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Chronic infection with Schistosoma mansoni parasites is associated with reduced allergic sensitization in humans, while schistosome eggs protects against allergic airway inflammation (AAI) in mice. One of the main secretory/excretory molecules from schistosome eggs is the glycosylated T2-RNAse Omega-1 (ω1). We hypothesized that ω1 induces protection against AAI during infection. Peritoneal administration of ω1 prior to sensitization with Ovalbumin (OVA) reduced airway eosinophilia and pathology, and OVA-specific Th2 responses upon challenge, independent from changes in regulatory T cells. ω1 was taken up by monocyte-derived dendritic cells, mannose receptor (CD206)-positive conventional type 2 dendritic cells (CD206+ cDC2), and by recruited peritoneal macrophages. Additionally, ω1 impaired CCR7, F-actin, and costimulatory molecule expression on myeloid cells and cDC2 migration in and ex vivo, as evidenced by reduced OVA+ CD206+ cDC2 in the draining mediastinal lymph nodes (medLn) and retainment in the peritoneal cavity, while antigen processing and presentation in cDC2 were not affected by ω1 treatment. Importantly, RNAse mutant ω1 was unable to reduce AAI or affect DC migration, indicating that ω1 effects are dependent on its RNAse activity. Altogether, ω1 hampers migration of OVA+ cDC2 to the draining medLn in mice, elucidating how ω1 prevents allergic airway inflammation in the OVA/alum mouse model., Competing Interests: HS receive research grants from the Lung foundation Netherlands and the Dutch Research Council and is a board member of the Netherlands Respiratory Society., (Copyright: © 2024 Patente et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources., Competing Interests: We declare that none of the authors have competing financial or non-financial interests., (Copyright © 2022 Marije E. Kuipers et al.)

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni's α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp&#95;089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with smp&#95;089290's female biased expression. Spatial localisation of smp&#95;089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp&#95;089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp&#95;089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp&#95;089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds., Competing Interests: The authors have declared that no competing interests exist.

Schistosomiasis remains a worldwide public health problem, especially in sub-Saharan Africa. The World Health Organization targets the goal for its elimination as a public health problem in the 2030 Neglected Tropical Diseases (NTDs) Roadmap. Concerted action and agile responses to challenges will be necessary to achieve the targets. Better diagnostic tests can accelerate progress towards the elimination by monitoring disease trends and evaluating the effectiveness of interventions; however, current examinations such as Kato-Katz technique are of limited power to detect light-intensity infections. The point-of-care circulating cathodic antigen (POC-CCA) test shows a higher sensitivity compared to the reference standard, Kato-Katz technique, but it still lacks sufficient sensitivity with low infection intensity. In this study, we examined antibody reactions against recombinant protein antigens; Schistosoma mansoni serine protease-inhibitor (SmSerpin) and RP26, by enzyme-linked immunosorbent assay (ELISA) in plasma samples with light-intensity infection. The sensitivity using the cocktail antigen of recombinant SmSerpin and RP26 showed 83.7%. The sensitivity using S. mansoni soluble egg antigen (SmSEA) was 90.8%, but it showed poor specificity (29.7%), while the cocktail antigen presented improved specificity (61.4%). We conclude that antibody detection to the SmSerpin and RP26 protein antigens is effective to detect S. mansoni light-intensity infections. Our study indicates the potential of detecting antibody against recombinant protein antigens to monitor the transmission of schistosomiasis in low endemicity contexts., (Copyright © 2021. Published by Elsevier B.V.)

Helminth parasites secrete a wide variety of immunomodulatory proteins and lipids to dampen host immune responses. Many of these immunomodulatory compounds are modified with complex sugar structures (or glycans), which play an important role at the host-parasite interface. As an example, the human blood fluke Schistosoma mansoni produces highly fucosylated glycan structures on glycoproteins and glycolipids. Up to 20 different S. mansoni fucosyltransferase (SmFucT) genes can be found in genome databases, but thus far only one enzyme has been functionally characterized. To unravel the synthesis of highly fucosylated N-glycans by S. mansoni, we examined the ability of ten selected SmFucTs to modify N-glycans upon transient expression in Nicotiana benthamiana plants. All enzymes were localized in the plant Golgi apparatus, which allowed us to identify the SmFucTs involved in core fucosylation and the synthesis of complex antennary glycan motifs. This knowledge provides a starting point for investigations into the role of specific fucosylated glycan motifs of schistosomes in parasite-host interactions. The functionally characterized SmFucTs can also be applied to synthesize complex N-glycan structures on recombinant proteins to study their contribution to immunomodulation. Furthermore, this plant expression system will fuel the development of helminth glycoproteins for pharmaceutical applications or novel anti-helminth vaccines.

Schistosomiasis treatment relies on the use of a single drug, praziquantel, which is insufficient to control transmission in highly endemic areas 1 . Novel medicines and vaccines are urgently needed 2,3 . An experimental human model for schistosomiasis could accelerate the development of these products. We performed a dose-escalating clinical safety trial in 17 volunteers with male Schistosoma mansoni cercariae, which do not produce eggs (clinicaltrials.gov NCT02755324), at the Leiden University Medical Center, the Netherlands. The primary endpoints were adverse events and infectivity. We found a dose-related increase in adverse events related to acute schistosomiasis syndrome, which occurred in 9 of 17 volunteers. Overall, 5 volunteers (all 3 of the high dose group and 2 of 11 of the medium dose group) reported severe adverse events. Worm-derived circulating anodic antigen, the biomarker of the primary infection endpoint, peaked in 82% of volunteers at 3-10 weeks following exposure. All volunteers showed IgM and IgG1 seroconversion and worm-specific cytokine production by CD4 + T cells. All volunteers were cured with praziquantel provided at 12 weeks after exposure. Infection with 20 Schistosoma mansoni cercariae led to severe adverse events in 18% of volunteers and high infection rates. This infection model paves the way for fast-track product development for treatment and prevention of schistosomiasis.

