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Department of Medical Microbiology and Immunology, University of Göteborg, Göteborg, Sweden;
Centre d’Immunologie et de Biologie Parasitaire, Institut National de la Santé et de la Recherche Médicale U167, Institut Pasteur de Lille, Lille, France;
Department of Biology, Addis Ababa University, Addis Ababa, Ethiopia; and
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Institut National de la Santé et de la Recherche Médicale U364, Nice, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-3, and IL-5 by liver cells was also markedly reduced after i.n.
treatment of CTB-Sm28GST, whereas IL-4 production was not impaired.
Intranasal treatment of infected mice with CTB-Sm28GST increased IgG1-,
IgG2a-, IgA-, and IgE-Ab-forming cell responses in liver in comparison
with treatment with CTB-OVA, or free Sm28GST. Most importantly, mucosal
treatment with CTB-Sm28GST significantly reduced animal mortality when
administered to chronically infected mice. Our results suggest that it
may be possible to design a therapeutic vaccine against schistosomiasis
that both limits infection and suppresses parasite-induced
pathology. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Earlier studies have indicated that parenteral immunization with several S. mansoni Ags or with irradiated cercariae could partially prevent infection after challenge with schistosomes. It would be a great achievement if vaccines could also control inflammatory immune-mediated tissue damage. One of the primary goals in developing therapies against diseases caused by tissue-damaging inflammatory reactions, such as granulomatous reactions induced by schistosome eggs, is to specifically abrogate or decrease to an acceptable level the intensity of such untoward immune reactions without affecting the remainder of the immune system. Induction of tolerance or suppression of mature pathogenic T lymphocytes represents an ideal form of specific immunotherapy. In this regard, mucosal administration of Ags, so-called oral tolerance, has been considered as a simple means to suppress systemic T cell-driven inflammatory responses (1, 2). A major advantage of this approach is that under certain conditions, peripheral tolerance can be induced without affecting the capacity of the host to mount a mucosal immune response (3). Although mucosal administration of Ags, e.g., by the oral or intranasal (i.n.)3 route, offers a convenient way for simultaneously inducing mucosal immune responses and systemic tolerance, its medical potential has remained limited by several problems. Unless very large doses of Ags are administered repeatedly, local mucosal immune responses and systemic tolerance are usually difficult to induce and/or are of short duration, and most importantly, systemic tolerance is more readily induced in the naïve as compared with the immune host (4). This contrasts with the needs for immunotherapeutic vaccines against inflammatory diseases caused by persistent microorganisms, which for obvious reasons should be effective in situations in which potentially pathogenic lymphocytes already exist, such as the case in patients already infected with schistosomes.
We have previously demonstrated that mucosal administration of several Ags coupled to cholera toxin B subunit (CTB) can induce vigorous mucosal immune responses (5). More recently, we have described that mucosal administration of a variety of soluble or particulate Ags coupled to CTB can also induce peripheral tolerance or suppression in systemically sensitized animals (6). For instance, oral or i.n. administration of CTB linked to pertinent autoantigens has been shown to protect disease-prone rodents against allergic encephalomyelitis, spontaneous type I diabetes, and autoimmune arthritis (7, 8). On the other hand, it is widely documented that schistosome Ag, S. mansoni 28-kDa GST (Sm28GST) displays a protective activity in various animal models, including primates by a reduction of worm burden or parasite fecundity in animals infected with S. mansoni (9, 10, 11).
These considerations have led us to examine whether mucosal administration of a protective vaccine candidate Sm28GST linked to CTB could on the one hand protect against parasite infestation and on the other hand suppress systemic T cell-mediated granulomatous reactions. The conjugate was administered i.n., a route effective for inducing systemic tolerance, but also for inducing an immune response in various mucosal tissues, including the airway mucosa, a major site of parasite attrition in mice vaccinated with irradiated S. mansoni cercariae (12).
In this study, we found that i.n. administration of minute amounts of CTB-Sm28GST could effectively affect both parasite development and granuloma formation associated with changes in Ag-specific cellular and humoral immune reactions, even when the treatment was started after parasite infestation. Importantly, such treatment significantly reduced animal mortality in chronically infected mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice (ALAB, Sollentuna, Sweden), aged 6–8 wk, were infected percutaneously (p.c.) with 70–100 S. mansoni (Puerto Rico strain) cercariae obtained from Biomphalaria glabrata-infected snails, as described (13). In some experiments, to induce a more prolonged chronic infection, mice were infected with 30 cercariae administered s.c. At various times after infection, total worm counts were determined after whole body perfusion with heparinized saline. Liver egg counts were determined after digestion of preweighed tissues with 4% potassium hydroxide (14). Egg hatching was determined, as described previously (15).
