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Infection and Immunity, October 2004, p. 6112-6124, Vol. 72, No. 10
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.10.6112-6124.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Nucleic Acid Vaccination with Schistosoma mansoni Antioxidant Enzyme Cytosolic Superoxide Dismutase and the Structural Protein Filamin Confers Protection against the Adult Worm Stage

Rosemary M. Cook,1 Claudia Carvalho-Queiroz,1 Gregory Wilding,2 and Philip T. LoVerde1*

Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences,1 Department of Biostatistics, State University of New York, Buffalo, New York2

Received 21 May 2004/ Returned for modification 28 June 2004/ Accepted 7 July 2004


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ABSTRACT
 
Schistosomiasis remains a worldwide endemic cause of chronic and debilitating illness. There are two paradigms that exist in schistosome immunology. The first is that the schistosomule stages are the most susceptible to immune killing, and the second is that the adult stage, through evolution of defense mechanisms, can survive in the hostile host environment. One mechanism that seems to aid the adult worm in evading immune killing is the expression of antioxidant enzymes to neutralize the effects of reactive oxygen and nitrogen species. Here, we challenge one paradigm by targeting adult Schistosoma mansoni worms for immune elimination in an experimental mouse model using two S. mansoni antioxidants, cytosolic superoxide dismutase (SmCT-SOD) and glutathione peroxidase (SmGPX), and a partial coding sequence for a structural protein, filamin, as DNA vaccine candidates. DNA vaccination with SmCT-SOD induced a mean of 39% protection, filamin induced a mean of 50% protection, and SmGPX induced no protection compared to controls following challenge with adult worms by surgical transfer. B- and T-cell responses were analyzed in an attempt to define the protective immune mechanism(s) involved in adult worm killing. SmCT-SOD-immunized mice presented with a T1 response, and filamin-immunized mice showed a mixed T1-T2 response. We provide evidence for natural boosting after vaccination. Our results demonstrate that adult worms can be targeted for immune elimination through vaccination. This represents an advance in schistosome vaccinology and allows for the development of a therapeutic as well as a prophylactic vaccine.


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INTRODUCTION
 
Schistosoma mansoni is a eukaryotic intravascular parasite that is a cause of schistosomiasis, a chronic and debilitating disease (23). Even though extensive research into the control of schistosomiasis has been ongoing for the past four decades, with some success, this disease remains an endemic problem in many regions worldwide (7, 55). Morbidity correlates with an inflammatory response to deposited eggs, and because the adult worm does not replicate in the vertebrate host, many researchers agree that a vaccine aimed at reducing worm burden and/or egg production would be the most effective and cost-efficient way to control schistosomiasis (4-6). It has been determined that a vaccine resulting in at least a 40% reduction in worm burden would significantly reduce morbidity and transmission rates (4-6).

To date, vaccine research has focused on the larval stages of schistosomes, primarily the lung stage schistosomule (11, 20). Approaches using animal models and studies on human immune responses to infection in areas of endemicity have demonstrated that while larval stages are susceptible to immune elimination, adult schistosomes have adapted several defense mechanisms to survive and flourish in the hostile environment of the host bloodstream for years (30, 33, 44, 51, 52). For example, investigators have shown the effectiveness of cells that release reactive oxygen species such as monocytes, macrophages, eosinophils, and platelets against schistosomule stages of S. mansoni in an antibody-dependent manner (13, 30). In vitro cytotoxicity assays as well as passive transfer experiments have demonstrated the importance of these cells in association with immunoglobulin E (IgE) and certain isotypes of IgG in rats, primates, and humans on the larval stages (10, 11, 13). A common defense mechanism against immune attack is the expression of antioxidant enzymes (9, 12, 30, 35). In general, these enzymes work to protect an organism from oxidative damage caused by the reactive oxygen species and other molecules associated with host toxic responses. Several antioxidant enzymes have been identified in S. mansoni. Our laboratory, among others, has identified and characterized a cytosolic Cu/Zn superoxide dismutase (SmCT-SOD) (28) and a glutathione peroxidase (SmGPX) (34, 36). The expression and activity of these enzymes increase as the parasite undergoes development from the schistosomulum to the adult worm (35). These data coincide with in vitro studies on antibody-dependent cell cytotoxicity that show the schistosomule stages of the parasite being most susceptible to oxidant damage, while the adult stage is the least susceptible (30, 37, 38).

Because S. mansoni is a multicellular eukaryote with a complex life cycle, including several larval stages within the vertebrate host, finding a specific immune mechanism that would effectively decrease worm burden has been difficult, and protection would likely involve both humoral and cell-mediated responses (57). DNA-based vaccines are therefore promising in that they are able to express and present antigen in native conformation to both humoral and cellular immune effectors (41, 46). Several independent experiments using DNA vaccination in an experimental mouse model of S. mansoni, using the open reading frames for SmCT-SOD and SmGPX, resulted in average worm burden reductions of 54 and 43% percent, respectively, compared to vector controls. These experiments employed naked DNA vaccination and a DNA-recombinant vaccinia virus prime-boost regime in both C57BL/6 and BALB/c mice (49). The experimental mice used in this model were challenged with cercariae, the infective larval stage.

As antioxidant enzymes are most abundant at the host-parasite interface of the adult worm, the stage that is least susceptible to immune killing, and immunization with S. mansoni antioxidant enzymes confers protection, the question of whether or not the adult stage of S. mansoni is a target for immune elimination with antioxidant enzymes as vaccine candidates was addressed.


