Bge snail cell-line antigens: ineffectiveness as antischistosomal vaccine in mice.

Mice and rabbits were immunized with antigens derived from Bge cells, Biomphalaria glabrata hemolymph, or Schistosoma mansoni. Antisera from mice given molluscan antigens did not form immunoprecipitates with soluble antigen from adult worms, but their binding to surfaces of sporocysts, cercariae, and schistosomules suggests the presence of cross-reacting determinants. In vitro, cell-mediated immune responses to Bge antigens were not demonstrable in infected nor in immunized mice. Mice immunized with Bge cell-line antigens and challenged with S. mansoni cercariae showed no reduction in worm burden when compared with control mice.     

Isolation and partial characterization of an antigen shared between Schistosoma mansoni, Fasciola hepatica, and Biomphalaria glabrata

An antigen was isolated and partially characterized from adult Schistosoma mansoni which immunologically cross-reacted with Fasciola hepatica and Biomphalaria glabrata, a schistosome intermediate snail host. The antigen was isolated from a solubilized freeze-thaw preparation containing 0.5% Triton X-100 by passage through a CNBr-activated Sepharose 4B column coupled with rabbit IgG prepared against a homogenate of B. glabrata hepatopancreas. The eluted antigen (designated as SMw-53P) stained with Coomassie Brilliant Blue R250, but not with Nile Blue A or periodic acid-Schiff's stains. The antigen did not bind to a Concanavalin A affinity column. SMw-53P was determined to have a molecular weight of 53,000 daltons by SDS-polyacrylamide gel electrophoresis. Western blotting, using sera from mice infected with S. mansoni, revealed the presence of the antigen in whole worm preparations of both S. mansoni and F. hepatica. The serodiagnostic potential of the antigen was evaluated by ELISA utilizing sera from S. mansoni-infected mice, or rabbits injected with homogenates of F. hepatica or S. mansoni whole worm, or B. glabrata hepatopancreas. SMw-53P was shown to strongly react with all antisera, but not normal mouse or rabbit sera. These data suggest a limited value for the antigen for the specific immunodiagnosis of schistosomiasis mansoni, but do suggest a possible potential as a general screening tool for detecting trematode infections. Further studies regarding the antigen's potential as a vaccine are indicated.     

A Schistosoma mansoni 62-kDa band is identified as an irradiated vaccine T-cell antigen and characterized as calreticulin

The Schistosoma mansoni soluble adult worm antigen (SAWA) bands of 62/60 kDa were found to contain immunodominant T-cell immunogen(s) in irradiated cercariae-immunized Swiss and C57BL/6 mice. In the present study, spleen T cells of BALB/c mice immunized twice with ultraviolet light-irradiated cercariae proliferated and produced interleukin 4 in response to the 62/60-kDa SAWA bands in T-cell western assays. To characterize the 62/60-kDa bands, an adult S. mansoni worm cDNA expression library constructed in lambdagt11 was immunoscreened with serum of mice immunized with the 62/60-kDa antigens, and the immunoreactive cDNA inserts were sequenced. Purified 62- and 60-kDa proteins were used for amino acid microsequencing and for immunization studies in Swiss, C57BL/6, and BALB/c mice and rabbits. Taken together, the data indicated that the 60-kDa molecules are poorly immunogenic in mice and rabbits, whereas the 62-kDa species identified as S. mansoni calreticulin, is a good T- and B-cell antigen and represents a potential vaccine candidate.     

Influence of intramolluscan larval stages of Echinostoma liei on the infectivity of Schistosoma mansoni cercariae

During co-infection of Biomphalaria glabrata with Schistosoma mansoni and Echinostoma liei, S. mansoni cercariae released before the complete resorption of S. mansoni sporocysts show a very strong decrease of their infectivity in mice. Under conditions of high interspecific competition (i.e. when the snails are infected by E. liei 8 days after infection by S. mansoni), the mean overall worm return is five times lower than that of the control experiment. A marked decrease of the infectivity of cercariae is also noted when snails, infected exclusively with either sporocysts of S. mansoni or rediae of E. liei, are associated in the same tank.     