Schistosoma mansoni venom allergen-like proteins (SmVALs) are part of a diverse protein superfamily partitioned into two groups (group 1 and group 2). Phylogenetic analyses of group 1 SmVALs revealed that members could be segregated into subclades (A-D); these subclades share similar gene expression patterns across the parasite lifecycle and immunological cross-reactivity. Furthermore, whole-mount in situ hybridization demonstrated that the phylogenetically, transcriptionally and immunologically-related SmVAL4, 10, 18 and 19 (subclade C) were all localized to the pre-acetabular glands of immature cercariae. Our results suggest that SmVAL group 1 phylogenetic relationships, stage-specific transcriptional profiles and tissue localization are predictive of immunological cross-reactivity., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Baboons vaccinated with radiation-attenuated cercariae develop high levels of protection against schistosome infection, correlating to high antibody titres towards schistosome antigens with unknown molecular identity. Using a microarray consisting of glycans isolated from different life-stages of schistosomes, we studied the anti-glycan immunoglobulin (Ig) G and IgM responses in vaccinated and challenged baboons over a time course of 25 weeks. Anti-glycan IgM responses developed early after vaccination, but did not rise in response to later vaccinations. In contrast, anti-glycan IgG developed more slowly, but was boosted by all five subsequent vaccinations. High IgM and IgG levels against O-glycans and glycosphingolipid glycans of cercariae were observed. At the time of challenge, while most antibody levels decreased in the absence of vaccination, IgG towards a subset of glycans containing multiple-fucosylated motifs remained high until 6 weeks post-challenge during challenge parasite elimination, suggesting a possible role of this IgG in protection., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Core β-1,2-xylose and α-1,3-fucose are antigenic motifs on schistosome N-glycans, as well as prominent IgE targets on some plant and insect glycoproteins. To map the association of schistosome infection with responses to these motifs, we assessed plasma IgE and IgG reactivity using microarray technology among Ugandans from rural Schistosoma mansoni (Sm)-endemic islands (n = 209), and from proximate urban communities with lower Sm exposure (n = 62). IgE and IgG responses to core β-1,2-xylose and α-1,3-fucose modified N-glycans were higher in rural versus urban participants. Among rural participants, IgE and IgG to core β-1,2-xylose were positively associated with Sm infection and concentration peaks coincided with the infection intensity peak in early adolescence. Responses to core α-1,3-fucose were elevated regardless of Sm infection status and peaked before the infection peak. Among urban participants, Sm infection intensity was predominantly light and positively associated with responses to both motifs. Principal component and hierarchical cluster analysis reduced the data to a set of variables that captured core β-1,2-xylose- and α-1,3-fucose-specific responses, and confirmed associations with Sm and the rural environment. Responses to core β-1,2-xylose and α-1,3-fucose have distinctive relationships with Sm infection and intensity that should further be explored for associations with protective immunity, and cross-reactivity with other exposures.

CRISPR/Cas9-based genome editing has yet to be reported in species of the Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as proof of principle. This secreted ribonuclease is crucial for Th2 polarization and granuloma formation. Schistosome eggs were exposed to Cas9 complexed with guide RNA complementary to ω1 by electroporation or by transduction with lentiviral particles. Some eggs were also transfected with a single stranded donor template. Sequences of amplicons from gene-edited parasites exhibited Cas9-catalyzed mutations including homology directed repaired alleles, and other analyses revealed depletion of ω1 transcripts and the ribonuclease. Gene-edited eggs failed to polarize Th2 cytokine responses in macrophage/T-cell co-cultures, while the volume of pulmonary granulomas surrounding ω1-mutated eggs following tail-vein injection into mice was vastly reduced. Knock-out of ω1 and the diminished levels of these cytokines following exposure showcase the novel application of programmed gene editing for functional genomics in schistosomes., Competing Interests: WI, VM, SK, AC, GR, GS, AC, TT, CK, PP, MM, CC, PD, NH, AT, RR, BE, CH, KH, MB, PB No competing interests declared

Schistosoma mansoni is a parasitic helminth that infects millions of people mostly in tropical parts of the world. Different life cycle stages of S.mansoni, that infect or develop in the human host, promote distinct immune responses and are known for their ability to modulate host immune responses. However, the molecular mechanisms through which the parasites interact with, and modulate the host immune system remain incompletely understood. Despite the well-known ability of various lipids to modulate immune responses, a comprehensive analysis of the lipidome of the different life cycle stages has not been performed. Using three complementary MS-based platforms to detect and quantify around 350 lipid species, we here characterized the lipid profiles of S. mansoni cercariae, worms and eggs, as well as extracts and excretory/secretory (ES) products of different life cycle stages of S. mansoni. We identified life cycle stage specific signatures of lipid classes of which cercariae were found to have the most distinct profile. Moreover, we detected several immunolomodulatory oxylipids in the different life cycle stages. Specifically, prostaglandins were found to be most highly enriched in egg preparations, while resolvins were specifically detected in cercariae. Together, the generation of this detailed lipid database of the different life cycle stages of S. mansoni will not only be important for a better understanding of the biology of the parasite itself but also of host-parasite interactions and how that could result in immunomodulation., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

While antigens from Schistosoma schistosomula have been suggested as potential vaccine candidates, the association between antibody responses with schistosomula antigens and infection intensity at reinfection is not well known. Schistosoma mansoni-infected individuals were recruited from a schistosomiasis endemic area in Uganda (n = 372), treated with 40 mg/kg praziquantel (PZQ) and followed up at five weeks and at one year post-treatment. Pre-treatment and five weeks post-treatment immunoglobulin (Ig) E, IgG1 and IgG4 levels against recombinant schistosomula antigens rSmKK7, rSmLy6A, rSmLy6B and rSmTSP7 were measured using ELISA. Factors associated with detectable pre-treatment or post-treatment antibody response against the schistosomula antigens and the association between five-week antibody responses and one year post-treatment reinfection intensity among antibody responders were examined. Being male was associated with higher pre-treatment IgG1 to rSmKK7, rSmLy6a and AWA. Five weeks post-treatment antibody responses against schistosomula antigens were not associated with one year post-treatment reinfection intensity among antibody responders' antibody levels against rSmKK7, rSmLy6B and rSmTSP7 dropped, but increased against rSmLy6A, AWA and SEA at five weeks post-treatment among antibody responders. S. mansoni-infected individuals exhibit detectable antibody responses to schistosomula antigens that are affected by treatment. These findings indicate that schistosomula antigens induce highly varied antibody responses and could have implications for vaccine development., (© 2018 The Authors. Parasite Immunology Published by John Wiley & Sons Ltd.)

Larvae of Schistosoma (schistosomula) are highly susceptible to host immune responses and are attractive prophylactic vaccine targets, although cellular immune responses against schistosomula antigens in endemic human populations are not well characterized. We collected blood and stool from 54 Schistosoma mansoni-infected Ugandans, isolated peripheral blood mononuclear cells and stimulated them for 24 hours with schistosome adult worm and soluble egg antigens (AWA and SEA), along with schistosomula recombinant proteins rSmKK7, Lymphocyte Antigen 6 isoforms (rSmLy6A and rSmLy6B), tetraspanin isoforms (rSmTSP6 and rSmTSP7). Cytokines, chemokines and growth factors were measured in the culture supernatants using a multiplex luminex assay, and infection intensity was determined before and at 1 year after praziquantel (PZQ) treatment using the Kato-Katz method. Cellular responses were grouped and the relationship between groups of correlated cellular responses and infection intensity before and after PZQ treatment was investigated. AWA and SEA induced mainly Th2 responses. In contrast, rSmLy6B, rSmTSP6 and rSmTSP7 induced Th1/pro-inflammatory responses. While recombinant antigens rSmKK7 and rSmLy6A did not induce a Th1/pro-inflammatory response, they had an association with pre-treatment infection intensity after adjusting for age and sex. Testing more schistosomula antigens using this approach could provide immune-epidemiology identifiers necessary for prioritizing next generation schistosomiasis vaccine candidates., (© 2018 The Authors. Parasite Immunology Published by John Wiley & Sons Ltd.)

