Interleukin-12 as an Adjuvant for an Antischistosome Vaccine Consisting of Adult Worm Antigens: Protection of Rats from Cercarial Challenge

Abstract

Our group previously demonstrated that a detergent extract (fraction S3) prepared from immature (4-week) Schistosoma mansoni parasites can induce partial, serum-transferable immunity to challenge infection in rats when administered as an alum precipitate. In the present study, we examined whether S3 prepared from adult (7-week) worms could similarly induce protection and whether immunity could be positively influenced by treatment with interleukin-12 (IL-12). IL-12 coadministered to Fischer rats and C57BL/6 mice at the time of S3 vaccination altered the prechallenge kinetics of S3-specific antibody titers in both species, ultimately leading to a stable enhancement of titers (relative to those in animals vaccinated without IL-12) in mice but not rats. Immunoblot analysis of prechallenge immune sera demonstrated that IL-12 treatment was associated with changes in the S3 antigen recognition profile in each species. Isotyping of specific antibodies in S3- plus IL-12-vaccinated mice prior to challenge infection revealed a moderate elevation in immunoglobulin G1 (IgG1) responses, strongly enhanced IgG2a and IgG2b responses, as well as diminished total serum IgE responses compared to those in mice given S3 only. In vaccinated rats, IL-12 profoundly suppressed specific IgG1 and enhanced IgG2b responses but did not affect IgG2a responses. S3- plus IL-12-vaccinated rats also produced less total IgE upon challenge infection. Enumeration of worm burdens revealed that vaccination with S3 plus IL-12 conferred 50% protection from cercarial challenge to rats, whereas rats given S3 only were not protected; mice were not protected by S3 vaccination regardless of IL-12 coadministration. The protection observed in S3- plus IL-12-vaccinated rats could not be transferred with serum, suggesting participation of an activated cellular component in the expression of immunity.

Immune responses to schistosome infections, as in several other models of infectious disease, have been shown to be profoundly affected by certain subpopulations of T-helper (Th) cells, which exert a major influence on the development of protective responses in animal models (reviewed in references 41 and 47). In the murine model of irradiated cercarial vaccination, immunity generated by a single vaccine dose is largely dependent on CD4⁺ T cells (22, 46, 56) and requires the T-helper type 1 (Th1)-associated cytokine gamma interferon (IFN-γ) (48, 50). Immunity can be augmented in this model by the coadministration of interleukin-12 (IL-12) (60, 61), a cytokine which has been shown to be a potent inducer of IFN-γ in vivo (15, 44). IL-12 has also been shown to induce Th1-associated immune responses and to confer protection to mice vaccinated with soluble lung-stage antigens (39).

Although much of the research involving IL-12 has focused on its role in promoting cell-mediated immune responses (5, 54, 58), the cytokine has been shown to bind to certain populations of B cells (57) and to function as a modest B-cell growth factor, acting in synergy with IL-2 to promote immunoglobulin secretion by polyclonally activated B cells (21). IL-12 has an upregulatory effect on the in vivo synthesis in mice of immunoglobulin G2a (IgG2a) (4, 6, 16, 20, 34, 38) and IgG2b (16, 20), which are associated with responses of the Th1 phenotype (7, 13, 30, 51, 52). Somewhat surprisingly, in light of its Th1-promoting effects, IL-12 treatment can also serve as a positive stimulus for the synthesis of T-helper type 2 (Th2)-associated isotype IgG1 in mice (4, 6, 16). Furthermore, IL-12 has been shown to heighten protective humoral responses that develop upon multiple exposures to irradiated Schistosoma mansoni cercariae, enhancing parasite-specific IgG1, IgG2a, and IgG2b responses while reducing total serum IgE responses (61). Thus, in addition to its well-established role in promoting cellular immunity, IL-12 can be envisioned as an adjuvant with potential utility for the enhancement of protective humoral responses in models of antischistosome vaccination.

Despite the fact that the irradiated cercarial vaccine has been quite effective in experimental settings and has proven to be an invaluable model for studying antischistosome immunity, a vaccination protocol with live parasites would be impractical for use in humans. For this reason, most efforts have focused on nonliving vaccines (reviewed in references 3, 9, 29, 49, and 53). Our laboratory has produced an experimental vaccine (fraction S3) which consists of antigens prepared by detergent extraction of worms (1). When administered intramuscularly to rats as an alum precipitate, S3 prepared from immature (4-week) parasites induces partial protective immunity (28 to 36%, depending on the dose) which is largely transferable with serum (26). S3 from adult (7-week) worms has not previously been evaluated for protective efficacy, although it is known that this stage can serve as a source of protective antigens, inasmuch as surgical transfer of adult mouse-derived parasites to the mesenteric vein has been shown to confer protection to rats (25) and soluble antigens from adults have been used to protectively vaccinate mice (19). Accordingly, the aim of the research described here was to evaluate the protective efficacy of 7-week S3 in rats and mice. Furthermore, because IL-12 has been successfully used as an adjuvant in both the irradiated cercarial and the lung-stage antigen vaccine models, we evaluated the cytokine as an adjuvant for vaccination with S3. The effects of IL-12 on humoral immunity in rats have not been previously characterized in a model of vaccination against infectious disease; thus, an additional objective was to examine the effects of the cytokine on this aspect of the immune response.