Vaccination of mice with a 30 kDa Schistosoma antigen with and without human adjuvant induces high protection against S. mansoni infection

A 30 kDa antigen was characterized as a hydrophobic polypeptide containing 16 amino acids and evaluated as a potential candidate vaccine against infection by Schistosoma mansoni. CD1 albino mice immunized at 0, 14, and 21 days with 25 or 50 microg of the 30 kDa antigen per mouse with and without alum developed high levels of IgG antibodies (predominantly IgG2a and IgG2b isotypes). When immunized mice were infected with 200 S. mansoni cercariae, the highest protection levels (61% and 65% reduction in worm burden in two separate experiments) were obtained using the 50-microg antigen without alum adjuvant. The granuloma size decreased to 10%, a non-significant level in mice immunized using alum adjuvant. The results demonstrate the ability of the 30 kDa antigen with and without alum adjuvant to protect mice against S. mansoni infection.     

Glycotope sharing between snail hemolymph and larval schistosomes: larval transformation products alter shared glycan patterns of plasma proteins

Recent evidence supports the involvement of inducible, highly diverse lectin-like recognition molecules in snail hemocyte-mediated responses to larval Schistosoma mansoni. Because host lectins likely are involved in initial parasite recognition, we sought to identify specific carbohydrate structures (glycans) shared between larval S. mansoni and its host Biomphalaria glabrata to address possible mechanisms of immune avoidance through mimicry of elements associated with the host immunoreactivity. A panel of monoclonal antibodies (mABs) to specific S. mansoni glycans was used to identify the distribution and abundance of shared glycan epitopes (glycotopes) on plasma glycoproteins from B. glabrata strains that differ in their susceptibilities to infection by S. mansoni. In addition, a major aim of this study was to determine if larval transformation products (LTPs) could bind to plasma proteins, and thereby alter the glycotopes exposed on plasma proteins in a snail strain-specific fashion. Plasma fractions (< 100 kDa/> 100 kDa) from susceptible (NMRI) and resistant (BS-90) snail strains were subjected to SDS-PAGE and immunoblot analyses using mAB to LacdiNAc (LDN), fucosylated LDN variants, Lewis X and trimannosyl core glycans. Results confirmed a high degree of glycan sharing, with NMRI plasma exhibiting a greater distribution/abundance of LDN, F-LDN and F-LDN-F than BS-90 plasma (< 100 kDa fraction). Pretreatment of blotted proteins with LTPs significantly altered the reactivity of specific mABs to shared glycotopes on blots, mainly through the binding of LTPs to plasma proteins resulting in either glycotope blocking or increased glycotope attachment to plasma. Many LTP-mediated changes in shared glycans were snail-strain specific, especially those in the < 100 kDa fraction for NMRI plasma proteins, and for BS-90, mainly those in the > 100 kDa fraction. Our data suggest that differential binding of S. mansoni LTPs to plasma proteins of susceptible and resistant B. glabrata strains may significantly impact early anti-larval immune reactivity, and in turn, compatibility, in this parasite-host system.     

Screening of murine monoclonal antibodies against living schistosomula of Schistosoma mansoni by radioimmunoassay

A radioimmunoassay was developed to screen supernatants of murine monoclonal antibodies against surface antigens of living schistosomula of Schistosoma mansoni. Of 196 clones screened, 10% bound schistosomula. Of these, 74% bound only schistosomula. The remaining molecules also reacted with soluble adult worm antigens and soluble egg antigens as determined by enzyme-linked immunosorbent assay. Immunoblot analysis demonstrated that monoclonal antibody 204-3E4 reacted with a 68 kDa protein, a glycoprotein that induces substantial resistance against S. mansoni infection. Recognition of an 18 kDa antigen by 204-3F1 antibody was stage-specific with the antigen being expressed in cercariae, 3- and 24-h-old parasites but not 4-day, lung stage or adult worms. Monoclonal antibody 204-4E3 reacted with purified S. mansoni paramyosin. These data indicate that radioimmunoassay using living schistosomula is a rapid alternative method to identify murine hybridomas that secrete antibodies which react with surface antigens of S. mansoni.     