Preparation of conjugate vaccines
Recombinant Sm28GST was expressed in Escherichia coli (strain TGE901) containing the plasmid pTG54 and purified as described earlier by affinity chromatography on a glutathione column (16).
rCTB was produced by a mutant strain of Vibrio cholerae 01 deleted of the CT genes and transfected with a multicopy plasmid encoding CTB, and purified from the culture medium by a combination of salt precipitation and chromatographic methods, as described (17).
For preparation of conjugates, rSm28GST was covalently coupled to rCTB using N-succinimidyl-3-(2-pyridyl)dithio)propionate (SPDP; Pharmacia-Upjohn, Uppsala, Sweden) as bifunctional coupling reagent (18). The resulting CTB-Sm28GST conjugate was purified by fast pressure liquid chromatography on a Superdex S-200 column and characterized for GM1 ganglioside receptor binding and serological reactivities by a solid-phase ELISA using immobilized GM1 ganglioside as capture system and enzyme-linked Abs to CTB and to Sm28GST as detection reagents, respectively (6). The purified conjugate contained approximately equal amounts (w/w) CTB and Sm28GST. For control purposes, a conjugate of CTB and OVA (approximate ratio, 1:1) was prepared and characterized in the same way.
Mucosal immunization regimens
Unless otherwise mentioned, ether-anesthetized mice received three consecutive doses of CTB-Sm28GST, each dose consisting of 10 µg of either CTB-conjugated Sm28GST, control (CTB-OVA) conjugate, or free GST in 25 µl pyrogen-free saline, given by i.n. instillation on days 14, 21, and 28 after infection. For comparative purposes, additional groups of mice were given the same conjugates administered three times intragastrically using a pediatric feeding tube, each dose consisting of 50 µg conjugate in 0.5 ml antacid buffer (0.35 M NaHCO3).
Induction of pulmonary granuloma formation
Synchronous lung granuloma formation was induced as described previously (19). Briefly, eggs were extracted from the livers of infected hamsters and enriched for mature eggs. Mice infected 12 wk earlier with 30 cercariae were injected i.v. with 1500 eggs. Animals were sacrificed after 6 days, and the total lung weight was recorded. The left lung was processed for histopathological examination, and lung granuloma sizes were determined as described below.
Histopathology
Animals were sacrificed at various times after infection, and livers and lungs were excised. The ventral median lobe of the liver and the left lung were fixed in 4% phosphate-buffered paraformaldehyde, pH 7.4, and dehydrated in graded (70%, 95%, 99%) solutions of ethanol. Specimens were then embedded in paraffin wax. Serial 8-µm-thick sections were prepared, stained with hematoxylin-eosin, and mounted in xylene.
The surface area of individual granulomas (a granuloma being defined as
containing a single egg) was determined using a computer-assisted image
analysis device (Leitz, Dresden, Germany). All granulomas were measured
in two sections through the respective organs selected to be
sufficiently distant from each other (
100 µm) to ensure that
granuloma was not measured twice. In addition, the relative areas of
tissue displaying inflammation were determined using the same sections.
Data were expressed as mean granuloma size (µm2
x 10-3) and as mean percentage of inflamed
liver or lung surface area, respectively.
Induction and measurement of systemic DTH reactions
DTH reactivity to Sm28GST was evaluated both in infected mice and in uninfected mice immunized with Sm28GST. Infected animals were challenged with 20 µg of Sm28GST injected into the left footpad, 4 wk after p.c. infection with 80 cercariae. Uninfected mice were first primed by injecting 100 µg Sm28GST together with Freund’s complete adjuvant (Difco, Detroit, MI) at the base of the tail, and challenged in the left footpad 12 days later. Footpad thickness was measured immediately before, 3 and 24 h after footpad challenge, using a dial gauge caliper (Oditest, Essen, Germany). Footpad swelling was determined for each individual animal by subtracting the prechallenge footpad thickness from that obtained at 3 and 24 h after challenge. From these values were subtracted the mean nonspecific footpad-swelling responses determined on a group of control (uninfected and unprimed) animals 3 and 24 h after footpad challenge with Sm28GST. The resulting value, referred to as specific DTH footpad thickness increment, was expressed in units of cm x 10-3.
