Immunoaffinity-isolated antigens induce protective immunity against larval Strongyloides stercoralis in mice

Experimental Parasitology 100 (2002) 112–120 www.academicpress.com

Immunoaffinity-isolated antigens induce protective immunity against larval Strongyloides stercoralis in mice De’Broski R. Herbert,a Thomas J. Nolan,b Gerhard A. Schad,b Sara Lustigman,c and David Abrahama,* b

a Department of Microbiology and Immunology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA Department of Pathobiology, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104, USA c The Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67th Street, New York, NY 10021, USA

Received 20 November 2001; accepted 28 January 2002

Abstract The objective of this study was to identify soluble protein antigens that would induce protective immunity against infective-stage larvae (L-3) of Strongyloides stercoralis in mice. Deoxycholate (DOC)-soluble proteins derived from L-3, adsorbed to aluminum hydroxide, induced protective immunity in BALB/c mice. The immunized mice generated parasite-specific IgG that could transfer passive immunity to na€ıve animals. The protective antibodies bound to parasite antigens found in the muscles and nerve cords of the L-3. An IgG affinity chromatography column generated with IgG from the sera of DOC-immunized mice was used to purify specific larval antigens. Proteins were eluted from the affinity column with sizes of 80, 75, 61, 57, 43, and 32 kDa. This antigen pool stimulated both proliferation and IL-5 production by splenocytes recovered from mice immunized with live L-3. Vaccination of mice with the immunoaffinity-isolated antigens led to significant protective immunity, with 83% of challenge larvae killed. This study demonstrates that IgG-isolated proteins are candidate antigens for a vaccine against larval S. stercoralis. Ó 2002 Elsevier Science (USA). All rights reserved.

1. Introduction Strongyloides stercoralis, a nematode parasite of humans, causes disease in the gastrointestinal system and skin of immunocompetent individuals and can cause fatal hyperinfection in immunocompromised individuals (Grove, 1996). Diagnosis of this infection is accomplished either by observing larvae in the stool or by immunodiagnosis (Conway et al., 1995; Grove, 1996). Mass chemotherapy of infected and uninfected individuals, selective chemotherapy of individuals diagnosed with the infections, and targeted chemotherapy directed at individuals at risk of infection have all been suggested as possible means to effectively control S. stercoralis (Conway et al., 1995). An additional method to supplement chemotherapy would be the use of a vaccine. Advantages of a vaccine include long-lasting protection

*

Corresponding author. Fax: +215-923-9248. E-mail address: [email protected] (D. Abraham).

of the individual following immunization and the prophylactic nature of the treatment. Protective immunity against nematode infections has been induced using a variety of approaches (Knox, 2000). These include immunization with irradiated larvae (Miller, 1971; Wong et al., 1974; Klei et al., 1982; Yates and Higashi, 1985; Abraham et al., 1988, 1989; Lange et al., 1993), excretory and secretory antigens (Kazura and Davis, 1982; Ghosh et al., 1996; McKeand, 2000), and recombinant antigens (Li et al., 1993; Taylor et al., 1995; Ghosh et al., 1996; Jenkins et al., 1996; Ghosh and Hotez, 1999; Abraham et al., 2001). An alternative approach has been to identify specific molecules derived from the parasite, which are then used in a vaccine. The rationale for this approach is to use native antigens that have a high probability of inducing protective immunity and to then limit the number of antigens in the vaccine pool using a specific selection process. An effective means of making the specific selection has been to use antibodies to identify potentially protective antigens (Silberstein and Despommier, 1984;

0014-4894/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 1 4 - 4 8 9 4 ( 0 2 ) 0 0 0 0 8 - 5

