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.     

Isolation and characterization of surface antigens from Schistosoma mansoni schistosomula

Surface antigens of Schistosoma mansoni schistosomula were isolated using antibodies produced in rat and human schistosomiasis. Three immunoreactive surface proteins of 40 000, 37 000 and 32 000 daltons were identified by SDS—PAGE analysis of immune complexes formed by incubation of a detergent extract of surface labelled schistosomula with infected rat sera. Surface antigens of similar molecular weight were also isolated when using sera of patients with schistosomiasis. Binding of schistosomula surface antigens to specific antibodies was substantially inhibited by components released by adult worms. The results suggest that these schistosomula surface antigens could be involved in the immune response against challenge infection but their protective role in immunity still remains to be determined.     

Enzyme-linked crossed immunoelectrophoresis: a technique for detection of allergens and their respective antibodies in human schistosomiasis

A method is described which allows the demonstration of allergens in complex antigens and their respective antibodies. Schistosoma mansoni antigens are separated in agarose by 2-dimensional electrophoresis, using an anti-S. mansoni serum from goats in the second dimension. After extensive washing test serum is spread over the gel and allowed to bind to the precipitated antigens. After further extensive washing, horseradish peroxidase-coupled anti-IgE antibodies are put on the plate and allowed to react. Bound antiserum is visualized with tetramethylbenzidine as a substrate.In pooled sera from schistosomiasis patients at least 7 antigen fractions of adult S. mansoni and 2 of cercarial antigen reacted with IgE antibodies. No reaction was found in normal sera.     

Schistosoma mansoni anticomplementary antigens (SACA): Detection in schistosomula and adult worms

In previous studies we have shown that schistosomula of Schistosoma mansoni are able to activate complement (C), in the absence of antibodies, by both the classical (CP) and the alternative C pathway (AP). In the present work we have demonstrated that certain factors present in the antigen extract of schistosomula and adult worms also showed an anticomplementary activity. These schistosome anticomplementary antigens (SACA) were found in the low molecular weight fraction (< 35,000 daltons) of the whole extract of adult schistosomes and were able to deplete C through both the CP and the AP.     

Identification of a major T cell immunogen in the anti-schistosome response of adult residents in an area endemic for Schistosoma mansoni

Vaccine-induced immunity to Schistosoma mansoni infection depends on the specific priming of certain T cell subsets and on the recall of this response by natural infections months or years after vaccine administration. Thus, those schistosome proteins that activate T cells in individuals stimulated by natural infections are potential candidate vaccine antigens. In the present study, we identified and purified one such T cell-stimulating antigen and evaluated its immunological properties in subjects living in an area endemic for Schistosoma mansoni. Chromatography fractions (gel filtration, followed by ion exchange chromatography) of soluble extracts of schistosomula were screened for their ability to stimulate schistosome-specific T cell clones derived from a subject sensitized by natural infection. A fraction stimulating most clones was identified and characterized. A few nanograms of this fraction, containing a major 9-10-kDa component, stimulated the T helper cells of most adults living in an endemic area of Brazil, and was able to trigger a strong cutaneous immediate hypersensitivity reaction. In contrast, children reacted weakly to this antigen preparation both in blastogenesis and in skin tests, although they mounted a significant reaction to crude larval antigen preparations. In conclusion, this work identifies a schistosomula antigen that induces a strong T cell response in adults sensitized by natural infections. This T cell response develops gradually in children and adolescents, is apparently not restricted by the HLA haplotypes common in the study area, and allows the production of parasite-specific IgE antibodies. Thus, this T cell response has some features of the immune response that is believed to protect chronically exposed humans from reinfection.     

Use of recombinant calreticulin and cercarial transformation fluid (CTF) in the serodiagnosis of Schistosoma mansoni

Schistosomiasis is traditionally diagnosed by microscopic detection of ova in stool samples, but this method is labour intensive and its sensitivity is limited by low and variable egg secretion in many patients. An alternative is an ELISA using Schistosoma mansoni soluble egg antigen (SEA) to detect anti-schistosome antibody in patient samples. SEA is a good diagnostic marker in non-endemic regions but is of limited value in endemic regions, mainly because of its high cost and limited specificity. Here we assess seven novel antigens for the detection of S. mansoni antibody in an endemic region (the Northern Nile Delta). Using recombinant S. mansoni calreticulin (CRT) and fragments thereof, anti-CRT antibodies were detected in the majority of 97 patients sera. The diagnostic value of some of these antigens was, however, limited by the presence of cross-reacting antibody in the healthy controls, even those recruited in non-endemic areas. Cercarial transformation fluid (CTF), a supernatant that contains soluble material released by the cercariae upon transformation to the schistosomula, is cheaper and easier to produce than SEA. An ELISA using CTF as the detection antigen had a sensitivity of 89.7% and an estimated specificity of 100% when used in non-endemic regions, matching the performance of the established SEA ELISA. CTF was substantially more specific than SEA for diagnosis in the endemic region, and less susceptible than SEA to cross-reacting antibody in the sera of controls with other protozoan and metazoan infections.     

