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Infection and Immunity, May 2000, p. 2503-2512, Vol. 68, No. 5
Department of Microbiology and Immunology,
The Medical School, University of Newcastle, Newcastle upon Tyne NE2
4HH,1 and Department of Biology,
University of York, York YO1 5DD,3 United
Kingdom, and INSERM U167, Institut Pasteur de Lille,
F-59019 Lille Cedex, France2
Received 25 August 1999/Returned for modification 28 September
1999/Accepted 14 February 2000
Tetanus toxoid has been used widely as an adjuvant. The atoxic
fragment C from tetanus toxin (TetC) is potently immunogenic when
expressed in Salmonella vaccine strains and has been used as a fusion partner for antigens (Ag). However, there has been no
formal comparison of the immunomodulatory impact of TetC on its fusion
partners. In this study, we have addressed this important issue. The
protective 28-kDa glutathione S-transferase (GST) from Schistosoma haematobium (Sh28GST) was expressed either as a
fusion to TetC or as the full-length Sh28GST alone in a nonvirulent
aroA-attenuated strain of Salmonella enterica
serovar Typhimurium. The Sh28GST proteins were soluble and stably
expressed in Salmonella, as evaluated by Western blotting
with TetC and/or Sh28GST antisera. Mice were immunized orally with a
single dose of the live recombinant Salmonella. The
constructs were stable in mice but, dramatically, only the strain
expressing the TetC-Sh28GST fusion elicited significant antibody (Ab)
responses directed against Sh28GST as determined by enzyme-linked
immunosorbent assay. An analysis of the isotype profiles showed that
these mice also produced anti-Sh28GST immunoglobulin A and
GST-neutralizing assays revealed high levels of neutralizing Abs in
sera. These are important correlates of protection in schistosomiasis. In addition, stimulation of spleen cells from immunized mice with Sh28GST Ag showed that both strains, expressing Sh28GST alone or the
TetC-Sh28GST fusion, were able to stimulate the secretion of
Th1-related cytokines (gamma interferon and interleukin 2) to
comparable levels. Thus, TetC has modulated the immune responses generated against its fusion partner, Sh28GST, by markedly enhancing the Ab responses elicited. These results have important implications in
the rational development of live vaccines.
Human schistosomiasis remains a
major global health problem with 200 million individuals currently
infected (41). Chemotherapy can be used to control
schistosomiasis, but rapid reinfection demands frequent retreatment,
which makes this approach expensive. Moreover, the number of cases in
the world has not been reduced over the last 20 years, and the recent
observations of strains with decreased drug susceptibility (15,
23) emphasize the need for the development of vaccines for a more
long-term approach.
A prime candidate in the development of such a vaccine is the 28-kDa
glutathione S-transferase (GST) (3, 9, 31). This antigen is present in the adult, egg, and larval stages of the parasite
and is thought to detoxify electrophilic compounds generated by the
host (38). Purified and recombinant GST from different schistosome species has been successfully used to elicit protective immunity in animal models (2, 4, 5, 8, 37). For a vaccine to
be successful, it does not necessarily need to induce sterile immunity.
A vaccine inducing partial resistance and/or affecting fecundity and
egg viability would be of value in controlling infection intensity and
disease severity (12). A significant body of research on the
development of schistosomiasis vaccines has dealt with the 28-kDa GST
from Schistosoma mansoni (Sm28GST); however, phase I
clinical trials have recently been initiated with the Schistosoma
haematobium 28-kDa GST (Sh28GST) (9). The Sh28GST has
been selected because resistance to S. haematobium is
correlated with immune system-mediated inhibition of worm fecundity in
humans and also because a noninvasive assessment of pathology to
evaluate the vaccine efficacy can be easily applied to urinary schistosomiasis (9).
In humans and in animal models an important correlate in protection is
the presence of antibodies which neutralize the enzymatic activity of
the GST (42). Intriguingly, this effect appears to be
mediated by immunoglobulin A (IgA) (17, 18). This
observation has important implications in the rational development of
effective vaccines. Thus, a vaccine that can be delivered to a mucosal
surface may be capable of eliciting the desired immune responses.