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Pearce et al., 1986; McGillivery et al., 1992; Jasmer et al., 1993; Jacobs et al., 1999). Protective immunity to larval S. stercoralis has been studied in mice to determine how the response is regulated and to identify the essential components. Protective immunity was induced in mice by immunization with either live or dead larvae (Abraham et al., 1995) and the immunity was shown to be dependent on IL-4 and IL-5, thus demonstrating a dependency on a Th2 response (Rotman et al., 1997; Herbert et al., 2000). Passive transfer of immunity was accomplished only with the IgM isotype of antibody and a dependency on complement activation was observed (Brigandi et al., 1996). Eosinophils were associated with the killing process as they were elevated in the microenvironment of the killed larvae in immunized mice and they were shown to contain products capable of directly killing the parasite (Abraham et al., 1995; Rotman et al., 1996; Herbert et al., 2000). The antigenic composition of larval Strongyloides sp. has been studied using a variety of different methods to solubilize the worms. Included in these methods were solubilization in phosphate-buffered saline (PBS) and in the anionic detergent, sodium deoxycholate (DOC). Differences were seen quantitatively and qualitatively in the antigen profiles generated after solubilization in these agents. Depending on the study, diagnostic antigens were either predominately found in the PBS or in the DOC pool of antigens (Northern and Grove, 1988; Sato et al., 1990a,b; Conway et al., 1993). The goal of the present study was to identify soluble antigens from the third-stage infective filariform larvae (L-3) of S. stercoralis that would induce protective immunity in mice. The approach taken was to solubilize the larvae in PBS or in DOC and then immunize mice with the different antigen pools adsorbed to the adjuvant alum. Alum was selected as the adjuvant since it preferentially induces Th2 responses (Kenney et al., 1989; Forsthuber et al., 1996; Yip et al., 1999). The specific antibody isotype found to transfer protective immunity was then used to isolate the putatively protective antigens. These antigens were then tested for their ability to stimulate T cell responses and to induce protective immunity.

2. Materials and methods 2.1. Experimental animals and parasites Male BALB/cByJ mice 6–8 weeks of age were obtained from Jackson Laboratories (Bar Harbor, ME) and housed in the Laboratory Animal Sciences facility at Thomas Jefferson University in filter-top microisolator boxes under light- and temperature-controlled conditions.

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S. stercoralis L-3 were obtained from the fresh stools of a laboratory dog infected with the parasite according to methods previously described (Abraham et al., 1995). Larvae were collected from charcoal cultures and washed by centrifugation and resuspension in sterile RPMI with 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.1 mg/ml gentamicin (antibiotics) (Sigma Chemical Co., St. Louis, MO). 2.2. L-3 solubilization L-3 were separated from fecal contamination by being placed for 20 min in a 2% solution of low-melt agarose (Type I-A: Low EEO); (Sigma). After solidification of the agar, PBS supplemented with antibiotics was added and larvae that migrated into the PBS solution were harvested and stored at )80 °C in glycerol. Soluble L-3 antigen was prepared by homogenizing the L-3 for 1 h on ice in the presence of a protease inhibitor cocktail (2 mM leupeptin, 2 lg=ml pepstatin A, 28 lg=ml aprotinin, and 5 mM ethylenediamine tetraacetate (Sigma)) and followed by sonication for 12 min. The larval material was then incubated at 4 °C for 18 h to obtain the PBS-soluble proteins (PBS-Ag). DOCsoluble proteins (DOC-Ag) were extracted from the PBS-insoluble fraction by incubation for 24 h with 20 mM Tris–Cl/0.5% deoxycholic acid (Sigma) in the presence of the same protease inhibitor mixture. The DOC-Ag was then dialyzed against PBS for 18 h, passed through a 0:2-lm syringe filter, quantitated by a Micro BCA Protein Assay (Pierce, Rockford, IL), and stored at )80 °C. 2.3. Immunization and challenge protocol Mice were immunized with soluble antigens using the following protocol: 2% aluminum hydroxide lowviscosity re-hydragel (alum) (Reheis, Inc., Berkeley Heights, NJ) was diluted 1:10 with PBS containing variable concentrations of the soluble larval extracts. Mice were injected with 200 ll of the solution in the nape of the neck on Day 0 and Day 14 followed on Day 28 by a challenge infection consisting of L-3 contained within a diffusion chamber. Preparation of diffusion chambers followed previously published methods (Abraham et al., 1995). Briefly, 14-mm Lucite rings (Millipore, Bedford, MA) were covered with 2.0-lm Isopore membranes (Millipore). The membranes were attached to the rings with cyanoacrylate adhesive (Super Glue Corp., Nollis, NY), the Lucite rings were cemented to each other with a compound consisting of equal parts of 1,2-dichloroethane (Fisher Scientific, Pittsburgh, PA), and acryloid resin (Rohm and Hass Co., Philadelphia, PA), and the completed diffusion chambers were sterilized in 100% ethylene oxide. Diffusion chambers containing 50 L-3 were