Isolation of tissue leukocytes
Liver and lung leukocytes were isolated as described earlier with slight modifications (20). Briefly, tissue specimens were cut into small slices (1 x 1 mm) and incubated under magnetic stirring in RPMI medium supplemented with 1 mg/ml of collagenase/dispase (Boehringer Mannheim, Mannheim, Germany) and 2.5% DNase at 37°C for 30 min. When necessary, this extraction was repeated. After low speed centrifugation (400 rpm for 5 min) to remove undigested tissues and debris, single cell suspensions were washed twice with PBS and cell pellets were resuspended in culture medium supplemented with 5% FCS, 5 x 10-5 M 2-ME, 1% L-glutamine, and antibiotics. Splenocytes were prepared by teasing the spleens through a nylon screen and lysing RBC by osmotic shock.
Lymphocyte proliferation
Spontaneous and Ag-induced proliferative responses were determined on triplicate cultures of liver or splenic leukocyte suspensions. Cells were seeded at 4 x 105 cells per flat-bottom microculture well. After incubation at 37°C for 72 h in the presence or absence of Sm28GST (10 µg/ml), cultures were pulsed for another 16-h period with 1 µCi of [3H]thymidine. Cultures were harvested onto glass filters using a semiautomatic cell harvester (Skatron, Lier, Norway), and the extent of radioactive thymidine incorporated was measured with a beta scintillation counter (Tricarb, Packard, Bandhagen, Sweden).
Enumeration of cytokine-secreting cells
Cells secreting IL-3, IL-4, IL-5, and IFN- production were
detected by reverse ELISPOT assays (21) using pairs of
unconjugated and biotinylated rat mAbs to mouse IL-3, IL-4, or IL-5
(PharMingen, San Diego, CA), and IFN- Abs (Genzyme, Cambridge, MA).
Briefly, liver leukocytes (5 x 105
cells/well) or splenic mononuclear cells (106
cells/well) were incubated at 37°C in an atmosphere with 5%
CO2 for 24 h in the presence or absence of
Sm28GST (10 µg/ml) in nitrocellulose-bottom wells (Millipore,
Bedford, MA) previously coated with Abs to the desired cytokine and
blocked with 5% FCS medium. Plates were then washed with PBS
containing 0.05% Tween 20, and individual wells were exposed to the
homologous biotinylated anticytokine reagent, appropriately diluted in
PBS-Tween. After consecutive incubations with HRP-conjugated
anti-biotin Abs (Vector Laboratories, Burlingame, CA), and
chromogen substrate (H2O2
and 3-amino-9-ethylcarbazole;
H2O2-AEC)
(22), plates were thoroughly washed with tap water and
examined for the presence of brown spots, which were enumerated under
low magnification.
Enumeration of Ab-secreting cells (ASC)
Liver and spleen cell suspensions were assayed for numbers of Sm28GST-specific IgG1-, IgG2a-, IgA-, and IgE-ASC by the ELISPOT technique (22). Briefly, cells were incubated for 16 h at 37°C in 100 µl of 5% FCS-RPMI medium in nitrocellulose-bottom 96-well plates previously coated with 1 µg of Sm28GST per well. Zones of solid phase-bound Igs secreted by individual ASC were revealed as spots by stepwise addition of biotinylated Abs to mouse IgG1, IgG2a, or IgA (Southern Biotechnology Associates, Birmingham, AL), followed by HRP-labeled avidin (Sigma, St. Louis, MO), and H2O2-AEC substrate. For detection of Ag-specific IgE-ASC, the wells were developed by sequential addition of biotinylated anti-mouse IgE (Southern Biotechnology Associates), streptavidin conjugated to alkaline phosphatase, and chromogen substrate consisting of bromochloroindolyl phosphate and nitroblue tetrazolium salt (22).
Measurement of serum Ab levels
Serum levels of IgG and IgA Abs to Sm28GST were analyzed by ELISA (23). Briefly, 96-well polystyrene plates (Dynatech Laboratories, Chantilly, VA) were coated with Sm28GST (0.5 µg in 50 µl PBS per well) for 3 h at 37°C. After three washes with PBS-Tween (0.05%), sera were serially diluted in PBS-Tween with 0.5% (w/v) gelatin and incubated overnight at 4°C. Following washes with PBS-Tween, plates were incubated for 2 h at 37°C with HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, or IgA (Southern Biotechnology Associates) diluted in PBS-Tween. Following washing and addition of enzyme substrate, solid phase-bound HRP activity was monitored spectrophotometrically. The titer of a serum was defined as the reverse of the highest dilution yielding an absorbance value twice that of a pool of control sera.