We report here that murine IL-12 is capable of inducing protective immunity to cercarial challenge in rats but not mice vaccinated with 7-week S3. The effects of IL-12 on humoral immunity are described for each species, and possible explanations for the differential protective outcomes are discussed.

MATERIALS AND METHODS

Animals and parasites.

Six-week-old female C57BL/6 mice were purchased from Taconic Laboratories (Germantown, N.Y.). Male Fischer rats (40 to 60 g) were obtained from Charles River Laboratories (Wilmington, Mass.). S. mansoni cercariae for challenge infection were shed from infected Biomphalaria glabrata snails obtained from the Biomedical Research Institute (Rockville, Md.) under National Institute of Allergy and Infectious Diseases supply contract AI 052590.

Adult worm subfraction S3.

Washed worms from 7-week infected female CD-1 mice were frozen-thawed twice in phosphate-buffered saline (PBS; 137 mM NaCl, 1.5 mM monobasic potassium phosphate, 8 mM dibasic sodium phosphate, 2.7 mM KCl; pH 7.4) containing the protease inhibitor phenylmethylsulfonyl fluoride (Sigma, St. Louis, Mo.) at 2 mM, homogenized on ice with a Tissue Tearor (Biospec Products Inc., Racine, Wis.), and centrifuged at 100,000 × g for 1.5 h at 4°C. Following two PBS washes, the pellet was resuspended in PBS-phenylmethylsulfonyl fluoride containing 0.5% (wt/vol) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) zwitterionic detergent (Sigma) and incubated at room temperature (RT) with stirring for 1 h. This suspension was centrifuged at 15,000 × g for 30 min at 4°C. The resulting supernatant (antigen fraction S3) was removed and stored frozen at −80°C until use.

Cytokine.

Recombinant murine IL-12 was a generous gift from Genetics Institute (Cambridge, Mass.). Recombinant rat IL-12 is not yet available; however, the mouse and rat IL-12 genes are highly homologous (23, 33), and murine IL-12 has been shown to have in vivo activity in rats (17).

Immunization and challenge infection.

Prior to injection, S3 was diluted (along with IL-12, when used) in a vehicle consisting of filter-sterilized injection buffer (100 mM NaCl, 24 mM monobasic sodium phosphate, 24 mM dibasic sodium phosphate; pH 7.2) containing 1% heat-inactivated (30 min at 56°C) syngeneic mouse or rat serum. Prior to injection, the diluted antigen solution was mixed with an equal volume of RT Rehsorptar 2% aluminum hydroxide gel (Intergen Co., Purchase, N.Y.) (alum). Control animals received injections of the vehicle mixed with alum. Dosage amounts, routes, and schedules are indicated below (see Tables 1 and 2 and Fig. 1 to 4). At various times, animals were challenged by percutaneous exposure of the tail (mice) or shaved abdomen (rats) as previously described (28). Serum from twice-infected Fischer hyperimmune rats (F-2× rat serum) was prepared as previously described (32). For passive transfer, rats (four per group) were vaccinated as in the active-vaccine trial and exsanguinated 4 weeks after the second immunization. Pooled sera were stored at −80°C until use.

TABLE 1.

Challenge infection of vaccinated mice

Vaccinationa	Worm burdenbc	Liver egg burdenc	Liver hydroxyproline level (μmol/104 eggs)c
Alum control	30 ± 3 (10)	18,060 ± 3,212	4.74 ± 0.45
S3	27 ± 2 (9)	20,933 ± 2,319	4.31 ± 0.33
S3 + IL-12	26 ± 3 (10)	22,115 ± 2,085	4.51 ± 0.32

^aMice in experimental groups were vaccinated subcutaneously at weeks 0, 3, and 6 with 100 μg of worm fraction S3 in alum (plus 0.5 μg of recombinant IL-12, when used). 

^bAnimals were exposed to 50 cercariae at week 10 and perfused at week 18. 

^cValues are means ± standard errors of the means (number of mice). No significant differences were observed between treatment groups for each measured parameter. 

TABLE 2.

Challenge infection of passively immunized rats

Seruma	Worm burdenb	% Reductionc	Pd
Alum control	93 ± 4 (5)
S3	93 ± 10 (7)	0	NS
S3 + IL-12	85 ± 8 (7)	8	NS
F-2×	55 ± 6 (6)	40	0.003

^aRats were injected subcutaneously on day 3 postchallenge with 2 ml of pooled serum prepared from animals immunized as in the active-vaccine trial (Fig. 1) or F-2× rat serum. The F-2× used was from a lot that has previously been found to confer 41 to 48% protection (unpublished data). 

