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.     

Changes in glucose metabolism and cyanide sensitivity in Schistosoma mansoni during development

Schistosoma mansoni was studied by biochemical and electrophysiological techniques to follow the physiological changes occurring during transformation in the mammalian host. Volume conducted electrical potentials and measurement of CO₂, evolution indicate that 3 h post-transformational schistosomula are highly sensitive to cyanide. By 24 h after transformation, evolution of CO₂ under control conditions is reducted by 77% from 3 h levels, while lactate excretion rises by 84%. Cyanide does not affect the frequency or magnitude of endogenous electrical transients, but does eliminate 83% of the already reduced levels of CO₂ evolved in 24 schistosomula. Electrophysiological analyses indicate that the timecourse of metabolic changes in skin- and mechanically transformed schistosomula are similar, and incubation of schistosomula in 200 μg ml^?1 puromycin does not alter the onset of cyanide insensitivity. The adult parasite evolves a low level of CO₂ which is reduced by 88% in the presence of 1 mM cyanide. No significant Pasteur effect is detected, however, and endogenous electrical activity as well as mechanical responses of the adult musculature are unaffected by cyanide exposure. Our results indicate that schistosomula continue to rely on cyanide-sensitive respiratory components for at least 3 h after transformation; by 24 h, however, the parasites are metabolically similar to the adult stage, i.e., they depend on lactate fermentation for most of their energy requirements.     

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.     

Cytotoxicity of human and baboon mononuclear phagocytes against schistosomula in vitro: induction by immune complexes containing IgE and Schistosoma mansoni antigens.

Normal human blood monocytes, pre-incubated at 37 degrees C with sera from patients infected with Schistosoma mansoni, strongly adhered to S. mansoni schistosomula in vitro, whereas no significant adherence was induced by sera from uninfected individuals. Comparable adherence occurred with normal baboon blood monocytes or peritoneal macrophages when these cells were incubated with sera from S. mansoni-infected baboons. Adherence of macrophages to schistosomula was associated with damage to the larvae, as estimated by a 51Cr release technique. Neither adherence nor cytotoxicity was induced by pre-incubation of the schistosomula, instead of the monocytes, with immune serum. The relevant factor in immune serum was heat-labile, but was not a complement component. Absorption and ultracentrifugation experiments showed that immune complexes, containing S. mansoni-specific IgE antibody and soluble parasite antigens, produced monocyte or macrophage adherence and cytotoxicity. Similar observations have been reported previously in the rat model. Since the production of large amounts of IgE is a predominant feature of schistosome infections in man and experimental animals, it is possible that this new mode of mononuclear phagocyte activation could act as an immune effector mechanism against S. mansoni.     

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.     

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.     

The exposed carbohydrates of schistosomula of Schistosoma mansoni and their modification during maturation in vivo

Lectins labeled with 125I or conjugated with fluorescein were employed to study the carbohydrates on the surface of different stages of schistosomula of Schistosoma mansoni. Newly transformed schistosomula were shown to bind concanavalin A; the 60 000 and 120 000 dalton agglutinins from Ricinus communis; the fucose-binding protein from Lotus tetragonolobus; wheat germ agglutinin and peanut agglutinin. Soybean agglutinin, Ulex europaeus agglutinin and Dolichos biflorus agglutinin, on the other hand, failed to bind to the schistosomulum surface. The binding of peanut and soybean agglutinin was unaffected by pretreatment of the parasites with neuraminidase. Binding of concanavalin A, the 120 000 dalton agglutinin from Ricinus communis, wheat germ agglutinin and peanut agglutinin to the surface of 5-day schistosomula, recovered from the lungs of mice, was also demonstrated. In each case, however, the level of binding was approximately 70% less than that observed with newly transformed schistosomula and the binding of the fucose-binding protein from L. tetragonolobus practically disappeared. In contrast with newly transformed schistosomula, lung stage schistosomula, pretreated with neuraminidase, displayed a significant increase in the binding of peanut and soybean agglutinin. The results indicate that a significant alteration in the surface carbohydrates of S. mansoni occurs during in vivo maturation of the parasite. This change may contribute to the organism's ability to survive in the vertebrate host.     

Freshly isolated and cultured human monocytes obtained by plasmapheresis kill schistosomula of Schistosoma mansoni.