Statistics
The data are expressed as means ± SD for different treatment groups. Statistical significance of differences between groups regarding worm burden, egg counts, granuloma size, and inflamed area, as well as proliferative responses was calculated by Student’s t test, and by Wilcoxon’s rank test for the results of cytokine-producing cells, DTH reactions, and Sm28GST-specific ASCs. The mortality results were analyzed by the ANOVA method; and *, **, and *** denote the levels of significant differences from the comparison groups as p < 0.05, p < 0.01, and p < 0.001, respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In preliminary experiments, we observed that i.n. or p.o. administration of Sm28GST alone before or a few weeks after infection with S. mansoni did not appreciably affect either the worm burden, or egg counts, or the extent of liver granuloma formation in infected animals (not shown). We therefore turned our attention to testing whether Sm28GST conjugated to CTB would be more effective.
In a first type of experiment (prevention experiment in Table I
), groups of mice received three doses
of either CTB-Sm28GST, the control conjugate CTB-OVA, or no treatment
at 4, 3, and 2 wk before S. mansoni infection. When the
animals were sacrificed and examined 7 wk after infection, the group
that had received CTB-Sm28GST displayed a significant reduction in both
the mean granuloma size and total inflamed liver area as compared with
either the untreated or the placebo conjugate-treated group (Table I).
The CTB-Sm28GST treatment also tended to reduce the worm burden and egg
counts, but this effect was not statistically significant (Table I).
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) evaluated whether
administering CTB-Sm28GST conjugate even after the parasite infection
had been established could affect liver pathology and/or the extent of
infection. Groups of mice received three i.n. doses of 10 µg each or
three p.o. doses of 50 µg each of CTB-Sm28GST at 2, 3, and 4 wk after
infection, and were compared with control groups receiving no treatment
after infection (experiment I) or i.n. treatment with CTB-OVA
(experiment II). Examination of animals sacrificed 7 wk after the
initial infection demonstrated significant reductions, by 42–61%, in
both granuloma size and total area of liver inflammation in animals
treated with CTB-Sm28GST conjugate, as compared with untreated mice, or
a control mice treated with the conjugate CTB-OVA (Table I).
Animals who had received treatment with CTB-Sm28GST also had reductions
in worm burden and liver egg counts compared with the untreated or
CTB-OVA-treated control groups, although this anti-infection effect
was less consistent than the effect on liver inflammation (Table I).
Furthermore, i.n. treatment with CTB-Sm28GST led to a decrease (by
almost 60%, p < 0.05) in liver egg viability compared
with the animals treated with CTB-OVA (data not shown).
No correlations could be observed between worm burden or egg counts and
liver granuloma sizes, suggesting that the suppression of granulomatous
reactions achieved by mucosal treatment with CTB-Sm28GST was not the
mere result of a reduction in worms and/or eggs: r =
0.14, p > 0.20 for regression analysis of worm burden
vs granuloma size, and r = -0.21, p >
0.20 for liver egg counts vs granuloma size. Consistent with this
notion, p.o. treatment with CTB-Sm28GST, albeit having only a marginal
effect on the worm burden and no suppressive effect at all on liver egg
counts, was almost as effective in suppressing granuloma formation and
associated liver inflammation as the i.n. treatment with the same
conjugate (Table I, treatment experiment I).
Intranasal treatment with CTB-Sm28GST suppresses lung granuloma formation in S. mansoni-infected mice
To further ascertain that suppression of liver granuloma formation
in infected animals by mucosal treatment with CTB-Sm28GST was
independent of any effect of treatment on worm burden or fecundity, the
effect of i.n. administration of CTB-Sm28GST on synchronized lung
granuloma formation was evaluated. Mice were first infected p.c. with
30 cercariae, and then treated with CTB-Sm28GST (10 µg) or PBS at
weeks 8, 9, and 10, i.e., at a stage in which parasites are no longer
vulnerable to immune attack. Two weeks after the last dose of
CTB-Sm28GST, mice received an i.v. injection of 1500 eggs and the sizes
of lung granulomas were determined 6 days later. The results showed
that treatment with CTB-Sm28GST decreased lung granulomatous responses
by 40–60% (p < 0.01), as compared with
infected but sham-treated animals given an identical egg challenge
(Fig. 1).