^bAnimals were challenged with 460 cercariae and perfused after 4 weeks. Values are means ± standard errors of the means (number of rats). 

^cRelative to values for the alum control group. 

^dCompared to the alum control group. NS, not significant. 

FIG. 1.

Challenge infection of rats vaccinated with worm fraction S3. Groups of 10 rats were vaccinated intramuscularly at weeks 0 and 5 with 50 μg of S3 in alum and with or without 0.5 μg of IL-12. Control animals received injections of the vehicle in alum. At week 10, rats were challenged percutaneously with 430 cercariae; worms were recovered by portal perfusion 4 weeks later. Each triangle indicates the number of worms recovered from an individual animal. Thick horizontal bars denote the mean worm burden of each group, and percent reduction (relative to the alum control mean) is indicated. Brackets indicate statistically significant differences between treatment groups.

FIG. 4.

Isotype profile of prechallenge (week-10) anti-S3 humoral responses in vaccinated mice (A) and rats (B). Animals were vaccinated as described in the legend to Fig. 2. Left panels show S3-specific IgM and IgG subclasses in sera from vaccinated animals. Values for alum control mice and rats were typically <10 and <50, respectively. Right panels show total serum IgE concentrations (values for vaccinated rats are from week 12 postchallenge). Broken lines indicate typical values for alum controls. Values for protective F-2× rat serum are included in rat data panels for reference. All values are means ± standard errors of the means, and an asterisk indicates statistical significance versus the S3-only-vaccinated group. Anti-S3 units (mice) and titers (rats) were calculated as described in Materials and Methods.

Measurement of protection from challenge infection.

Eight weeks after the challenge infection, worms were recovered from vaccinated mice by use of a 45-μm-pore-size Nitex screen (Tetko Inc., Briarcliff Manor, N.Y.) following portal perfusion performed as previously described (27). Worms were recovered from rats in a similar manner at 4 weeks by use of a 25-μm-pore-size screen. Parasite eggs were recovered from mouse livers by overnight digestion of minced tissue at 37°C in 4% KOH. Hepatic fibrosis was assessed by the chemical measurement of hydroxyproline levels in liver samples by an adaptation of method B of Bergman and Loxley (2). The adapted hydroxyproline method and the egg recovery protocol were kindly provided by Allen Cheever at the National Institutes of Health.

Measurement of schistosome-specific antibody responses by enzyme-linked immunosorbent assay (ELISA).

Serum was prepared from blood samples taken from mice and rats via the orbital plexus at various times. S3-specific antibody titers were determined as follows. Microtiter plates containing 100 μl of S3 (10 μg/ml in 50 mM carbonate-bicarbonate coating buffer) per well were incubated overnight at 4°C. Prior to blocking, plates were incubated for 1 h at 37°C, the contents were decanted, and plates were rinsed four times with distilled water. Plates were then blocked for 1 h at RT with 1% (wt/vol) nonfat dry milk in coating buffer. Following blocking and each subsequent step, plates were rinsed five times with PBS containing 0.05% Tween 20 (PBS-T). Serum samples were serially diluted in PBS-T to a final volume of 100 μl per well and incubated for 1 h at 37°C. S3-specific antibodies were then detected with horseradish peroxidase (HRP)-labeled goat anti-mouse or anti-rat immunoglobulin G (IgG) (heavy- and light-chain) antibodies (Cappel Laboratories, Cochranville, Pa.) that had been diluted 1:1,000 in PBS-T and incubated for 30 min at 37°C. Bound HRP-labeled antibodies were detected by the addition of 100 μl of ABTS substrate solution [0.1% (wt/vol) 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) in 0.1 M citrate buffer (pH 5.0)–0.03% H₂O₂] to each well. After 20 min of incubation at RT, the A₄₁₀ was recorded with a Dynatech MR4000 microplate reader and antigen-specific titers were calculated by interpolating the highest dilution giving an A₄₁₀ of 0.100 following subtraction of the background. All values were normalized to a positive standard (7-week infected mouse or F-2× rat serum, depending on the species being assayed) included in each assay to control for day-to-day variations.

Antibody isotype analysis by ELISA.

The isotype distribution of S3-specific antibodies in mice was determined with a Mouse MonoAB ID/SP kit (Zymed Laboratories, South San Francisco, Calif.) with the following modifications to the protocol described in the preceding section. S3-coated plates were incubated for 1 h at 37°C with dilutions of mouse serum that had yielded A₄₁₀ values of approximately 1.0 in the titration ELISA. Following a PBS-T wash, biotinylated isotype-specific secondary antibodies were added and plates were incubated for 1 h at 37°C. This step was followed by 30 min of incubation at 37°C with streptavidin-conjugated HRP (SA-HRP; Zymed) diluted as described in the kit instructions. Fifty-microliter volumes were used throughout the isotyping assay, except for the final ABTS substrate solution step (100 μl). A₄₁₀ values were determined at 40 min, and arbitrary units for antigen-specific antibody isotypes were calculated by determining the value for each isotype as a fraction of the total A₄₁₀ in the isotyping ELISA and then multiplying this value by the total antigen-specific titer for the animal at that time point.