The efficacy of human peripheral blood monocytes (PBM) in killing of schistosomula is controversial. The purpose of this study was to determine the schistosomulacidal activity of human monocytes isolated by two different techniques. Peripheral blood monocytes were obtained either by venipuncture (PBMv) or plasmapheresis (PBMp), purified on Ficoll-Paque, and cultured briefly. The cells then were incubated with schistosomula (cell parasite ratio of 10(4):1) for 16 to 18 hours with or without interferon-gamma IFN-gamma (600 U/ml) or sera from patients with schistosomiasis as a source of antischistosomal antibodies (HASA). Freshly isolated PBMv treated with IFN-gamma or HASA did not kill schistosomula. Freshly isolated PBMp alone killed 22 +/- 13% (mean +/- standard deviation [SD]; n = 9) of worms over background and after incubation with IFN-gamma and HASA, 30 +/- 17%. PBMp cultured in vitro for 7 days killed 50 +/- 15% (mean +/- SD; n = 12) of the schistosomula. Pretreatment of the cells with IFN-gamma and incubation with HASA did not significantly enhance the parasite killing beyond this level. Electron microscopy showed that freshly isolated PBMp attached to the worms and fused occasionally with the outer tegumental membrane. Granules constituted 1.4% of the cytoplasmic volume. Degranulation onto the parasite surface was not observed. Peripheral blood monocytes obtained by plasmapheresis accumulated glycogen during in vitro culture with the parasite and released threefold more H2O2 than PBMv after exposure to phorbol myristate acetate. Thus plasmapheresis increases the schistosomulacidal activity of PBM, enhances the generation of H2O2 and promotes the accumulation of glycogen.     

Human monocyte-derived macrophages are lysed by schistosomula of Schistosoma mansoni and fail to kill the parasite after activation with interferon gamma.

In this study was examined the interaction between schistosomula of Schistosoma mansoni and human monocyte-derived macrophages activated with interferon gamma (IFN-gamma). Peripheral blood monocytes were matured for 6 days and activated by further culture with IFN-gamma (600 U/ml). These IFN-gamma-treated monocyte-derived macrophages are cytotoxic for the tumor cell line K562, which is not killed by nonactivated monocyte-derived macrophages. Activated monocyte-derived macrophages were incubated with schistosomula at ratios of 10(3):1 and 10(4):1 in the presence of serum pooled from patients with schistosomiasis. This antiserum promoted an increased adherence of cells to the parasite. However, the activated monocyte-derived macrophages failed to kill the schistosomula under all conditions tested. On the contrary, the monocyte-derived macrophages were killed by schistosomula in a time-dependent and antibody-dependent manner, which was most evident at a lower effector/target ratio, 200:1. Electron microscopy showed that monocyte-derived macrophages were lysed on the surface of schistosomula. Further, both monocyte-derived macrophages and contaminating blood platelets fused with the parasite surface membrane, so that the cell plasma membrane and the outer tegumental membrane formed a hybrid membrane. The results indicate that matured human monocyte-derived macrophages activated by IFN-gamma are unable to kill schistosomula. Instead, the effector cells fuse with the parasites and are lysed by them.     

Specific binding of human low-density lipoprotein to the surface of schistosomula of Schistosoma mansoni and ingestion by the parasite.

Low-density lipoproteins (LDL) may be important in human schistosomiasis because LDL bound to the surface of the parasite inhibits the binding of anti-schistosomal antibodies. Low-density lipoproteins also may serve as a source of lipids for the parasite membrane synthesis. Here LDL fluorescently labeled with carbocyanine dye (DiI-LDL) was used to measure the specificity of binding of LDL to the surface of schistosomula of Schistosoma mansoni and to examine the distribution of the LDL particles over time. DiI-LDL binding was saturable and specific, with strong inhibition by unlabeled LDL and apoB but not by apoA1, bovine serum albumin, or IgG, and only weak inhibition by high-density lipoproteins. Half of the bound DiI-LDL was displaced by unlabeled LDL. DiI-LDL remained bound on the surface of schistosomula for up to 36 hours in culture. However parasites also ingested both DiI-LDL and a second fluorescent LDL, Bodipy-LDL. Over time, both fluorophores appeared throughout the worm tissues, suggesting the LDL particles were breaking down and that the fluorophores and lipids originally contained within the LDL particle were partitioning throughout the worm. Thus human LDL appears to bind to the surface of schistosomula specifically. Ingested LDL appears to be broken down and may serve as a source of host lipids for the parasite.     