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MATERIALS AND METHODS
 
Parasites and parasite antigens. The S. mansoni (NMRI strain) life cycle was maintained with Biomphalaria glabrata snails and golden hamsters. Adult worms (45 days old) and 21- to 23-day-old worms were obtained by perfusion of hamsters with an established infection (22). These worms were washed and maintained in sterile prewarmed (37°C) RPMI containing HEPES (10 mM), lactalbumin (0.5%), penicillin-streptomycin (500 U/ml and 100 µg/ml, respectively), and fetal bovine serum (10%).

Adult worm NP-40 extract (WE) and soluble egg antigen (SEA) were obtained as previously described (18). The entire open reading frame of SmCT-SOD was cloned from the pcDNAI/AMP vector (49) into the pMALc2x (New England Biolabs, Beverly, Mass.) and pET14b (Novagen, Madison, Wis.) expression vectors. The entire open reading frame of SmGPX and the 1.7-kb fragment of S. mansoni filamin were cloned into the pGEX-4T-1 and pGEX-3X vectors (Amersham Biosciences, Piscataway, N.J.), respectively. Recombinant protein was expressed in Escherichia coli by using the above-described expression systems and purified by column elution (15). Recombinant His-SmCT-SOD, maltose binding protein (MBP)-SmCT-SOD, glutathione S-transferase (GST)-SmCT-SOD, GST-SmGPXm (a mutated form of SmGPX in which the selenocysteine [TGA] has been changed to cysteine [TCT] [36]), thrombin-cleaved SmGPXm, and GST-filamin were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting prior to use in enzyme-linked immunosorbent assay (ELISA). Protein concentrations of all samples were determined by using a bicinchoninic acid protein assay kit (Pierce, Rockford, Ill.).

Preparation of the antioxidant DNA vaccines. cDNA containing the entire open reading frames of SmCT-SOD, SmGPX, and SmGPX in the reverse orientation (XPG) (vector control) were previously cloned into the eukaryotic expression vector pcDNAI/Amp (pc) (49). A partial coding sequence of filamin consisting of 1.7 kb was cloned into the same vector (M. R. Mohamed et al., submitted for publication) (accession number AY463158). Each plasmid preparation was then purified by double-gradient centrifugation in CsCl2 (47), dialyzed against phosphate-buffered saline (PBS) to remove the CsCl2, ethanol precipitated, and resuspended in sterile sucrose (25% in PBS) to aid in the increased expression of the injected vaccine in the mouse muscle (56).

Immunization of mice with DNA constructs. In each vaccination experiment, groups of female BALB/c mice (Harlan Sprague-Dawley, Indianapolis, Ind.), age 4 to 5 weeks, received 100 µg of purified pcDNA via two injections with a 26-gauge needle into the gastrocnemius muscle of each leg. Four weeks later, each group of mice received a second 100-µg dose of the respective DNA. The animals were then rested for six weeks prior to challenge.

Whole blood was collected from the retro-orbital sinus of each mouse at week 0 (preimmune [PI]), week 10 (prechallenge [PC]), and week 14 (preperfusion [PP]) of the experiment. Sera were used in antibody analyses. All samples were stored at –20°C until use.

Challenge of mice and determination of vaccine efficacy. Surgical transfer of adult worms into the mesenteric veins of mice was performed as previously described (17) with some modification. Mice were challenged with 50 to 75 S. mansoni worms, age 21 to 23 days, perfused from infected golden hamsters and washed as described above. The worms were counted and drawn into a 1-ml syringe fitted with a 26-gauge, 3/4-in. stainless steel needle (Becton Dickinson). Mice were anesthetized and maintained with Isoflurane (1 to 1.5%). Following surgical preparation of the skin, a ~3-cm transverse incision was made through the skin and muscle layer along the linea alba to prevent unnecessary bleeding. The intestine was then gently drawn out to expose the mesenteric vein that resides medial to the cecum. The worms were injected into this vein in 0.5 ml of sterile medium. Following injection, bleeding was stopped by using Gelfoam (Johnson & Johnson) sponges. The intestines were carefully placed back into the abdominal cavity and flushed with warm (37°C) sterile saline. The muscle layer was sutured with a simple-interrupted stitch, using 4-0 Dexon (Davis & Geck), followed by subcutaneous suturing of the skin. The mice were allowed to recover under a heat lamp and administered 0.15 mg of Buprenex (buprenorphine HCl; Reckitt & Colman Pharmaceuticals, Richmond, Va.) per ml for pain relief as well as fluids subcutaneously.

Approximately 4 weeks postchallenge, the mice were sacrificed and perfused via the hepatoportal system to recover the parasites. To ensure that all worms were recovered, the liver was cut into sections and pressed between two glass slides for microscopic examination. The worms were counted, and percent recovery was determined by comparing the number of worms that successfully transferred into the vein to the number of worms recovered. As an indicator of efficacy, the mean percent protection observed in the experimental groups was determined as the decrease in the worm burden of each group compared to the mean percent recovery of the control groups (XPG vaccinated and unvaccinated).

Analysis of specific antibody responses by ELISA. Serum samples collected as described above were serially diluted and used to determine total IgG and Ig isotype responses by ELISA (8, 15). Goat anti-mouse IgG- or goat anti-rabbit IgG-alkaline phosphatase conjugate (Sigma) was then added to each well, and the pNPP phosphatase substrate system (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) was used to detect antigen-specific antibody by absorbance (405 and 595 nm). Isotype analysis was performed similarly, with the additional step of adding rabbit anti-mouse isotype-specific antibodies to the wells from a Mouse-Typer isotyping panel kit (Bio-Rad) following incubation with vaccinated-mouse sera.