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implanted in a subcutaneous pocket created on the lower flank of the mice and were removed on Day 1 or Day 5. The surviving live parasites, as determined by morphology and motility, were quantitated using a dissecting microscope. 2.4. Passive transfer of immunity Sera were collected from control and immunized mice at the time that the diffusion chambers were recovered. The serum from immunized mice was passed through a Gammabind Plus protein G Sepharose column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) to separate IgM, IgA, and IgE, which flowed through the column, from the IgG, which bound to the beads. The IgG fraction was eluted from the column using 0.5 M acetic acid, pH 3.0, which was immediately neutralized with saturated Tris–HCl, pH 9.8. The IgM, IgA, and IgE fractions were further separated by sequential passage through anti-mouse IgE and anti-mouse IgA affinity columns prepared as previously described (Brigandi et al., 1996). One hundred microliters of serum from control and immunized mice was diluted to 200 ll with PBS and then transferred into the subcutaneous pocket, in which a diffusion chamber was inserted for 24 h. Experiments were also performed to determine which specific antibody isotypes had the ability to passively transfer immunity. An enzyme-linked immunosorbent assay (ELISA) determined the quantity of each isotype found in 100 ll of serum. This quantity of antibody was diluted into 200 ll of PBS and injected into na€ıve mice as described above. 2.5. Antigen purification by affinity chromatography IgG from mice immunized with DOC-Ag was then used on a column to purify those antigens that would bind to the IgG (IgG specific-Ag); 5 mg of IgG from mice immunized with DOC-Ag was linked to CNBractivated Sepharose. A nonspecific IgG column was also generated with normal mouse IgG (Sigma). DOC-Ag was first passed over the nonspecific IgG column, to remove those antigens that would nonspecifically bind to the antibody (Brigandi et al., 1997), and the flowthrough was collected and added to the IgG-specific column. Following washing with binding buffer (15 mM NaCl, 19 mM NaH2 PO4 , 81 mM NaH2 PO4 ), proteins were eluted using 0.5 M acetic acid, pH 3.0, which was immediately neutralized with saturated Tris–Cl, pH 9.8. Two fractions were obtained: IgG specific-Ag and the flow-through antigens (IgG non-bound-Ag). The proteins were dialyzed overnight against PBS, concentrated in Centricon Centrifugal Concentrators (Millipore), and quantitated by the Micro BCA Protein Assay (Pierce).