Schistosoma mansoni: Detection and characterization of schistosome derived antigens by inhibition enzyme-linked immunosorbent assay (IELISA) utilizing monoclonal antibodies

Monoclonal antibodies were used in an inhibition enzymelinked immunosorbent assay (IELISA) to detect a variety of schistosome derived antigens. Preparations were obtained from various stages ofSchistosoma mansoni and from the eggs ofS. japonicum. Using appropriate titers of monoclonal antibodies it was possible to detect less than 0.01 g/ml of schistosome antigens. The sensitivity of IELISA was dependent upon the type and concentration of the monoclonal antibody used, as well as upon the source of the antigens. Specificity studies showed that some of the monoclonal antibodies recognized species specific antigenic determinants, while others reacted against genus specific antigens. Furthermore, certain antibodies interacted with antigens which were neither genus or species specific. Fractions ofS. mansoni andS. japonicum egg antigen extracts, which have been previously considered to be relatively pure, were compared using certain monoclonal antibodies. The results indicate that these fractions are very heterogenous with regard to the unique antigenic specificities. For example, most antigenic determinants in crude soluble egg antigen are not retained on concanavalin-A lectin affinity columns and major serologic antigens have more than one determinant with different distributions. The expression of these antigenic determinants appears to be a function of both the concentration of the specific antigen and the mode of expression of the moiety within the antigenic matrix.     

Surface proteins of Schistosoma mansoni and their expression during morphogenesis

Surface components of Schistosoma mansoni have been identified by lactoperoxidase-catalyzed iodination. Cercariae have a simple labeling pattern in comparison to schis- tosomula. Transformation of cercariae to schistosomula results in the loss of a low molecular weight material which may be the glycocalyx, and the appearance of many more labeled proteins. Mechanical conversion of cercariae to schistosomula requires subsequent incubation at 37 °C for more than 1 h to give the full surface-labeling pattern of schistosomula. The majority of proteins found on schistosomula appear to be present throughout the remaining part of the developmental cycle, although adult male worms had only low levels of these antigens, and female worms had virtually no detectable surface antigens. The low level of expression of schistosome antigen could be caused by adsorbed host antigen, although no evidence for adsorbed host protein was found, or by a reduced level of antigens present on the worm surface. The low level of schistosome antigen could have a role in the resistance of adult worms to the host's immune response.     

Characterization of the Glycoprotein Antigens Which Mediate the Schistosoma japonicum Circumoval Precipitin Reaction

The circumoval precipitin test is a serological test used for diagnosis of schistosomiasis japonica. Soluble egg antigens of Schistosoma japonicum block the formation of the circumoval precipitin by serum from infected humans. Consequently, circumoval precipitin inhibition was used to monitor purification of the soluble egg antigens of S. japonicum. Crude egg antigens were separated into protein and glycoprotein fractions by lectin chromatography on concanavalin A Sepharose. The glycoprotein fraction produced two intense precipitin lines upon immunodiffusion analysis with human chronic infection sera. The protein fraction produced two faint precipitin lines which did not cross-react with those of the glycoprotein fraction. The glycoprotein fraction contained 90% of the circumoval precipitin inhibitory activity. Isoelectric focusing of ¹²⁵I-labeled concanavalin A Sepharose fractions revealed at least four groups of potential S. japonicum antigens, termed JAG I, II, and III, and a JAG IV complex. These had isoelectric points ranging from 3.2 to 6.7. In these respects, the S. japonicum egg antigen glycoproteins are similar to those of Schistosoma mansoni. The glycoproteins were further separated by diethylaminoethyl ion-exchange chromatography. On immunodiffusion analysis it was found that one of the strong Ouchterlony precipitin lines was associated with glycoproteins that did not adsorb to diethyl-aminoethyl columns, whereas the second Ouchterlony precipitin was heterodisperse, being present in the first, second, and third of the four peaks eluted from the diethylaminoethyl column. Immunoelectrophoresis of the diethylaminoethyl fractions demonstrated that the antigen present in highest concentration in soluble egg antigen glycoproteins, JAG II, was extremely heterodisperse in its behavior on diethylaminoethyl columns. This is unlike the S. mansoni antigens which can be easily separated by diethylaminoethyl ion-exchange chromatography.     

