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Schistosomiasis is a serious parasitic disease that infects over 200 million people worldwide and causes an estimated 500,000 deaths per year. Despite the fact that its global distribution has changed significantly in the past 50 years, particularly in regions where control strategies have been successfully employed, the disease remains endemic in over 70 developing countries (22). Chemotherapy, although effective, does not prevent reinfection, and in addition, partial drug resistance to the most commonly used chemotherapeutic agent against schistosomiasis, praziquantel (7, 11), has been reported. Immunological intervention in the form of a vaccine would contribute to the success of present efforts if added to existing control strategies (22). At present, there are no practical vaccines against any human helminth infection (1, 2, 5). In the case of schistosomiasis, even a partially protective vaccine would be beneficial, since the parasite does not reproduce in the mammalian host and morbidity is correlated with worm burden (23, 24). It has been demonstrated that injection into experimental animals of live cercariae attenuated by sublethal doses of radiation results in high levels of resistance against subsequent reinfection (21). However, though effective, such an irradiated vaccine is not a practical approach, and the search continues for defined antigen vaccines that would protect from initial infection and/or egg-granuloma-associated pathology (5). Development of either recombinant or synthetic peptide vaccines would be a suitable approach for vaccine production based on protective components of the parasite.

Peptide vaccines against many microbial agents have been widely investigated in the past two decades (4) and have been shown to induce partial protection in vivo, e.g., in the cases of measles and influenza (3, 13). In the realm of parasite vaccines, efforts have been made to prepare synthetic peptide vaccines against malaria (6, 17). Against schistosomiasis, efforts are being made to direct specific B-cell and T-helper-cell responses by identifying different epitopes in protective antigens such as Sm23 and triose-phosphate isomerase (19, 20). A cytotoxic T-cell C-terminal lipopeptide has been derived from the vaccine candidate Sm28GST (16) and employed as well in the form of multiple antigen peptide (8).

In our laboratory, we have purified an antigen from Schistosoma mansoni extract by affinity chromatography on a highly protective monoclonal antibody (MAb), 152-66-9B. This antigen, denoted 9B-Ag, is 450 kDa in its native form but migrates as a 200-kDa band in the presence of sodium dodecyl sulfate and, under reducing conditions, exhibits two main subunits, of 45 and 30 kDa (27). We have previously demonstrated that vaccination of mice with 9B-Ag resulted in high levels of protection, ca. 45%, against challenge infection (14, 15, 27). It was of interest, therefore, to explore whether peptide segments of this antigen are capable of inducing a protective effect.

Herewith we report that a 14-residue peptide, corresponding to the N-terminal region of the 9B-Ag 45-kDa subunit and denoted 9B-peptide1, was capable of inducing a significant level of protection of mice (30 to 50%) against challenge infection. A similar peptide, denoted 9B-peptide2 and differing in only the residue in position 7, showed no protective activity. These results are discussed in view of the potential of such a simple, relatively short peptide to serve as a vaccine candidate against schistosomiasis.

Parasite. A Puerto Rican strain of S. mansoni was maintained in outbred CD1 mice and Biomphalaria glabrata snails. S. mansoni cercariae and schistosomula were prepared as described previously (15). Adult worms were obtained by liver perfusion from chronically infected mice at 6 to 7 weeks postinfection, as previously described (23).

Antisera. The following mouse sera were obtained from CD1 and C57BL/6J mice: normal mouse serum and serum from acutely infected mice taken 9 weeks after exposure to 300 cercariae. In addition, mouse ascitic fluid was obtained for preparation of the monoclonal antibody (152-66-9B) (27). Individual human serum samples (a gift from Zvi Bentwich, The Kaplan Hospital, Rehovot, Israel) were obtained from Ethiopian immigrants to Israel who had been exposed previously to schistosomiasis. All patients suffered from relatively mild chronic infections, and the samples were obtained before any treatment was applied.

RIA. Solid-phase radioimmunoassay (RIA) was performed essentially as described by Pierce and Klinman (18), with a slight modification: the antigens were stuck to the plate in the presence of 2% glutaraldehyde-phosphate-buffered saline solution at a concentration of 5 µg of 9B-peptide1 or 9B-peptide2.