2.6. Protein analysis PBS-Ag, DOC-Ag, IgG specific-Ag, IgG non-boundAg, normal mouse IgG, and protein standards (Bio-Rad Corp.) were separated in a one-dimensional SDS–PAGE using 12% polyacrylamide 0.5-mm slab gels with 4% polyacrylamide stacking gel in a Mini-Protean II apparatus (Bio-Rad). All electrophoreses were performed at 30 mA constant current and the proteins were stained using a Silver Stain Plus Kit(Bio-Rad). Relative molecular masses (Mr ) were determined after calculation of the RF value for the standard proteins as previously described (Shapiro et al., 1967). 2.7. Electron microscopy L-3 were fixed for 30 min in 0.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, containing 1% sucrose, and were then processed for immunoelectron microscopy. Thin sections of embedded worms were incubated with purified IgG from control and DOC-Ag immunized mice, followed by incubation with a suspension of 15nm gold particles coated with protein A, as previously described (Lustigman et al., 1991, 1996; Irvine et al., 1994). 2.8. Proliferation assay Spleens were removed from control mice and from mice immunized with live L-3 after recovery of the challenge infection. Red blood cells were lysed with ammonium chloride Tris, pH 7.3, and the recovered lymphocytes were stimulated with 10 lg=ml of PBS-Ag, DOC-Ag, IgG non-bound-Ag, or IgG specific-Ag. Four days following antigen stimulation, cells were pulsed overnight with 1 lCi per well of ðH3 Þ TdR (Amersham Pharmacia Biotech AB), harvested onto glass filters, and counted on a liquid scintillation counter to determine the amount of incorporated thymidine. 2.9. IL-5 Elispot assay Lymphocytes, prepared as above, were stimulated with the four antigen pools and the number of cells producing IL-5 was determined. Multiscreen plates (Millipore) were coated with capture antibody TRFK-5 at a concentration of 10 lg=ml in borate buffer, pH 8.5, overnight at 4 °C. Plates were then washed and blocked with 5% BSA for 2 h at room temperature. Cells were plated in triplicate wells in RPMI 1640 supplemented with 10% fetal calf serum and 10 U/ml recombinant IL-2 (NCI, Biological Resources Branch, Frederick, Mo). Following a 72-h incubation at 37 °C, cells were removed by washing with PBS/0.1% Tween 20 and incubated with the appropriately matched biotinylated anti-mouse secondary antibodies diluted in PBS 1%

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BSA (pH 7.3). Plates were washed and incubated with alkaline phosphatase (Sigma) for 2 h at room temperature. Finally, plates were washed with PBS Tween 20 before developing the colorimetric assay by the addition of Sigma Fast BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/Nitroblue tetrazolium) tablets for 30 min. The plates were then washed with distilled water and airdried. Spots were quantitated by examination under a dissecting microscope. 2.10. ELISA Nunc Maxisorp 96-well plates (Nunc Inc., Naperville, IL) were coated with 50 ll of 10 lg=ml of DOC-Ag overnight at 4 °C. Plates were blocked with borate blocking solution (0.17 M boric acid, 0.12 M NaCl, 0.05% Tween 20, 0.25% BSA, 1 mM EDTA, pH 8.5) at 37 °C for 1 h. Wells were washed with distilled water and test samples, diluted in PBS containing 1% BSA (pH 7.3), were placed in wells at serial dilutions and incubated at 37 °C for 2 h. Biotinylated goat anti-mouse IgM or IgG (Pharmingen) was added and plates were incubated at 37 °C for 2 h. Plates were then washed and avidin peroxidase (Sigma) was added for 30 min followed by the peroxidase substrate 2,20 -azino-di(3-ethylbenzthiazoline-6-sulfonate) (ABTS; Kirkgaard and Perry Laboratories, Inc., Gaithersburg, MD). ABTS color reaction was measured at 410 nm on a Dynatech MR5000 microplate reader (Dynatech Laboratories Inc., Chatilly, VA). 2.11. Statistical analysis Experiments consisted of five mice per group and all experiments described were performed at least twice. Statistical analysis of the data was performed using multifactorial ANOVA. Probability values of less than 0.05 ðP < 0:05Þ were considered significant.

3. Results 3.1. Immunization with deoxycholate-soluble antigens induces protective immunity Experiments were performed to determine whether PBS- or DOC-soluble extracts of S. stercoralis L-3 would induce protective immunity in mice to the infection. Mice were immunized with 25 lg of PBS-Ag or DOC-Ag adsorbed to alum and injected subcutaneously into na€ıve mice on Day 0 and Day 14. This was followed by a challenge infection with 50 L-3 contained in a diffusion chamber on Day 28. Other mice were injected with either live L-3 or alum without antigen as positive and negative controls for the immunization. The percentage of live larvae found in the diffusion