Live salmonella vaccines have been used as carriers for the mucosal
delivery of heterologous antigens from viruses, bacteria, and parasites
to the immune system. These recombinant Salmonella strains
were able to induce cellular, humoral, and secretory immune responses
to the recombinant antigens (1, 29, 32, 34). Tetanus toxoid
has been used widely as an adjuvant. The atoxic fragment C from tetanus
toxin (TetC) is potently immunogenic when expressed in
Salmonella vaccine strains. We have previously described the
expression of antigens from worms and viruses as a fusion to TetC. This
has resulted in the development of multivalent Salmonella vaccines which have elicited protective immunity (10).
This expression system has been used with great success. This system
has also served to allow the expression of antigens from worms and
bacteria which had proved otherwise difficult to express in
Salmonella spp., a process which has been referred to as
"expression rescue" (16, 24). In a previous study
(24), we have described the expression and immunogenicity of
Sm28GST as a fusion to TetC. However, there has been no formal
comparison of the immunomodulatory impact of TetC on its fusion
partners. In this study, we have addressed this important issue and
have selected Sh28GST, which is undergoing clinical vaccine trials,
rather than Sm28GST. The Sh28GST was expressed either as a fusion to
TetC or as the full-length Sh28GST alone. Mice were immunized orally
with a single dose of the live Aro-attenuated Salmonella
vaccine strains harboring each construct, and the ensuing immune
responses are described.
Bacterial strains, plasmids, oligonucleotides, and DNA
sequencing.
Bacterial strains and plasmids used in this study are
summarized in Table 1. Bacteria were
cultured in Luria-Bertani (LB) broth and on LB agar with ampicillin (50 µg/ml), when appropriate.
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Tetanus Toxin Fragment C Expressed in Live
Salmonella Vaccines Enhances Antibody Responses to Its
Fusion Partner Schistosoma haematobium Glutathione
S-Transferase
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Strains and plasmids used in this study
SDS-PAGE and Western blotting. Expression of the TetC-Sh28GST fusion and Sh28GST alone was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting as described previously (10). Cells growing in mid-log phase, with antibiotic selection, were harvested by centrifugation, resuspended in phosphate-buffered saline (PBS) containing 1% Triton X-100, and disrupted on ice for 10 s at 10 µ with a sonicator (Soniprep 150; MSE). The lysates were fractionated by SDS-10% PAGE, as described by Laemmli (28). The proteins were transferred to a nitrocellulose membrane by electroblotting and reacted with either a polyclonal rabbit antiserum directed against recombinant TetC (rTetC; Boehringer Mannheim, Sussex, United Kingdom) or a polyclonal mouse antiserum directed against recombinant Sh28GST (rSh28GST). The blots were then probed with horseradish peroxidase (HRP) conjugated to either goat anti-rabbit Ig or goat anti-mouse Ig antibodies (Dako, Bucks, United Kingdom) and developed with 4-chloro-1-naphthol (Sigma, Dorset, United Kingdom).
Animals, immunizations, and viable counting on organ
homogenates.
Female BALB/c mice were purchased from Harlan Olac
(Blackthorn, Bicester, United Kingdom) and used when at least 8 weeks
of age. Bacteria were grown in LB broth supplemented with 50 µg of ampicillin/ml as required. For oral immunization, 4 × 1010 to 5 × 1010 CFU of each construct in
200 µl of PBS was given once intragastrically by gavage tube to
anesthetized mice. The immunization doses were checked by viable counts
on LB agar plates. The mice were bled at 3, 5, 7, 9, and 11 weeks after
immunization, and sera were stored individually at 
20°C.