Schistosoma mansoni polypeptides immunogenic in mice vaccinated with radiation-attenuated cercariae

We compared the humoral immune response of mice protected against Schistosoma mansoni by vaccination with radiation-attenuated cercariae to that of patently infected mice, and we identified antigens that elicit a greater, or unique, immune response in the vaccinated mice. These comparisons were based upon radioimmunoprecipitations and immunodepletion of [35S]methionine-labeled schistosomular and adult worm polypeptides, followed by one- and two-dimensional polyacrylamide gel analyses. The humoral responses of patently infected mice and of mice vaccinated once were remarkably similar and were directed against schistosome glycoproteins ranging in molecular size from greater than 300 to less than 10 kDa. Exposing mice to a second vaccination resulted in a marked change in the immune response, to one predominantly directed toward high molecular size glycoproteins. Sequential immunodepletion techniques identified five schistosomular and seven adult worm antigens that showed a greater or unique immunogenicity in vaccinated mice as compared with patently infected mice. These adult worm antigens were purified by preparative sequential immunoaffinity chromatography and used to prepare a polyclonal antiserum, anti-irradiated vaccine. This antiserum bound to the surface of live newly transformed and lung-stage schistosomula, as assessed by immunofluorescence assays, and was reactive with a number of 125I-labeled schistosomular surface polypeptides, including a doublet of 150 kDa that was also recognized by sera of vaccinated mice but not by sera of patently infected mice.     

Isolation and partial characterization of shared antigens of Biomphalaria glabrata and Schistosoma mansoni and their evaluation by the ELISA and the EITB

Serum from humans infected with Schistosoma mansoni, when reacted with a Biomphalaria glabrata soluble hepatopancreas antigen extract (BgSHA) yields 2 lines of precipitation by gel diffusion and 1 by immunoelectrophoresis. The IgG from the serum of a human infected with S. mansoni was coupled to CNBr-activated Sepharose 4B. BgSHA (8.0 mg) was then filtered through the gel and the bound antigens, denoted BgSm, eluted with HCl-glycine, pH 2.6. These bound antigens comprised 2.8% of the total BgSHA. BgSm was then applied to an anti-Fasciola hepatica column as above. The drop through in PBS (38% yield) and containing the BgSm antigens depleted of cross-reactivity with F. hepatica was then tested by the ELISA to evaluate its serodiagnostic potential. These antigens detected a primary S. mansoni infection by 4 wk but were less sensitive than SmSEA in the detection of a primary infection with S. mansoni. However, the BgSm-specific antigens were more specific than SmSEA and showed less cross-reactivity with the serum of mice infected with F. hepatica. At least 16 peptides were seen by silver staining following SDS-PAGE with 5-20% gradient gels. The 2 more prominent bands obtained were estimated to have molecular weights of 62 and 66 kd. Nitrocellulose strips blotted with BgSHA were incubated with the serum of mice infected with S. mansoni for 12 wk and developed 6 bands with molecular weights of 66, 57, 55, 50, 48 and 32 kd.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vaccination in murine schistosomiasis with adult worm-derived antigens: variables influencing protection in outbred mice

Previous studies have shown that both permissive (mouse) and partially permissive (rabbit) hosts develop high levels of resistance against Schistosoma mansoni infection after vaccination with a multiple antigen extract (SE) obtained by incubation of living adult worms in saline, plus bacterial adjuvant. To investigate variables influencing SE-induced protection in murine schistosomiasis, a series of distinct vaccination protocols were performed focussing on the immunization dose, carrier systems, route, site and amplitude of challenge infection, and time between immunization and challenge. In addition, a new approach was adopted to evaluate SE protective activity, by means of population analysis of worm burden frequency distributions in a large scale study of vaccination in outbred Swiss mice. Distinct curves of frequency and a drastic difference in worm burden distribution of frequencies from SE-vaccinated x non-vaccinated mice were found. It was shown that SE could generate 75% mean protection in outbred mice even in the absence of adjuvant. In addition SE immunization was also able to induce full protection against lethal infection. SE-induced protection could be modulated by such parameters as dose of SE immunization/challenge interval, and route of cercariae injection. These data show that SE yields very high protective activity in outbred mice, and may provide a further insight for rational design of a vaccine in experimental schistosomiasis.     

