Mansoni Antigens Research Articles

Induction of protective immunity and modulation of granulomatous hypersensitivity in mice using PIII, an anionic fraction of Schistosoma mansoni adult worm.

This study was performed in order to define Schistosoma mansoni antigens that are able to function as modulator agents in the granulomatous hypersensitivity to parasite eggs in BALB/c and C57BL/6 mice. A fraction of S. mansoni, designated PIII, derived from adult worm antigen preparation (SWAP) was obtained using anion-exchange chromatography on an FPLC system. Immunization of mice with PIII in the presence of Corynebacterium parvum and Al(OH)3 as adjuvant induced an immune response in this animals as determined by ELISA and spleen cell proliferation assays against S. mansoni antigens SEA, SWAP and PIII. In addition, PIII caused a significant degree of protection against a challenge infection in immunized mice as observed by the decrease on worm burden recovered from the portal system. We also showed that PIII profoundly inhibited the vigorous anamnestic granulomatous response to eggs in the liver and lungs. This suppression correlated with a significant decrease in granuloma size. From these results we conclude that the PIII preparation contains antigens that can mediate protective anti-parasite immunity and downregulate granulomatous hypersensitivity to S. mansoni eggs.

Molecular Characterization of a 20.8-kDaSchistosoma mansoni Antigen: SEQUENCE SIMILARITY TO TEGUMENTAL ASSOCIATED ANTIGENS AND DYNEIN LIGHT CHAINS

Survival of Schistosoma mansoni within the infected host requires the parasite to actively maintain its protective tegument. The components responsible for this maintenance are therefore attractive targets for immunoprophylaxis or chemotherapy. Here we report the molecular characterization of a 20.8-kDa tegumental antigen with sequence similarity to dynein light chains and tegumental associated antigens. A cDNA encoding the 20.8-kDa polypeptide contains an open reading frame of 181 amino acids and predicts an isoelectric point of 7.27. Expression of the 20.8-kDa antigen is developmentally regulated, with the highest concentration found in cercariae. Our data show that the 20.8-kDa polypeptide specifically interacts with a S. mansoni 10.4-kDa dynein light chain that we have previously described (Hoffmann, K. F., and Strand, M. (1996) J. Biol. Chem. 271, 26117-26123). Velocity sedimentation analysis of a parasite extract demonstrated that this 10.4-kDa dynein light chain and the 20.8-kDa polypeptide were present in a complex that sedimented at 4.4 Svedberg units. We have also shown by antibody cross-reactivity that a 20.8-kDa homolog of the S. mansoni antigen is present in Schistosoma japonicum, but not in Schistosoma hematobium or Fasciola hepatica. Because the 20.8-kDa polypeptide displays ideal characteristics of a potential vaccine candidate, including (i) expression in the tegument, (ii) significant divergence from mammalian brain cytoplasmic dynein, and (iii) a conserved homolog in S. japonicum, we are currently evaluating its immunoprophylactic efficacy.

Polyvinyl alcohol-glutaraldehyde as solid-phase in ELISA for schistosomiasis.

Soluble adult Schistosoma mansoni antigen preparation (SWAP) was covalently fixed onto polyvinyl alcohol-glutaraldehyde discs and an enzyme linked-immunosorbent assay (ELISA) was set up. The best conditions for the assay were established and it was found that small amount of antigen such as 1.5 micrograms was required. A comparison between this procedure and the conventional ELISA was proceeded. A reliable method of antigen immobilization was achieved and the low prices of the employed reagents are economically attractive.

Cloning, heterologous expression and antigenicity of a schistosome cercarial protease.

A gene coding for the 30 kDa Schistosoma mansoni cercarial protease was amplified using the polymerase chain reaction (PCR) from genomic DNA templates. Cloning and sequencing of several independent PCR clones revealed the presence of an intron additional to the one described in the original cloning of the gene. The 3 exons were cloned into expression vectors so that they could be expressed as separate glutathione-S-transferase (GST) translational fusions. Recombinant bacteria carrying these expression plasmids expressed the fusion proteins at high levels. Western blotting of bacterial lysates with sera raised against the native S. mansoni cercarial protease showed that all 3 exons were recognized. Thus we have produced recombinant bacteria capable of providing large amounts of an S. mansoni antigen for immunological studies and evaluation as a candidate vaccine.