Partial isolation of a membrane antigen which induces the formation of antibodies lethal to schistosomes cultured in vitro

Fractions of adult schistosomes (S. mansoni) were tested for their ability to absorb an IgG antibody activity which appears in the sera of rats and Rhesus monkeys infected with the parasite and is lethal to schistosomula cultured in vitro. The same fractions were tested for their immunogenicity in raising lethal antibody when administered to rats in Freund's adjuvant. The results of these experiments indicated that an antigen which both absorbs and induces the formation of lethal antibody is present almost entirely in the water-insoluble portion of adult schistosome homogenates. The antigen appears to be tightly bound to this membrane fraction since the use of strong anionic detergents is necessary in its extraction. Membranes solubilized in sodium dodecyl sulphate were fractionated by gel filtration on Sephadex columns equilibrated with the detergent. The same specific Sephadex fraction was shown to absorb both rat and Rhesus monkey lethal antibodies as well as induce the antibody when administered to rats. Further purification and analysis suggested that the immunogen was one of four proteins with molecular weights estimated between 21,000 and 33,000 Daltons.

Rats vaccinated with partially purified antigen developed levels of lethal antibody similar to those found in animals immune to challenge through previous exposure to the parasite, yet were themselves only marginally resistant to infection. These results indicate that while clearly toxic to schistosomula grown in vitro, lethal antibody operating on its own in vivo is incapable of mediating the rejection of schistosome infections.

     

Protection by phospholipids of Schistosoma mansoni schistosomula against the action of cytotoxic antibodies and complement

Schistosoma mansoni schistosomula cultured in the presence of phospholipids showed a decreased sensitivity to the lethal complement-mediated action of anti-schistosome antibodies. Phosphatidyl choline, sphingomyelin and phosphatidyl ethanolamine had a protective action on the schistosomula transformed in vitro by passage through the skin or by a mechanical procedure. Phosphatidyl choline acted regardless of its fatty acid composition. Phosphatidyl serine and phosphatidic acid did not protect. Thus, it appears that phospholipids can play a role in parasite resistance to immune attack by cytotoxic antibodies and complement, and that this role is specific to certain phospholipid types.     

Human lipoprotein binding to schistosomula of schistosoma mansoni. Displacement by polyanions, parasite antigen masking, and persistence in young larvae.

It was previously shown by the authors that the binding of human low-density lipoprotein (LDL) to the surface of schistosomula inhibits the binding of human anti-schistosomal antibodies and is inhibited by suramin. Here, three questions were considered. 1) Are LDLs bound to schistosomula displaced from the membrane by polyanions? 2) Does bound LDL mask or hide antigens recognized by human anti-schistosomal antibodies? 3) Is LDL, binding capability present when the larvae enter the blood stream? The first question was tested by measuring the percentage of the schistosomular surface membrane covered by LDL after exposure to LDL with or without dextran sulfate or suramin. The bound LDL was visualized with polyclonal goat anti-human apolipoprotein B (anti-apo B) antibodies and peroxidase-conjugated secondary antibodies. After overnight culture in 20 micrograms/300 microliters LDL, 84.0% +/- 0.3% of the parasite surface was covered by LDL reaction product. When the polyanions suramin or dextran sulfate were added to the cultures for 30 minutes, only 59.7% +/- 4.9% of the surface was covered by reaction product, demonstrating that the LDL was partially displaced from the membrane by these compounds. The second question was tested by measuring the binding of human and mouse monoclonal anti-schistosomal antibodies before and after exposure to LDL, with or without partial removal of the bound LDL by suramin. LDL partially inhibited antibody binding in a reversible fashion. The LDL clearly masked parasite antigens, most probably by steric hindrance. However, there may be competitive inhibition of antibody binding by the LDL as well, because human anti-schistosomal antibodies inhibited LDL binding to worms and both human anti-schistosomal antibody and LDL binding to schistosomula were inhibited by suramin. Finally, the third question was tested by quantitative immunofluorescence. The LDL binding capability persisted and nearly doubled by 72 hours after transformation from cercariae. These experiments demonstrated that LDL bound to the surface of schistosomula through the time they enter the blood stream. LDL bound to the parasite surface may help the parasite to evade antibody-dependent cytotoxic reactions by masking parasite antigens.     