ELISA. For enzyme-linked immunosorbent assay (ELISA), whole-parasite sonicate (10 µg/well), peptide-protein conjugates (5 µg/well) in sodium carbonate buffer (pH 9.6), or free 9B-peptide1 or 9B-peptide2 in 2% glutaraldehyde-phosphate-buffered saline solution served as antigen adsorbed to the plates.

Immunization and protection studies. Groups of 8 to 10 mice (C57BL/6J) were immunized intradermally and in the foot pads with bovine serum albumin (BSA)-peptide conjugate (50 µg/100 µl), as follows. The first injection was in complete Freund adjuvant (CFA), into the foot pads and in the base of the tail, with a booster injection with the same conjugate in incomplete Freund adjuvant (IFA) by intraperitoneal or subcutaneous injection. Four weeks after the last booster, infection with 70 to 90 cercaria was performed by the ring method (23). Control groups included one of the following: carrier alone (BSA or ovalbumin [OVA]) or adjuvant alone, injected by the same regimen as described above.

Complement-dependent cytotoxicity assay. The susceptibility of schistosomula to antibodies and complement was determined as previously described (27).

Statistical analysis. Statistical analysis was performed with the Stat View II program (Abacus Concepts Inc., Berkeley, Calif.).

Peptide sequences and preparation. N-terminal sequencing analysis was performed on samples, obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of the 45-kDa subunit of 9B-Ag (~20 µg) after blotting to polyvinylpyrrolidone paper. Sequences were determined by the Edman degradation method in a 475A Protein Microsequencer (Applied Biosystems) with a 120PTH analyzer and a 900A control data analysis module. The 14-residue peptides were synthesized by M. Fridkin of the Department of Organic Chemistry at The Weizmann Institute by the solid-phase method with a multipeptide synthesizer (Abimed AMS 422) and were purified by high-performance liquid chromatography. Repeated sequencing of this 45-kDa subunit showed two sequences with a difference in one amino acid, at position 7. Accordingly, two peptides were synthesized: 9B-peptide1 (GFTTNEERYNVFAE) and 9B-peptide2 (GFTTNEPRYNVFAE). The peptides were coupled to BSA or OVA by using the water-soluble carbodiimide reagent EDCI for protein conjugation (26). A 1:38 molar ratio of carrier to peptide was used. The lack of lysine residues in either peptide favors binding to the carrier and not cross-linking to itself.

Recognition of the 9B peptides by the 155-66-9B MAb. The two synthetic peptides of the N-terminal region of the 45-kDa subunit of 9B-Ag, namely 9B-peptide1 and 9B-peptide2, were bound in their free forms (5 µg/well) or as BSA conjugates to RIA plates and incubated with different concentrations of MAb 152-66-9B immunoglobulin (Ig) over a range of dilutions: 1/10 to 1/1,280 for free peptide and 1/50 to 1/20,000 for the conjugates. Quantitation with ¹²⁵I-goat anti-mouse Ig showed that the peptides are recognized by the MAb to similar levels (Fig. 1a) and that the binding obtained with the conjugated 9B peptides was higher than that of the free peptides (Fig. 1b).

View larger version (31K): [in this window] [in a new window]   FIG. 1.   9B peptide recognition by MAb 152-66-9B (a and b) and by sera from acutely infected mice (c and d). Recognition was measure by monitoring antibody binding to free 9B-peptide1 (circles) or 9B-peptide2 (squares) (a and c) or to 9B peptides coupled to OVA (b and d). Binding of nonrelated MAb anti-2,4-dinitrophenol to 9B-peptide1 () or to 9B-peptide2 () is also shown. Results in panels c and d are presented after subtraction of background with control serum.

