Schistosoma mansoni: fractionation of polysaccharide egg antigens by lectin affinity chromatography.

Crude polysaccharide antigen was extracted from Schistosoma mansoni egg homogenate by 44% aqueous phenol. The aqueous soluble polysaccharide extract was subjected to affinity chromatography with concanavalin A-Sepharose 4B. Two fractions (bound and unbound) were obtained; both of them gave precipitin lines with serum obtained from mice infected with S. mansoni. These precipitin lines gave partial identity. Further fractionation with wheat germ agglutinin-sepharose of the unbound fraction resulted in three antigenic fractions. These different antigens were eluted with different N-acetylglucosamine molarities (0, 0.05) and 0.5) and gave lines of identity when reacted against infected mouse serum. When the four antigenic materials were subjected to polyacrylamide gel electrophoresis and stained for proteins and polysaccharides no migration bands were observed. The chemical analysis of the two initial fractions showed a small percentage of amino acids in both fractions. Sugar analysis with gas-liquid chromatography showed different sugar composition of the two initial fractions.     

Acquisition of human blood group antigens by Schistosoma mansoni.

Juvenile forms of Schistosoma mansoni (schistosomula) have been cultured in human blood of various specificities and tested for the presence of blood group substances on their surfaces. The tests employed were survival following transfer into rhesus monkeys immunized against human blood substances, mixed agglutination reactions, and immunofluorescence. A, B, H AND Lewisb+ antigens were expressed at the surface when the parasites were cultured in blood of appropriate specificities. Rhesus, M N S, AND Duffy antigens could not be detected on the parasite surface following culture. The evidence suggests that the expressed blood group antigens are of host origin and are acquired by the parasite during culture, probably in the form of glycolipids or megaloglycolipids. It is likely that these substances are also acquired by parasites in the bloodstream of man. They may serve to mask surface parasite antigens, and so enable schistosomes to evade parasite-specific humoral or cellular immune responses.     

Detection of antibodies against ovum antigens in Schistosoma mansoni bilharziasis

Fifty-five sera from bilharziasis patients from West Indies and thirty-five control sera (from normal subjects and patients with hydatidosis and fascioliasis) were examined by ELISA for antibodies reacting with S. mansoni soluble egg antigen MSA1 . MSA1 antigen was prepared according to Pelley and coupled to isothiocyanate substituted plastic discs. Sensitivity and specificity of reaction were good. Moreover, in experimental infection in mice, sera of mice cured with praziquantel became negative. The sera were also examined by a whole egg antigen in counter immunoelectrophoresis. The results of the two different techniques correspond well although ELISA using purified antigen prove to be more specific and sensitive.     

Schistosomal granuloma modulation. I. Schistosoma mansoni worm antigens CAA and CCA prime egg-antigen-induced hepatic granuloma formation

Adult Schistosoma mansoni worms can positively modulate soluble egg antigen (SEA)-induced granulomas formed around SEA-coupled beads implanted in the liver. In this study, our aim was to further unravel the immunopathological characteristics of S. mansoni-worm-derived antigens in vivo. (a) Adult worm antigen (AWA)-coupled Sepharose beads, implanted into the liver, induced granulomas, containing numerous eosinophilic granulocytes and elicited marked periparticular fibrosis (composed of interstitial matrix proteins and basement membrane components). (b) Quantitative morphological analysis demonstrated that in naive mice, AWA-induced hepatic granuloma formation peaked in volume 16 days after injection of the beads. An accelerated response against AWA-coupled particles (peak volume at 8 days) was observed in mice carrying a single-sex, male S. mansoni infection. (c) When the granuloma volume induced by SEA-coupled beads in unisexually S. mansoni infected mice was compared to granulomas induced by beads laden with both SEA and AWA in unsensitized mice, no significant differences in granuloma volume were seen, indicating the existence of in vivo egg/worm antigen cross-sensitization. (d) Naive mice, sensitized with the worm antigens circulating anodic antigen (CAA) or circulating cathodic antigen (CCA), mounted a strongly accelerated response towards SEA-coupled beads implanted in the liver. We infer that, in vivo, worm antigens cross-sensitize with egg antigens and have both granulomogenic and fibrogenic characteristics. The S. mansoni soluble worm antigens CCA and CAA prime hepatic egg-antigen-induced granuloma formation possibly through the presence of immunogenic carbohydrates. These mechanisms lead to an accelerated response against SEA. Received: 10 April 1998 / Accepted: 25 June 1998     