Measurement of antibody responses. Antibody responses were measured by enzyme-linked immunosorbent assay (ELISA) as described previously (11). Briefly, 96-well microtiter plates (Nunc, Paisley, United Kingdom) were coated overnight at 4°C with either 0.1 µg of rTetC (produced in Escherichia coli; Boehringer Mannheim) or 0.1 µg of rSh28GST (produced in yeast; F. Trottein, Institut Pasteur de Lille) diluted in 0.1 M carbonate buffer (pH 9.6) per well. Twofold serial dilutions of antisera in PBS-1% bovine serum albumin were added, and the plates were incubated for 90 min at 37°C. HRP-conjugated antibodies specific to mouse Ig (Dako) and each isotype (Zymed, Cambridge Bioscience, Cambridge, United Kingdom) were added according to the manufacturer's instructions. For IgA determination, biotinylated goat anti-mouse IgA antibodies diluted 1:1,000 and streptavidin-HRP diluted 1:500 (Sigma) were used. The plates were developed with o-phenylene diamine (Sigma) prepared according to the manufacturer's instructions. After 20 min at 37°C, the reaction was stopped with 3 M H2SO4 and plates were read at 490 nm.
For antibody titration, serial twofold dilutions of pooled sera were assessed by ELISA as described above. Titers were defined as the highest dilution of the antisera that gave an absorbancy three times higher than background (26).Purification of Sh28GST from recombinant salmonellae. Recombinant salmonellae were grown overnight in LB broth supplemented with 50 µg of ampicillin/ml, harvested, washed once with PBS, resuspended in PBS containing 1% Triton X-100, and disrupted on ice for 2 min. After sonication, the lysates were clarified by centrifugation at 25,000 × g for 20 min at 4°C. The soluble fraction was recovered and diluted in equilibration buffer (PBS containing 1 mM EDTA and 0.5 mM phenylmethylsulfonyl fluoride). Glutathione (GSH)-agarose beads (Sigma) were suspended in equilibration buffer for 2 h before use, packed into a column (10 by 50 mm), and equilibrated with the same buffer. The protein sample was applied at a flow rate of 1 ml/min. After extensive washing with equilibration buffer, Sh28GST was eluted with 5 mM GSH (Sigma) in elution buffer (50 mM Tris-HCl, pH 8.0). Fractions of 1 ml were collected and analyzed for the presence of Sh28GST by SDS-PAGE and Coomassie blue staining. Fractions containing the protein were pooled, concentrated by ultrafiltration on a Centricon-10 concentrator (Amicon Ltd., Gloucestershire, United Kingdom), and dialyzed against PBS. The protein concentration was determined using the bicinchoninic acid protein assay reagent kit (Pierce, Chester, United Kingdom), according to the manufacturer's instructions.
Enzymatic activity of Sh28GST. The enzyme activity of the Sh28GST purified from recombinant Salmonella was measured as described by Habig et al. (19), with the following modifications. In 820 µl of reaction buffer (100 mM potassium phosphate [pH 6.5] containing 1 mM 1-chloro-2,4-dinitrobenzene [Sigma] and 5 mM GSH), 10 µl of enzyme (10 µg/ml) was added. The enzymatic reaction was monitored spectrophotometrically at 340 nm for 60 min.
The neutralizing activity of the anti-Sh28GST antisera was analyzed as described by Kremer et al. (26). Briefly, in a total reaction volume of 430 µl containing 400 µl of reaction buffer, 30 µl of enzyme mixture was added. This enzyme mixture contained 10 µl of enzyme (10 µg/ml) incubated with 20 µl of potassium phosphate (pH 6.5) or of antisera for 1 h at 37°C, followed by 1 h at 4°C. The reaction was started by the addition of the enzyme mixture and was monitored continuously for 20 min at 340 nm.Cytokine assays.
Cytokine levels in spleen cell culture
supernatant were measured after antigenic stimulation. Fifteen weeks
after immunization, two mice from each of the immunized groups were
killed, and the spleens were aseptically removed and placed in RPMI
1640 (Sigma; HEPES modification). Single-cell suspensions were prepared
by mashing the spleens through a sieve with a syringe plunger and washing them in RPMI 1640 and complete RPMI 1640 (CRPMI; RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 50 µM
2-mercaptoethanol, 2 mM glutamine, 100 µg of streptomycin/ml, and 100 U of penicillin/ml). Erythrocytes were lysed by the addition of 5 ml of
Gey's solution per spleen and incubated on ice for 4 min. The cells
were then washed twice before being resuspended in CRPMI and counted.
Cell cultures were set up in round-bottom 96-well plates (Corning Glass
Works, Corning, N.Y.) at 5 × 105 cells/well in 200 µl and incubated at 37°C in 95% humidity with 5% CO2.