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In other experiments, mice were infected with 30 S.
mansoni cercariae, an infection dose known to give rise to a
relatively slowly progressing chronic infection. Starting 8 wk after
the infection, i.e., at a stage when granuloma formation is maximal,
the animals were treated with 10 µg of either CTB-Sm28GST, CTB-OVA,
or Sm28GST alone, given i.n. on three occasions, 1 wk apart. Treatment
with CTB-Sm28GST significantly prolonged the survival of infected mice
as compared with treatment with unconjugated Sm28GST or with a CTB-OVA
conjugate (p < 0.05, analyzed by the ANOVA
method) (Fig. 2). The reduction in
mortality was associated with a significant suppression of liver
granuloma formation and overall liver inflammation in mice treated by
CTB-Sm28GST, as evident from assessment of liver pathology from
subgroups of mice examined 12 wk after infection, i. e., 2 wk after the
last treatment dose (Table I, chronic experiment).
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The effect of i.n. treatment with CTB-Sm28GST on DTH reactivity
was assessed both after sensitization by injected Sm28GST Ag and after
infection. In the first type of experiment, mice were
sensitized by s.c. injection of Sm28GST in Freund’s complete
adjuvant. Three and six days after the immunization, animals
received i.n. treatment with either CTB-Sm28GST, unconjugated Sm28GST,
or control conjugate CTB-OVA. One week after the last dose, animals
were challenged with Sm28GST injected in the footpad, and specific
footpad-swelling responses were determined 3 and 24 h later. As
can be seen in Table II (experiment I),
i.n. treatment with CTB-Sm28GST significantly suppressed both the early
and late footpad-swelling responses to Sm28GST, whereas treatment with
comparable doses of free Sm28GST had no appreciable effect. In the
second set of experiments, mice were instead first infected with 80
S. mansoni cercariae and then treated with two i.n. doses of
CTB-Sm28GST, Sm28GST alone, or CTB-OVA, given 2 and 3 wk after
infection. One week after the last treatment, animals were challenged
in the footpad with Sm28GST, and footpad-swelling responses were
examined. Treatment with i.n. CTB-Sm28GST significantly suppressed the
DTH reactivity to Sm28GST in these infected animals, whereas treatment
with free Sm28GST had no effect (Table II, experiment II).
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When examined 7 wk after initial infection with 80 cercariae, the
proliferative responses of spleen mononuclear cells to in vitro added
Sm28GST were significantly (p < 0.01 by
Student’s t test) suppressed in animals who had been i.n.
treated with CTB-Sm28GST at 2, 3, and 4 wk after infection as compared
with animals treated with control CTB-OVA conjugate (Fig. 3A).
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B). Intranasal administration of CTB-Sm28GST differentially affects cytokine production by liver and spleen cells from S. mansoni-infected mice
In infected mice treated i.n. with CTB-Sm28GST, the numbers of
liver leukocytes spontaneously producing IL-3, IL-5, or IFN- were
markedly reduced as compared with infected mice treated with CTB-OVA
conjugate, while the number of cells producing IL-4 was not reduced
(Fig. 4). For spleen cells on the other
hand, IL-3, IL-5, and IL-4 responses remained unaltered, whereas the
IFN- response to in vitro exposure to Sm28GST was increased in
CTB-Sm28GST-treated animals (data not shown).
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Intranasal treatment with CTB-Sm28GST increased the serum IgG Ab
response to Sm28GST, in contrast to treatment with free Sm28GST or with
control CTB-OVA conjugate, which did not appreciably change the Ab
response to infection. Mice treated with the CTB-Sm28GST conjugate had
significantly higher levels of IgG1 as well as IgG2b-specific Ab titers
compared with the other treated groups. Serum IgG2a and IgA Ab titers
on the other hand did not differ from those of mice treated with
Sm28GST alone or CTB-OVA (Table III).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study demonstrates that mucosal administration of a protective Sm28GST linked to CTB can both partly inhibit parasite development and even more strikingly and consistently suppress liver granuloma formation, the latter being a pathological hallmark of S. mansoni infection. This dual effect was accompanied by a marked decrease in host mortality in chronically infected animals.