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chambers was determined on Days 1 and 5 postinfection. Protective immunity was seen on Day 1 postinfection in mice immunized with live larvae but not in mice immunized with soluble antigens (data not shown). At Day 5 post-infection immunization with DOC-Ag induced statistically significant levels of protective immunity, whereas PBS-Ag did not. The level of protective immunity induced by DOC-Ag was, however, significantly less than that induced by immunization with live L-3 (Table 1). Differential cell analysis was performed on the cells migrating into the diffusion chambers and it was determined that the percentage of eosinophils was elevated in all mice immunized with soluble antigens or with live L-3 (Table 1). Mice were then immunized with either 12.5, 25, or 50 lg of DOCAg; protective immunity was induced at equal levels with all three doses, with reductions in parasite survival ranging from 54 to 68%. 3.2. Protective immunity is dependent on IgG that recognizes unique L-3 antigens IgG, IgA, and IgM were separated from a pool of sera collected from mice immunized with DOC-Ag, through passage over respective isotype-specific affinity columns. Levels of IgE following purification were too low to allow further analysis. Passive transfer experiments were performed to determine which immunoglobulin isotypes would confer immunity to na€ıve mice. The concentration of transferred purified immunoglobulin was equivalent to that found in 100 ll of intact serum, as determined by ELISA. Unfractionated na€ıve and immune sera were used as controls. Naive mice were subcutaneously implanted with diffusion chambers containing 50 L-3 and simultaneously injected in the same location with the fractionated immunoglobulin isotypes or unfractionated whole sera. Recovery of diffusion chambers was performed 24 h later and remaining live larvae were quantitated. IgG from Table 1 Effect of immunization of mice with live L-3, PBS-soluble antigens from L-3 (PBS-Ag), and deoxycholic acid-soluble antigens (DOC-Ag) from L-3 Treatment

Live recovery %

Reduction %

Eosinophils %

Alum control Live L-3 PBS-Ag DOC-Ag

42  10 4  5 35  9 21  5

—

32 15  3 8  4 13  9

90 17 50

Note. Results listed are the percentage of live parasites recovered from control and immunized mice 5 days postinfection and the percentage of cells found in the diffusion chambers which were eosinophils. Percentage reduction is the percentage difference between the live recovery of larvae from control and that from immunized mice. Data represent the mean and the standard deviation of five animals per group. * Statistical difference from control value (P < 0:05).

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Table 2 Effect of passive transfer of serum from na€ıve mice and mice immunized with L-3 antigens soluble in deoxycholic acid (DOC-Ag) and the transfer of IgM, IgA, and IgG from immunized mice on the survival of larvae in na€ıve mice Transfer

Live recovery %

Reduction %

Na€ıve serum Immune serum Immune IgM Immune IgA Immune IgG

36  8 40  15 38  19 48  11 18  7

—

0 0 0 50

Note. Percentage reduction is the percentage difference between the live recovery of larvae from control and that from immunized mice. Data represent the mean and the standard deviation of five animals per group. * Statistical difference from control value (P < 0:05).

DOC-Ag-immunized mice was the only isotype capable of inducing a statistically significant decrease in larval survival. Injection of unfractionated immune sera did not cause larval death, although it contained the same quantity of immune IgG transferred in the purified immunoglobulin fraction (Table 2). Immunoelectron microscopy was performed to determine where the IgG from DOC-Ag-immunized mice, which was shown to function in passive transfer of immunity, bound to the L-3. It was determined that the antibodies bound to regions in the muscles and nerve cords and not to the cuticle of the larvae (Fig. 1). IgG from DOC-Ag-immunized mice was also used to construct affinity chromatography columns. DOCAg was passed through the column to purify the antigens recognized by the protective antibodies. The proteins that bound to the column and were then eluted (IgG specific-Ag) were then compared to proteins found in PBS-Ag, DOC-Ag, and the DOC proteins that did not bind to the column. Based on the number of bands seen in the silver-stained polyacrylamide gel it was clear that both PBS and DOC solubilized many different proteins and that unique proteins were found in both the PBS-Ag (Fig. 2, lane 1) and the DOC-Ag (Fig. 2, lane 2). DOC-Ag proteins that did not bind to the column were found to have molecular weights of 90, 82, 77, 47, 32, and 24 kDa (Fig. 2, lane 3). Proteins which did bind to the column and which were then eluted had molecular weights of 80, 75, 61, 57, 43, 32, and 21 kDa. (Fig. 2, lane 5). As a control for the mouse IgG that would be eluted off of the column when the bound antigen was recovered, normal mouse IgG was run on the gel (Fig. 2, lane 4). At least one band at 75 kDa is shared between the IgG specific-Ag pool of proteins and the normal mouse IgG, thus suggesting that this band may not be derived from parasite antigens. Therefore, it appears that a small number of antigens, weighing 80, 61, 57, 43, and 21 kDa, were selectively enriched in the IgG specific-Ag pool.