Antigens were added in triplicate. At intervals, supernatants were
collected in flat-bottom 96-well plates and frozen at 80°C until
use in cytokine ELISAs. rTetC and rSh28GST were the same as those used
for antibody determinations and were diluted in CRPMI at concentrations
of 5 and 10 µg per well, respectively. For Salmonella
antigens, a whole-cell extract designated C5 lysate was prepared as
described previously (40). The protein concentration of C5
lysate was determined by the bicinchoninic acid method.
), and IL-4 were assayed
by ELISA as described previously (11), using a pair of
specific monoclonal antibodies (capture and detection) against each
cytokine and dilutions of a recombinant cytokine for the construction
of standard curves (Pharmingen, San Diego, Calif.).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of pTECH2-Sh28 and pTECH10-Sh28.
A Sh28GST
expression cassette was synthesized by PCR using the Sh28GST cDNA clone
H89.2 (39) as a template. Oligonucleotide primers P1 and P2
were designed to amplify the gene, beginning with the start codon and
terminating with the stop codon. In addition, forward and reverse
primers were tailored with BamHI and SpeI, respectively, to allow directional cloning into pTECH2. The PCR product
was gel purified and digested with BamHI and SpeI
and then cloned into pTECH2, which had been digested with the same enzymes and subsequently gel purified. The construct pTECH2-Sh28 directed the expression of a TetC-Sh28GST fusion protein. The vector
pTECH10-Sh28, expressing Sh28GST alone without the TetC fusion partner,
was constructed by inverse PCR using pTECH2-Sh28 as the template DNA.
The inverse PCR was conducted with high-fidelity thermostable
Pfu DNA polymerase and primers P3 and P4. The PCR product
was gel purified and ligated, resulting in the complete removal of the
TetC gene (Fig. 1).
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Expression of Sh28GST in salmonella.
The recombinant plasmids
were first transformed into E. coli TG2, and expression on
cells harboring the constructs was evaluated by SDS-PAGE and Western
blotting. The TetC-Sh28GST fusion protein and Sh28GST remained soluble
and reacted with antisera to TetC and Sh28GST, respectively. Single
bands of the expected molecular masses for the TetC-Sh28GST fusion
protein (80 kDa) and Sh28GST alone (28 kDa) were recognized in several
independent transformants, and DNA restriction analysis of the plasmid
DNA confirmed their identity (data not shown). The constructs were
initially transformed into the intermediate Salmonella
strain SL5338 (r
m+) to increase the
efficiency of electroporation into the vaccine strain SL3261
(r+ m+) and finally transformed into the
vaccine strain SL3261. The resulting recombinant strains were
designated as in Table 1. The identity of the expression plasmids
pTECH2-Sh28, isolated from JJ503 and JJ703, and pTECH10-Sh28, isolated
from JJ504 and JJ704, was further verified by DNA sequencing using
sequencing primers. The expression of the recombinant proteins in
SL5338 and SL3261 genetic backgrounds was judged by SDS-PAGE and
Western blotting. Western blot analysis showed that the TetC-Sh28GST
fusion and Sh28GST alone were similarly expressed in both background strains (Fig. 2). However, it was
estimated by densitometer that the expression level of Sh28GST alone in
JJ504 and JJ704 was approximately fivefold higher than that of the
TetC-Sh28GST fusion in JJ503 and JJ703 (Fig. 2B).
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Stability of the plasmid constructs in vivo and immunization of mice. BALB/c mice were immunized orally with approximately 4 × 1010 to 5 × 1010 CFU of Salmonella enterica serovar Typhimurium strains SL3261, JJ702, JJ703, and JJ704. Viable counts of homogenates of liver and spleen were conducted at day 14 after immunization. The recombinant constructs grew but persisted in the tissues at a level 1 log unit lower than that of parental strain, SL3261. Viable counts of JJ703 and JJ704 for media with and without ampicillin were very similar, indicating that the plasmid was not being lost in vivo. In addition, when colonies recovered from livers and spleens were analyzed, all of them retained the constructs and Western analysis showed that recombinant proteins were still well expressed (data not shown).