For rats, the antigen-specific titer of individual antibody isotypes was determined as follows. S3-coated plates were incubated with serially diluted sera in 1% milk–PBS-T for 1 h at 37°C. This step was followed by 1 h of incubation at 37°C with biotinylated isotype-specific monoclonal antibodies (PharMingen, San Diego, Calif.) diluted 1:250 in 1% milk–PBS-T. SA-HRP diluted 1:4,000 in PBS-T (no milk) was added after washing, and plates were incubated for 1 h at 37°C. Following the final PBS-T wash, ABTS substrate solution was added and A₄₁₀ values were determined after 40 min. The antigen-specific titer of antibody isotypes was calculated as described for total specific titers in the preceding section.

Total serum IgE was assayed by use of monoclonal rat anti-mouse IgE capture antibody (Zymed) or monoclonal mouse anti-rat IgE (PharMingen) plated at 2 μg/ml in coating buffer (plates had been incubated and blocked as described above). Serum samples were diluted 1:10 to 1:50 in 1% milk–PBS-T and incubated for 2 h at 37°C. Plates were then incubated for 1 h at 37°C with biotinylated monoclonal anti-mouse Ig-kappa light-chain (Zymed) or biotinylated monoclonal anti-rat IgE (PharMingen) diluted 1:250 in 1% milk–PBS-T. SA-HRP and substrate steps were conducted as described above, and A₄₁₀ readings were taken at 10 min. The concentration of IgE in serum samples was determined with purified mouse IgE (AbProbe International, Portland, Maine) or rat IgE (Zymed) as a standard.

Electrophoresis and Western immunoblotting.

S3 samples were boiled for 5 min in sample buffer (25 mM Tris [pH 6.8], 5% [wt/vol] sodium dodecyl sulfate, 50% glycerol, 6 M urea) and separated on a 4% stacking–12.5% separating minigel (10 μg of S3/well). Following equilibration of the gel in Towbin transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol), proteins were transferred to a Hybond nitrocellulose membrane (Amersham Life Science Inc., Arlington Heights, Ill.) in a semidry transfer cell (Bio-Rad) for 30 min at 10 V. The membrane was fixed for 10 min at RT in a solution of 7% acetic acid, 40% methanol, and 3% glycerol, washed three times (10 min per wash) with Tris-buffered saline (20 mM Tris, 137 mM NaCl [pH 7.6]) containing 0.1% Tween 20 (TBS-T), and allowed to air dry at RT for at least 3 h. The membrane was then blocked overnight in 5% [wt/vol] dry nonfat milk–1% bovine serum albumin–1% ovalbumin–0.01% sodium azide. On the following day, the membrane was washed with TBS-T, cut into strips, and incubated in diluted pooled immune mouse or rat sera (in 5% milk–TBS-T) for 1.5 h at RT. Following a TBS-T wash, HRP-conjugated goat anti-mouse or rat IgG (heavy and light chains) at a 1:5,000 dilution in 5% milk–TBS-T was added, and the membrane was incubated for 1 h at RT. Following a TBS-T wash, the membrane was treated with enhanced chemiluminescence (ECL) reagents (Amersham) mixed in accordance with the manufacturer’s protocol and was exposed to Hyperfilm ECL detection film (Amersham). Exposure times were chosen to maximally visualize individual bands.

Statistical analyses.

Significance testing was conducted by analysis of variance with Fisher’s protected least significant difference for multiple group comparisons, with P values of <0.05 being considered significant. All experimental and control groups contained 10 animals unless otherwise indicated.

RESULTS

S3 plus IL-12 partially protects rats, but not mice, from cercarial challenge.

We previously reported that two intramuscular immunizations of 4-week S3 in alum (dosage range, 20 to 200 μg) were sufficient to convey to Fischer rats partial protective immunity from cercarial challenge (26). Therefore, to evaluate the protective efficacy of 7-week S3, rats were immunized with 50 μg of S3 in alum at weeks 0 and 5. For animals receiving murine IL-12, 0.5 μg of the cytokine was mixed with diluted S3 prior to emulsion in alum. At week 10, rats were challenged percutaneously with 430 cercariae, and after 4 weeks (week 14), their worm burdens were enumerated. Rats vaccinated with S3 plus IL-12 were shown to have a highly significant, 50% reduction in mean worm burden compared to alum controls, whereas rats given S3 without IL-12 had only a nonsignificant, 5% reduction (Fig. 1).