Following initial invasion of Schistosoma mansoni cercariae, schistosomula reside in the skin for several days during which they can interact with the dermal immune system. While murine experiments have indicated that exposure to radiation-attenuated (RA) cercariae can generate protective immunity which is initiated in the skin stage, contrasting non-attenuated cercariae, such data is missing for the human model. Since murine skin does not form a reliable marker for immune responses in human skin, we used human skin explants to study the interaction with non-attenuated and RA cercariae with dermal innate antigen presenting cells (APCs) and the subsequent immunological responses. We exposed human skin explants to cercariae and visualized their invasion in real time (initial 30 min) using novel imaging technologies. Subsequently, we studied dermal immune responses and found an enhanced production of regulatory cytokine interleukin (IL)-10, pro-inflammatory cytokine IL-6 and macrophage inflammatory protein (MIP)-1α within 3 days of exposure. Analysis of dermal dendritic cells (DDCs) for their phenotype revealed an increased expression of immune modulators programmed death ligand (PD-L) 1 and 2, and increased IL-10 production. Ex vivo primed DDCs suppress Th1 polarization of naïve T-cells and increase T-cell IL-10 production, indicating their regulatory potential. These immune responses were absent or decreased after exposure to RA parasites. Using transwells, we show that direct contact between APCs and cercariae is required to induce their regulatory phenotype. To the best of our knowledge this is the first study that attempts to provide insight in the human dermal S. mansoni cercariae invasion and subsequent immune responses comparing non-attenuated with RA parasites. We reveal that cercariae induce a predominantly regulatory immune response whereas RA cercariae fail to achieve this. This initial understanding of the dermal immune suppressive capacity of S. mansoni cercariae in humans provides a first step toward the development of an effective schistosome vaccine.

To accelerate the development of novel vaccines for schistosomiasis, we set out to develop a human model for Schistosoma mansoni infection in healthy volunteers. During natural infections, female schistosomes produce eggs that give rise to morbidity. Therefore, we produced single-sex, male Schistosoma mansoni cercariae for human infection without egg production and associated pathology. Cercariae were produced in their intermediate snail hosts in accordance with the principles of good manufacturing practice (GMP). The application of GMP principles to an unconventional production process is a showcase for the controlled production of complex live challenge material in the European Union or under Food and Drug Administration guidance.

Infection with the helminth Schistosoma (S.) mansoni drives the development of interleukin (IL)-10-producing regulatory B (Breg) cells in mice and man, which have the capacity to reduce experimental allergic airway inflammation and are thus of high therapeutic interest. However, both the involved antigen and cellular mechanisms that drive Breg cell development remain to be elucidated. Therefore, we investigated whether S. mansoni soluble egg antigens (SEA) directly interact with B cells to enhance their regulatory potential, or act indirectly on B cells via SEA-modulated macrophage subsets. Intraperitoneal injections of S. mansoni eggs or SEA significantly upregulated IL-10 and CD86 expression by marginal zone B cells. Both B cells as well as macrophages of the splenic marginal zone efficiently bound SEA in vivo, but macrophages were dispensable for Breg cell induction as shown by macrophage depletion with clodronate liposomes. SEA was internalized into acidic cell compartments of B cells and induced a 3-fold increase of IL-10, which was dependent on endosomal acidification and was further enhanced by CD40 ligation. IPSE/alpha-1, one of the major antigens in SEA, was also capable of inducing IL-10 in naïve B cells, which was reproduced by tobacco plant-derived recombinant IPSE. Other major schistosomal antigens, omega-1 and kappa-5, had no effect. SEA depleted of IPSE/alpha-1 was still able to induce Breg cells indicating that SEA contains more Breg cell-inducing components. Importantly, SEA- and IPSE-induced Breg cells triggered regulatory T cell development in vitro. SEA and recombinant IPSE/alpha-1 also induced IL-10 production in human CD1d+ B cells. In conclusion, the mechanism of S. mansoni-induced Breg cell development involves a direct targeting of B cells by SEA components such as the secretory glycoprotein IPSE/alpha-1.

Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein's Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.