Antibody response induced after immunization with recombinant
salmonellae in mice.
Tail bleeds were taken at weeks 3, 5, 7, 9, and 11 from all eight mice per group, and all antisera were assessed
simultaneously by ELISA against TetC (Fig.
3A) and Sh28GST (Fig. 3B). A single oral
dose of 4 × 1010 CFU of recombinant
Salmonella was sufficient to generate specific immune
responses against TetC and Sh28GST. The antibody responses against TetC
and Sh28GST were detected as early as week 3 and peaked at week 5 after
immunization, with no significant decrease over the time of the study.
Although all mice immunized with JJ702 and JJ703 expressing TetC alone
or the TetC-Sh28GST fusion invoked a strong antibody response to TetC,
the antibody titer in sera from mice immunized with JJ703 was much
higher than that in sera from mice immunized with JJ702. As expected no
anti-TetC antibodies were detected in sera from mice immunized with
SL3261 or JJ704 (Fig. 3A). Mice immunized with JJ703 developed a
significant anti-Sh28GST response. In contrast, mice immunized with
JJ704 developed a weaker response (Fig. 3B). While a peak anti-Sh28GST
antibody titer of 1/51,200 was elicited in mice immunized with JJ703, a
peak titer of 1/3,200 was elicited in mice immunized with JJ704.
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Purification and enzymatic activity of Sh28GST expressed in
salmonella.
As the strain expressing Sh28GST alone, JJ704,
elicited only a weak antibody response against Sh28GST, it is possible
that the recombinant protein may have misfolded significantly and lost its immunogenicity. Furthermore, it has been reported that protective immunity is associated with antibodies directed against the active site
of Sm28GST (42). Therefore, it may be important that the active site of the recombinant Sh28GST expressed in
Salmonella also remain intact. These possibilities were
investigated by purification of Sh28GST using affinity chromatography
and by determining the enzymatic activity of the purified recombinant
Sh28GST expressed in JJ704. As shown in Fig.
5A, the Sh28GST produced in S. enterica serovar Typhimurium strain JJ704 harboring the
pTECH10-Sh28 construct was purified in a single step by affinity
chromatography on a GSH-agarose column. Eluted fractions were monitored
by measuring the optical density at 280 nm, and the fractions
containing protein were pooled and then analyzed by SDS-PAGE. The
protein purified from S. enterica serovar Typhimurium strain
JJ704 comigrated exactly with the protein purified from yeast, which
was used as a positive control. These results suggest that the Sh28GST
expressed in Salmonella folds properly and binds to GSH in a
reversible manner, since Sh28GST can be competitively eluted with free
GSH.
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Neutralization of the Sh28GST enzymatic activity by sera from
immunized mice.
Since the induction of neutralizing antibodies has
been related to protection against schistosomiasis in humans
(31), the ability of the anti-Sh28GST antisera to neutralize
the enzymatic activity of Sh28GST was also investigated using an
enzymatic inhibition assay. As shown in Table
2, only the group of mice immunized with
JJ703 expressing the TetC-Sh28GST fusion produced significant levels of
neutralizing antibodies. The inhibition value was as high as 85.4% 3 weeks after a single oral immunization and gradually diminished with
time. In contrast, no significant inhibition was observed with sera
obtained from mice immunized with SL3261, JJ702, and JJ704 expressing
Sh28GST alone.
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Cellular response induced in mice immunized with recombinant salmonella. To investigate the cellular immune responses induced in mice immunized with the recombinant Salmonella, spleen cells obtained 15 weeks after immunization were stimulated in vitro with C5 lysate, rTetC, and rSh28GST antigens. Cytokine levels in cell culture supernatants were measured at various time points after antigenic stimulation.
