCBS)
ampicillin. Results are shown as means + standard deviations
(error bars) of log10 CFU/organ.
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Induction of anti-TT antibody response and in vivo protection after
immunization with IEM101-delivered TetC.
Nonanesthetized
BALB/c mice received four doses of ca. 5 × 108
CFU of either IEM101 or IEM101(pTETnir15), grown without
aeration, by the intranasal route. A third group of mice
received the same dose of heat-inactivated IEM101(pTETnir15).
Levels of humoral anti-TT IgG were analyzed by ELISA. When killed
bacteria were used for immunization, no anti-TT IgG response was
detected in the sera of the immunized mice. After three immunizations,
mice immunized with live bacteria mounted an anti-TT humoral response, which was boosted following the fourth immunization (Fig.
6). An anti-TT IgA response could be also
detected in nasal washes, indicating that stimulation of local
responses occurred (Fig. 7).
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FIG. 6.
Anti-TT serum antibody responses. Anti-TT serum antibody
responses in 10 BALB/c mice after receiving one to four doses of 5 × 108 CFU of IEM101(pTETnir15) are presented. Results
are shown as individual values of log10 anti-TT IgG.
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FIG. 7.
Anti-TT mucosal IgA response. Anti-TT IgA antibody
responses in nasal washes of five BALB/c mice after receiving four
doses of 5 × 108 CFU of either IEM101 or
IEM101(pTETnir15) are presented. Results are shown as mean titers,
and the error bar indicates the standard deviation from the mean
titer.
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To verify whether the induced anti-TT antibodies were able to protect
immunized mice against a lethal challenge with tetanus toxin, mice were
challenged with 10 × LD50 of tetanus toxin on day 70 and observed for mortality for 7 days after the challenge. The results,
reported in Fig. 8, show that all mice
immunized with IEM101(pTETnir15) survived the challenge, whereas
control animals, immunized with IEM101, died within 36 h,
demonstrating that the immune response raised by
Vibrio-delivered TetC was able to neutralize the toxin in
vivo and confer protection.
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FIG. 8.
Mouse survival after challenge with TT. Eight mice were
immunized with four doses of 5 × 108 CFU of either
IEM101 or IEM101(pTETnir15). At day 70, mice were challenged with
10 × LD50 of tetanus toxin and observed for mortality
for 7 days after challenge.
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In vivo protection following immunization with
IEM101-delivered Tcf.
BALB/c animals received four doses of
ca. 5 × 108 CFU of either IEM101 or
IEM101(pNIR-tcf) by the intranasal route. We were unable to detect
specific anti-Tcf responses in the sera and mucosal washes of immunized
mice, either by ELISA or Western blotting. Purified recombinant
MalE-Tcf and partially purified Tcf from B. pertussis were
used as antigens for the assays, but sera of mice immunized with IEM101
also recognized such preparations. These background signals could be
due to a response to endogenous MalE from IEM101 or cross-reaction with
proteins copurified with Tcf.
Although it was impossible to detect a specific anti-Tcf antibody
response, we asked whether mice immunized with
IEM101-delivered Tcf were protected from tracheal
colonization by B. pertussis, since it has been described
that a tcf mutant B. pertussis strain showed
decreased tracheal colonization, implying a possible role for this
protein in tracheal colonization (15). On day 70, immunized animals
were challenged via the intranasal route with BP18323 Smr.
Mice were sacrificed 14 days after the challenge, and the bacterial colonization of their tracheas and lungs was evaluated. The results, shown in Fig. 9, indicate that mice
immunized with IEM101(pNIR-tcf) had significantly less bacteria in
their tracheas than did the control animals immunized with IEM101. No
significant difference between the numbers of bacteria isolated
from the lungs of the two groups of mice was observed.
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FIG. 9.
B. pertussis colonization in trachea and lung
following intranasal infection. Six mice were immunized with four doses
of 5 × 108 CFU of either IEM101 or
IEM101(pNIR-tcf). At day 70, mice were challenged intranasally with
106 CFU of BP18323. Fourteen days after challenge, the mice
were sacrificed and colonization in lungs and tracheas was determined.
Results are expressed as geometrical means of bacterial counts. Values
are significantly different, with a P of <0.03. NS, values
not significantly different.
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DISCUSSION |
The construction of attenuated V. cholerae strains
as live oral vaccines for cholera raises the possibility of using such strains to deliver heterologous antigens and to develop multivalent vaccines. We have thus expressed two antigens from nonenteropathogens in a naturally attenuated strain of V. cholerae,
IEM101, and investigated its potential as a delivery system.
TetC was expressed in IEM101 by using pTETnir15 (29).