During the complex lifecycle of Schistosoma mansoni, a large variety of glycans is expressed. To many of these glycans, antibodies are induced by the infected host and some might be targets for vaccines or diagnostic tests. Spatial changes in glycan expression during schistosome development are largely unexplored. To study the surface-exposed glycans during the important initial stages of infection, we analyzed the binding of a panel of anti-glycan monoclonal antibodies (mAbs) to cercariae and schistosomula up to 72 h after transformation by immunofluorescence microscopy. The mAb specificity toward their natural targets was studied using a microarray containing a wide range of schistosomal N-glycans, O-glycans and glycosphingolipid glycans. With the exception of GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F), mono- and multifucosylated GalNAcβ1-4GlcNAc (LDN)-motifs were exposed at the surface of all developmental stages studied. Multifucosylated LDN-motifs were present on cercarial glycocalyx-derived O-glycans as well as cercarial glycolipids. In contrast, the Galβ1-4(Fucα1-3)GlcNAc (Lewis X) and LDN-F-motifs, also expressed on cercarial glycolipids, and in addition on a range of cercarial N- and O-glycans, became surface expressed only after transformation of cercariae to schistosomula. In line with the documented shedding of the O-glycan-rich cercarial glycocalyx after transformation these observations suggest that surface accessible multifucosylated LDN-motifs are mostly expressed by O-glycans in cercariae, but principally by glycosphingolipids in schistosomula. We hypothesize that these temporal changes in surface exposure of glycan antigens are relevant to the interaction with the host during the initial stages of infection with schistosomes and discuss the potential of these glycan antigens as intervention targets., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoni induce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles of glycans and antigenic glycan-motifs during a range of critical stages of the complex schistosome lifecycle. We performed a longitudinal profiling study covering schistosome glycosylation throughout worm- and egg-development using a mass spectrometry-based glycomics approach. Our study revealed that during worm development N-glycans with Galβ1-4(Fucα1-3)GlcNAc (LeX) and core-xylose motifs were rapidly lost after cercariae to schistosomula transformation, whereas GalNAcβ1-4GlcNAc (LDN)-motifs gradually became abundant and predominated in adult worms. LeX-motifs were present on glycolipids up to 2 weeks of schistosomula development, whereas glycolipids with mono- and multifucosylated LDN-motifs remained present up to the adult worm stage. In contrast, expression of complex O-glycans diminished to undetectable levels within days after transformation. During egg development, a rich diversity of N-glycans with fucosylated motifs was expressed, but with α3-core fucose and a high degree of multifucosylated antennae only in mature eggs and miracidia. N-glycan antennae were exclusively LDN-based in miracidia. O-glycans in the mature eggs were also diverse and contained LeX- and multifucosylated LDN, but none of these were associated with miracidia in which we detected only the Galβ1-3(Galβ1-6)GalNAc core glycan. Immature eggs also exhibited short O-glycan core structures only, suggesting that complex fucosylated O-glycans of schistosome eggs are derived primarily from glycoproteins produced by the subshell envelope in the developed egg. Lipid glycans with multifucosylated GlcNAc repeats were present throughout egg development, but with the longer highly fucosylated stretches enriched in mature eggs and miracidia. This global analysis of the developing schistosome's glycome provides new insights into how stage-specifically expressed glycans may contribute to different aspects of schistosome-host interactions., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Upon infection with Schistosoma, antibody responses are mounted that are largely directed against glycans. Over the last few years significant progress has been made in characterising the antigenic properties of N-glycans of Schistosoma mansoni. Despite also being abundantly expressed by schistosomes, much less is understood about O-glycans and antibody responses to these have not yet been systematically analysed. Antibody binding to schistosome glycans can be analysed efficiently and quantitatively using glycan microarrays, but O-glycan array construction and exploration is lagging behind because no universal O-glycanase is available, and release of O-glycans has been dependent on chemical methods. Recently, a modified hydrazinolysis method has been developed that allows the release of O-glycans with free reducing termini and limited degradation, and we applied this method to obtain O-glycans from different S. mansoni life stages. Two-dimensional HPLC separation of 2-aminobenzoic acid-labelled O-glycans generated 362 O-glycan-containing fractions that were printed on an epoxide-modified glass slide, thereby generating the first shotgun O-glycan microarray containing naturally occurring schistosome O-glycans. Monoclonal antibodies and mass spectrometry showed that the O-glycan microarray contains well-known antigenic glycan motifs as well as numerous other, potentially novel, antibody targets. Incubations of the microarrays with sera from Schistosoma-infected humans showed substantial antibody responses to O-glycans in addition to those observed to the previously investigated N- and glycosphingolipid glycans. This underlines the importance of the inclusion of these often schistosome-specific O-glycans in glycan antigen studies and indicates that O-glycans contain novel antigenic motifs that have potential for use in diagnostic methods and studies aiming at the discovery of vaccine targets., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Schistosomiasis is a serious health problem especially in developing countries and affects more than 243 million people. Only few anthelmintic drugs are available up to now. A major obstacle for drug treatment is the different developmental stages and the varying host compartments during worm development. Anthelmintic drugs have been tested mainly on adult schistosomes or freshly transformed cercariae. Knowledge concerning the larval stages is lacking., Methodology/principal Findings: In this study, we used in vitro-grown schistosomula (aged between 2 to 14 days) to investigate drug effects of the three anthelmintics praziquantel, artemether, and oxamniquine. Further, we analyzed the antibody accessibility of two exemplary schistosome antigens SmCD59a and SmKK7, before and after drug treatment. Our results demonstrated that praziquantel applied at a concentration of 1 μM inhibited development of all life stages. Application of 10 μM praziquantel led to dramatic morphological changes of all schistosomula. Artemether at 1 and 10 μM had differential effects depending on whether it was applied to 2-day as compared to 7- and 14-day schistosomula. While 2-day schistosomula were not killed but inhibited from further development, severe morphological damage was seen in 7- and 14-day schistosomula. Oxamniquine (1 and 10 μM) led to severe morphological impairment in all life stages. Analyzing the accessibility of the antigens SmCD59a and SmKK7 before drug treatment showed no antibody binding on living intact schistosomula. However, when schistosomula were treated with anthelmintics, both antigens became exposed on the larvae. Oxamniquine turned out to be most effective in promoting antibody binding to all schistosomula stages., Conclusion: This study has revealed marked differences in anthelmintic drug effects against larvae. Drug treatment increases surface antigen presentation and renders larvae accessible to antibody attack.

The Schistosoma mansoni venom allergen-like (SmVAL) protein family consists of 29 members, each possessing a conserved α-β-α sandwich tertiary feature called the Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain. While the SmVALs have been found in both excretory/secretory (E/S) products and in intra/sub-tegumental (non-E/S) fractions, the role(s) of this family in host/parasite relationships or schistosome developmental processes remains poorly resolved. In order to begin quantifying SmVAL functional diversity or redundancy, dissecting the specific activity (ies) of individual family members is necessary. Towards this end, we present the characterisation of SmVAL9; a protein previously found enriched in both miracidia/sporocyst larval transformation proteins and in egg secretions. While our study confirms that SmVAL9 is indeed found in soluble egg products and miracidia/sporocyst larval transformation proteins, we find it to be maximally transcribed/translated in miracidia and subsequently down-regulated during in vitro sporocyst development. SmVAL9 localisation within sporocysts appears concentrated in parenchymal cells/vesicles as well as associated with larval germinal cells. Furthermore, we demonstrate that egg-derived SmVAL9 carries an N-linked glycan containing a schistosome-specific difucosyl element and is an immunogenic target during chronic murine schistosomiasis. Finally, we demonstrate that recombinant SmVAL9 affects the expression of extracellular matrix, remodelling matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) gene products in both Biomphalaria glabrata embryonic cell (BgMMP1) and Mus musculus bone marrow-derived macrophage (MmMMP2, MmMMP9, MmMMP12, MmMMP13, MmMMP14, MmMMP28, TIMP1 and TIMP2) in vitro cultures. These findings importantly suggest that excreted/secreted SmVAL9 participates in tissue reorganisation/extracellular matrix remodelling during intra-mammalian egg translocation, miracidia infection and intra-molluscan sporocyst development/migration., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Schistosoma mansoni eggs have to cross the endothelium and intestinal wall to leave the host and continue the life cycle. Mechanisms involved in this essential step are largely unknown. Here we describe direct binding to the S. mansoni eggshell of von Willebrand factor and other plasma proteins involved in haemostasis. Using deletion-mutants, we demonstrated that it is the A1 domain of von Willebrand factor that binds to the eggshell. Our results suggest that binding of plasma proteins to the eggshell promotes binding to the endothelium, initiating the passage of the egg through the blood-vessel wall to be excreted in the end., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Host lectin-like recognition molecules may play an important role in innate resistance in Biomphalaria glabrata snails to larval schistosome infection, thus implicating parasite-expressed glycans as putative ligands for these lectin receptors. While host lectins may utilize specific glycan structures for parasite recognition, it also has been hypothesized that the parasite may use this system to evade immune detection by mimicking naturally-expressed host glycans, resulting in reduced immunorecognition capacity. By employing immunocytochemical (ICC) and Western blot assays using schistosome glycan-specific monoclonal antibodies (mABs) we sought to identify specific glycan epitopes (glycotopes) shared in common between larval Schistosoma mansoni and B. glabrata hemocytes, the primary immune effector cells in snails. Results confirmed the presence of selected larval glycotopes on subpopulations of hemocytes by ICC and association with numerous hemocyte proteins by Western blot analyses, including a trimannosyl core N-glycan (TriMan), and two fucosylated lacdiNAc (LDN) variants, F-LDN and F-LDN-F. Snail strain differences were seen in the prevalence of constitutively expressed F-LDN on hemocytes, and in the patterns of protein immunoreactivity with these mABs. In contrast, there was little to no hemocyte reactivity with mABs for Lewis X (LeX), LDN, LDN-F or LDN-DF. When intact hemocytes were exposed to larval transformation products (LTPs), distinct cell subpopulations displayed weak (LeX, LDN-DF) to moderate (LDN, LDN-F) glycotope reactivity by ICC, including snail strain differences in the prevalence of LDN-reactive cellular subsets. Far-Western blot analyses of the hemocytes following exposure to larval transformation proteins (LTPs) also revealed multiple mAB-reactive hemocyte protein bands for LeX, LDN, LDN-F, and LDN-DF. These results demonstrate the existence of complex patterns of shared larval glycan constitutively expressed on hemocytes and their proteins, as well as the ability of hemocytes to acquire shared glycans by the selective binding of parasite-released LTP. Unraveling the functional significance of these naturally expressed and acquired shared glycans on specific hemocyte populations represents an important challenge for future investigations., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Omega-1, a glycosylated T2 ribonuclease (RNase) secreted by Schistosoma mansoni eggs and abundantly present in soluble egg antigen, has recently been shown to condition dendritic cells (DCs) to prime Th2 responses. However, the molecular mechanisms underlying this effect remain unknown. We show in this study by site-directed mutagenesis of omega-1 that both the glycosylation and the RNase activity are essential to condition DCs for Th2 polarization. Mechanistically, we demonstrate that omega-1 is bound and internalized via its glycans by the mannose receptor (MR) and subsequently impairs protein synthesis by degrading both ribosomal and messenger RNA. These experiments reveal an unrecognized pathway involving MR and interference with protein synthesis that conditions DCs for Th2 priming.