The IFN- responses upon stimulation with the different antigens are
shown in Fig. 6. Stimulation with
S. enterica serovar Typhimurium C5 lysate induced similar
responses in all immunized groups and the responses remained stable
with no significant decrease over the time of the study (data not
shown). The IFN- response upon stimulation with rTetC and rSh28GST
varied with the strains used for immunization. rTetC stimulation of
cells from mice immunized with JJ702 and JJ703 expressing TetC or the
TetC-Sh28GST fusion resulted in IFN- responses that were most
apparent at 6 days after stimulation, although IFN- responses at
weeks 4 and 8 were also statistically significant (P < 0.01). Furthermore, the response to rTetC obtained with
JJ703-immunized mice was much greater than that obtained with
JJ702-immunized mice (Fig. 6A). Stimulation with rSh28GST elicited
significantly (P < 0.01) greater production of IFN-
in splenocytes from mice immunized with JJ703 and JJ704 expressing
TetC-Sh28GST and Sh28GST alone than in those from mice immunized with
JJ702 expressing TetC alone, 8 days poststimulation (Fig. 6B).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Tetanus toxoid has been universally exploited as a potent adjuvant for chemically coupled antigens. Fragment C from tetanus toxin is highly immunogenic when expressed in Salmonella. In order to enhance the immune response, precise genetic fusions of recombinant antigens with TetC have been constructed in live Salmonella vaccines (25). This strategy has received considerable attention; however, a formal proof of the immunomodulatory nature of TetC is urgently required. A number of carrier proteins have been used in live vaccines for the presentation of heterologous epitopes from antigens including flagellin, E. coli heat-labile toxin B, outer membrane protein A (OmpA), phage lambda receptor (LamB), maltose binding protein E (MalE), and hepatitis B core antigen (13, 14, 29, 30, 35, 36). In a number of cases, these carrier molecules have been shown to enhance the immune responses to otherwise nonimmunogenic heterologous epitopes when expressed in Salmonella. However, little is known of the immunological impact these carrier molecules on full-length antigens. The results presented in this study for the first time provide strong evidence to suggest that TetC can enhance the immune responses to a fusion partner, in this case Sh28GST. This molecule has been expressed either as the native protein or as a fusion to TetC. Both TetC-Sh28GST and Sh28GST are stably expressed in S. enterica serovar Typhimurium strain SL3261. However, Sh28GST alone in JJ704 was expressed to approximately fivefold greater levels than the TetC-Sh28GST fusion in JJ703. The expression plasmids in both strains JJ703 and JJ704 were stably retained in vitro in the absence of drug selection. Furthermore, viable counts performed on organ homogenates of livers and spleens in groups of mice immunized orally with JJ703 and JJ704 indicated that the recombinant strains persist in tissues equally well. In addition, after in vivo passage the strains retained the ability to express the respective recombinant proteins, implying that the constructs were stable in vivo and not prone to genetic rearrangements.
However, after a single oral inoculation the immune responses generated by these constructs were very different. The antibody responses to Sh28GST that were generated were much greater in mice inoculated with JJ703 expressing the TetC-Sh28GST fusion than in mice inoculated with JJ704 expressing Sh28GST alone. Thus, despite the significantly lower in vitro expression of the fusion TetC-Sh28GST than of Sh28GST alone, JJ703 was even more effective at invoking antibody responses against Sh28GST. The low antibody responses generated by JJ704 expressing Sh28GST alone cannot be attributed to misfolding of the molecule, as Sh28GST retains full enzymatic activity. In addition, JJ703 was capable of eliciting antibody responses to TetC greater than those obtained with JJ702. This observation is consistent with the anti-TetC responses to TetC-antigen fusions observed previously (10, 11, 24, 25).
Adjuvants are capable of modulating antibody subclasses against specific epitopes of antigen (22). The analysis of anti-Sh28GST antibody subclasses in mice immunized with recombinant salmonellae showed the presence of IgG1, IgG2a, and IgG2b antibodies. However, there were no apparent qualitative differences in the antibody subclasses elicited by JJ703 and JJ704. Thus, it appears that the greater antibody responses generated by TetC to its fusion partner Sh28GST cannot be attributable to a particular antibody subclass. It has been shown that different routes of immunization elicit different antibody isotype profiles directed against the S. mansoni Sm28GST (26) and the S. haematobium Sh28GST (27) in mice immunized with rBCG. The intranasal and intravenous immunizations led to an initial production of high titers of IgG2a and low titers of IgG1 and IgG2b. Immediately after boosting, high titers of IgG2b also emerged. The results obtained by using the pTECH system have consistently shown that high levels of IgG1 antibodies can be elicited against several recombinant antigens, delivered by different Salmonella vaccine strains and by both oral and intravenous immunizations (10, 11). The results obtained in this study with a single oral immunization of recombinant Salmonella show that the main antibody subclasses directed against Sh28GST were IgG1 and IgG2a, with lower levels of IgG2b antibodies. Interestingly, notable levels of specific IgA antibodies were also detected in the sera from mice immunized with JJ703. Thus TetC enhanced the IgA response to Sh28GST. This is of particular interest since it has been suggested that IgA antibodies might participate in the effector immune responses, which protect against schistosomiasis in experimental models (17) and humans (18). The lack of detection of IgM was puzzling; either it is not induced or more likely appeared and disappeared below detectable level by week 3.