Immunoglobulin A response in murine schistosomiasis: stimulatory role of egg antigens.

The immunoglobulin G (IgG) and IgA antibody responses to different Schistosoma mansoni antigens have been determined in chronically infected mice as well as in unisexually infected animals. With a panel of enzyme-linked immunosorbent assays (ELISAs), soluble antigens from furcocercariae, adult worms, and eggs were probed with sera collected at 3-week intervals. Bisexually infected animals developed significant IgG and IgA antibody responses to the antigens tested, which increased after egg deposition. In unisexual infections no significant differences were recorded in the IgG antibody profile for furocercaria and adult worm antigens, whereas the IgA antibody response was impaired. Both the IgA and IgG antibody responses toward egg antigens were reduced compared with those in a bisexual infection. Furthermore, a specific mucosal IgA antibody response was observed only in the bisexually infected animals. Histological analysis performed on bisexually infected mice led to the observation of eggs and granulomatous lesions within the Peyer's patch follicles, which are essential sites for the induction of mucosal immunity in the intestine. These data suggest a relationship between egg deposition and the induction of the IgA antibody response toward schistosomes.

Sm480: a high molecular weight Schistosoma mansoni antigen associated with protective immunity.

Rabbit antisera were raised against an antigen present in Schistosoma mansoni adult worms and eggs, and were shown to yield a single immunoprecipitin arc in immunoelectrophoresis and immunodiffusion against S. mansoni egg and worm antigen extracts. The antisera conferred partial but significant protection (22-30%) against a S. mansoni challenge when transferred to mice five and six days after the mice had been infected percutaneously with 200 cercariae. The egg and the worm forms of the antigen were immunologically cross-reactive, but the egg antigen possessed peptidolytic activity that could be inhibited with serine protease inhibitors. In indirect immunofluorescence the rabbit antisera reacted with surfaces of cercariae, five-day old lung-stage schistosomula, miracidia and praziquantel-treated adult worms. Gel-filtration chromatography demonstrated a relative molecular size of approximately 480 kDa for both the egg and worm forms of the antigen, and lectin-affinity chromatography indicated both were glycosylated. The serine protease activity and large relative molecular size of egg Sm480 were confirmed by a combination of radiolabelling with tritiated di-isopropyl fluorophosphate, immunoelectrophoresis and polyacrylamide gel electrophoresis.

Dot-ELISA for the detection of IgM and IgG antibodies to Schistosoma mansoni worm and egg antigens, associated with egg excretion by patients.

Human schistosomiasis, caused by Schistosoma mansoni, is highly prevalent in Brazil and usually diagnosed by time consuming stool analysis. Serological tests are of limited use in this disease, mainly for epidemiological studies, showing no discrimination between previous contact with the parasite and active infections. In the present study, we standardized and compared a Dot-ELISA for IgM and IgG antibodies against S. mansoni antigens from eggs and worms with a routine IgG and IgM immunofluorescence assay using similar antigens, in the study of sera from 27 patients who had quantified egg stool excretion. The positivity obtained for IgG Dot-ELISA was 96.3% and 88.9% for IgM Dot-ELISA with worm antigen and 92.6% and 90.9% with egg antigen. The IFI presented similar positivities using worm antigen, 92.6% (IgG) and 96.3% (IgM), and lower results with egg antigen, 77.8%(IgG and IgM). The patients studied were divided into two groups according to their egg excretion, with greater positivity of serological tests in higher egg excreters. When comparing the quantitative egg excretion and the serological titers of the patients, we detected a correlation only with IgM Dot-ELISA, with r = 0.552 (p = 0.0127). These data show that Dot-ELISA can be used for the detection of specific antibodies against S. mansoni in sera from suspected patients or in epidemiological studies and, with further purification of egg antigen and larger samples, IgM Dot-ELISA could be a possible tool for rough estimates of parasite burden in epidemiological studies.

Analysis of the primary structure and post-translational modifications of the Schistosoma mansoni antigen Smp28 by electrospray mass spectrometry.