T-cell proliferation assays. Splenocytes of two mice from each group (2.5 x 105 cells) and syngeneic irradiated antigen-presenting cells (5 x 105 cells) were stimulated with medium alone, concanavalin A (ConA) (1 µg/ml), or crude or recombinant antigens (5 to 10 µg/ml) in 96-microwell plates (Nunc) at 37°C in humidified air containing 5% CO2. The final volume was 200 µl/well in culture medium (RPMI 1640 supplemented with 100 U of penicillin G per ml, 100 µg of streptomycin per ml, 10 µg of polymyxin B per ml, and 10% fetal bovine serum). One microcurie of [3H]methylthymidine (NEN) was added to each well 78 to 80 h after the start of the assay, and 16 to 18 h later, the thymidine incorporation was determined in a beta scintillation counter (Wallac). Results were expressed as the stimulation index, i.e., the ratio of the mean counts per minute of triplicate culture cells taken up in the presence of the antigen to those obtained with medium alone.

Flow cytometry and intracellular cytokine staining. Splenocytes were analyzed for intracellular cytokine expression pattern in a four-color flow cytometric analysis as described previously (3, 53, 54) with some modifications. Briefly, aliquots of 107 cells from each group were cultured with or without crude and recombinant antigens (final concentration, 25 µg/ml) for 5 h and with phorbol myristate acetate-ionomycin (used as a positive control) for 4 h at 37°C in humidified air containing 5% CO2. Brefeldin A (BFA) (10 µg/ml) (Sigma) was added 4 h before the end of the culture. The cells were blocked with 200 µg of normal rat IgG (Caltag) per ml and stained with surface rat anti-mouse antibodies anti-CD3{varepsilon}-peridinin chlorophyll protein (145-2C11), anti-CD8{alpha}-fluorescein isothiocyanate (53-6.7), and anti-CD4-phycoerythrin (RM4-5) (BD-PharMingen) for 30 min at 4°C. Samples were fixed with 500 µl of 2.0% ultrapure formaldehyde (Polysciences) in PBS per tube and washed twice with filtered PBS-0.5% bovine serum albumin-0.1% NaN3. Aliquots of 106 splenocytes were stained with 20 µl of one of the following rat anti-mouse cytokine antibodies labeled with allophycocyanin per well: anti-interleukin-4 (IL-4) (11B11), anti-IL-5 (TRFK5), anti-IL-10 (JES5-16E3), anti-gamma interferon (IFN-{gamma}) (XMG1.2), anti-tumor necrosis factor alpha (TNF-{alpha}) (MP6-XT22). The antibodies were diluted in Fix & Perm permeabilization reagent (Caltag), and staining was in the dark for 30 min at room temperature. After washing with PBS-0.5% bovine serum albumin-0.1% NaN3, cells were fixed with 200 µl of 2.0% ultrapure formaldehyde in PBS per well and acquired in a FACSCalibur flow cytometer with CELLquest software (Becton Dickinson) with a minimum of 30,000 events. In each experimental run, unstained, isotype-matched, and standard stained cells were included as controls to assess the amount of nonspecific binding and for quality control of the acquisition in order to verify the corrected performance of the instrument. The multiparameter acquired data were analyzed by using the WinList 3D 4.0 software (Verity Software House, Topsham, Maine) (3, 53). The lymphocyte gate R1 was defined based on forward light scatter and side light scatter characteristics of the cells. Subsequent gates were generated to quantify total, cytokine-positive, and cytokine-negative splenic cells. The data were expressed as frequency of the gated cell population and cytokine-positive cells/gated cell population [for instance, (CD4+ cytokine+ cells/CD4+ cells) x 100] after short-term stimulation with antigens. The aim was to normalize the differences in the diminished total frequencies of the lymphocytes after surgical transfer (data not shown), consistent with the reduction observed previously by other groups after infection with S. mansoni cercariae (24, 31, 32). The frequency of cytokine-producing cells was then calculated by using the mean percentage in cultures with antigen (from two independent experiments with two pooled mice splenocytes at each of the PC and PP time points). The mean percentage in cultures with medium alone and with fusion partners (GST, His, or MBP) was subtracted from the experimental means.

Soluble-cytokine analysis. Cells were isolated from the spleens of two mice from each experimental and control group at the PC and PP time points. These cells were stimulated in vitro with various antigens (10 µg/ml), along with medium alone (negative control), ConA (12.5 µg/ml; positive control), WE, and background controls. The cells were incubated in complete T-cell medium at 37°C with 5% CO2, and 1-ml samples of supernatants were collected at 48 and 144 h. The samples were frozen at –80°C and sent to Roswell Park Cancer Institute (Buffalo, N.Y.) for cytokine analysis with the cytometric bead array (CBA). Briefly, the CBA uses spectrally discrete particles that can capture soluble analytes. The analyte (cytokine) is then measured by using a fluorescence-based detection mechanism and flow cytometric analysis (Luminex). Levels of secreted IFN-{gamma}, TNF-{alpha}, IL-6, and IL-10 were measured (29).

Statistical analysis. To test for differences between treatment groups, a mixed-model analysis of variance was used, with the group as the fixed effect and the experiment as the random effect. This model fits average percent protection as a function of treatment group. One of the assumptions when using a traditional analysis of variance model is that observations are independent. To control for the dependence which exists in observations obtained from the same experiment, an experiment effect was also added to the model. A mixed-model approach allows us to treat this experimental effect as randomly coming from a population of possible experimental effects rather than as a fixed effect of interest. Pairwise comparisons between groups was done by using Tukey's adjustment for multiple comparisons. A two-sample t test assuming unequal variances was used to compare changes in antibody response and frequency of both individual- and combined-cytokine-producing cells observed in control group individuals to those in the experimental animals.