James and Pearce have demonstrated that in mice immunized with soluble adult worm antigen fractions, subcutaneous inoculation is superior to the intramuscular route for the induction of protective immunity (18). Accordingly, mice were vaccinated subcutaneously with 100 μg of S3 in alum (plus 0.5 μg of IL-12, when used) three times at 3-week intervals. Four weeks after the third immunization (week 10), the mice were challenged percutaneously with 50 cercariae. Eight weeks after infection (week 18), worm burdens, liver egg burdens, and liver hydroxyproline levels were evaluated (Table 1). No significant differences were observed between any of the treatment groups for the measured parameters.

IL-12 affects the kinetics and magnitude of S3-specific humoral responses.

In agreement with previously reported findings obtained with 4-week S3 (26), mice and rats vaccinated with 7-week S3 developed vigorous antigen-specific humoral responses (Fig. 2). Vaccinated mice rapidly developed specific antibody titers that reached steady-state levels by week 6 (Fig. 2, left panel). A third immunization failed to boost anti-S3 titers in these animals; we previously reported a similar result for mice with 4-week S3 (26). The inclusion of IL-12 in the S3 vaccine negatively affected the kinetics of anti-S3 responses during the first 4 weeks of the immunization period, with mice given S3 plus IL-12 having more than twofold-lower titers than mice vaccinated with S3 only (P, ≤0.02). This diminution was found to be temporary, as specific titers in S3- plus IL-12-vaccinated mice became equivalent to those in S3-only-vaccinated animals by week 6. Furthermore, specific titers in S3- plus IL-12-vaccinated mice continued to increase following the third immunization, reaching a threefold-higher level than that in S3-only-vaccinated mice (P, <0.0001) by the time of challenge infection at week 10. Titers in S3- plus IL-12-vaccinated mice remained enhanced by two- to threefold relative to those in S3-only-vaccinated animals throughout the infection (the P value was ≤0.007 for all time points postinfection), despite dropping somewhat at week 17. In alum control mice, anti-S3 titers rapidly increased from the background beginning at 2 weeks postinfection (week 12) and became equivalent to those in S3-only-vaccinated mice (but significantly lower than those in S3- plus IL-12-vaccinated mice; P, 0.004) by the time of perfusion at week 18.

FIG. 2.

Kinetics of total S3-specific humoral immune responses in vaccinated mice and rats, as assayed by an ELISA. Mice (left panel) were vaccinated at weeks 0, 3, and 6 with 100 μg of S3 in alum and with or without 0.5 μg of IL-12. Rats (right panel) were vaccinated at weeks 0 and 5 with 50 μg of S3 and with or without 0.5 μg of IL-12. At week 10, mice and rats were challenged percutaneously with 50 and 430 cercariae, respectively. Values are the means ± standard errors of the means for 10 animals, and an asterisk indicates statistical significance versus the S3-only-vaccinated group. Times of immunization, challenge infection, and perfusion are indicated at the top of each panel (challenge is also indicated by the vertical line). For reference, the anti-S3 titer of F-2× rat serum (protective hyperimmune rat serum) is indicated by the hatched bar in the right panel.

In vaccinated rats, antigen-specific titers increased rapidly in the 5 weeks following the first immunization (Fig. 2, right panel) and continued to rise gradually following the second dose at week 5, reaching steady-state levels by the time of challenge at week 10. As in mice, IL-12 altered the kinetics of humoral responses in vaccinated rats, but in a dissimilar manner: S3- plus IL-12-vaccinated rats showed enhanced responses for the first 6 weeks of the immunization period compared to animals immunized with S3 only (the P value was ≤0.025 at each time point). This effect was temporary; by week 8 the two groups had become equivalent, and they remained so for the remainder of the experiment. Both groups of vaccinated rats developed considerably higher antigen-specific titers than their mouse counterparts by the time of challenge infection (Fig. 2, compare left and right panels). As an illustration, at week 10 titers in S3-only-vaccinated rats were >17-fold higher than those in S3-only-vaccinated mice (P, <0.0001), and those in S3- plus IL-12-vaccinated rats were 6-fold higher than those in S3- plus IL-12-vaccinated mice (P, <0.0001). At the time of challenge, both groups of vaccinated rats had anti-S3 titers which were threefold higher than those of protective F-2× hyperimmune rat serum (bar in Fig. 2, right panel).

IL-12 affects the antigen recognition profile of vaccinated animals.

S3 is a complex mixture of many protein antigens having a wide range of molecular weights (data not shown). Immunoblot analysis was conducted to determine which of these were immunogenic in vaccinated animals and to examine any effect of IL-12 treatment on the S3 antigen recognition profile. Sera were analyzed at week 8, when groups of each species receiving S3 and S3 plus IL-12 had equivalent specific antibody titers (Fig. 2). Pooled sera from S3-vaccinated mice were shown to react with a limited subset of S3 antigens, primarily two major species that colocalized with the 80- and 108-kDa molecular mass markers (Fig. 3, left panel). Sera from mice vaccinated with S3 plus IL-12 also recognized these two antigens (the ca. 80-kDa antigen was more intensely recognized than in S3-vaccinated mice), as well as a group of less intense bands in the 50- to 70-kDa range, a faint smear at ca. 30 kDa, and a band of <17 kDa.