The Schistosoma mansoni glutathione-S-transferase with an apparent molecular mass of 28 kDa, Smp28, has a blocked N-terminus which has been elucidated with the aid of the cDNA sequence combined with mass spectrometry and amino acid composition analysis of the N-terminal tryptic peptide. The blocked N-terminal tryptic peptide (m/z 695.8) contained an equimolar ratio of E, G, H, A, I and K3 upon amino acid composition analysis in agreement with its expected sequence AGEHIK, and showed a delta m = +41.7 Da compared to the predicted mass, which is consistent with the N-terminal alanine being acetylated (delta m = +42.0 Da). The mass of the complete molecule (23,744.5 +/- 3.3 Da) determined by electrospray mass spectrometry showed a further mass increase of 14 Da with respect to Smp28 containing an N-acetylated alanine. This result is consistent with one of the seven methionines being present as a methionine sulfoxide in ca. 90% of the Smp28 molecules in this preparation. Tryptic mapping of Smp28 showed five of the seven methionines to be partially oxidized by mass spectrometry. This is indicative of the ease with which this modification occurs. Two minor components were detected along with the intact molecule, corresponding to modified forms of the molecule, originating from reaction of the only cysteine residue either with itself forming a covalent dimer or with glutathione. On-line liquid chromatography-mass spectrometry has been compared with the off-line complete tryptic map of Smp28 confirming 97% of the primary structure in less than 2 h.

Developmentally regulated localization and phosphorylation of SmIrV1, a Schistosoma mansoni antigen with similarity to calnexin.

Potential molecular targets of a protective humoral immune response against schistosomiasis have previously been identified based on their enhanced immunogenicity in mice vaccinated with irradiated cercaria as compared to chronically infected mice. One of these antigens, IrV1, has been molecularly cloned and its sequence shown to be similar to the molecular chaperone calnexin. In this investigation, we partially characterized IrV1 from different developmental stages of the schistosome. Immunoprecipitation studies with antibodies raised against a portion of recombinant IrV1 demonstrated its presence in cercaria, schistosomula, and adult worms with an apparent molecular mass on SDS-polyacrylamide gel electrophoresis of 90 kDa. There was an approximate 6-fold increase in protein expression level during the cercaria to schistosomula transformation. Consistent with a potential role as a molecular chaperone, IrV1 was associated with several metabolically labeled proteins in co-immunoprecipitation studies with the adult worm tegumental fraction. Similar to calnexin, IrV1 was metabolically labeled with 32P in adult worms on serine and threonine residues and was one of the major phosphoproteins of this stage. This phosphorylation was developmentally regulated and coincided with the transformation of cercaria into schistosomula. The localization was also stage-specific as IrV1 was transported from internal regions of cercaria to the outer tegumental layer of schistosomula. The presence of IrV1 on the surface of schistosomula, an unprecedented localization for this family of endoplasmic reticulum proteins, supports additional studies of the immunoprophylactic potential of this molecule.

Molecular characterization of two Schistosoma mansoni proteins sharing common motifs with the vif protein of HIV-1.

We have previously described a rat mAb directed against a peptide derived from the vif protein of HIV-1 that recognized two Schistosoma mansoni (Sm) antigens with a major band at 65 kDa. Epitope mapping of this mAb using overlapping hexapeptides derived from the vif peptide revealed that the motif recognized was PLPSVT. The screening of a Sm cDNA library led to the identification of two clones, Sm70 and Sm65. The two deduced protein sequences did not share any common structural features apart from the epitope recognized by the mAb (see below), and did not show significant identity to sequences present in the data bases. However, the N terminus of the deduced sequence of the Sm70 protein exhibits a consensus sequence known to be an ATP/GTP binding site. Furthermore, the C terminus of the deduced Sm65 protein sequence was found to contain a conserved hexapeptide with a consensus sequence LPETGE reported to be an important motif of the surface proteins of gram-positive cocci. Both proteins exhibit a peptide sequence (PLRSVT for Sm70 and PVGSVT for Sm65) similar to the epitope recognized by the mAb anti-vif. Western blotting experiments showed that the mAb anti-vif reacted with both proteins. However, only Sm65 was recognized by sera from HIV-1-seropositive individuals, whereas both proteins were recognized by S. mansoni-infected patients.