Acetylcholinesterase of Schistosoma mansoni: antigenic cross-reactivity with Electrophorus electricus and its functional implications

Acetylcholinesterase (AChE) of the parasite Schistosoma mansoni cross-reacts with rabbit antibodies against AChE from the electric eel. Cross-reactivity was demonstrated by radioimmunoassay with sonicate, as well as soluble, preparations from various stages of the parasite life cycle. On sucrose density gradient, the parasite enzyme migrates as an 7.5 S form, whereas an 9.5 S peak is observed with the enzyme-antibody complex. Immunofluorescence microscopy shows specific staining of intact schistosomula as well as of adult worms. Moreover, the interaction of these antibodies resulted in a marked complement-dependent cytotoxicity towards intact schistosomula, indicating that AChE is an antigen of significance on the surface of the parasite.     

Local Antiglycan Antibody Responses to Skin Stage and Migratory Schistosomula of Schistosoma japonicum

Schistosomiasis is a tropical disease affecting over 230 million people worldwide. Although effective drug treatment is available, reinfections are common, and development of immunity is slow. Most antibodies raised during schistosome infection are directed against glycans, some of which are thought to be protective. Developing schistosomula are considered most vulnerable to immune attack, and better understanding of local antibody responses raised against glycans expressed by this life stage might reveal possible glycan vaccine candidates for future vaccine research. We used antibody-secreting cell (ASC) probes to characterize local antiglycan antibody responses against migrating Schistosoma japonicum schistosomula in different tissues of rats. Analysis by shotgun Schistosoma glycan microarray resulted in the identification of antiglycan antibody response patterns that reflected the migratory pathway of schistosomula. Antibodies raised by skin lymph node (LN) ASC probes mainly targeted N-glycans with terminal mannose residues, Galβ1-4GlcNAc (LacNAc) and Galβ1-4(Fucα1-3)GlcNAc (LeX). Also, responses to antigenic and schistosome-specific glycosphingolipid (GSL) glycans containing highly fucosylated GalNAcβ1-4(GlcNAcβ1)_n stretches that are believed to be present at the parasite''s surface constitutively upon transformation were found. Antibody targets recognized by lung LN ASC probes were mainly N-glycans presenting GalNAcβ1-4GlcNAc (LDN) and GlcNAc motifs. Surprisingly, antibodies against highly antigenic multifucosylated motifs of GSL glycans were not observed in lung LN ASC probes, indicating that these antigens are not expressed in lung stage schistosomula or are not appropriately exposed to induce immune responses locally. The local antiglycan responses observed in this study highlight the stage- and tissue-specific expression of antigenic parasite glycans and provide insights into glycan targets possibly involved in resistance to S. japonicum infection.     

A lung model of schistosome immunity in mice.

When mice are challenged intravenously with schistosomula of Schistosoma mansoni, host cell reaction and parasite attrition proceed entirely in the lung, where these events can be followed by quantitative histology and worm recovery. In nonimmune animals the destruction of schistosomula in the lungs proceeds gradually, resulting in the elimination of about 80% of the challenge organisms after 6 days. Cell reaction begins promptly, as evidenced by the appearance of neutrophilic foci around many of the lung schistosomula within 30 minutes after injection, and results in increasing numbers of damaged organisms and residual inflammatory foci 24 hours and 6 days later, respectively. In contrast, when schistosomula are injected into mice immune by virtue of an established S. mansoni infection, parasite destruction is augmented and accelerated, a process already evident by 24 hours. By the sixth day, 98% of the challenge organisms have been eliminated, a substantially greater reduction in parasite survival than that occurring in the normal host. This increased attrition of schistosomula is also reflected in the decreased numbers of parasites recovered from minced lung tissue of immune mice 6 days after challenge. Immune cellular inflammatory reactions to schistosomula are, likewise, greatly intensified and can be readily distinguished from those of normal mice by the proportions of parasites involved and by the large numbers of eosinophils surrounding them. In some instances, degranulation of eosinophils onto the parasite tegument is observed. Schistosomula cultured for 24 or 44 hours in a medium containing mouse red blood cells elicit significantly less cellular reaction and show greater survival in the lungs of immune animals than do freshly derived schistosomula. It would therefore appear that the susceptibility of maturing schistosomes to immune cellular attack is limited to the first day or two after their metamorphosis from cercariae. These observations form the framework of a new in vivo model for analyzing the dynamics of the cellular and humoral processes involved in the immune destruction of a metazoan parasite. The model also lends itself to studies of the immunologic interrelationships between innate and acquired resistance to infection with schistosomes, as well as the mechanisms by which these parasites evade the host immune response.     