FIG. 3.

Analysis of prechallenge S3-specific humoral immune responses by ECL Western immunoblotting. (Left) Immunoblot of S3 antigens with pooled immune mouse serum from each group (20-min exposure). (Right) Blot performed as for the left panel with immune rat serum from each of the three groups as well as protective F-2× rat serum; however, a 1-min exposure was sufficient to visualize the bands. All vaccine sera were from week 8, a time at which the S3-only-vaccinated and S3- plus IL-12-vaccinated animals of each species had statistically similar anti-S3 titers (Fig. 2). All sera were diluted 1:1,000. S3 was used at 10 μg in all lanes, and molecular mass markers (in kilodaltons) are indicated to the right of each panel. Open triangles indicate bands of interest discussed in the text.

Consistent with their higher antibody titers (Fig. 2), sera from rats vaccinated with S3 in the presence or absence of IL-12 recognized considerably more antigens in S3 than did those from either group of vaccinated mice (Fig. 3, compare left and right panels). Both groups of vaccinated rats reacted most strongly with an antigen of >90 kDa, which was subsequently shown to correspond to Sm97 paramyosin (data not shown), an antigen that in purified or recombinant form induces partial protective immunity in mice (45). Strong reactivity to Sm97 paramyosin could be detected in both groups of vaccinated rats as early as week 3, at which time it was the predominant antigen recognized (data not shown). Sera from S3- plus IL-12-vaccinated rats had an overall pattern similar to that of sera from S3-only-vaccinated rats; however, some differences were noted. These included the antigens of <19 kDa and ca. 26 kDa (less intensely recognized by S3- plus IL-12-vaccinated rat serum), as well as that of ca. 31 kDa and a doublet of ca. 50 kDa (more intensely recognized). Both groups of S3-vaccinated rats had considerable qualitative and quantitative differences in their antigen recognition profiles compared to protective F-2× rat serum. For example, F-2× rat serum recognized more S3 antigens in the 17- to 33-kDa molecular weight range, while reacting less strongly with Sm97 paramyosin and the ca. 26-kDa band.

IL-12 has differential effects on the isotype distributions of specific antibodies in rats and mice.

Prechallenge (week-10) immune sera from vaccinated animals were analyzed to evaluate IL-12-mediated effects on antibody isotype distributions (Fig. 4). In both groups of vaccinated mice, IgG1 was found to be the dominant S3-specific isotype detected (Fig. 4A), with S3- plus IL-12-vaccinated mice having a twofold-higher response than S3-only-vaccinated mice (P, <0.01). Compared to IgG1, specific IgG2a and IgG2b responses in S3-only-vaccinated mice were low; however, inclusion of IL-12 enhanced the IgG2a response by 40-fold and the IgG2b response by 14-fold (the P value was <0.0001 in both cases). The enhancement of specific IgG2a and IgG2b responses in S3- plus IL-12-vaccinated mice persisted throughout the subsequent challenge infection (data not shown). Unlike the other measured isotypes, specific IgM responses were unaffected by IL-12 treatment. Analysis of prechallenge total serum IgE levels demonstrated that IL-12 diminished the concentration of IgE in vaccinated mice by threefold (P, <0.002).

Rats vaccinated with S3 in the absence of IL-12 produced vigorous antigen-specific IgG1 responses (Fig. 4B) which were comparable to those observed in protective F-2× rat serum. In contrast to the results obtained for mice, exogenous IL-12 had a strong negative impact on specific IgG1 titers in vaccinated rats, reducing the titers of this isotype by 30-fold (P, <0.0001). In further contrast to the results obtained for mice, IL-12 had no enhancing effect on specific IgG2a titers in rats, which were induced in both vaccinated groups at levels comparable to those observed in F-2× rat serum. IL-12 enhanced specific IgG2b titers by more than 20-fold (P, <0.0001), making this isotype the dominant one in S3- plus IL-12-vaccinated rats. The enhancement of IgG2b titers and the suppression of IgG1 titers by IL-12 persisted throughout the subsequent challenge infection (data not shown). Antigen-specific IgM was a relatively minor component in both groups of vaccinated rats, and (as observed in mice) IL-12 had no effect on this isotype. Likewise, prechallenge IgG2c titers were low in both groups (<100; data not shown). Total serum IgE was induced in rats at low levels (≤40 ng/ml) by S3 vaccination, with exogenous IL-12 having no effect on IgE prior to challenge. However, within 2 weeks of infection, the concentration of IgE, while increasing considerably in both vaccinated groups, had become threefold higher in rats vaccinated without IL-12 (Fig. 4B) P, 0.0004). The postchallenge levels of IgE observed in both groups of vaccinated rats were considerably lower than those elicited by protective F-2× rat serum.