Schistosoma mansoni: Analysis of monoclonal antibodies reactive with gut-associated antigens

The analysis of a series of monoclonal antibodies (mAbs) developed in our laboratory against gutassociated antigens ofSchistosoma mansoni is described. It was found that mAbs that recognized epitopes of antigens in the gut and on the eggshell were mainly of the IgM isotype; these epitopes are likely to be carbohydrate in composition. Of a number of mAbs that were reactive with antigens important to the human humoral immune response, 75% appeared to be reactive with the circulating cathodic antigen.     

Human hybridoma generation by hypo-osmolar electrofusion: characterization of human monoclonal antibodies to Schistosoma mansoni parasite antigens.

Human monoclonal antibodies which bind Schistosoma mansoni worm and egg antigens were identified and characterized from hybridomas generated using the hypo-osmolar electrofusion technique of somatic cell fusion. Splenocytes from S. mansoni infected individuals were mitogen-activated in vitro and subsequently fused by electrofusion. The greatest number of HAT resistant hybridomas per helical fusion chamber was obtained with unfrozen splenocytes cultured for 4-6 days after introduction of mitogen. Hybridomas secreting IgG antibodies recognizing parasite antigens were identified by ELISA. Twenty-one cloned cell lines secreting IgG antibody were maintained for at least 6 months. Characterization of antigen reactivity by Western blot analysis of nien cloned cell lines revealed antibodies which bound stage specific parasitic antigens. The data show that the technique of hypo-osmolar electrofusion produces stable, antibody producing hybridomas. The human monoclonal antibodies screened represent candidate molecules useful in the investigations of the human pathogen S. mansoni.     

Immunochemical characterisation of Schistosoma mansoni glycolipid antigens.

The aim of this study was to investigate the occurrence, distribution and immunochemical properties of antibody-defined carbohydrate epitopes in neutral glycolipid fractions of Schistosoma mansoni eggs, cercariae and adults. The amount of extractable, antigenic, neutral glycolipids was lowest in adult worms, increasing consecutively in cercariae and eggs. The immunoreactivity of the glycolipids resided in the carbohydrate moiety in that it was periodate-sensitive. Serological reactivity, and monosaccharide component analysis, anomeric configuration and methylation-linkage analyses indicated that there were two dominant epitopes, which could be partially defined immunologically. The first epitope was detected on egg, cercarial and adult glycolipids. It was strongly recognised by mouse chronic infection sera and rabbit hyperimmune sera raised against specific egg antigens, and was defined by the monoclonal antibody M2D3H (Bickle QD, Andrews BJ. Characterisation of Schistosoma mansoni monoclonal antibodies which block in-vitro killing: failure to demonstrate blockage of immunity in vivo. Parasite Immunol 1988;10:151-168). M2D3H appeared to have the same epitope specificity as monoclonal antibody 128C3/3 (Weiss J, Magnani JL, Strand M. Identification of Schistosoma mansoni glycolipids that share immunogenic carbohydrate epitopes with glycoproteins. J Immunol. 1986;136:4275-82). The internal epitope was defined structurally by the presence of fucose 3-linked to 3,4-disubstituted N-acetylglucosamine, which was itself partially substituted by a second fucose residue, to yield the determinant -4[Fucalpha1,2Fucalpha3]GlcNAcbeta1-. The second epitope was defined by the anti-LewisX monoclonal antibody 4D1 and was found primarily on cercarial glycolipids. It was chemically characterised as the LewisX epitope of Galbeta1,4[Fucalpha1,3]GlcNAcbeta1- in a terminal position. The removal of fucose greatly diminished the binding of the anti-LewisX and M2D3H monoclonal antibodies, as well as the polyclonal chronic infection sera, to glycolipids of all three life-cycle stages and thus revealed the epitopic importance of fucose.     