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RESULTS
 
In vitro expression of DNA constructs. To ensure antigen expression from the pcDNAI/AMP vector in a eukaryotic system, BSC40 (monkey kidney fibroblast) cells were transfected with the various pcDNA constructs. Both SmCT-SOD and filamin could be detected in crude protein extracts from transfected cells by Western blot analysis with specific anti-GST-SmCT-SOD or anti-GST-filamin polyclonal rabbit serum (data not shown). Expression of SmGPX was detected by immunostaining pcGPX-transfected COS-7 cells with rabbit anti-GST-SmGPX sera and labeling with goat anti-rabbit-Alexafluor-568 (data not shown).

DNA vaccination of BALB/c mice and determination of vaccine efficacy. In order to test the hypothesis that DNA vaccination with candidate antioxidant enzymes will target adult S. mansoni worms, immunized BALB/c mice were challenged by surgical transfer of adult worms into the mesenteric circulation, and the worm burden was determined for each animal (see Materials and Methods).

In all three experiments, control group (unvaccinated and pcXPG vaccinated) worm burdens were consistent within and between control groups (Table 1), verifying that this animal vaccination model gave efficient and consistent results. Mice vaccinated with pcCT-SOD demonstrated a consistent decrease in worm burden, with averages of 42.8, 35.9, and 38.0% protection in three independent experiments (Table 1). The overall percent protection of the mice in the three experiments was found to be significantly different over that of controls (P < 0.0001). However, vaccination with pcGPX in two separate experiments failed to confer a statistically significant amount of protection in experimental mice (Table 1).


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  		TABLE 1. Worm burden and protection data from three independent experiments

A fragment encoding part of a protein homologous to a structural human actin-binding protein, filamin, proved effective when used as a vaccine candidate. When delivered in the pcDNA vector, filamin was able to significantly decrease adult worm burden compared to controls, giving 57 and 44% protection in two independent experiments (Table 1).

Analysis of antibody responses to DNA vaccination. Total IgG antibody responses of sera from vaccinated mice were analyzed for specificity to target antigens and for intensity of the specific response by ELISA. Figure 1A is representative of the total IgG responses of pcCT-SOD- and pcXPG-vaccinated mice to GST-SmCT-SOD, MBP-SmCT-SOD, and His-SmCT-SOD recombinant proteins. At the PP (week 14) time point, mice vaccinated with pcCT-SOD had a statistically significantly higher total IgG response (P = 0.002) than the pcXPG control group (Fig. 1A). This increase in the SmCT-SOD-specific response following challenge with adult parasites was seen in all three experimental groups (data not shown). The relatively high variability in response between individuals depicted by the error bars was found to be consistent in all animal groups in every experiment. As a control, antibody responses to WE and SEA in pcCT-SOD and pcXPG groups were also analyzed (Fig. 1A, inset). As expected, a marked increase in total IgG in response to WE and SEA was observed at the PP time point for both groups, while no significant response was noted prior to introduction of the parasite (Fig. 1A, inset). Next, isotype analysis of pooled sera specific for His-SmCT-SOD from each time point was performed. As seen in Fig. 1B, the only serum that gave an absorbance over the cutoff value, as well as being over 1.8 times greater than that in control sera, was IgG2a pooled sera from pcCT-SOD-vaccinated mice at the PP time point. IgG2a antibodies from pooled pcCT-SOD PP sera were consistently higher than those from pcXPG controls in all sera tested (Fig. 1B and data not shown). The IgG1-, IgG2b-, and IgG3-specific antibodies were never higher than those in mice vaccinated with control plasmid. Levels of specific IgA, and IgE failed to increase over preimmune levels in control or experimental animals in all groups tested (data not shown).



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  		FIG. 1. (A) Total IgG antibody response of pcXPG-and pcCT-SOD-vaccinated groups to recombinant GST-SmCT-SOD. Sera from individual pcXPG (n = 12)- and pcCT-SOD (n = 11)-vaccinated mice were analyzed at a 1:100 dilution, and the results represent the averages for the individuals from each group ± SEMs. Differences between pcXPG-vaccinated controls and pcCT-SOD-vaccinated animals were analyzed at each time point: *, P = 0.002. The inset shows the total IgG responses of pcXPG- and pcCT-SOD-vaccinated mice to SEA and WE; sera from individual mice were analyzed at a 1:200 dilution. (B) IgG isotype analysis of sera from pcCT-SOD-vaccinated mice. Sera from pcXPG- and pcCT-SOD-vaccinated mice were pooled, and specific IgG isotype responses to His-SmCT-SOD recombinant protein were analyzed by ELISA. Results represent the averages of triplicate absorbance readings, and the sera were diluted 1:100. The dotted lines represent the cutoff values average absorbance of preimmune sera plus three standard deviations.

Figure 2A illustrates total IgG responses of pc-filamin- and pcXPG-vaccinated mice to the GST-filamin fusion protein. Background GST responses of each individual mouse were subtracted from their respective GST-filamin absorbance readings. Like for the pcCT-SOD-vaccinated mice, the increase in specific total IgG response in pc-filamin-vaccinated animals was found to be statistically significant (P = 0.05) compared to that in pcXPG-vaccinated animals at the PP time point. Again, total IgG in response to WE and SEA was measured, and the expected increase in antibody response was observed at the PP time point, with no response observed prior to exposure to the parasite (Fig. 2A, inset). As seen in Fig. 1A and 2A (insets), the control groups (pcXPG) had greater total IgG in response to WE and SEA than the pcCT-SOD and pc-filamin groups, possibly representing a more polarized Th2 response in the control animals in response to worm and egg antigens. Next, the antibody isotypes of pooled sera specific for filamin from the pcXPG and pc-filamin groups were analyzed. As seen in Fig. 2B, pc-filamin sera had over a two-times-greater increase in both IgG2a and IgG2b over pcXPG controls at the PP time point, and the absorbance readings for these two antibody isotypes were over the cutoff values at the PC and PP time points. IgG1 antibody specific for filamin was also higher in the experimental group than in controls, with only PP sera giving absorbance readings over the cutoff value. Levels of IgG3, IgA, and IgE never increased over levels in preimmune sera for any group tested (Fig. 2B and data not shown).