Transfer of serum from rats vaccinated with S3 plus IL-12 does not protect naive rats from cercarial challenge.

Because of the highly significant protection induced in rats by S3 plus IL-12 (Fig. 1) and in accordance with previously published data demonstrating the induction of serum-transferable immunity in rats vaccinated with 4-week S3 (26), we conducted a passive transfer experiment. Rats were immunized as in the active-vaccine trial, and pooled immune sera were used to passively immunize naive rats following challenge infection. With the exception of the F-2× rat serum positive control, no significant differences in worm burden were observed between any of the treatment groups (Table 2).

DISCUSSION

In this study, we have demonstrated that recombinant murine IL-12, a cytokine previously shown to enhance vaccine-induced protective immunity to schistosomes in mice, can be effectively used as an adjuvant in rats for immunization with adult S. mansoni detergent-soluble antigens. While IL-12 has been used to augment protective immunity in a rat tumor model (42), to our knowledge this is the first report of the cytokine being used as an adjuvant for vaccination against infectious disease in rats. Furthermore, we demonstrate that IL-12 treatment of S3-vaccinated rats and mice has profound, but somewhat different, effects on humoral immune responses in each species.

Primary schistosome infections in rats induce a predominantly Th2-associated cytokine pattern (10); however, little is known concerning the role of Th1-associated immunity to the parasite in rats. We show that the Th1-inducing cytokine IL-12 can confer protection to rats vaccinated with antigen fraction S3 (Fig. 1) and that the IL-12-mediated effects on antigen-specific IgG1 and IgG2b (Fig. 4B) are consistent with the induction of IFN-γ (17). Additionally, the suppression of postchallenge IgE observed in IL-12-treated rats is suggestive of a Th1 phenotype akin to that observed in protectively vaccinated mice (39, 60, 61). In fact, the strong suppression of IgG1 in IL-12-treated rats reported in this study (which was not observed in S3- plus IL-12-vaccinated mice; Fig. 4) suggests that rats may have developed a more “Th1-polarized” response than mice. Finally, murine IL-12 is known to induce Th1 responses in rat models of autoimmune disease (31, 43). It is thus reasonable to hypothesize that in rats vaccinated with S3, exogenous IL-12 induced or enhanced protective Th1-associated immune responses. Further studies examining the role of cytokines and various cell types in S3- plus IL-12-vaccinated rats will be necessary to evaluate this hypothesis.

As shown in Fig. 1, vaccination with 7-week S3 in the absence of IL-12 did not significantly protect rats from challenge infection. Furthermore, despite the highly significant protection observed in S3- plus IL-12-vaccinated rats (Fig. 1), we were unable to passively transfer protection to naive rats with vaccine sera (Table 2). These findings are in contrast to previous work demonstrating that serum from rats protectively vaccinated with 4-week S3 (in alum without other adjuvants) was sufficient to transfer immunity to naive recipients (26). It is conceivable that uncharacterized differences in 4-week S3 and 7-week S3 may account for the disparity; further experiments will be necessary to address this possibility. The lack of serum-transferable immunity does not definitively exclude a protective role for antibodies in rats given S3 plus IL-12; however, it suggests that if protective humoral responses were generated, they may require an activated cellular component to operate effectively. Parasite-specific IgG2a antibodies have been shown to mediate protective immunity in rats vaccinated with irradiated cercariae (14), and both groups of vaccinated rats in this study produced prechallenge IgG2a titers that were comparable to that of protective F-2× rat serum (Fig. 4B). However, immunoblot analysis indicated that the overall S3 antigen recognition profile of vaccinated rats was less extensive than that of F-2× rat serum (Fig. 3). Furthermore, vaccinated rats produced very little IgE prior to challenge infection; IgE also mediates protection in rats (8, 14, 55). These differences may explain the lack of serum-transferable immunity in rats protectively vaccinated with S3 plus IL-12.

While the effects of IL-12 on humoral immunity in mice have been the subject of several studies (see above), to date very little work of this kind has been performed with rats. As reported here, the effects of exogenous IL-12 on IgG1 and IgG2b (Fig. 4B) are in general agreement with the results of another study with rats that used a class I major histocompatibility complex alloantigen administered via blood transfusion (17). Moreover, that study demonstrated a lack of IL-12-mediated effects on antigen-specific IgG2a in rats, a finding also in agreement with the data presented here. We are unaware of any published study examining the effects of IL-12 on total IgE levels in rats; however, the rat data (Fig. 4B) are concordant with our findings for mice (Fig. 4A) as well as those of other groups using the murine model (39, 59, 61). The altered isotype distribution observed in S3- plus IL-12-vaccinated rats may also provide some explanation for the changes observed in the relative signal intensities of certain bands in the immunoblot analysis, compared to the data for S3-only-vaccinated rats (Fig. 3).