Schistosome eggs and egg-derived molecules are potent immunomodulatory agents. There is increasing evidence that the interplay between egg glycoproteins and host C-type lectins plays an important role in shaping immune responses during schistosomiasis. As most experiments in this field so far have been performed using complex protein/glycoprotein mixtures or synthetic model glycoconjugates, it is still largely unclear which individual moieties of schistosome eggs are immunologically active. In this review we will discuss molecular aspects of Schistosoma mansoni egg glycoproteins, their interactions with C-type lectins, and the relevance to schistosome egg immunobiology., (Copyright © 2011 Elsevier Inc. All rights reserved.)

During infection with the blood fluke Schistosoma mansoni, glycan motifs present on glycoproteins of the parasite’s eggs mediate immunomodulatory effects on the host. The recognition of these glycan motifs is primarily mediated by C-type lectin receptors on dendritic cells and other cells of the immune system. However, it is not yet known which individual glycoproteins interact with the different C-type lectin receptors, and which structural components are involved. Here we investigated the structural basis of the binding of two abundant egg antigens, kappa-5 and IPSE/a1, by the C-type lectin receptor dendritic cell-specific ICAM3-grabbing non-integrin, macrophage galactose-type lectin and mannose receptor. In the natural soluble form, the secretory egg glycoprotein IPSE/a1 interacts with dendritic cells mainly via mannose receptors. Surprisingly, in plate-based assays mannose receptors preferentially bound to mannose conjugates, while in cell-based assays, IPSE/a1 is bound via the fucosylated Galb1-4(Fuca1-3)GlcNAc (LeX) motif on diantennary N-glycans. Kappa-5, in contrast, is bound by dendritic cells viaall three C-type lectin receptors studied and for a minor part also via other, non-C-type lectin receptors.Kappa-5 interacts with macrophage galactose-type lectins via the GalNAcb1-4GlcNAc antenna present on its triantennary N-glycans, as well as the GalNAcb1-4(Fuca1-3) GlcNAc antennae present on a minor N-glycan subset. Dendritic cell-specific ICAM3-grabbing non-integrin binding of kappa-5 was mediated via the GalNAcb1-4(Fuca1-3)GlcNAc antennae, whereas binding of mannose receptors may involve either GalNAcb1-4(Fuca1-3)GlcNAc antennae or the fucosylated and xylosylated chitobiose core. This study provides a molecular and structural basis for future studies of the interaction between C-type lectin receptors and other soluble egg antigen glycoproteins and their effects on the host immune response., (2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Schistosomiasis (bilharzia) is a chronic and potentially deadly parasitic disease that affects millions of people in (sub)tropical areas. An important partial immunity to Schistosoma infections does develop in disease endemic areas, but this takes many years of exposure and maturation of the immune system. Therefore, children are far more susceptible to re-infection after treatment than older children and adults. This age-dependent immunity or susceptibility to re-infection has been shown to be associated with specific antibody and T cell responses. Many antibodies generated during Schistosoma infection are directed against the numerous glycans expressed by Schistosoma. The nature of glycan epitopes recognized by antibodies in natural schistosomiasis infection serum is largely unknown., Methodology/principal Findings: The binding of serum antibodies to glycans can be analyzed efficiently and quantitatively using glycan microarray approaches. Very small amounts of a large number of glycans are presented on a solid surface allowing binding properties of various glycan binding proteins to be tested. We have generated a so-called shotgun glycan microarray containing natural N-glycan and lipid-glycan fractions derived from 4 different life stages of S. mansoni and applied this array to the analysis of IgG and IgM antibodies in sera from children and adults living in an endemic area. This resulted in the identification of differential glycan recognition profiles characteristic for the two different age groups, possibly reflecting differences in age or differences in length of exposure or infection., Conclusions/significance: Using the shotgun glycan microarray approach to study antibody response profiles against schistosome-derived glycan elements, we have defined groups of infected individuals as well as glycan element clusters to which antibody responses are directed in S. mansoni infections. These findings are significant for further exploration of Schistosoma glycan antigens in relation to immunity.

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.