Fig. 1. Ultrastructural localization by immunoelectron microscopy of parasite antigens recognized by IgG from mice that developed protective immunity after immunization with DOC-Ag. Thin sections of S. stercoralis larvae were incubated first with IgG from immunized mice (A) or with IgG from normal mice (B) and then with rabbit antimouse Ig antibodies followed by protein A coupled to 15-nm gold particles for indirect antigen localization (bar, 500 nm). Note the labeling in the regions of the muscles (mu) and the nerve cord (nc) and an absence of label in the cuticle (cu). Labeling was not observed in sections incubated in normal mouse serum (B).

3.3. IgG-specific Ag induce protective immunity The capacity of the proteins selected by immune IgG to induce T cell responses was next determined. Splenocytes used in these studies were recovered from control mice and from mice immunized with live L-3. Live L-3 were injected on Day 0 and Day 14 followed by a challenge infection on Day 21. The percentage of live larvae recovered on Day 22 was 75  10% for control and 9  3% for immunized mice. Splenocytes, recovered on Day 22, were analyzed for T cell proliferation and IL-5 production in response to PBS-Ag, DOC-Ag, IgG non-bound antigens, and IgG specific-Ag. DOC-Ag and IgG specific-Ag were equally successful at inducing T cell proliferation (Fig. 3A) and IL-5 production (Fig. 3B). In contrast, little activity was observed from stimulation with PBS-Ag or IgG non-bound antigen pools. Immunization experiments were performed with the IgG specific-Ag to determine whether these antigens would induce protective immunity against a challenge infection. Mice were immunized with 25 lg of DOC-Ag, 10 lg of IgG non-bound antigens, or 10 lg of IgG

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Table 3 Effect of immunization of mice with alum, deoxycholate acid soluble antigens (DOC-Ag), DOC-Ag that did not bind to the immune IgG column (Non-bound Ag) and antigens that bound to the immune IgG and that were eluted (IgG Specific-Ag) Treatment

% Live recovery

% Reduction

IgG ELISA

Alum Control DOC-Ag Non-Bound Ag IgG Specific Ag

70  6 50  12 39  12 12  8

29 44 83

0:09  0:01 0:48  0:04 0:45  0:10 0:66  0:14

Results listed are the percentage of live parasites recovered from control and immunized mice and the optical densities for IgG responses in an ELISA measuring responses to DOC-Ag. Percentage reduction is the percentage difference between the live recovery of larvae from control and that from immunized mice. Data represent the mean and the standard deviation of five animals per group. * Statistical difference from control value. ** Statistically different from all other groups (P < 0:05).

Fig. 2. Silver-stained SDS–PAGE separation of PBS-soluble antigens (lane 1), deoxycholic acid-soluble antigens (DOC-Ag) (lane 2), DOCAg that did not bind to the immune IgG column (lane 3), normal mouse IgG (lane 4), and antigens that bound to the immune IgG and that were eluted (lane 5).

and was comparable to the levels achieved with live L-3 immunization (Table 3). Finally, it was observed that the high levels of protective immunity seen in mice immunized with IgG specific-Ag correlated with increased IgG reactivity to DOC-Ag in these mice (Table 3).

specific-Ag. Injection of mice with 10 lg IgG specific-Ag consistently induced significant larval killing that exceeded the levels observed in DOC-Ag-immunized mice

4. Discussion

Fig. 3. T cell proliferation (A) and IL-5 production as measured by Elispot (B) of splenocytes recovered from mice immunized with live larvae in response to PBS soluble antigens (PBS-Ag), deoxycholic acidsoluble antigens (DOC-Ag), IgG non-bound antigens (Non-bound), and IgG-specific antigens (IgG Specific). * Statistical difference from PBS-Ag and Non-Bound P ¼< 0:05.