Identification of Schistosoma mansoni Antigens Recognized by T Cells of C57BL/6 Mice Immunized with Gamma-Irradiated Cercariae

Immunization of C57Bl/6 mice with gamma-irradiated Schistosoma mansoni cercariae induces highly significant protection against subsequent challenge with unattenuated living cercariae. The antigens that evoke T cell-mediated immunity in this model are vaccine antigen candidates since T lymphocytes mediate antibody-independent immune responses and control the production of most antibodies. To identify these antigens, spleen cells of mice immunized twice with gamma-irradiated cercariae were tested for proliferative responsiveness and production of interleukins (Il) and interferon-gamma (IFN-gamma) following incubation with separated soluble schistosomular (SSA) and adult worm (SAWA) antigens in T cell western assays. Data obtained at 3, 4, 5, and 6 wk post-secondary immunization in 2 experiments indicated that SSA and SAWA bands of 62-60, 50, and 45 kDa reproducibly elicited T cell proliferation and production of Il-2, Il-4, and IFN-gamma by spleen cells from immunized, but not unimmunized, mice. Bands of 72-68, 29.5, and 28 kDa elicited proliferation and production of Il-2 and Il-4, but not IFN-gamma. Bands of 35-33 and 24 kDa induced either proliferation or Il production at some intervals.

Recognition of Different Schistosoma mansoni Antigens by Specific Human T Cell Clones

Human T lymphocyte clones (TLC) sensitized to Schistosoma mansoni soluble egg antigens (SEA) were developed from chronic schistosomiasis mansoni patients in order to investigate the regulatory mechanisms involved with in vitro granulomatous hypersensitivity to this parasite. All clones studied displayed CD4 + phenotype and required antigen-presenting cells in antigen-driven proliferation and granuloma formation. Each T lymphocyte clone has been shown to proliferate and generate in vitro granulomas in response to SEA, adult worm antigens (SWAP), or cercaria antigens (CAP). In contrast, no proliferation was observed in any of these T cell clones when unrelated S. mansoni antigens were used. Some SEA-generated TLC were not able to proliferate in the presence of SEA; however. S. mansoni SEA-reactive ones were able to recognize epitopes in Schistosoma japonicum SEA, indicating cross-reactivity between these two species. Using IFN-γ ELISA, it was shown that TLC cells stimulated with SEA can secrete appreciable amount of this lymphokine. Further in vitro studies with these SEA-TLC will help us to understand in more depth the role of regulatory T cells in the human granulomatous hypersensitivity to S. mansoni eggs.

Analysis of Recombinant Schistosoma mansoni Antigen rSmp28 by On-Line Liquid Chromatography-Mass Spectrometry Combined with Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

A recombinant Schistosoma mansoni antigen produced in Saccharomyces cerevisiae and purified by glutathione-Sepharose affinity chromatography was analyzed by tryptic peptide mapping using on-line reversed-phase high-performance liquid chromatography pneumatically assisted electrospray mass spectrometry confirming the complete primary structure. Partial covalent modification of the single cysteine in the protein with glutathione as well as partial dimerization of the Cys-containing tryptic peptide was observed. Combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and tryptic digestion of the monomeric protein in the gel slice revealed that dimerization was occurring during enzymatic digestion. Furthermore, part of the Cys-containing fragment was covalently modified with one moiety of β-mercaptoethanol by the electrophoresis sample buffer and five of the seven methionine-containing pep-tide fragments were partially oxidized to the respective sulfoxides. The use of capillary columns provided a complete peptide map of rSmp28 on 7 pmol of tryptic digest after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

An IgG (Fc gamma)-binding protein of Taenia crassiceps (Cestoda) exhibits sequence homology and antigenic similarity with schistosome paramyosin.