Bacteria administered intranasally to mice induced a serum vibriocidal response and anti-TetC antibodies and stimulated local responses to the
foreign protein. An anti-TT response able to confer protection against
lethal challenge with TT was achieved.
Interestingly, immunization with heat-inactivated bacteria did not
raise antibodies against TetC, although there was induction of
vibriocidal activity, at levels comparable to those induced by live
bacteria. Bacterial viability was shown to be necessary for
V. cholerae interaction with M cells in rabbit
intestine (28). It is probable that bacterial viability is
also needed for interaction with M cells from respiratory mucosa;
therefore, live bacteria would interact with M cells more efficiently
than inactivated bacteria and consequently induce a stronger immune
response. Persistence of IEM101 in the respiratory tract after
intranasal inoculation (for at least 24 h) could also provide a
more effective stimulation of the immune system. Plasmid retention was
considerably high; thus, in vivo expression of the heterologous antigen
could potentially occur. However, no significant differences in
vibriocidal antibody titers in sera from animals receiving live and
heat-inactivated Vibrio bacteria were observed; it is
possible that the assay used to measure vibriocidal activity was not
sensitive enough to detect such differences.
The nirB promoter was also able to drive the expression of
tcfA from B. pertussis in IEM101. Tcf was
processed and exposed on the IEM101 surface, although only a low
proportion of the population was highly positive in the FACScan assay.
This could be due to the low level of Tcf production; alternatively,
Tcf could have a different conformation or exposure on the
V. cholerae surface, by interaction with and/or
sterical hindrance by outer membrane structures. However, it should be
noted that only 31% of the B. pertussis population was
recognized by the anti-MalE-Tcf antiserum used to probe Tcf. This could
mean that the antibodies directed against the MalE-Tcf fusion protein
do not bind efficiently to the Tcf protein in its native form.
Mouse immunization with Vibrio bacteria expressing Tcf
resulted in a protective effect against Bordetella infection
at the tracheal level, with approximately 10-fold reduction in
colonization. These results corroborate previous observations that
tcf mutants are less able to colonize the mouse trachea
after aerosol infection than is the wild-type strain (15).
The nature of the protective immunity induced by
Vibrio-delivered Tcf is not known, as we were not able to
detect a specific anti-Tcf immune response in the sera of immunized
mice. Tcf shares an RGD motif with filamentous hemagglutinin and
pertactin. The RGD motifs from both filamentous hemagglutinin
(20) and pertactin (23) have been shown to be involved in adherence to host cells. Therefore, antibodies against Tcf
could provide protection, since they could bind to Tcf on the
Bordetella surface, preventing efficient bacterial adhesion to trachea mucosa; in addition, they could have bactericidal or opsonic
activity. On the other hand, since cell-mediated responses play an
important role in protection against Bordetella infection in
the murine model (27, 32), the possibility that
V. cholerae-delivered Tcf induced cellular responses
should also be considered.
We did not observe protection at the lung level, and this result agrees
with the observation that tcf mutants present the same
pattern of lung colonization after aerosol infection as does wild-type
Bordetella (15). Both observations could be due
to lack of Tcf expression at that particular environment by
Bordetella and/or the expression of other adhesion factors
with more relevant roles in lung colonization.
In conclusion, we demonstrated the potential of V. cholerae as a delivery system for heterologous antigens, since two
model antigens from nonenteropathogens were delivered by live
Vibrio, and protective responses were elicited in both
cases. We also showed that mouse intranasal inoculation with
V. cholerae can be used for preliminary
evaluation of immunogenicity. In order to enhance the
immunogenicity of heterologous product, improvements in expression
level and stability should be achieved by investigating different
promoters; a promising alternative is the use of promoters from in
vivo-induced (ivi) genes (7).
| |
ACKNOWLEDGMENTS |
This work was supported in part by EC grant TS3*-CT93-0255.
We thank Sandra Nuti and Domenico Rosa for FACScan analysis; Franco
Giovannoni for the TT challenge; Fabrizio Zappalorto for animal
handling; Samuele Peppoloni, Gill Douce, Duan Guancai, and Vincenzo
Scarlato for sera, protein, and bacterial strains; Roberto Manetti for
technical advice; Giorgio Corsi for artwork; and Giuseppe Del Giudice
for helpful discussion.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: IRIS,
Chiron Vaccines Immunobiological Research Institute in Siena, Via
Fiorentina, 1, 53100 Siena, Italy. Phone: 39-577-243414. Fax:
39-577-243564. E-mail: Rappuoli{at}IRIS02.BIOCINE.IT.
Editor: J. T. Barbieri
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Top
Abstract
Introduction