Reactivity of 9B peptides with infected sera from mouse or human origin. Figure 1c and d demonstrate the recognition of both peptides by infected mouse sera: free 9B-peptide1 is better recognized by the infected mouse sera than 9B-peptide2 (Fig. 1c). The results are in counts per minute of the goat anti-mouse Ig antibody bound to the infected serum after subtraction of counts per minute obtained with the noninfected serum. Similar results were obtained when peptides conjugated to OVA were used as the antigen. Again, 9B-peptide1-OVA is better recognized than 9B-peptide2-OVA by infected mouse sera (Fig. 1d). Free 9B-peptide1 and 9B-peptide2 are also recognized by individual sera from human patients infected with S. mansoni (n = 12), as detected by ELISA. All of the human sera at a 1:80 dilution showed significant binding to the two peptides (Fig. 2). Nonrelated peptides (a commercial mixture of human immunodeficiency virus peptides used for serum evaluation) did not show any binding even when tested with nondiluted serum samples (data not shown).

View larger version (43K): [in this window] [in a new window]   FIG. 2.   9B peptide recognition by sera from infected humans. Recognition was quantitated by monitoring antibody binding to free 9B-peptide1 (a) or free 9B-peptide2 (b) by ELISA. Bars represent sera from infected individuals at a 1/80 dilution. S(N1) and B(N2) are controls from noninfected individuals. O.D., optical density.

Protection conferred by 9B peptides. The most important issue was whether the synthetic peptides could induce protection against challenge infection with the parasite. To analyze the protective properties of the 9B peptides, we immunized mice with 50 µg of the BSA-conjugated peptides twice at a 1-month interval and challenged them 1 month later by the ring assay with 70 to 90 cercariae. As shown in Table 1, worm burden was reduced by an average of 45% ± 3% after immunization with 9B-peptide1 conjugate (P, 0.0001 to 0.005) and by only 5% ± 3% after immunization with 9B-peptide2 conjugate, as compared with control mice that either received only carrier or adjuvant alone or were not immunized at all. Sera from vaccinated mice showed significant antibody titers against the respective peptides, as monitored by ELISA with OVA-conjugated peptides as matrix-bound antigen. Anti-9B-peptide1 antisera gave higher titers than anti-9B-peptide2 antisera. No binding to the OVA carrier alone was detected (Fig. 3a).

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Antibody titers and complement-dependent cytotoxicities of sera from mice vaccinated with BSA conjugates of 9B-peptide1 or 9B-peptide2. (a) Antibody binding, quantitated by ELISA, of anti-9B-peptide1-BSA antibody with OVA conjugates of 9B-peptide1 () and 9B-peptide2 (), as well as unconjugated OVA (), and 9B-peptide2-BSA antibody with OVA conjugates of 9B-peptide1 () and 9B-peptide2 (), as well as unconjugated OVA (). (b) Complement-mediated cytotoxic effects on schistosomula. Sera from vaccinated mice were added to 200 schistosomula and incubated for 30 min. The susceptibility to complement lysis was determined in the presence of guinea pig complement. Results are expressed as percent net killing of schistosomula, namely, the difference between mortality in the presence of active (filled bars) and of heat-inactivated (open bars) complement for anti-9B-peptide1-BSA and anti-9B-peptide2-BSA. CFA, negative-control sera from mice immunized with CFA alone; 300CA, positive-control sera from mice infected with 300 cercaria.

Complement-dependent cytotoxicity of anti-9B-peptide1. Antisera produced by vaccinated mice against 9B-peptide1 or 9B-peptide2, were investigated for their capacities to mediate complement-dependent killing of schistosomula. Sera from mice immunized with CFA alone or from normal, untreated mice were used as negative controls, and sera from infected mice were used as positive controls. Experiments were performed with fresh guinea pig serum as a source of complement and with heat-inactivated serum as an internal control. The extent of killing was taken as the difference between the percentages of dead schistosomula in the presence of active and of inactivated complement. The results (Fig. 3b) show that anti-9B-peptide1 antibodies induced significant levels of cytotoxicity (40% of net killing). This value is close to the level of cytotoxicity caused by the positive controls (an average of 55% of killing). The toxic effect of anti-9B-peptide1 mouse antibody was manifested also by tegumental blebbing and granulation of the parasite in the presence of fresh guinea pig complement. In contrast, in the presence of anti-9B-peptide2 antiserum, only 20 to 24% of killing was observed, similarly to the nonspecific killing by control (CFA-injected) serum. Similar low-level killing was observed in the internal controls in the presence of heat-inactivated serum. These results corroborate the protective effect of the 9B-peptide1 conjugate described above.