Previous studies from this laboratory have suggested the presence of antibody-binding molecules on the surface of Taenia crassiceps metacestodes. We now report the purification of a T. crassiceps Fc gamma-binding protein which has an equivalent molecular size (96 kDa), is antigenically similar and exhibits significant amino acid sequence homology to schistosome paramyosin. The similarities in molecular weight of the T. crassiceps protein, Schistosoma mansoni paramyosin and antigen B of T. solium, the close amino acid sequence homologies between the T. crassiceps protein and S. mansoni paramyosin and between S. mansoni paramyosin and antigen B of T. solium, and the antigenic similarity of the T. crassiceps protein with paramyosin indicates that this family of platyhelminth proteins are closely related. The known characteristics of T. solium antigen B (collagen binding affinity; disruption of complement function) and the Fc gamma-binding activity of the T. crassiceps molecule suggests that this class of proteins may be multifunctional, fulfilling not only a structural role but also as components interacting with the host immune system to increase parasite survival.

Molecular cloning and expression of SmIrV1, a Schistosoma mansoni antigen with similarity to calnexin, calreticulin, and OvRal1.

Protective immunity against schistosomiasis induced by vaccination of mice with irradiated cercaria can be passively transferred to uninfected mice with sera or IgG. Antigens that are uniquely or more strongly recognized by such protective sera compared with sera from infected unprotected mice have been identified previously. Two genes, SmIrV1 and SmIrV5, were selected from an adult worm cDNA library by screening with antibodies raised against these candidate vaccine proteins. Active immunization with the SmIrV5 protein induces high levels of protection in mice. We report here the molecular cloning and sequencing of SmIrV1 which contains a deduced amino acid sequence of 582 residues with similarity to three proteins: calnexin, calreticulin, and OvRal1, a surface antigen of the filarial nematode Onchocerca volvulus. SmIrV1 can be divided into three regions: a neutral N-terminal region with a putative signal sequence, followed by a proline- and tryptophan-rich P region in which two sets of sequences are repeated four times and a C-terminal region which is highly acidic with an isoelectric point of 4.7. We expressed the P and C regions of SmIrV1 and showed that this polypeptide reacts with sera of immunized as well as chronically infected mice.

Seroepidemiology and serodiagnosis of schistosomiasis in Kenya using crude and purified egg antigens of Schistosoma mansoni in ELISA

The performance of antibody detection for the diagnosis of schistosomiasis has been evaluated in Kenya. Approximately 1500 blood samples from 3 areas with endemic schistosomiasis ( Schistosoma mansoni only, S. haematobium only, and a mixed infection area), and from a non-endemic control area, were tested for their antibody reactivity in an enzyme-linked immunosorbent assay (ELISA). The results were compared with infection status determined by parasitological examination. Two test antigens were used: unfractionated S. mansoni egg homogenate (SEA), and CEF6, a previously described, partially purified fraction of SEA containing 2 cationic antigens. The antigens prepared from eggs of Kenya and Puerto Rico S. mansoni isolates gave very similar results. Bloods from patients with S. haematobium infection cross-reacted significantly with the two S. mansoni antigen preparations, but reactivity against CEF6 appeared more specifically indicative of S. mansoni infection. Of 254 blood samples from schoolchildren in the non-endemic area, 100% gave ELISA optical density readings at 492 nm (OD 492) <0·20 against SEA, and 98% were <0·20 against CEF6. With 887 blood samples from subjects of all ages in the area endemic for S. mansoni alone, using an ELISA OD 492 cut-off point of 0·20, SEA and CEF6 had sensitivities of 94% and 97% respectively, and specificities of 64% and 59% respectively. Increasing the OD 492 cut-off value reduced the sensitivity and increased the specificity of both test antigens. Specificity of both antigens was poor with samples from 234 children in an area endemic for both S. mansoni and S. haematobium (<20% for both antigens at an OD 492 cut-off value of 0·20). It is suggested, with supporting evidence, that one reason for the apparently poor specificity of the serological testing in the endemic area is the inherently poor sensitivity of parasitological examinations of small volumes of stool. There was a significant positive correlation between blood elisa results and the number of eggs excreted by infected subjects in the area endemic for S. mansoni only. Highest correlation coefficients were obtained in children aged under 10 years, and CEF6 gave marginally higher correlation coefficients than SEA. The graphs of prevalence and intensity of schistosome infection drawn from serological results were similar in shape to the graphs of these 2 quantities based on parasitological results, and the results indicate that serology merits wider use as an epidemiological tool for determining infection status in schistosomiasis.