Schistosoma mansoni: comparison of a Caribbean and African strain and experimental crossing based on compatibility with intermediate hosts and Rattus rattus

To elucidate changes relative to compatibility with intermediate and definitive hosts affecting Schistosoma mansoni since it was introduced to the New World, the compatibility of S. mansoni from Africa (the Cameroons), from the Caribbean (Guadeloupe), and those resulting from experimental crosses with the gastropods Biomphalaria glabrata and B. pfeifferi has been studied. Results show that S. mansoni, regardless of its origin or its usual snail host, always infects B. pfeifferi more successfully than B. glabrata. The success rate with B. pfeifferi is 100% with 5 miracidia of S. mansoni from Guadeloupe and 97% with 5 miracidia from the Cameroons. On the other hand, in B. glabrata infection rate was 54% with 5 miracidia from Guadeloupe and 0% with 5 miracidia from the Cameroons (a rate of 19% is reached when using 10 miracidia). Hybrid miracidia infect B. pfeifferi and B. glabrata with a success rate of 60 and 86%, respectively, which are intermediate between those of the parent strains. Studies of S. mansoni development in Rattus rattus show that there is better infectivity and survival for the Caribbean strain than the Cameroon strain: the percentage worm recovery 4 weeks after exposure in 34% for S. mansoni from Guadeloupe, 14% for S. mansoni from the Cameroons, and 31% for the hybrids. The mortality rate between 4 and 12 weeks after exposure is 51% for S. mansoni from Guadeloupe, 87% for S. mansoni from the Cameroons, and 31% for the hybrids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes induced in Biomphalaria glabrata (Say, 1818) following trials for artificial stimulation of its internal defense system

Biomphalaria glabrata can react through different pathways to Schistosoma mansoni miracidium penetration, according to the degree of resistance/susceptibility presented by different snail strains, which is a genetically determined character, resistance being the dominant feature. However, it has been observed that previous susceptible snail strain may change its reactive behavior along the course of infection, exhibiting later a pattern of cercarial shedding and histopatopathological picture compatible with high resistance. Such observation suggests the possibility of B. glabrata to develop a sort of adaptative immunity face a schistosome infection. To explore on this aspect, the present investigation looked for the behavior of S. mansoni infection in B. glabrata previously subjected to different means of artificial stimulation of its internal defense system. Snails previously inoculated with irradiated miracídia (Group I); treated with S. mansoni antigens (Group II) or with a non-related parasite antigen (Group III) were challenged with 20 viable S. mansoni miracidia, and later looked for cercarial shedding and histopathologic changes at different times from exposition. Nodules of hemocyte accumulations were found at the site of antigen injection. These nodules resembled solid granulomas, and were larger and more frequent in snails injected with S. mansoni products as compared to those injected with Capillaria hepatica. However, the presence of such granulomas did not avoid the S. mansoni challenge infection from developing in a similar way as that seen in controls. The data are indicative that hemocytes are able to proliferate locally when stimulated, such capacity also remaining localized, not being shared by the population of hemocytes located elsewhere within the snail body.     

Molecular and functional characterization of a tandem-repeat galectin from the freshwater snail Biomphalaria glabrata, intermediate host of the human blood fluke Schistosoma mansoni

In the present study, a tandem-repeat type galectin was characterized from an embryonic cell line (Bge) and circulating hemocytes of the snail Biomphalaria glabrata, intermediate host of the human blood fluke Schistosoma mansoni. The predicted B. glabrata galectin (BgGal) protein of 32 kDa possessed 2 carbohydrate recognition domains, each displaying 6 of 8 conserved amino acids involved in galactoside-binding activity. A recombinant BgGal (rBgGal) demonstrated hemagglutinating activity against rabbit erythrocytes, which was specifically inhibited by galactose-containing sugars (lacNAc/lac>galNAc/gal). Although native galectin was immunolocalized in the cytoplasm of Bge cells and the plasma membrane of a subset of snail hemocytes (60%), it was not detected in cell-free plasma by Western blot analysis. The findings that rBgGal selectively recognizes the schistosome-related sugar, lacNAc, and strongly binds to hemocytes and the tegument of S. mansoni sporocysts in a sugar-inhibitable fashion suggest that hemocyte-bound galectin may be serving as a pattern recognition receptor for this, or other pathogens possessing appropriate sugar ligands. Based on molecular and functional features, BgGal represents an authentic galectin, the first to be fully characterized in the medically-important molluscan Class Gastropoda.     