The goal of this project was to identify a limited pool of native antigens from S. stercoralis L-3 that would function effectively in a vaccine. Two different agents, PBS and DOC, were used to solubilize the larvae. It was determined that protective immunity was only induced in mice exposed to the DOC-soluble antigens and not in mice immunized with the PBS-soluble antigens. Eosinophil influx, one of the hallmarks of protective immunity induced by live larvae (Abraham et al., 1995; Rotman et al., 1996; Herbert et al., 2000), was seen in mice immunized with either PBS or DOC antigens and not in control mice exposed only to alum. This finding suggests that a Th2 immune response was induced by both pools of antigens, but a protective response was induced only by the antigens solubilized by DOC. The composition of these two pools of antigens was seen to differ both quantitatively and qualitatively, as was previously reported (Northern and Grove, 1988; Sato et al., 1990a,b; Conway et al., 1993). It therefore appears that protective antigens were successfully solubilized by DOC and were not solubilized by PBS. Studies with the L-3 of Strongyloides ratti have shown that DOC will strip off surface antigens (Northern et al., 1989). It is therefore possible that the difference between these two pools of antigens might be in the presence of surface antigens, antigens that surely come into contact with the components of the immune response. Evidence in the present study, however, suggests that the protective antigens are not found on the surface of the larvae.

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The dichotomy between the two pools of antigens was further evidenced in the response of T cells recovered from mice immunized with live larvae. Only the DOCsoluble antigens were capable of stimulating the T cells to replicate and to produce IL-5. This finding differs from another study in which it was shown that PBSsoluble antigens induced greater cellular reactivity, whereas DOC-soluble antigens from Brugia pahangi induced higher antibody reactivity (Lammie et al., 1990). DOC-soluble antigens of S. stercoralis L-3 were clearly superior to PBS-soluble antigens at inducing cellular responses. The cells used in the proliferation and Elispot assays were derived from mice immunized with live L-3. The findings from the present study thus show that the DOC-soluble antigens were recognized by mice immunized with live larvae and suggest that the same antigens are effective in both models. There is also evidence from this study that there are significant differences between immunity induced with live larvae and that with DOCAg. Killing of the challenge larvae in mice immunized with live larvae was seen as early as 24 h postinfection, while protective immunity induced with DOC-Ag was only seen at 5 days post-infection thus suggesting that the two immunization protocols induced immunity through different mechanisms. Support for this hypothesis comes from the observation that IgM was protective in mice immunized with live larvae (Brigandi et al., 1996), whereas IgG was protective in mice immunized with DOC-Ag. A similar observation was reported in a study comparing immunization of mice with Ancylostoma duodenale L-3 or the recombinant protein ASP-1 adsorbed to alum. Parasite-specific IgM was the predominant isotypic response to live L-3 immunization, whereas IgG1was the primary response induced following immunization with the ASP-1 adsorbed to alum (Ghosh and Hotez, 1999). It must be emphasized, however, that IgG from mice immunized with DOC-Ag was capable of killing larvae when transferred into na€ıve mice within 24 h. It is possible that IgM in live immunized mice and IgG in DOC-Ag-immunized mice require different amounts of time to kill the larvae and that the extended time required by IgG might be related to the time required to attain sufficient concentrations in the microenvironment of the parasite. This conclusion is based on the observation that rapid killing occurs when the antibody is placed directly into the microenvironment of the larvae. IgG purified from serum recovered from mice immunized with DOC-Ag was able to passively transfer immunity to na€ıve mice while whole serum which served as the source of the purified IgG could not. This finding suggests that there were serum components inhibitory of immune IgG in whole serum, which were removed during IgG purification. It has been reported that whole serum from humans with chronic infections of Schisto-