Glycans present on glycoproteins from the eggs of the parasite Schistosoma mansoni are mediators of various immune responses of the human host, including T-cell modulation and granuloma formation, and they are the target of glycan-specific antibodies. Here we have analyzed the glycosylation of kappa-5, a major glycoprotein antigen from S. mansoni eggs using a targeted approach of lectin purification followed by mass spectrometry of glycopeptides as well as released glycans. We demonstrate that kappa-5 has four fully occupied N-glycosylation sites carrying unique triantennary glycans composed of a difucosylated and xylosylated core region, and immunogenic GalNAcβ1-4GlcNAc (LDN) termini. Furthermore, we show that the kappa-5 specific IgE antibodies in sera of S. mansoni-infected individuals are directed against the core region of the kappa-5 glycans. Whereas two previously analyzed immunomodulatory egg glycoproteins, IPSE/alpha-1 and omega-1, both express diantennary N-glycans with a difucosylated core and one or two Galβ1-4(Fucα1-3)GlcNAc (Lewis X) antennae, the kappa-5 glycosylation appears unique among the major soluble egg antigens of S. mansoni. The distinct structural and antigenic properties of kappa-5 glycans suggest a specific role for kappa-5 in schistosome egg immunogenicity.

In schistosomiasis, the majority of symptoms of the disease is caused by the eggs that are trapped in the liver. These eggs elicit an immune reaction that leads to the formation of granulomas. The eggshell, which is a rigid insoluble structure built from cross-linked proteins, is the site of direct interaction between the egg and the immune system. However, the exact protein composition of the insoluble eggshell was previously unknown. To identify the proteins of the eggshell of Schistosoma mansoni we performed LC-MS/MS analysis, immunostaining and amino acid analysis on eggshell fragments. For this, eggshell protein skeleton was prepared by thoroughly cleaning eggshells in a four-step stripping procedure of increasing strength including urea and SDS to remove all material that is not covalently linked to the eggshell itself, but is part of the inside of the egg, such as Reynold's layer, von Lichtenberg's envelope and the miracidium. We identified 45 proteins of which the majority are non-structural proteins and non-specific for eggs, but are house-keeping proteins that are present in large quantities in worms and miracidia. Some of these proteins are known to be immunogenic, such as HSP70, GST and enolase. In addition, a number of schistosome-specific proteins with unknown function and no homology to any known annotated protein were found to be incorporated in the eggshell. Schistosome-specific glycoconjugates were also shown to be present on the eggshell protein skeleton. This study also confirmed that the putative eggshell protein p14 contributes largely to the eggshell. Together, these results give new insights into eggshell composition as well as eggshell formation. Those proteins that are present at the site and time of eggshell formation are incorporated in the cross-linked eggshell and this cross-linking does no longer occur when the miracidium starts secreting proteins., (Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Soluble egg antigens (SEA) of the human parasite Schistosoma mansoni are among the strongest natural stimuli of Th2 responses. Omega-1, a major glycoprotein in SEA, initiates these characteristic Th2 responses through conditioning of dendritic cells (DCs). In view of the reported immunomodulatory potential of SEA glycans, we have investigated omega-1 glycosylation, using an approach combining mass spectrometric techniques and enzyme treatments at the glycopeptide level. We demonstrate that omega-1 has two fully occupied N-glycosylation sites, each mainly carrying core-difucosylated diantennary glycans with one or more Lewis X motifs in the antennae. Using a specific approach of nanoscale LC-MS(/MS) and MALDI-TOF(/TOF) MS in combination with exoglycosidase treatments of tryptic glycopeptides, we were able to provide a detailed, site-specific glycosylation analysis of a single, native S. mansoni glycoprotein. The obtained knowledge of the glycans present on omega-1 contributes to a full understanding of the mode of action of this immunomodulatory glycoprotein.

Fucosylated carbohydrate epitopes (glycotopes) expressed by larval and adult schistosomes are thought to modulate the host immune response and possibly mediate parasite evasion in intermediate and definitive hosts. While previous studies showed glycotope expression is developmentally and stage-specifically regulated, relatively little is known regarding their occurrence in miracidia and primary sporocysts. In this study, previously defined monoclonal antibodies were used in confocal laser scanning microscopy, standard epifluorescence microscopy and Western blot analyses to investigate the developmental expression of the following glycotopes in miracidia and primary sporocysts of Schistosoma mansoni: GalNAcbeta1-4GlcNAc (LDN), GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDN-F), Fucalpha1-3GalNAcbeta1-4GlcNAc (F-LDN), Fucalpha1-3GalNAcbeta1-4(Fucalpha1-3)GlcNAc (F-LDN-F), GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF), Fucalpha1-2Fucalpha1-3GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (DF-LDN-DF), Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis X) and the truncated trimannosyl N-glycan Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAcbeta1-Asn (TriMan). All but Lewis X were variously expressed by miracidia and sporocysts of S. mansoni. Most notably, alpha3-fucosylated LDN (F-LDN, F-LDN-F, LDN-F) was prominently expressed on the larval surface and amongst glycoproteins released during larval transformation and early sporocyst development, possibly implying a role for these glycotopes in snail-schistosome interactions. Interestingly, Fucalpha2Fucalpha3-subsituted LDN (LDN-DF, DF-LDN-DF) and LDN-F were heterogeneously surface-expressed on individuals of a given larval population, particularly amongst miracidia. In contrast, LDN and TriMan primarily localised in internal somatic tissues and exhibited only minor surface expression. Immunoblots indicate that glycotopes occur on overlapping but distinct protein sets in both larval stages, further demonstrating the underlying complexity of schistosome glycosylation. Additionally, sharing of specific larval glycotopes with Biomphalaria glabrata suggests an evolutionary convergence of carbohydrate expression between schistosomes and their snail host.

Soluble egg antigens of the parasitic helminth Schistosoma mansoni (S. mansoni egg antigen [SEA]) induce strong Th2 responses both in vitro and in vivo. However, the specific molecules that prime the development of Th2 responses have not been identified. We report that omega-1, a glycoprotein which is secreted from S. mansoni eggs and present in SEA, is capable of conditioning human monocyte-derived dendritic cells in vitro to drive T helper 2 (Th2) polarization with similar characteristics as whole SEA. Furthermore, using IL-4 dual reporter mice, we show that both natural and recombinant omega-1 alone are sufficient to generate Th2 responses in vivo, even in the absence of IL-4R signaling. Finally, omega-1-depleted SEA displays an impaired capacity for Th2 priming in vitro, but not in vivo, suggesting the existence of additional factors within SEA that can compensate for the omega-1-mediated effects. Collectively, we identify omega-1, a single component of SEA, as a potent inducer of Th2 responses.

Current diagnosis of schistosomiasis is still not ideal. In the present study we evaluated a targeted affinity approach using mAb 114-4D12, reactive with a unique Schistosoma mansoni-specific glycan epitope, combined with matrix-assisted laser-desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. For nine of 11 urine samples (1ml) from Egyptian individuals with different intensities of infection, a characteristic MALDI-TOF mass spectrum was observed, representing a series of fuco-oligosaccharides that are produced by schistosome eggs. The identification of these small molecule markers may lead to a new egg-load-related assay for light infections in schistosomiasis.