Previous studies have indicated that prior mucosal administration of Sm28GST could confer partial protection against murine schistosomiasis. In this regard, oral administration of liposomes containing Sm28GST (25) or of recombinant live Bordetella pertussis-expressing Sm28GST (26) has been shown to induce parasite-specific IgA Ab responses and to evoke partial protection against infection. Although the precise mechanism of action of Sm28GST-specific IgA Abs is still obscure, passive transfer of Sm28GST-specific mAbs (27) can protect animals against an ongoing S. mansoni infection and leads to reductions in egg burden and viability. Furthermore, studies with sera from humans infested with S. mansoni have indicated that IgA was capable of neutralizing Sm28GST enzymatic activity as well as reducing female worm fecundity in vitro (28). The results of our study are consistent with these findings, and indicate that mucosal administration of Sm28GST linked to CTB can decrease worm burden as well as tissue egg counts and viability, although this anti-infectious effect was less constant than the suppressive effects found on granuloma formation and inflammation.
Intranasal treatment with CTB-Sm28GST increased the serum Ab responses to Sm28GST, but this increase was mainly accounted for by IgG1 and IgG2b Abs. Although specific serum IgA Abs were barely detectable, Sm28GST-specific IgA Ab production was markedly increased in the spleen and liver of mice treated with CTB-Sm28GST conjugate. This apparent discrepancy can easily be explained by the fact that a large proportion of serum IgA in mice is in a polymeric form and is very rapidly catabolized by the hepato-biliary route (29).
The finding that a reduction in worm burden and egg counts could be obtained even when i.n. treatment with CTB-Sm28GST was initiated as late as 2 wk after initial infestation, i.e., at a stage when most schistosomulae have already left the lung vasculature, suggests that protection in those cases was affecting a more mature stage of parasite development. The latter suggestion is consistent with earlier observations indicating that young worms appear to be vulnerable to immune attack (30) and with a recent report showing that mucosal vaccination of mice with rGST can induce damage to adult worms after a challenge infection with Schistosoma japonicum (31). The finding that i.n. but not oral administration of CTB-Sm28GST could affect worm and egg burdens, although both routes are known to be effective at inducing secretory IgA Ab responses at mucosal sites, suggests that the intensity and the site(s) of expression of such immune response may be critical. In this respect, recent studies in humans and in rodents have indicated that mucosal IgA responses appear to be superior with respect to magnitude, duration, and tissue distribution after i.n. as compared with p.o. administration of CTB (32).
A most striking observation was the finding that i.n. treatment with CTB-Sm28GST suppressed leukocyte infiltration into the liver of S. mansoni-infected mice. The latter observation is in keeping with the results of our recent studies in animal models of inducible or spontaneous autoimmune diseases, in which feeding tiny doses of autoantigen linked to CTB protected animals against clinical disease and also suppressed leukocyte infiltration in the target organ (7, 8). The marked reduction in liver inflammation observed after i.n. or oral treatment with CTB-Sm28GST does not seem to be the mere result of a decrease in parasite burden, or tissue egg deposition or viability. First, not only the overall area of liver inflammation was reduced but also, and usually to about the same degree, the size of individual granulomas reflecting the extent of inflammation around each deposited egg. Second, no correlation was found between worm or egg numbers and liver inflammation, an interpretation that is also supported by the finding that oral treatment with CTB-Sm28GST, albeit ineffective in reducing worm and egg numbers, was effective in suppressing granuloma formation. Moreover, the finding that i.n. treatment with CTB-Sm28GST could suppress lung granuloma formation after systemic embolization of schistososme eggs further supports the notion that mucosal treatment with CTB-Sm28GST promotes antiparasite immunity and suppression of liver inflammation by independent effector mechanisms.
A previous study by Weinstock and coworkers (33) has demonstrated that enteric (cecal) administration of whole S. mansoni eggs could reduce granuloma size in both liver and gut of S. mansoni-infected mice. More recently, preliminary studies have indicated that i.n. treatment with a Con A-binding S. mansoni soluble egg Ag linked to CTB had very similar effects to those seen with CTB-Sm28GST, reducing not only liver granuloma formation, but also worm and egg burdens (Sun et al., unpublished results). Such effects may be related to the presence of GST in the S. mansoni soluble egg Ag preparation used and/or to the presence of additional protective Ags.