Molecular characterization and identification of Th1 epitopes of a Schistosoma japonicum protein similar to prosaposin

The tegument of schistosomula contains T cell antigens that might simulate the protective mechanisms of the radiation-attenuated vaccine in a mouse model of schistosomiasis. Immune mechanisms mediated by the CD4+ Th1 response are important in the RAV model. To rapidly identify Th1 epitopes in molecules from the Schistosoma japonicum schistosomula tegument, this study analyzed S. japonicum proteomics data. Preliminary experiments identified a protein similar to prosaposin (SjPSAP) from the tegument of schistosomula. We confirmed that SjPSAP was present in the tegument of the parasite using an indirect immunofluorescence assay. We then identified Th cell epitopes in SjPSAP using in silico prediction combined with experimental validation. From the SjPSAP sequence, we used several algorithms to predict 11 promiscuous Th cell epitopes that might bind to both murine and human MHC class II molecules. To validate the in silico predictions, proliferation and cytokine production profiles of spleen lymphocytes from BALB/c mice immunized with the 11 predicted peptides were measured in vitro using a modified methyl thiazolyl tetrazolium assay and flow cytometry. The results showed that 4 of the 11 predicted peptides induced a recall CD4+ Th1 response in vitro. We measured direct binding of the four peptides predicted to induce a response to antigen-presenting cells from BALB/c mice using a fluorometric method and found that the peptides bound to both I-Ad and I-Ed mouse molecules. These results demonstrated that potentially protective Th1-type epitopes in SjPSAP molecules could be identified rapidly by combining in silico prediction with experimental validation. This strategy could be a fast method for identifying Th1 epitopes in a schistosoma antigen with features such as large size or poor expression of recombinant antigens.     

Surface antigens on Schistosoma mansoni. II. Adsorption of a Forssman-like host antigen by schistosomula

A study was made of the nature of mouse (host) antigens adsorbed by schistosomula of Schistosoma mansoni. Using the mixed antiglobulin test, extracts of a number of individual mouse tissues were tested for their ability to coat schistosomula. All were effective to some extent, with the greatest activity being found in extracts of the lung and spleen. Antibodies against the schistosomulum-coating antigen as well as surface host antigens of adult Schistosoma mansoni were removed by absorbing with erythrocytes from a number of Forssman-positive but not Forssman-negative animal species. These antibodies were also absorbed by Forssman-positive guineapig kidney extract and methanol soluble (Forssman-positive) but not insoluble fractions of sheep erythrocyte stromata and mouse lungs. Schistosomula could be coated in vitro with methanol soluble fractions of mouse lung and erythrocytes and sheep erythrocytes. Though both mouse and sheep coating antigens reacted with anti-mouse and anti-sheep antibodies, reactions were stronger with the homologous antiserum. It was concluded that schistosomula of Schistosoma mansoni adsorb from mice an antigen similar but not identical to the Forssman antigen of sheep erythrocytes, and that this antigen is also found on the surface of adult worms.     

Evidence that the 32, 38 and 20 kilodalton surface antigens of schistosomula and schistosomes are a family of conserved proteins

The structures of the 38, 32 and 20 kDa surface antigens isolated from schistosomula and adult worms of Schistosoma mansoni were compared by two-dimensional peptide mapping, by immunological analysis and by one- and two-dimensional electrophoresis. Peptide mapping showed a high degree of similarity between the isolated antigens from both parasite stages. The NIMP/M47 monoclonal antibody showed cross-reactivity between the 32 and the 20 kDa antigens under denaturating and non-denaturating conditions, as demonstrated by immunoprecipitation and Western blotting. It is concluded that these antigens constitute a homologous family of surface antigens.