Furthermore, the antibodies elicited by JJ703 expressing TetC-Sh28GST, but not JJ704 expressing Sh28GST alone, have significant GST-neutralizing activity. In humans and animal models, an important correlate of protection from schistosomiasis is the presence of GST-neutralizing antibodies. It has been reported that antisera with a GST-neutralizing activity of 60 to 70% result in a significant reduction in worm fecundity (G. J. Riveau, unpublished observations). In this study, we have reported inhibition values as high as 85.4% 21 days after a single immunization, which gradually fell with time. It may be that boosting helps to maintain such high levels of neutralizing antibodies. Hence not only can TetC enhance the antibody response to its fusion partner Sh28GST but also it can stimulate the induction of IgA and antibodies with significant GST neutralization activity.
Stimulation of splenocytes from immunized animals with rSh28GST and
rTetC resulted in clear IFN- and IL-2 responses. In contrast to the
significantly different antibody responses elicited by the strains,
splenocytes from mice immunized with either JJ703 or JJ704 produced
comparable amounts of both IFN- and IL-2 when stimulated with
Sh28GST. However, no specific IL-4 response could be detected, possibly
because whole spleen cells rather than purified CD4+ T
cells were assayed. This is consistent with cytokine patterns we have
observed previously (10, 11). Upon stimulation with rTetC,
splenocytes from mice immunized with JJ702 and JJ703 also secreted
similar levels of IFN- and IL-2. Thus, TetC does not appear to have
altered the secretion of the cytokines investigated after stimulation
of splenocytes from JJ703-immunized mice with Sh28GST antigen. Not only
can a vaccine adjuvant increase the potency of an antigen, it can also
modulate the humoral or cell-mediated immune responses generated. The
TetC fusion partner may increase the antibody responses to Sh28GST by
providing T-cell help, but the precise molecular mechanisms need
further investigation. It is also possible that the augmented antibody
responses have been influenced by the levels of other cytokines such as
IL-13 and IL-12. For example, cholera toxin, a potent mucosal adjuvant, has recently been demonstrated to elicit T-helper-cell type 2 responses
by inhibiting the production of IL-12 (6).
In summary, the results presented in this study demonstrate the immunomodulatory effects of TetC in raising the antibody responses to fused antigens. Thus, a single dose of JJ703 expressing TetC-Sh28GST is able to potently elicit antibodies against Sh28GST, in contrast to JJ704 expressing Sh28GST alone. Furthermore, IgA antibodies are produced and the sera have significant GST neutralization activity, all important correlates of protection against schistosomiasis in the mouse model. The ability of these constructs to confer protection against S. haematobium is currently under investigation. The approach presented in this investigation may represent a general strategy for eliciting antibody responses to recombinant antigens expressed in live vaccines. Thus TetC may be exploited as a potent tool to immunostimulate the production of antibodies to antigens in live vaccines when these are the protective responses required.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the European Union.
We thank David Dunne, University of Cambridge, for helpful comments.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, The Medical School, University of Newcastle, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom. Phone: 44 191 222 7066. Fax: 44 191 222 7736. E-mail: Anjam.Khan{at}ncl.ac.uk.
Present address: Department of Biochemistry, Imperial College of
Science, Technology, and Medicine, London SW7 2AZ, United Kingdom.
Editor: S. H. E. Kaufmann
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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