Publication of the transcriptomes of Schistosoma mansoni and Schistosoma japonicum, in conjunction with the sequencing and assembly of their genomes, has generated a comprehensive picture of Schistosoma transcriptional and genomic diversity. Subsequently, researchers who study conjugal and developmental biology, tegumental composition and larval or egg, secretory and excretory products have used these data, in combination with the latest '-omics' technologies, to extend large-scale screens of the schistosome transcriptome, proteome and glycome. In this article, we review these postgenomic investigations and contend that the generated datasets provide a plethora of novel drug, vaccine and immunomodulatory targets that might be useful for developing new antischistosome agents.

The eggs of Schistosoma mansoni express a plethora of glycoconjugate antigens. A specific subset of these antigens can be detected in the serum or urine of infected individuals by a diagnostic sandwich ELISA using the anti-carbohydrate monoclonal antibody (mAb) 114-4D12-A [Nourel Din MS, Nibbeling R, Rotmans JP, Polderman AM, Krijger FW, Deelder AM. Quantitative determination of circulating soluble egg antigen in urine and serum of Schistosoma mansoni-infected individuals using a combined two-site enzyme-linked immunosorbent assay. Am J Trop Med Hyg 1994;50:585-94]. We used affinity chromatography to isolate the 114-4D12-binding glycoprotein subset from soluble egg antigens (SEA) of S. mansoni. SEA and the isolated SEA-subset (SEA-4D12) were subjected to reductive beta-elimination and hydrazinolysis to release intact glycans and glycan fragments, respectively, from the protein backbones. The released glycans were characterised by matrix-assisted laser-desorption-ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS), liquid-chromatography (LC)-MS and gas chromatography (GC)-MS linkage analysis. Glycans released by reductive beta-elimination from SEA-4D12 were larger and more heavily fucosylated than glycans released from SEA. Most SEA-4D12 glycans contained a branched O-glycan core structure carrying up to 4 N-acetylhexosamines per chain which were substituted with maximum 12 fucose residues. Hydrazinolysis of SEA-4D12 resulted in the release of fucosylated antenna fragments. After 2-aminobenzamide (2AB)-labelling these fragments were subjected to 114-4D12-affinity purification. Normal phase (NP)-LC analysis of the flow-through and retained fractions indicated that the Fucalpha1-2Fucalpha1-3GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAcbeta1- element forms the epitope of mAb 114-4D12. Most O-glycans from SEA-4D12 contain this structural element. Epitope-bearing N-glycans have not been found. In terms of abundance in total SEA, only a minority of all glycans possesses the epitope. This multifucosylated motif has so far only been found in schistosomes, providing a structural basis for the high specificity of the diagnostic antibody.

In infections with Schistosoma mansoni the paired adult worms produce hundreds of eggs daily, of which many get trapped in various organs of the human host. The eggs produce complex and unique protein- and lipid-linked glycans, which are important activators and modulators of the host's immune response. The same parasite-derived glycoconjugates are also attractive immunodiagnostic targets in enzyme-linked immunosorbent assays (ELISAs), which detect circulating antigens in serum or urine of the host. Here, we report for the first time that in addition to glycoprotein and glycolipid antigens, schistosome eggs also excrete unique unconjugated oligosaccharides. Employing the schistosome-specific anti-carbohydrate monoclonal antibody 114-4D12 in an affinity purification approach, a specific set of free oligosaccharides was detected by matrix-assisted laser-desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF MS) in human S. mansoni infection urine as well as in egg-incubation medium, but not in worm-culture medium. Nano-scale reverse-phase liquid chromatography-mass spectrometry (nano-RP-LC-MS) analysis of the purified egg-derived oligosaccharides indicated that the captured compounds form a series of multi-fucosylated multimeric N-acetylhexosamine chains with a non-reducing terminal Fucalpha1-2Fucalpha1-3GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAcbeta1- (DF-LDN-DF) sequence which forms the epitope of mAb 114-4D12. Since fucosylated (egg) glycoconjugates have been shown to harbour immunogenic properties, we anticipate that these unconjugated oligosaccharides also play a role in the immunobiology associated with schistosome eggs. Moreover, our data indicate that mass spectrometric detection of a set of signature molecules in urine has potential as a new approach for the diagnosis of schistosomiasis and possibly other helminth infections.

Sex-specific gene expression by Schistosoma mansoni worms has been demonstrated at the transcriptome as well as the proteome levels. In view of the important role of glycans in the biology of schistosomes and the interaction with their human host, we have investigated the sex-specific protein glycosylation. Mass spectrometric profiling and structural characterization of PNGase F-released N-glycans revealed the following gender-specific glycosylation patterns: Complex-type N-glycans of females mainly carried Gal(beta1-4)GlcNAc (LacNAc) and Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc (Lewis x) antennae structures, whereas GalNAc(beta1-4)GlcNAc- (LacdiNAc; LDN) and GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc (LDN-F) were prevalent in N-glycans from males. LDN(-F) motifs were found to occur as repeats on the antennae of large N-glycans that contained up to seven LDN(-F) units. The female complex-type glycans were mostly di-antennary and tri-antennary, whereas male structures were predominantly of the mono-antennary and di-antennary type. Oligomannosidic N-glycans were expressed at similar levels in females and males. The localization of the sex-biased glycan motifs was studied by immunofluorescence microscopy using defined anti-glycan monoclonal antibodies (mAbs). The Lewis x element was strongly expressed in the gut of both males and females, but with respect to tegument localization, the females expressed this structure, while Lewis x seemed to be almost completely absent from the male tegument. The expression of LDN-F was predominantly detected in the parenchyma of both male and female worms as well as in the tegument of the male ventral cavity facing the female. LDN was detected in the tegument of male and female worms at similar levels. The sex-specific expression and differential localization of these antigenic glycan motifs in schistosomes may play a role in male-female interactions during conjugal biology and may lead to a differential immune reaction of the host to the two sexes.

Schistosomiasis is characterised by periovular granuloma formation within the portal tract and presinusoidal venules. As inflammation wanes, continued attempts to wall off and repair hepatic injury, lead to the development of extensive fibrosis. The codependence of chronic inflammation and angiogenesis is a well-known phenomenon. Neovascularisation is a complex process of endothelial cell proliferation and remodelling of the extracellular matrix. Previous studies demonstrated the ability of schistosome soluble egg antigens (SEAs) to stimulate endothelial cell activation in vitro. In the present study, we investigated the angiogenic potential of SEA in Swiss and BALb/c mice, after infection with Schistosoma mansoni or S. haematobium and by implanting SEA-coated beads into the murine liver. Anti-CD34 and anti-Ki-67 immunohistochemical stainings demonstrated newly formed blood vessels within and at the periphery of the granulomas. However, in one third of the granulomas the pre-existing portal stroma was not destroyed by the granulomatous inflammation, angiogenesis was minimal or absent and further growth of the granuloma was prevented. In C57BL/6J and C3H/HeN inbred mice, this polarisation was even more pronounced. In 91% of the granulomas in C57BL6/J mice the portal stroma was preserved. These mice had significantly smaller granulomas, less fibrosis and less mortality as compared to the high pathology C3H/HeN mice, where 87% of the granulomas were of the angiogenic type with destruction of the pre-existing stroma, leading to more severe chronic pathology. Thus, host's genetic mechanisms regulating the degree of angiogenesis and fibrosis, determine the severity of schistosome-induced pathology.