Serodiagnosis of imported schistosomiasis by a combination of a commercial indirect hemagglutination test with Schistosoma mansoni adult worm antigens and an enzyme-linked immunosorbent assay with S. mansoni egg antigens

A commercial indirect hemagglutination (IHA) test using erythrocytes coated with Schistosoma mansoni adult worm antigens (WA) and an enzyme-linked immunosorbent assay (ELISA) with S. mansoni egg antigens (SEA) were assessed for their use in serodiagnosis of imported schistosomiasis (hereafter these tests are designated WA/IHA and SEA/ELISA, respectively). The sensitivity of the tests was evaluated with sera from 75 patients with proven S. mansoni infection, 25 with proven S. haematobium infection, and 10 with clinical Katayama fever. The specificity was assessed with sera from 283 patients with various parasitic, bacterial, viral, and fungal infections and sera containing autoimmune antibodies. Sensitivities of the WA/IHA with a cutoff titer of 1:160 (WA/IHA(160)) in detecting S. mansoni, S. haematobium, S. mansoni and S. haematobium combined, and clinical Katayama fever were 88.0, 80.0, 86.0, and 70.0%, respectively, with a specificity of 98.9%. The WA/IHA with a cutoff of 1:80 (WA/IHA(80)) showed sensitivities of 94.7, 92.0, 94.0, and 90.0%, respectively, with a specificity of 94.7%. The comparable values of SEA/ELISA were 93.3, 92.0, 93.0, and 50.0%, respectively, with a specificity of 98.2%. Combined use of ELISA and WA/IHA(80) gave sensitivities of 100% for S. mansoni, S. haematobium, and S. mansoni and S. haematobium combined and 90% for Katayama fever. The specificity of this combination in detecting schistosomiasis was 92.9%. Combination of SEA/ELISA with WA/IHA(160) gave sensitivities of 98.7, 96.0, 98.0, and 80% with a specificity of 97.2%. Our findings suggest that WA/IHA and SEA/ELISA are each sensitive and specific serological tests that are easy to use for the diagnosis of imported schistosomiasis. The combined use of these two tests enabled the serological diagnosis of schistosomiasis to be achieved with very high degrees of both sensitivity and specificity.     

The occurrence of proteolipid antigens in Schistosoma mansoni

Adult Schistosoma mansoni were shown to synthesize a peptide containing lipid against which an antiserum could be raised in rabbits. The proteolipid purified by silicic acid chromatography was soluble in chloroform/methanol mixtures, it was very hydrophobic and contained fatty acids in its molecule, as well as other unidentified neutral lipids.     

Immunity in Schistosoma mansoni using antigens of Fasciola hepatica isolated by concanavalin A affinity chromatography.

Antigens of Fasciola hepatica adult worms were chromatographed using concanavalin A-Sepharose 4B. Two unbound peaks appeared in the inclusion volume (DT-1 and DT-2), and one peak was eluted with alpha-methylglucoside (E1-1). At least seven peaks were obtained by isoelectric focusing of E1-1. The largest of these peaks, with an average pI of 4.0, contained the antigens reactive with antibodies to Schistosoma mansoni. Mice immunized with DT-2 or E1-1 and challenged with S. mansoni cercariae developed 39 to 82% fewer worms than controls. DT-1 had no protective effect. Combining DT-1 and DT-2 abolished this protection. These experiments demonstrate that F. hepatica glycoprotein antigens induce in mice significant protection to infection with S. mansoni and offer an interesting approach to the study of vaccines in experimental schistosomiasis.     

Surface antigens on Schistosoma mansoni. I. Demonstration of host antigens on schistosomula and adult worms using the mixed antiglobulin test

Using the mixed antiglobulin test it was possible to demonstrate mouse-like antigens on the surface of schistosomula and adult Schistosoma mansoni, but not cercariae. The results indicated that schistosomula incubated with mouse tissue in vitro (newborn mouse extract) or in vivo (peritoneal cavity) adsorb mouse antigens onto their surfaces. Mouse antigens were also demonstrated on the surfaces of adult worms. In contrast, no mouse antigens could be demonstrated on cercariae or cercarial tails which had been incubated with mouse antigens, or on schistosomula or cercariae which had not been exposed to mouse antigens. Adsorption of mouse antigens by formalin-fixed, and therefore, non-viable schistosomula suggested that host surface antigens are passively adsorbed by schistosomula and are not actively produced by the parasite.     