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  		FIG. 2. (A) ELISAs detecting total IgG antibodies against recombinant GST-filamin in mice vaccinated with pcXPG (n = 4), or pc-filamin (n = 5). Sera from individual animals were analyzed at a 1:100 dilution, and the results represent the averages for the individuals from each group ± SEMs. Differences between pcXPG-vaccinated controls and pc-filamin-vaccinated animals were analyzed: *, P = 0.05. The inset shows the total IgG responses of pcXPG- and pc-filamin-vaccinated mice to SEA and WE. Sera from individual mice were analyzed at a 1:200 dilution. (B) IgG isotype analysis of pc-filamin-vaccinated mice. Sera from the pcXPG- and pc-filamin-vaccinated mice were pooled, and specific IgG isotype responses to GST-filamin recombinant protein were analyzed by ELISA. Results represent the averages of triplicate absorbance readings, and the sera were diluted 1:100. The dotted lines represent the cutoff values as describe in the legend to Fig. 1. Background GST absorbance readings were subtracted from all serum responses analyzed.

The levels of total IgG in pcGPX-vaccinated mice in response to SmGPX were similar to those in pcXPG groups in both experiments at all time points, with absorbance readings similar to those of background controls (data not shown). Sera from unvaccinated controls from all three experiments were also tested. The absorbance readings from these mice at the PI and PP time points were similar to pcXPG mice for each specific antigen (data not shown).

T-cell proliferation assays. We further evaluated whether vaccination of BALB/c mice with pcCT-SOD, pcGPX, pc-filamin, and control pcXPG DNA was able to elicit T-cell proliferation after specific stimulation in a recall experiment, 6 weeks after vaccination (PC) and 4 weeks after challenge with transferred worms (PP). Splenocytes derived from each group were stimulated with ConA and S. mansoni antigens (Fig. 3A). Spleen cells from all groups proliferated against ConA, showing their viability (data not shown). After vaccination (PC), splenocytes from pcCT-SOD- and pcGPX-vaccinated mice proliferated following stimulation with WE, confirming the presence of SmCT-SOD and SmGPX antioxidants in the adult stage. After challenge (PP), spleen cells from all groups exhibited an increase in proliferation in response to WE stimulation compared to medium alone (the proliferative response of the pc-filamin group against WE was not determined). Cells from the protected pcCT-SOD and pc-filamin groups also proliferated in response to their respective recombinant antigen, while cells from the unprotected pcGPX group did not.



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  		FIG. 3. Proliferation and cytokine production from pooled spleen cells. (A) T-cell proliferation assay of splenocytes after vaccination with pcCT-SOD, pcGPX, pc-filamin, and pcXPG control DNA, before (PC) and after (PP) challenge with S. mansoni worms. Results are expressed as stimulation index (S.I.) and are representative of individual experiments. N.D., not done. (B) Dot blot representations of cytokine-positive and cytokine-negative splenic cells demonstrate the placement of R2 (CD4+ lymphocytes), R3 (CD8+ lymphocytes), R4 (CD4+ cytokine+ cells), and R5 (CD8+ cytokine+ cells) gates. The R1 gate was based on the forward scatter versus side scatter (not shown). The data were expressed as frequencies of CD4+ cytokine+/CD4+ and CD8+ cytokine+/CD8+ cells, after short-term stimulation with antigens, described in Materials and Methods. PE, phycoerythrin; FITC, fluorescein isothiocyanate; APC, allophycocyanin.

Analysis of intracytoplasmic cytokine levels in an in vitro recall assay. To determine the nature of the active immune response taking place in these vaccinated groups before and after challenge, we performed an analysis of the frequency of cytokine-expressing lymphocytes at a single-cell level for TNF-{alpha}, IFN-{gamma}, IL-4, IL-5, and IL-10 after antigenic stimulation, before and after challenge. A multiparameter analysis was performed, as exemplified in Fig. 3B and described in Materials and Methods.

First, the cytokines were grouped based on the type 1 (T1) (TNF-{alpha} and IFN-{gamma})- and type 2 (T2) (IL-4, IL-5, and IL-10)-associated immune responses by using the mean percentage of cytokine-expressing cells after antigenic stimulation and background subtraction. Table 2 shows the mean (± standard error of the mean [SEM]) frequency of T1 or T2 cytokine-producing cells after stimulation with recombinant antigens (SmCT-SOD, filamin, and SmGPX) from vaccinated and pcXPG control mice, before (PC) and after (PP) challenge with surgically implanted S. mansoni worms. Before challenge, no statistical differences between the vaccinated groups and the pcXPG group were found (Table 2). After surgical implant of parasite worms, the frequencies of both CD4+ T2 (P = 0.006) and CD8+ T2 (P = 0.048) cytokine-positive cells were significantly decreased in the protected pcCT-SOD group after stimulation with SmCT-SOD compared to the control pcXPG group. We failed to show any significant difference in the pc-filamin and pcGPX groups compared to the pcXPG group following specific antigenic stimulation, both PC and PP.