The influence of adjuvant on humoral responses to glutathione-S-transferase fusion proteins

Mice were immunized with one of two Schistosoma mansoni antigens, Sm20 and Sm50, expressed as fusion proteins with Schistosoma japonicum 26,000 Dalton glutathione-S-transferase (GST) or with GST alone. Antibody responses to GST were shown to be critically dependent on the adjuvant used. In Sm20-GST immunized mice, we noted a striking bias to respond either to Sm20 or to GST according to the adjuvant used and responses to both in the same individual were rare. Fusion with Sm50, which is relatively non-immunogenic, appeared to down-regulate responses to GST.

Immune-dependent chemotherapy of schistosomiasis

Host immune responses have been shown to enhance the efficacy of several schistosomicidal drugs. The evidence derives mainly from experiments on Schistosoma mansoni infections in the mouse with their immune status variously modulated; this review emphasises praziquantel (PZQ), which is now the main drug used for treatment of human schistosomiasis. Electron microscopy and indirect immunofluorescence indicate that PZQ disrupts the integrity of the surface membranes of S. mansoni, particularly those covering the dorsal tubercles of adult male worms, and this causes antigens which are the targets of antibody attack to be revealed. We review the evidence that two S. mansoni antigens in particular are implicated in the immune-dependent action of PZQ: a 200 kDa glycoprotein and a 27 kDa antigen with non-specific esterase activity. Consistent with the involvement of the latter antigen, increased non-specific esterase activity was demonstrated histochemically on the surface of intact PZQ-treated male worms, and we describe a chromogenic substrate assay for quantifying the amount of esterase activity that is exposed after drug treatment. The potential relevance of these observations for enhancing the efficacy of drugs currently used to treat human schistosomiasis, and for devising novel therapeutic strategies, is discussed.

New approaches for the control and eradication of schistosomiasis in Venezuela.

Schistosomiasis in America with the exception of Brazil, behaves as a chronic mild disease with few clinical manifestations due to low parasite burden. These features restrict the clinical and parasitological diagnosis. The most commonly used stool examination method, Kato-Katz, becomes insensitive when the majority of individuals excrete less than 100 eggs/g of feces. In view that antigen-detecting techniques have not been able to reveal light infections, the antibody detecting assays remain as a very valuable diagnostic tool for epidemiological surveillance. The Venezuelan Schistosomiasis Research Group (CECOICE) has designed a mass chemotherapy strategy based on sero-diagnosis. Since blood sampling is one of the important limiting factors for large seroepidemiological trials we developed a simple capillary technique that successfully overcame most of the limitations of blood drawing. In this sense, ELISA seems to be the most adequate test for epidemiological studies. Soluble egg Schistosoma mansoni antigen (SEA) has been largely used in Venezuela. The sensitivity of ELISA-SEA in our hands is 90%, moreover its specificity reach 92% when populations from non-endemic areas but heavily infected with other intestinal parasites are analyzed. The Schistosomiasis Control Program is currently carrying out the surveillance of endemic areas using ELISA-SEA as the first screening method, followed by the Circumoval Precipitin test for validation assay. The results with these two serological techniques allowed us to defined the criteria of chemotherapy in populations of the endemic areas. On the search of better diagnostic technique, Alkaline Phosphatase Immunoenzyme Assay (APIA) is being evaluated in field surveys.

MHC‐restriction of antibody responses to an 86 kilodalton antigen of Schistosoma mansoni

The previously observed MHC-restriction of the antibody response to an 86kDa S. mansoni antigen has been investigated in more detail. The I-A locus of the H-2 complex has been implicated as conferring responder or non-responder status on mice expressing the k and b alleles respectively. Inheritance of responsiveness was dominant over non-responsiveness. An additional level of complexity was observed in the p86 antibody responder status of individuals within a responding inbred strain. This could not be accounted for directly by the level of patent infection, but showed an inverse correlation with the level of egg output in H-2k mice. Differential antibody responsiveness to other antigens, between individuals of the same strain, occurred independently of the differential responsiveness to p86.