Immunofluorescent reaction of antibodies directed against antigens from the intestinal epithelium of Schistosoma mansoni. III. Study of the reactivity of monoclonal antibodies

A monoclonal mouse antibody of IgM class was raised against an epitope of the gut epithelium of the adult worm and was applied to the detection of antigen in parasite infection. The antigen was found in urine from mice and hamsters infected with Schistosoma mansoni; a good correlation between the concentration of antigen and worm burden was observed. The antigen was thermostable, soluble in trichloracetic acid; it was not hydrolysed by proteinase K but it was destroyed by metaperiodate. The antigen was shown to be Schistosoma genus specific. It was found in different developmental stages of the parasite. High levels were detected in egg extracts.     

T-cell helper response to antigens of Schistosoma mansoni in CBA mice.

It is believed that a T-cell helper response against the schistosome surface is a necessary prerequisite for the development of protective immunity in schistosomiasis. Accordingly, the carrier effect has been used to assay eleven antigenic preparations of Schistosoma mansoni for their helper T-cell priming against surface components of the schistosomula. Three weeks after i.v. injection of the preparation, CBA mice were injected with schistosomula coated with trinitrophenol (TNP) and 4 days later, their spleens were assayed for plaque-forming cells (PFC) against TNP. Formalin-fixed schistosomula and crude adult worm tegumental membrane induced the highest response; only thirty schistosomula or 10mug of crude membrane protein were needed to generate a T-cell helper response equivalent to that induced by a living infection. All other antigenic preparations, including formalin-fixed cercariae, live miracidia, eggs and adult worm culture fluid, generated some response indicating the presence of shared carrier components.     

Cercarial glycocalyx of Schistosoma mansoni activates human complement.

Human complement activation by cercariae and schistosomula of the human parasite Schistosoma mansoni was studied in vitro. Cercariae are composed of tails which are shed after infection of the host and bodies which transform into the larvae or schistosomula after infection. After incubation in fresh normal human serum (NHS), cercarial tails bound more anti-C3 antibodies than did cercarial bodies (CB), and the tails were rapidly lysed, while the attached CB remained intact. Complement activation by cercariae was dependent on the alternative pathway but was independent of antibody, as shown by C3 deposition by hypogammaglobulinemic human sera. By transmission microscopy, the fibrillar glycocalyx on both CB and tails was stained by NHS but not by heat-inactivated serum (HI-NHS). The glycocalyx was labeled with periodate and tritiated borohydride, and parasites were incubated in NHS and HI-NHS. After solubilization, the labeled glycocalyx on organisms incubated in NHS but not HI-NHS bound anti-C3 antibodies. Of the CB incubated with eserine sulfate to prevent transformation, 78% +/- 10% were dead after culture for 24 h in NHS. In contrast, 21% +/- 12% of the CB were dead after culture in HI-NHS. Schistosomula incubated in NHS bound 37% of the amount of anti-C3 antibodies bound by cercariae but were not killed by NHS. In conclusion, the cercarial glycocalyx activated human complement, and schistosomula were less susceptible to killing than cercariae because they had less glycocalyx and activated less complement.     

Immunofluorescence test of antibodies directed against antigens of the intestinal epithelium of Schistosoma mansoni. II. In the bilharziasis patient

Specific IgM antibodies to antigens present in the epithelial cells of the gut of adult S. mansoni were measured in 2 230 persons living in areas of different levels of endemicity. Significant differences were found in the prevalence of IgM antibodies in the infected patients of the compared bilharziasis foci: lower prevalences were found in high endemic foci, involving the possibility of a tolerance phenomenon.     

Immunoglobulin A-specific serodiagnosis of acute human cytomegalovirus infection by using recombinant viral antigens.

The immunoglobulin A-specific reactivities of recombinant viral proteins from nine different reading frames of human cytomegalovirus were evaluated in enzyme-linked immunosorbent assay experiments. Antigen fragments of reading frames pUL32, pUL44, and pUL57 were identified as preferable antigens for immunoglobulin A serodiagnosis. Application of autologous fusion proteins which combine these polypeptides may be useful especially for the early detection of acute secondary human cytomegalovirus infection.     