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  		TABLE 2. Frequencies of cytokine-producing cells after stimulation with specific antigens from vaccinated mice compared to control pcXPG before (PC) and after (PP) challenge with surgically implanted S. mansoni worms

Once the overall T1- and T2-associated CD4+ and CD8+ cell responses upon antigenic stimulation were evaluated, we stratified the gated CD4+ and CD8+ cells in IL-4, IL-5, IL-10, TNF-{alpha}, and IFN-{gamma} cytokine-expressing cells before and after surgical challenge. All background control percentages (medium alone and fusion partner tags) were subtracted from antigen-stimulated percentages, and the results were expressed as mean percentage and SEM (Fig. 4). After vaccination, the pcCT-SOD and pc-filamin groups showed a high frequency of both CD4+ and CD8+ lymphocytes expressing IFN-{gamma}, IL-4, IL-5, and IL-10 after SmCT-SOD (for pcCT-SOD) and WE and filamin (for pc-filamin) antigenic stimulation (Fig. 4). After challenge, the frequencies of CD4+ cells expressing TNF-{alpha}, IL-4, and IL-5 (Fig. 4A) and of CD8+ cells positive for IFN-{gamma}, TNF-{alpha}, and IL-4 increased in the control pcXPG group after stimulation with WE compared to the pcCT-SOD and pc-filamin groups, while the pc-filamin-vaccinated group showed an increase in both CD4+ and CD8+ cells positive for IL-5. The levels of CD4+ and CD8+ IL-10-positive cells in the pcGPX group increased after challenge when stimulated with WE. No statistically significant differences were found between the frequencies of any particular cytokine-positive cells in the vaccinated groups and pcXPG control group when the cytokine responses were analyzed individually (P > 0.05).



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  		FIG. 4. Frequency of individual cytokine-producing cells against parasite antigens after vaccination with pcCT-SOD, pcGPX, pc-filamin, and pcXPG control DNA before (PC) and after (PP) challenge with surgically implanted S. mansoni worms. The data represent the mean percentages (± SEMs) of CD4+ (A) and CD8+ (B) cells expressing intracellular IL-4, IL-5, IL-10, TNF-{alpha}, and IFN-{gamma} cytokines within the gated cells after stimulation for 5 h minus the mean percentages of unstimulated cells and fusion partner controls. There were no statistical differences between the control pcXPG group and the vaccinated groups (P > 0.05).

Analysis of secreted-cytokine levels from splenic cells in an in vitro recall assay. After determining the cytokine expression profile of splenic T cells following antigenic stimulation, the secreted levels of IFN-{gamma}, TNF-{alpha}, IL-6, and IL-10 from spleen cells stimulated in vitro with various antigens were analyzed. As described in Materials and Methods, spleen cells from mice at the PC and PP time points were incubated, and 1-ml supernatant samples were taken for CBA analysis. Figure 5 illustrates the concentrations of the various cytokines secreted from spleen cells pooled from two mice from each group. As seen in Fig. 5, there were marked increases in IFN-{gamma} secretion from both the pcCT-SOD and pc-filamin cells compared to the pcXPG control cells when they were stimulated with WE and specific antigen. The increase in IFN-{gamma} secretion by pcCT-SOD spleen cells was noted following vaccination (PC) and was enhanced following challenge, suggesting a boost in response following introduction of the parasite. Spleen cells from pcGPX-vaccinated mice secreted levels of IFN-{gamma} comparable to those of the control group in response to WE, and the levels were only slightly increased in response to specific antigen compared to the protected groups (pcCT-SOD and pc-filamin). As with the number of cells producing TNF-{alpha} in the control group (Fig. 4), the amount of this cytokine secreted by pcXPG cells was increased compared to that secreted by pcCT-SOD cells stimulated with SmCT-SOD, while filamin failed to produce measurable amounts of this cytokine (Fig. 4 and 5). SmGPXm was able to stimulate TNF-{alpha} secretion from both pcXPG and pcGPX cells (Fig. 5). In response to WE and both antioxidant enzymes (SmCT-SOD and SmGPX), control cells secreted increased levels of IL-6 PP, followed by higher levels in the pcGPX group than in the pcCT-SOD and pc-filamin groups. Lastly, levels of IL-10 secreted from all experimental groups were increased PP following stimulation with WE compared to pcXPG cells.



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  		FIG. 5. Levels of secreted cytokines of spleen cells from pcXPG-, pcCT-SOD-, pc-filamin-, and pcGPX-vaccinated mice stimulated with antigen in vitro. Concentrations of IFN-{gamma}, TNF-{alpha}, IL-6, and IL-10 secreted from PC or PP cells of vaccinated mice stimulated in vitro with WE, recombinant MBP-SmCT-SOD, GST-filamin, and thrombin-cleaved SmGPXm (GPX) (x axis) are shown. At 48 and 144 h poststimulation, 1-ml samples of supernatants from cell cultures were collected and analyzed by CBA (see Materials and Methods). Background control values (medium alone and GST and MBP tags) were subtracted from experimental values.


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DISCUSSION
 
Two paradigms concerning host immune responses to schistosomes have existed. The first is that the schistosomule stages are the stages most susceptible to immune elimination, and the second is that the adult stage is least susceptible and is able to avoid immune killing through the evolution of several defense mechanisms (30, 33, 44, 51, 52). In vitro assays have shown the effectiveness of antibody-dependent cell cytotoxicity and reactive oxygen and nitrogen species in killing schistosomule stages of the parasite, with a minimal effect on adult worms (30). Our previous work with S. mansoni demonstrated that expression and activity levels of the antioxidant enzymes SmCT-SOD and SmGPX increase as the parasite develops from schistosomule to adult (35). Data from mice vaccinated with pcCT-SOD and pcGPX and challenged with cercariae showed protection of 54 and 43%, respectively (49). Based on these observations, we hypothesized that adult worms could be targeted for immune killing by using these antioxidant enzymes as vaccine candidates.