Identification of Schistosoma mansoni antigens by means of biologically active monoclonal antibodies

Two monoclonal antibodies of the IgE class (54.10) and of the IgG₁ class (27.21), that were shown previously to possess biological activity against Schistosoma mansoni larvae, were used for identification of surface antigens of the cercariae and schistosomula. This was performed by immunoprecipitation, immunoaffinity chromatography and immunoblotting. The epitope reactive with 27.21 mcIgG₁ is present on four polypeptides (60, 50, 27 and 19 kDa) derived from the parasite. The 60 kDa is specific to cercariae, whereas the 50 kDa is a glycoprotein shared both by cercariae and schistosomula. The antigen reactive with the 54.10 mcIgE was isolated by affinity chromatography on 54.10 column, and contained three major peptides of 125, 94 and 30 kDa. The 125 and 94 kDa band probably originate from the same protein, since they yield almost identical peptide maps. The isolated antigen retained its biological activity as demonstrated in the basophils degranulation assay.     

Partial characterization and kinetics of expression of Sm15, aSchistosoma mansoni tegumental antigen

Differential antibody screening of an adultSchistosoma mansoni cDNA expression library constructed in lambda gt11 identified a partial cDNA clone, A70. This cDNA encodes a fusion protein recognized by antibodies raised against highly irradiated schistosomula and adult worm tegumental membranes but not by anti-egg antibodies. Anti-tegumental membrane antisera affinity-purified on the A70 cDNA fusion protein were used for Western blotting analysis and indirect immunofluorescence, resulting in the identification of a 15-kDa protein (Sm15) in the tegument of adult worms. This is one of the principal tegumental antigens recognized by antibodies from mice protectively vaccinated with adult worm tegumental membranes. Sm15 is much smaller than the protein encoded by its gene, suggesting that it results from a highly processed precursor. It was found that Sm15 behaves as an integral membrane protein upon partitioning in Triton X-114 and that it is present in worms of 2 weeks or older but not in schistosomula or miracidia. The affinity-purified antibodies also revealed the presence of a 23-kDa antigen in whole-worm homogenates that is apparently coexpressed with Sm15. The 23-kDa antigen was not found associated with membranes and is probably a soluble protein. A further series of Western blots were undertaken using antibodies affinity-purified from serum raised against schistosomula. In this case, the 23- and 15-kDa products were not recognized, but rather soluble proteins ranging from 45- to 150-kDa were detected in almost all larval stages investigated. The results suggest that the precursor is differentially processed during maturation.     

Rapid Detection of a Schistosoma mansoni Circulating Antigen Excreted in Urine of Infected Individuals by Using a Monoclonal Antibody

Schistosoma circulating antigens were used to indicate the infection intensity and to assess cure. An immunoglobulin G2a (IgG2a) mouse monoclonal antibody was used in a fast dot-enzyme-linked immunosorbent assay (ELISA; FDA) for rapid and simple diagnosis of schistosomiasis in the field. Seven hundred Egyptians were parasitologically examined for Schistosoma mansoni and other parasitic infections. A rectal biopsy was done as a “gold standard” for individuals showing no S. mansoni eggs in their feces. Egg counts were obtained by the Kato smear method for only 100 of 152 individuals with eggs in their feces. Specific anti-schistosome IgG antibodies were evaluated in sera by ELISA. Urine samples from the 700 individuals were tested by FDA for detection of the circulating antigen. The assay showed a sensitivity of 93% among 433 infected individuals and a specificity of 89% among 267 noninfected individuals. FDA showed the highest efficiency of antigen detection (91%) compared with the efficiency of antibody detection by ELISA (75%) and stool analysis (60%). In addition, FDA detected infected patients with 20 eggs/g of feces. Also, the sensitivity of FDA ranged from 90 to 94% among samples from patients with different clinical stages of schistosomiasis. All the assay steps can be completed within 30 min at room temperature for 96 urine samples. The monoclonal antibody identified a 74-kDa antigen in different antigenic extracts of S. mansoni and Schistosoma haematobium and in the urine of infected individuals. In addition, a 30-kDa degradation product was identified only in the urine samples. On the basis of these results, FDA should be used as a rapid tool for the sensitive and specific diagnosis of Schistosoma infection.     