Signal transduction events in human peripheral blood mononuclear cells stimulated by Schistosoma mansoni antigens

Activation of protein tyrosine kinases (PTKs) is a common step of T cell stimulation. However, the relationship between PTKs and activation of peripheral blood mononuclear cells (PBMC) from intestinal chronic schistosomiasis patients has not been explored yet. In this study, we investigated the participation of Lck and ZAP-70 protein tyrosine kinases (PTKs), as well as PLC-gamma1 and Shc proteins in PBMC activation by Schistosoma mansoni antigens. PBMC were stimulated with SEA (soluble egg antigen) or SWAP (soluble worm preparation), lysed, precipitated with specific antibodies and the level of tyrosine phosphorylation evaluated. Our results show that Lck and Shc were phosphorylated upon stimulation of the cells with SWAP, as well as with SEA. However, the phosphorylation level was more pronounced in SWAP than in SEA-stimulated cells. Phosphorylation of ZAP-70 was observed only in SWAP stimulated cells. Additionally, PLC-gamma1 phosphorylation was not observed in PBMC stimulated with SEA. Together, these results indicate that SEA and SWAP induce PBMC proliferation through distinct intracellular signaling pathways. Moreover, the weaker response of PBMC to SEA compared to SWAP stimulation suggests down-regulation of cells from intestinal chronic schistosomiasis patients to SEA, which may occur during immunomodulation to S. mansoni response.     

Development and optimization of the FAST-ELISA for detecting antibodies to Schistosoma mansoni

The Falcon assay screening test (F.A.S.T.) system was used to develop a rapid, sensitive, and quantitative kinetic-based enzyme-linked immunosorbent assay (k-ELISA) for detecting antibodies against Schistosoma mansoni adult microsomal antigens (MAMAs). The FAST-ELISA uses polystyrene beads on sticks molded to the lid of a microtitration plate. The beads are coated with antigen. Reagents and sera are placed in microtitration plates and the beads exposed to reagents by immersion. The exposure time required for a single dilution of serum or other antibody source, conjugate, and substrate is 5 min each. Excluding preparation time, two plates can easily be assayed in 30 min. The optima for assay conditions, reproducibility, quantitative linearity, and sensitivity are delineated. A battery of sera from patients with both homologous and heterologous infections was tested, and a dilution series of a standard reference serum pool was included with each test. Results were expressed in number of units as calibrated against the standard reference sera pool. Antigen-coated bead storage studies were performed with untreated and three chemically treated antigens. The storage stability of MAMA, ability to perform the assay with minimal equipment, sensitivity, short assay time, and ease of operation make the FAST-ELISA ideal for field studies.     

Candidate vaccine antigens identified by antibodies from mice vaccinated with 15- or 50-kilorad-irradiated cercariae of Schistosoma mansoni.

In murine schistosomiasis, the highest levels of resistance to cercarial challenge are obtained by vaccination with radiation-attenuated cercariae. To identify candidate vaccine antigens relevant to the vaccine model, we examined parasite antigens recognized by antibodies from mice vaccinated with irradiated cercariae of Schistosoma mansoni. To optimize recognition of a wide spectrum of antigens, several factors that influence the level of protection in this model were varied; specifically, we examined the effect of (i) single versus multiple vaccinations with irradiated cercariae, (ii) the dose of irradiation (15 or 50 kilorads) administered to the cercariae, and (iii) the genetic background of mouse strains, high-responder (C57BL/6J) versus moderate-responder (CBA/J) mice. We found that the number of vaccinations did not alter antibody specificity but modified the relative antibody titers against particular antigens. The dose of irradiation used to attenuate the immunizing cercariae had a similar effect on antibody titers but in addition influenced antibody specificity. Only mice that had been vaccinated with moderately irradiated cercariae recognized cathepsin B (Sm31) and Sm32. Interestingly, when vaccinated mice of the two strains, C57BL/6J and CBA/J, were compared, differences in antibody responses to particular antigens were observed. Both strains recognized the integral membrane protein Sm23, glutathione S-transferase, and cathepsin B, whereas Sm32 and paramyosin were recognized only by CBA/J mice, and heat shock protein 70 was recognized exclusively by C57BL/6J mice. In this study, we conclusively identified six distinct antigens that are specifically recognized by the humoral immune response of vaccinated mice.