One advantage of using DNA vaccination against a multicellular eukaryote is the introduction of antigen in the native conformation (41, 46). Here, we applied nucleic acid vaccine technology to address the efficacy of our vaccine candidates, SmCT-SOD, SmGPX, and filamin, to eliminate liver stage schistosomes. In order to test our hypothesis, we used a surgical technique to transfer adult worms, 21 to 23 days old, directly into the mesenteric circulation of BALB/c mice (17) following DNA vaccination with our candidates. Challenging mice in this manner allowed us to study the effects of our vaccination on adults while bypassing all schistosomule stages and immune responses elicited by them. As seen in Table 1, there are other advantages of transferring worms into the mesenteric vein versus cercarial challenge. First, the exact number of worms that were successfully transferred is known, and second, the variability between individuals is less than it is with cercarial challenge.

In support of our hypothesis, mice that received two 100-µg doses of pcCT-SOD experienced highly significant and consistent decreases in worm burden (Table 1) (42.8, 35.9, and 38.0%) compared to control animals in three independent experiments. Surprisingly, mice immunized with pcGPX did not experience a significant decrease in worm burden compared to controls. Earlier data had shown significant protection in pcGPX-vaccinated mice challenged with cercariae (49). We are at a loss to explain the SmGPX results. As our previous immunolocalization studies examined only the 3-h schistosomula and the adult stage worms (35), we are currently testing (using immunofluorescence) the hypothesis that schistosomula begin to express SmGPX before they reach the hepatoportal circulation (perhaps at the late lung stage) and are thus subject to killing before they reach the liver.

The third candidate, pc-filamin, contains a 1.7-kb fragment encoding a protein with homology to a human actin-binding protein (Mohamed et al., submitted). Its expression, along with SmCT-SOD and SmGPX, was found to be associated with the adult worm tegument, the host-parasite interface (35; Mohamed et al., submitted). Our data demonstrate that vaccination with pc-filamin was protective against cercarial challenge, with an average of 43% protection, and, based on existing paradigms, it was hypothesized to target the larval stages (Mohamed et al., submitted). To our surprise, pc-filamin gave the best protection, i.e., 57.1 and 43.5%, in two independent experiments. This average 50% decrease in worm burden compared to controls was found to be statistically very significant (Table 1). These data offer a major advance in schistosome vaccine research. This work, along with our previous data on a murine model against cercarial challenge, suggest that both the larval and adult stages may be targeted for elimination when the immune response is primed with SmCT-SOD and filamin delivered in a eukaryotic DNA expression system. A vaccine that is able to target adult worms not only would decrease worm burden by prevention but might be able to reduce an existing infection as well. This, combined with the predicted longevity and stability of DNA vaccines, would reduce the need for drug therapy, prescreening, and follow-up treatment (55). We are currently investigating whether we can immunize and protect animals with established infections. The notion of adult worms as a target of immune elimination has been inferred for Schistosoma haematobium and Schistosoma bovis but not for S. mansoni or Schistosoma japonicum (1). However, in vitro studies have shown a transient reexpression of susceptibility to killing of post-lung stage S. mansoni worms (2 to 2.5 weeks old but not 4 to 6 weeks old) by cytokine-activated macrophages (42), which is postulated to be due to changes in parasite physiology during parasite development (2). Our data clearly demonstrate that 3-week or older liver stage worms are susceptible to elimination by vaccine-induced immunity.

In our murine model, we were able to show a consistent, statistically significant increase in total IgG in all three pcCT-SOD experimental groups to SmCT-SOD over pcXPG controls and a statistically significant response to filamin in pc-filamin-vaccinated mice compared to pcXPG controls PP, although at a relatively low titer (1:100). The route of DNA administration may be a factor affecting antibody titer, as it has been noted that injection in the ear pinna mediates a higher specific titer than intramuscular or subcutaneous injection (16, 48). Based on the specific responses to SmCT-SOD and filamin observed PP in the respective experimental groups, as well as the increase in response to WE in all groups, we suspect that introduction of the native antigen upon challenge boosts the response that was primed by vaccination. The proliferative response (Fig. 3) to WE by the experimental groups following vaccination and to specific antigen following challenge reinforce this notion. While it is probable that SmCT-SOD and filamin are present in WE, the specific antibody response to these antigens can only be detected following challenge. This "natural" boosting in IgG antibody response was also observed after challenge in rats immunized with DNA encoding Sm28GST (21). This would be advantageous in an area of endemicity, where long-lasting immunity would be boosted by natural exposure to the parasite. The overall greater response to WE and SEA by the control groups (pcXPG) may be due to a greater humoral response to other worm antigens in comparison to specific responses observed in the experimental groups. When secreted cytokines in the supernatant were investigated, there was an increase in IFN-{gamma} secretion from spleen cells following challenge in both pcCT-SOD- and pc-filamin-immunized animals in response to WE and specific antigen that was not observed in the unprotected groups (pcXPG and pcGPX), which may be influencing the polarity of the immune response (Fig. 5). When isotype analysis was performed on the pcCT-SOD and pc-filamin sera, levels of IgG2a were consistently higher than control values. This trend was also noted in mice vaccinated with Sm23-pcDNA (19), which experienced a 21 to 44% protection against cercarial challenge. IgG2a is a Th1 cytokine-derived antibody involved in enhanced phagocytosis, and evidence indicates that this isotype in rats, and the human homologue IgG1, are protective against schistosomula in an antibody-dependent cell-cytotoxic manner (13, 14, 21, 43). The consistent increase in SmCT-SOD-specific IgG2b in both control and pcCT-SOD-vaccinated mice indicates that the antigen stimulates a more T1-type antibody response. This is consistent with our previous results showing a T1 predominance when SmCT-SOD is used in various vaccine formulations (15). In contrast, for pc-filamin-vaccinated mice, a significantly increased frequency of CD4+ cells positive for T2 cytokines after WE stimulation (data not shown) correlates with the increased levels of filamin-specific antibodies in pc-filamin that indicates a more Th0- or mixed Th1-/Th2-type response. It remains to be seen if the specific antibodies we detected are involved in protection in our model, a question currently under investigation. In addition, our data suggest that different immune mechanisms may be involved in the eliminations of liver stage worms following vaccination with SmCT-SOD compared to vaccination with filamin.