Sm60, a mannose-binding protein from Schistosoma mansoni with inflammatory property

We demonstrate here that a mannose-binding protein from Schistosoma mansoni, termed Sm60, was recovered in the mannose-eluted fraction (Man(+)) upon affinity chromatography on immobilised mannose of the soluble antigen fraction from adult worm tegument and cercariae. Sm60 was detected in the Man(+) fraction as a prominent doublet with an apparent molecular mass of 60-66 kDa by SDS-PAGE and appeared as a single band with a pI of approximately 6.9 by isoelectrofocusing. Sm60 was also detected in preparations of schistosomula extract and soluble egg antigens using a mouse polyclonal anti-Sm60 serum on immunoblotting assay. This antiserum demonstrated that Sm60 was localised on the tegument of S. mansoni adult worm. In order to determine the role of Sm60 in host-parasite interactions, we showed that Sm60 induced in vitro migration of human neutrophil in a dose-dependent manner and in vitro mast cell degranulation. Sm60 triggered these activities through its carbohydrate-binding site, since these activities were selectively inhibited by 0.2 M D-mannose, but not by 0.2 M D-galactose. Furthermore, Sm60 induced in vivo neutrophil migration. In contrast, mast cell-depleted rats presented a significant reduction of the neutrophil migration induced by Sm60 as compared with non-depleted controls. These data suggest that in vivo neutrophil migration induced by Sm60 is modulated by mast cell-dependent mechanisms. Sm60 might play a key role in the host-parasite interaction, and its characterization opens perspective to examine the role of this molecule in the biology of S. mansoni.     

Antibody responses to the fucosylated LacdiNAc glycan antigen in Schistosoma mansoni-infected mice and expression of the glycan among schistosomes

Infections of animals with parasitic worms, such as Schistosoma mansoni, induce humoral immune responses to carbohydrate antigens, raising the possibility that such antigens might be useful targets for the development of vaccines and new diagnostic approaches. Here we describe the identification of fucosylated LacdiNAc (LDNF) [GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc-R] as a new carbohydrate antigen in S. mansoni that induces humoral immune responses in infected mice. The presence of antibodies was determined by ELISA using a neoglycoconjugate synthesized to express LDNF sequences. Sera from S. mansoni-infected, but not uninfected, mice contain IgM, IgG, IgA, and IgE antibodies to LDNF. The IgG antibodies are primarily of the IgG1 and IgG3 subclasses, with no detectable levels of the complement-fixing IgG2a and IgG2b isotypes. An IgM monoclonal antibody, designated SMLDNF1, was generated from the spleens of S. mansoni-infected mice, and the antibody exhibits specific recognition of LDNF sequences, but not other fucosylated glycans tested. Immunocytochemical analysis demonstrates that LDNF antigens are localized on the tegumental surface of adult S. mansoni. Western blot analysis indicates that LDNF sequences are expressed on numerous high-molecular-weight glycoproteins from the three major human schistosome species, as well as the bird schistosome Trichobilharzia ocellata. The identification of LDNF antigen on the tegumental glycoproteins of schistosomes and the ability to synthesize LDNF conjugates should aid in the development of glycan-based vaccines and immunodiagnostic tests for schistosomiasis and in determining the role(s) of the glycans in worm development and pathogenesis.     

Identification, comparison and partial characterization of glycoproteins in the hemolymph of Schistosoma mansoni (Trematoda)-susceptible and resistant Biomphalaria glabrata (Gastropoda).

1. Glycoproteins in the plasma (cell-free hemolymph) of Schistosoma mansoni-resistant and susceptible strains of Biomphalaria glabrata were identified, characterized and compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotting and a series of peroxidase-labeled lectins with different sugar specificities. 2. Schistosome-resistant snails possessed at least three glycoproteins (70, 116, 205 kDa) that were not present in plasma from susceptible snails. 3. Sporocysts (larval S. mansoni) preincubated in plasma from schistosome-resistant or susceptible B. glabrata adsorbed certain glycoproteins to their surface and some altered the binding of lectin probes to the parasite. 4. This study indicates that glycoproteins in the plasma of B. glabrata are associated with S. mansoni-susceptibility and resistance.     