Correlation between cell-mediated immunity and degree of infection in subjects living in an endemic area of schistosomiasis

Cell-mediated immunity to Schistosoma mansoni antigens, unrelated antigens and mitogens was evaluated in 50 subjects with the same degree of exposure to infection living in an endemic area of schistosomiasis. The degree of infection, assessed by the number of eggs/g of stool, was variable in this population (0-5604), suggesting differences in susceptibility to infection. Absence of lymphoproliferative response was observed in 56% of this group, despite having a response to purified protein derivative of tuberculin (PPD) and tetanus toxoid (TT) antigens and to pokeweed mitogen. The 50 subjects were divided into two groups, according to their degree of infection. The lymphoproliferative responses to schistosomula and adult worm antigens in the group with a low degree of infection (≤400 eggs/g of stool) were higher than the ones documented in patients with a high degree of infection (>400 eggs/g of stool), and these differences were statistically significant (p ≤0.001). An inverse correlation between the lymphocyte proliferation in response to S. mansoni antigens and the degree of infection was also observed (p = 0.02), indicating that subjects with a lower degree of infection have a higher lymphoproliferative response to schistosomula and adult worm antigens. No differences in the lymphocyte reactivity to other antigens (PPD and TT) were detected in these groups. An impairment of interferon-γ in vitro production was observed when the lymphocytes from these subjects were stimulated with S. mansoni adult worm antigen, although they produced gamma interferon in response to phytohemag-glutinin.     

Antigenic cross‐reactivity between Schistosoma mansoni and peanut: a role for cross‐reactive carbohydrate determinants (CCDs) and implications for the hygiene hypothesis

The antigenic reactivity of constituents of Schistosoma mansoni and peanut (Arachis hypogaea) was investigated to determine whether identical antigenic epitopes possessed by both organisms provided a possible explanation for the negative correlation between chronic schistosome infection and atopy to allergens. Aqueous extracts of peanuts were probed in Western immunoblots with rabbit IgG antibodies raised against the egg, cercarial and adult worm stages of S. mansoni. Several molecules in the peanut extract were antigenically reactive with antibodies from the various rabbit anti‐schistosome sera. A pair of cross‐reactive peanut molecules at ~30 000–33 000 molecular weight was purified and both proteins were identified by mass spectrometric analysis as the peanut allergen Ara h 1. Anti‐S. mansoni soluble egg antigen antibodies that were eluted off the peanut molecules reacted with two S. mansoni egg antigens identified by mass spectrometry as IPSE/α‐1 and κ‐5. Alignments of the amino acid sequences of Ara h 1 and either IPSE/α‐1 or κ‐5 revealed a low level of peptide sequence identity. Incubation of nitrocellulose paper carrying electrophoresed peanut molecules, six constituents of other allergic plants and S. mansoni egg antigens in a mild solution of sodium metaperiodate before probing with antibodies, inhibited most of the cross‐reactivities. The results are consistent with the antigenic cross‐reactive epitopes of S. mansoni egg antigens, peanut and other allergic plants being cross‐reactive carbohydrate determinants (CCDs). These findings are novel and an explanation based on ‘blocking antibodies’ could provide an insight for the inverse relationship observed between schistosome infection and allergies.     

Chronic intestinal nematode infection exacerbates experimental Schistosoma mansoni infection

Mixed-parasite infections are common in many parts of the world, but little is known of the effects of concomitant parasite infections on the immune response or on disease progression. We have investigated the in vivo effects of a chronic gastrointestinal nematode infection on the infectivity and development of the immune response against the common trematode helminth Schistosoma mansoni. The data show that mice carrying an established chronic Trichuris muris infection and coinfected with S. mansoni, had significantly higher S. mansoni worm burdens than mice without coinfection. The increase in S. mansoni worm burden was accompanied by a higher egg burden in the liver. Kinetic analysis of S. mansoni establishment indicate reduced trapping of S. mansoni larvae during skin-to-lung migration, with T. muris-induced alterations in lung cytokine expression and inflammatory foci surrounding lung-stage schistosomula, suggesting that the immunomodulatory effects of chronic T. muris infection elicited at the gut mucosal surface extend to other organs and perhaps specifically to other mucosal surfaces. The data show that a preexisting chronic gastrointestinal nematode infection facilitates the survival and migration of S. mansoni schistosomula to the portal system, and as a result, increases the egg burden and associated pathology of S. mansoni infection.     

The allergens of Schistosoma mansoni: I. Initial separation and comparative assay

Ten antigen fractions were prepared from adult Schistosoma mansoni by extraction into borate-buffered saline, precipitation at pH 4.6 and separation on Sephadex G-100. The allergic activity of these antigens was assayed by a modified Prausnitz—Kustner type reaction in rats; this test system was found to be sensitive and consistent, allowing differences in allergenicity between antigens to be accurately assessed.