A major question remaining from this study is the mode of action of
CTB-Sm28GST. Previous studies have indicated that mucosal
administration of large doses of Ags administered repeatedly can result
in deletion (34) or preferential anergy of Th1 cells
producing IL-2 and IFN- (35). On the other hand, others
have shown that relatively low doses of Ags administered repeatedly can
induce expansion of Th2-like regulatory cells (36) and/or
activation of Th3 cells (2) capable of producing IL-4,
IL-10, and/or TGF-ß, cytokines that are known to antagonize
Th1-driven immune responses. With regard to the latter scenario, CTB
has recently been shown to up-regulate TGF-ß1 activity
(37), a cytokine that is also known to promote IgA isotype
switching (38). Since we did not measure the production of
this cytokine, the possibility that the protective antiparasitic and
antiinflammatory effects of CTB-Sm28GST observed in this study were
mediated through the action of TGF-ß production remains opened.
In this study, i.n. treatment with CTB-Sm28GST suppressed egg-induced
granuloma formation as well as DTH responses to Sm28GST. Taken together
with the fact that IFN- and IL-2 production were decreased in the
liver of animals given the nasal CTB-Sm28GST conjugate, and that Th2
CD4+ cells appear to play a major role in
expanding granulomatous lesions and fibrosis in murine models
(39), these observations indicate that this form of immune
suppression can affect both Th1- and Th2-driven responses. The latter
results are consistent with a recent report showing that i.n.
administration of an allergen linked to E. coli heat-labile
enterotoxin B subunit, a GM1-binding analogue of CTB,
suppressed DTH and IgE Ab responses (40), the archetypes
of Th1 and Th2 responses, respectively. However, the fact that IFN-,
IL-2, IL-3, and IL-5 responses were decreased while production of IL-4
remained unaffected in the liver of animals treated with CTB-Sm28GST
indicates that this form of immune deviation involves mechanisms that
are likely to be more complex than a simple shift from Th1 to Th2
responses. A similar observation was made recently in a murine model of
Leishmania major infection, in which i.n. administration of
a protective parasite Ag conjugated to CTB led to decreased production
of IL-2 and IFN-, but intact IL-4 responses, and suppressed lesion
development (41). Although IL-4 has been regarded as a
major mediator in the development of L. major lesions
(41) and in schistosome egg-induced granuloma formation
(39, 42, 43, 44), these findings indicate that IL-4 per se may
not play a very critical role in the early stages of the inflammatory
process associated with such lesions. The lack of alteration of IL-4
levels associated with decreased production of IL-5, another Th2
cytokine, and IL-2, observed in the liver of CTB-Sm28GST-treated mice
may be explained by the fact that IL-5, but not IL-4, requires IL-2,
which is endogenously produced in schistosome granuloma
(45). Since eosinophils have been shown to be major
sources of IL-5 (and IL-3) (46) and to be abundant in
early schistosome granuloma lesions, decreased hepatic production of
IL-5 could be the result of reduced liver eosinophilia after nasal
CTB-Sm28GST treatment. Although IFN- production was decreased in the
liver of CTB-Sm28GST-treated animals, splenic IFN- production was
increased. This observation is similar to our previous studies in an
animal model of autoimmune encephalitis, in which oral administration
of CTB-conjugated myelin autoantigen enhanced IFN- production in
peripheral lymph nodes (7), but suppressed it in the
target organ (J.-B. Sun et al., manuscript in preparation). Given the
reported anti-inflammatory role of IFN- in schistosome-induced
granulomatous reactions (43, 47, 48, 49, 50), this observation
suggests that IFN- could exert its protective role in the systemic
compartment by limiting the expansion of precursors of inflammatory
cells from the systemic pool and/or by interfering with their migration
into the liver.
In conclusion, the results of this study suggest that it may be possible to design a combined parasite Ag-CTB vaccine that both limits infection and halts egg-induced pathology, and in more general terms, the results demonstrate the potential of such a noninvasive immunization strategy to block T cell-mediated disease processes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jia-Bin Sun, Department of Medical Microbiology and Immunology, University of Göteborg, 413 46, Göteborg, Sweden. E-mail address:
3 Abbreviations used in this paper: i.n., intranasal; ASC, Ab-secreting cells; CTB, cholera toxin B subunit; DTH, delayed-type hypersensitivity; p.c., percutaneous; p.o., postoral; Sm28GST, Schistosoma mansoni 28-kDa GST; ELISPOT, enzyme-linked immunospot.
Received for publication November 2, 1998. Accepted for publication May 4, 1999.
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