Specific tyrosine phosphorylation induced in Schistosoma mansoni miracidia by haemolymph from schistosome susceptible, but not resistant, Biomphalaria glabrata

Molecular interplay during snail-schistosome interactions is poorly understood and there is much to discover concerning the effect of snail host molecules on molecular processes in schistosomes. Using the Biomphalaria glabrata - Schistosoma mansoni host-parasite system, the effects of exposure to haemolymph, derived from schistosome-resistant and susceptible snail strains, on protein tyrosine phosphorylation in miracidia have been investigated. Western blotting revealed several tyrosine phosphorylated proteins in this larval stage. Exposure of miracidia to haemolymph from susceptible snails for 60 min resulted in a striking, 5-fold, increase in the tyrosine phosphorylation of a 56 kDa (p56) S. mansoni protein. In contrast, haemolymph from resistant snails had little effect on protein tyrosine phosphorylation levels in miracidia. Confocal microscopy revealed that tyrosine phosphorylation was predominantly associated with proteins present in the tegument. Finally, treatment of miracidia with the tyrosine kinase inhibitor genistein significantly impaired their development into primary sporocysts. The results open avenues for research that focus on the potential importance of phospho-p56 to the outcome of schistosome infection in snails, and the significance of protein tyrosine kinase-mediated signalling events to the transformation of S. mansoni larvae.     

The effects of temperature change on the infection rate of Biomphalaria glabrata with Schistosoma mansoni

The aim of this study was to investigate the influence of temperature on the development of Schistosoma mansoni infections in Biomphalaria glabrata. The snails were infected at 15, 20, and 30 degrees C, and the cercarial release was analyzed after 30 and 60 days post-infection. Our results showed that a decrease in the temperature has a substantial influence on the development of S. mansoni infection in B. glabrata, with significant differences (p < 0.05) between 15 and 30 degrees C. These data could provide a better understanding of the epidemiological aspects of schistosomiasis.     

Schistosoma mansoni: immunofluorescent detection of its antigen reacting with Biomphalaria glabrata amoebocytes

The reaction of amoebocytes in the hemolymph of the infected intermediate host, Biomphalaria glabrata, to Schistosoma mansoni antigens has been investigated using the indirect immunofluorescence antibody test. Monolayers of amoebocytes, prepared from hemolymph of infected and normal snails, were first fixed and then reacted with antisera obtained from mice infected for 7 to 9 weeks. Nonspecific and cross-reactions between the antisera and monolayers of amoebocytes were eliminated by absorbing the antisera with tissues from uninfected snails. The liberation of detectable schistosomal antigens in the hemolymph in soluble and particulate forms coincided with completion of the infection cycle 3 to 4 weeks after exposure to miracidia. The schistosomal antigens were demonstrable in the cytoplasm of amoebocytes and in the center of amoebocyte aggregates.     

Induction of protective immunity against Schistosoma mansoni by a nonliving vaccine is dependent on the method of antigen presentation

A preparation of nonliving parasite antigens containing both soluble and particulate components of frozen-and-thawed invasive larvae was used to immunize C57BL/6J mice against challenge Schistosoma mansoni infection. The method of antigen presentation was observed to be critical to the ability of this preparation to induce protective immunity, because intradermal administration in conjunction with a bacterial adjuvant (BCG) resulted in strong protection against challenge parasites (51% reduction in worm burden in six experiments), whereas i.v. injection of the same antigenic preparation was completely ineffective. Induction of resistance was accompanied by specific immune responsiveness toward schistosome antigens. Protection correlated more closely with sensitization for specific delayed hypersensitivity than with elicitation of circulating antibodies to larval surface antigens or immediate hypersensitivity in these models. These results suggest that it will be possible to design a defined vaccine against S. mansoni infection, but that identification of the route of antigen presentation that most effectively elicits relevant immune effector mechanisms will be crucial to the success of any vaccination protocol involving nonliving antigens.