In an attempt to identify a change in immune response related to the protection levels observed, the intracytoplasmic cytokine data were grouped based on the associated immune responses, i.e., T1 (TNF-{alpha} and IFN-{gamma}) or T2 (IL-4, IL-5, and IL-10), and the mean frequency of cytokine positive cells was calculated after background subtraction. When the frequencies of experimental groups were lower than those of unstimulated controls, negative frequency values were found. In Table 2, we chose not to reduce these values to zero after subtraction of control values in order to show the magnitude of the basal response compared to the stimulated one after immunization and after surgical challenge, as opposed to the case for Fig. 4, where only values above zero from individual cytokine are represented. Interestingly, after surgical implant of parasite worms, the frequencies of both CD4+ and CD8+ T2 cytokine-positive cells were significantly decreased in the protected pcCT-SOD group after stimulation with SmCT-SOD compared to the control pcXPG (Table 2). Although we failed to show any statistical significance when the contribution of the response of cells expressing individual cytokines was analyzed (Fig. 4), the significant increase in the IgG2a antibody levels and in the specific IFN-{gamma} secretion from spleen cells following challenge in both pcCT-SOD- and pc-filamin-immunized animals (which was not observed in the unprotected pcXPG and pcGPX groups) may point to a role for Th1, and possibly Tc1 (45), responses in the development of protection in this model.

Our data also indicate that in control mice, TNF-{alpha} and secreted IL-6 cytokines are elevated, likely due to responses to granuloma formation (43). The decreased levels of both CD4+ and CD8+ TNF-{alpha}-expressing cells and secreted TNF-{alpha} in the pcCT-SOD- and pc-filamin-vaccinated groups are likely to represent a suppression at both the macrophage (26) and lymphocyte levels, possibly through IL-4 and/or IL-10 (such as for the CD4+ and CD8+ cells producing IL-4 and IL-10 found in both protected groups) and/or transforming growth factor ß, although we did not investigate this possibility (25, 39, 40). It has been proposed that IL-10 is a key player in the polarization of both T1 and T2 responses in vaccination studies (27, 50, 57). However, the exact role of IL-10 in vaccination studies with defined antigens as reported here is yet to be defined.

Although phorbol myristate acetate-ionomycin stimulation was able to induce cytokine-expressing cells in the pcGPX-vaccinated group (data not shown), we could not detect lymphocytes that were cytokine positive after stimulation with specific antigens (with the exception of some IL-10), in contrast to the high levels of secreted TNF-{alpha}. Differences that were observed between the CBA and single-cell intracytoplasmic cytokine results could be due to differences in assay sensitivity; treatment with BFA for 4 h, which leads to cytokine accumulation in the cytoplasm as opposed to consumption during culture; or differences in the length of time the cells were exposed to antigens in culture before the assessment of cytokines (since the intracytoplasmic cytokines were evaluated after antigenic stimulation for 1 h plus 4 h with BFA, while secreted cytokines were investigated after 48 and 144 h of stimulation). In addition, as we focused on the frequency of CD4+ and CD8+ cytokine-positive lymphocytes, we cannot exclude the contribution of cytokine secretion by other cell types, such as monocytes, B cells, granulocytes, etc.

We have demonstrated that DNA vaccines encoding SmCT-SOD or filamin as vaccine candidates are able to significantly and consistently reduce worm burden by targeting adult S. mansoni worms (21 days and older) for immune elimination. The protection observed is specific, as mice immunized with pcGPX and challenged with adult worms were not significantly protected. We have also shown evidence suggesting that a T1 response (with specific IgG2a antibody and IFN-{gamma} secretion) may be involved in the protective response. Future experiments will be directed to further understand the immune mechanisms stimulated in our experimental model, as well as to determine whether the specific IgG antibody response observed is protective through passive transfer experiments. Taken together, the data presented here support the hypothesis that adult S. mansoni worms can be targeted for immune elimination. Furthermore, SmCT-SOD and filamin, when used as immune targets, are able not only to decrease the adult worm burden but to decrease it enough to possibly have an impact on the pathology and transmission of S. mansoni (4, 7), with the protection observed in the experimental groups meeting the World Health Organization criteria of 40% protection or better (5). These data also offer an advance over existing S. mansoni vaccine paradigms in that this is the first report of a vaccine successfully targeting adult worms for immune elimination. This finding offers the potential of developing a therapeutic as well as a prophylactic vaccine.


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ACKNOWLEDGMENTS
 
The technical assistance of Sharon Willard and assistance with analysis of flow cytometric data by Carleton Stewart are gratefully acknowledged.

This research was supported by NIAID grant AI18867.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, 138 Farber Hall, School of Medicine and Biomedical Sciences, SUNY, Buffalo, NY 14214. Phone: (716) 829-2459. Fax: (716) 829-2169. E-mail: loverde{at}buffalo.edu. Back

Editor: W. A. Petri, Jr.


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Infection and Immunity, October 2004, p. 6112-6124, Vol. 72, No. 10
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.10.6112-6124.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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