Skin-reactivity was detected in both acid-soluble and acid-insoluble fractions. Specific allergenicity was located in peak 3 of a G-100 separation of the acid-soluble fraction and in peaks 1 and 2 of a G-100 separation of the acid-insoluble fraction suggesting that the allergens of S. mansoni were of at least two types: (1) a protein of mol. wt above 150,000 precipitated at pH 4.6, and (2) a protein of mol. wt 20–30,000 remaining in solution at this pH.

It is suggested that both these allergens are glycoproteins. Non-specific histamine-releasing agents were found in peak 1 of the G-100 separation of the acid-soluble material.

     

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     

Received 14 May 1973; Relationship between Killer and Rosette-Forming Cells Reactive to H-2 478

Optimum conditions were selected for testing of direct and indirect H-2 reactive rosette-forming cells (RFC) with the use of sheep or mouse erythrocytes coated with soluble H-2 antigens. Rosette formation with mouse erythrocytes is shown to be an immunologically specific reaction and to be inhibited by the pretreatment of cells with the corresponding soluble H-2 antigen preparation. Rate of killer cell and RFC formation is different after primary and secondary immunization with an allogeneic tumor, Unlike killer cells. RFCs are inactivated without complement by rabbit antibodies against mouse γ-globulin (MGG). In contrast to killer cells, which are eliminated in the presence of complement by anti-θ antibodies but not by anti-plasma cell antigen (PC.1) and anti-MGG antibodies, direct RFCs are not inactivated by anti-θ but are eliminated partiallv by anti-MGG and anti-PC₁. The RFCs are shown to be concentrated in the low-density fractions of the discontinuous bovine serum albumin gradient, whereas cytotoxic lymphocytes are distributed less closely, being concentrated in the intermediate fraction, which does not differ morphologically from the initial cell suspension. Killer and rosette-forming cells reactive to the same H-2 antigens appear to be two non-overlapping populations of T and B cells, respectively, and no T-B cooperation is required for destruction of target cells.     

Dihydroartemisinin: a new story of an old drug against Schistosoma mansoni infection

Currently, praziquantel is the drug of choice for the treatment of human Schistosoma mansoni infections. It has not been proved until now that there is real praziquantel resistance, but there is decreased praziquantel sensitivity. A search for novel antischistosomal agents against the parasite has been given a high priority. Dihydroartemisinin, formerly identified as an antimalarial drug, has been shown to be active against both Schistosoma japonicum and S. mansoni in mice. Interestingly, dihydroartemisinin is found to be highly effective against the 14–28-day schistosomula of S. mansoni, and treatment with multiple low doses of the drug achieves a high efficacy with reduced toxicity to the host. The long time development from juveniles to adults allows adequate timing for treatment of this neglected tropical disease. It is supposed that dihydroartemisinin, a safe orally administered agent, may be used for the prevention and control of human S. mansoni infections, notably in areas with reduced praziquantel sensitivity or praziquantel resistance detected.     

Up-regulation of SUMO E3 ligases during lung schistosomula and adult worm stages

Small ubiquitin-like modifier (SUMO) conjugation of proteins occurs through a concert action of enzymes using a similar ubiquitination mechanism. After a C-terminal peptide is cleaved from the SUMO precursor by a protease to reveal a di-glycine motif, SUMO is activated by an E1 enzyme (Aos1/Uba2) and conjugated to target proteins by the sole E2 enzyme (Ubc9) guided to the appropriate substrates by the SUMO E3 ligase. Previous reports from our group showed that Schistosoma mansoni has two paralogs of SUMO: one E2 conjugation Ubc9 and two SUMO-specific proteases (SENPs). The differential gene expression profile observed for SUMO pathway genes throughout the S. mansoni life cycle attests for the distinct patterns of SUMO conjugates observed during parasite development particularly during the cercariae to schistosomula transition. To continue this investigation, we here analysed the repertoire of SUMO E3 ligases and their expression profiles during cercariae/schistosomula transition. In silico analysis through S. mansoni databases showed two conserved SUMO E3 ligases: protein inhibitor of activated STAT (PIAS) and Ran-binding protein 2 (RanBP2). Furthermore, expression levels of the SUMO E3 ligases were measured by qRT-PCR using total RNA from cercariae, adult worms and mechanically transformed schistosomula. Our data showed an up-regulation of expression in lung schistosomula and adult worm stages. In conclusion, the differential expression of SmPIAS and SmRanBP2 during schistosomula development was similar to the expression levels of all genes related to SUMO conjugation, thereby suggesting that the control of protein activity, localisation or stability during cercariae to schistosomula transition is SUMO-dependent.