soma mansoni did not transfer passive immunity to mice, whereas antibodies recovered from an antigen affinity column were effective (Jwo and LoVerde, 1989). Furthermore, antibodies capable of blocking the function of protective antibodies against S. mansoni have been reported in both humans (Khalife et al., 1986) and rats (Grzych et al., 1984). Although the present studies did not formally demonstrate blocking antibodies, it remains a possible explanation for the agent removed from the serum during purification. The protective IgG was conjugated to a CNBr-activated Sepharose column, to which DOC-Ag was allowed to bind and from which it was subsequently eluted. It was determined that five antigens weighing 80, 61, 57, 43, and 21 kDa were selectively enriched in the IgG specific-Ag pool. Previous reports have shown that protective IgM recovered from mice immunized with live larvae recognized proteins at 64, 60, 35, 34, 31, and 30 kDa in L-3 and proteins at 76, 61, 57, 55, and 42 kDa in mammalian-adapted L-3 (Brigandi et al., 1997). Many different antigens from S. stercoralis L-3 which have use in serological diagnosis of the infection in humans have been identified (Genta et al., 1987, 1988; Brindley et al., 1988; Sato et al., 1990a,b; Conway et al., 1993; Ramachandran et al., 1998). It is not possible to state that the antigens identified in this study are different from the antigens recognized by protective IgM from mice without more sensitive comparative analyses. The same is also true with regard to the comparison between antigens recognized by immunized mice and infected humans. It appears, however, based on molecular weight, that the majority of antigens recognized by IgG in DOC-Ag were not the same as the antigens used for diagnosis of the infection in humans. This raises the possibility that solubilization of L-3 by DOC releases antigens which are not normally seen in the course of infection in humans. Concealed antigens have been shown to have vaccine efficacy against other nematodes (Newton and Munn, 1999; Smith et al., 2000). The normally concealed antigens would then induce an immune response for which parasite evasion mechanisms have not evolved. The hypothesis that concealed antigens may be involved in the protective immune response is supported by the location of the binding of the protective IgG to the larvae. The antibodies were found to bind to regions in the muscles and in the nerve cords and not to the cuticle of the larvae. Internal antigens including muscle proteins have been shown to be capable of inducing immunity to other helminth infections (Pearce et al., 1988; Nanduri and Kazura, 1989; Jenkins et al., 1998). IgG specific-Ag induced protective immunity at a level equivalent to live immunization and significantly better than DOC-Ag. The concentration of IgG specificAg used for immunization was 10 lg per dose while immunization with DOC-Ag used 25 lg per dose. It is

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possible that 10 lg of IgG specific-Ag contained a higher concentration of the antigens relevant for protection than was present in 25 lg of the DOC-Ag pool. Doubling the dose of DOC-Ag used in the immunization did not, however, increase the level of protective immunity that was induced. Alternatively, there might be antigens in the DOC-Ag pool that suppressed the immune response to the protective antigens. This phenomenon was observed in mice immunized with a multivalent vaccine against Onchocerca volvulus (Abraham et al., 2001). The IgG specific-Ag were recognized by T cells recovered from L-3-immunized mice as seen in the proliferation and the IL-5 Elispot assays. The level of responses induced by the IgG specific-Ag was equivalent to those induced by the complete DOC-Ag. In addition, mice immunized with the IgG specific-Ag developed greater IgG responses to the DOC-Ag than mice immunized with DOC-Ag. The elevated IgG response was found to correlate with the increase in protective immunity. In conclusion, this study demonstrated that solubilization of L-3 with DOC released antigens which induced protective immunity in mice. The protective immune response was dependent on parasite-antigen-specific IgG and the IgG recognized a small set of antigens. These IgG-specific antigens were capable of stimulating T cells recovered from immunized mice and these antigens represented the dominant antigens for inducing the IgG response. Immunization of mice with the IgG-specific antigens resulted in an 80% reduction in the survival of challenge parasites. These purified proteins represent the pool of antigens from which vaccine candidates can be cloned.

Acknowledgments The authors acknowledge support from NIH Grants HL058723, HL60793, and AI 22662. We also thank Ann Marie Galioto, Laura Kerepesi, Ofra Leon, and Shalom Leon for expert technical assistance. Special thanks go to Yelena Oskov for assistance with the electron microscopy.

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