While S3 plus IL-12 was quite effective in the rat model, S3 vaccination with or without IL-12 did not lead to protection of C57BL/6 mice from cercarial challenge, as measured by worm burdens, liver egg burdens, or liver fibrosis (Table 1). Although the lack of protection with 7-week S3 without IL-12 is in agreement with previously published data for 4-week S3 (26), the inability of S3 plus IL-12 to induce protection in mice is in contrast to the results of other murine studies in which IL-12 was used to augment protective immunity. As an illustration, the effects of exogenous IL-12 on antigen-specific isotype distribution in mice (Fig. 4A) are similar to those of the study by Wynn et al. in which IL-12 was found to augment protective humoral responses induced by multiple doses of radiation-attenuated cercariae (61). However, that vaccination protocol generated a high level of protection in the absence of IL-12 (>70%) and, because of its infectious nature, represents a more “authentic” exposure to the parasite than the nonliving antigen fraction S3; this fact may explain the inability of the latter to induce protection in mice. Likewise, while it has been demonstrated by Mountford et al. that IL-12 induces immunity upon vaccination with a soluble lung-stage antigen preparation (SLAP) (39), SLAP may more closely mimic the antigen exposure of a radiation-attenuated infection (in which the parasite is arrested in the lungs and dies, releasing antigens) than vaccination with extracts of other life-cycle stages (40). Furthermore, an attempt to induce protection by use of two immunizations with 25 μg of 7-week S3 plus 1 μg of IL-12 without any other adjuvant was also unsuccessful (unpublished data), despite the fact that the protocol was similar to that used by Mountford et al. (39). These findings strongly indicate that the lack of protection in the S3- plus IL-12-vaccinated mice and the successful protective immunization with SLAP plus IL-12 are due to qualitative and/or quantitative differences between the antigens contained in S3 and SLAP.

In this study, prechallenge (week 8) sera from mice treated with IL-12 recognized a larger number of S3 antigens than did sera from untreated mice (Fig. 3), despite the fact that the two groups had comparable antibody titers at that point (Fig. 2). While the expanded antigen recognition profile in S3- plus IL-12-vaccinated mice may not have relevance from a protection standpoint, this finding nonetheless is interesting, as it suggests that IL-12 can be used to augment humoral responses to complex antigen mixtures. Metzger et al. have proposed a two-step model in which IL-12 acts on humoral immunity in mice by enhancing IgG2a in an IFN-γ-dependent manner as well as generally increasing IgG production independently of IFN-γ (36). In demonstrating highly enhanced IgG2a and moderately increased IgG1 production (Fig. 4A), our results are consistent with this model. Furthermore, the immunoblot data support the hypothesis that IgG secretion by certain clones of B cells reacting with “minor” bands is increased by IL-12 treatment above the threshold level for detection, a notion which is also concordant with the model of Metzger et al. (36).

We report here that mice vaccinated with S3 produced lower antibody titers than rats (Fig. 2) and that sera from mice recognized fewer antigens than did sera from rats in immunoblot analysis (Fig. 3), despite enhancement by IL-12 treatment. It is conceivable that such lower immunogenicity could explain the observed lack of protection in mice (Table 1). However, previous findings with 4-week S3 demonstrated that when rats and mice produced comparable anti-S3 titers following vaccination, only rats developed protective immunity (26). Aside from differential immunogenicity, the relative susceptibility of worms to immune attack in each species may provide an explanation for these findings. In both species, the lung is a major site of immune-mediated attrition of larval worms (35). However, rats are also known to eliminate primary schistosome infections from the liver starting on about day 28 (11); this event coincides with elevated IgE titers, recruitment of mast cells, and mast cell degranulation (12, 37), as well as liver eosinophilia (24).

Inasmuch as S3- plus IL-12-vaccinated rats were shown to produce less total IgE than S3-only-vaccinated rats 2 weeks after cercarial challenge (Fig. 4B) as well as on day 28 postinfection (data not shown), it is unlikely that IL-12 treatment enhanced IgE-mediated clearance mechanisms in the liver. However, we cannot discount the possibility of enhanced liver-stage worm attrition mediated by an IgE-independent mechanism that may be induced or enhanced by exogenous IL-12 in rats. It is also conceivable that lung-stage parasites are more susceptible to immune attack in rats than they are in mice; however, experiments involving heterologous transfer of infection sera do not support this hypothesis (32). Finally, it is possible that in the model of S3 vaccination, rats are more amenable to the protection-enhancing effects of exogenous IL-12 than are mice. Further studies are necessary to examine these possibilities.

In conclusion, we have extended the utility of IL-12 as an adjuvant to a rat model of antischistosome immunization by demonstrating that a vaccine consisting of IL-12, alum, and adult worm antigens is capable of protecting rats from cercarial challenge. The successful use of IL-12 in a species other than the mouse provides further evidence that the cytokine or adjuvants designed to induce it may ultimately be used to augment vaccine-induced immunity in humans.