Schistosomes are parasitic flatworms that infect millions of people in (sub)tropical areas around the world. Glycoconjugates of schistosomes play a critical role in the interaction of the different developmental stages of the parasite with the host. In particular, glycosylated components of the eggs produced by the adult worm pairs living in the bloodstream are strongly immunogenic. We have investigated the glycosylation of interleukin-4-inducing factor from schistosome eggs (IPSE/alpha-1), a major secretory egg antigen from Schistosoma mansoni that triggers interleukin-4 production in human basophils, by MS analysis of tryptic glycopeptides. Nanoscale LC-MS(/MS) and MALDI-TOF(/TOF)-MS studies combined with enzymatic degradations showed that monomeric IPSE/alpha-1 contains two N-glycosylation sites, which are each occupied for a large proportion with core-difucosylated diantennary glycans that carry one or more Lewis X motifs. Lewis X has been reported as a major immunogenic glycan element of schistosomes. This is the first report both on the expression of Lewis X on a specific schistosome egg protein and on a protein-specific glycosylation analysis of schistosome eggs.

N-Glycans from glycoproteins of the worm stage of the human parasite Schistosoma mansoni were enzymatically released, fluorescently labelled and analysed using various mass spectrometric and chromatographic methods. A family of 28 mainly core-alpha1-6-fucosylated, diantennary N-glycans of composition Hex(3-4)HexNAc(6-12)Fuc(1-6) was found to carry dimers of N,N'-diacetyllactosediamine [LacdiNAc or LDN; GalNAc(beta1-4)GlcNAc(beta1-] with or without fucose alpha1-3-linked to the N-acetylglucosamine residues in the antennae {GalNAc(beta1-4)[+/-Fuc(alpha1-3)]GlcNAc(beta1-3)GalNAc(beta1-4)[+/-Fuc(alpha1-3)]GlcNAc(beta1-}. To date, oligomeric LDN and oligomeric fucosylated LDN (LDNF) have been found only on N-glycans from mammalian cells engineered to express Caenorhabditis elegansbeta4-GalNAc transferase and human alpha3-fucosyltransferase IX [Z. S. Kawar et al. (2005) J Biol Chem280, 12810-12819]. It now appears that LDN(F) repeats can also occur in a natural system such as the schistosome parasite. Like monomeric LDN and LDNF, the dimeric LDN(F) moieties found here are expected to be targets of humoral and cellular immune responses during schistosome infection.

The immunogenic O-glycan of circulating anodic antigen (CAA) is a high-molecular-mass polysaccharide with the unique -->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GalpNAc-(1--> repeating unit. To obtain information at the molecular level about the specificity of monoclonal antibodies against CAA, the immunoreactivity of two series of bovine serum albumin-coupled synthetic oligosaccharides related to the CAA O-glycan was monitored using ELISA and surface plasmon resonance spectroscopy. The importance of the axial hydroxyl group of beta-D-GalpNAc for antibody binding was investigated using the following series of analogues: beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->O); beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O); and beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O). In the second series of analogues, beta-D-Glcp6S-(1-->3)-beta-D-GalpNAc-(1-->O), beta-D-GalpNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), and beta-D-Glcp6S-(1-->3)-beta-D-Gal-pNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), the native beta-D-GlcpA moiety was replaced by beta-D-Glcp6S to evaluate the influence of the nature of the charge on antibody recognition. Comparison of the immunoreactivity of these series with that measured for conjugates containing corresponding synthetic CAA fragments showed that the antibody binding levels can be correlated to the antibody specificity to CAA fragments. For the most reactive antibodies, the structural changes chosen (beta-D-GalpNAc replaced by beta-D-GlcpNAc, and beta-D-GlcpA replaced by beta-D-Glcp6S) completely eradicated the binding.

Complex multifucosylated oligosaccharides are structural elements of glycoprotein and glycolipid subsets of larval, egg, and adult stages of Schistosoma, the parasitic worms that cause schistosomiasis, a serious disease affecting more than 200 million people in the tropics. The fucosylated structures are thought to play an important role in the immunology of schistosomiasis. Defined schistosomal oligosaccharides that enable immunological studies are difficult to obtain from natural sources. Therefore, we have chemically synthesized spacer-linked GlcNAc, Fucalpha1-3GlcNAc, Fucalpha1-2Fucalpha1-3GlcNAc, and Fucalpha1-2Fucalpha1-2Fucalpha1-3GlcNAc. This series of linear oligosaccharides was used to screen a library of anti-schistosome monoclonal antibodies by surface plasmon resonance spectroscopy. Interestingly, the reactive antibodies could be grouped according to their specificity for the different oligosaccharides tested, showing that these oligosaccharides form different immunological entities based on the number and linkage of the fucose residues. Subsequently, the thus defined monoclonal antibodies were used to visualize the expression of the corresponding oligosaccharide epitopes by adult Schistosoma mansoni worms.

Pulmonary surfactant protein (SP)-D is an important component of the innate immune system of the lung, which is thought to function by binding to specific carbohydrates on the surface of viruses and unicellular pathogens. SP-D has been shown to have a relatively high affinity for the monosaccharides mannose, glucose, and fucose. However, there is limited information on SP-D binding to complex carbohydrate structures, and binding of SP-D to fucose in the context of an oligosaccharide has not yet been investigated. In this study, we used surface plasmon resonance spectroscopy to examine the potential of SP-D to bind to various synthetic fucosylated oligosaccharides, and identified Fucalpha1-3GalNAc and Fucalpha1-3GlcNAc elements as strong ligands. These types of fucosylated glycoconjugates are presented at the surface of Schistosoma mansoni, a parasitic worm that, during development, transiently resides in the lung. In line with the findings by surface plasmon resonance, we found that SP-D can bind to larval stages of S. mansoni, demonstrating for the first time that SP-D interacts with multicellular lung pathogens.

By the use of surface plasmon resonance spectroscopy, immunoglobulin G (IgG) subclass and IgM antibodies against three schistosome-derived carbohydrate structures, FLDN (Fucalpha1-3GalNAcbeta1-4GlcNAcbeta1-3Galalpha1), LDN-DF [GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAcbeta1], and LDNF [GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galalpha1], were measured in 184 previously unexposed Kenyan immigrants who moved into the Masongaleni area, where Schistosoma mansoni is endemic. They were sampled within their first year of exposure and again 2 years later. A cohort selected out of the original residents of the area, who had been exposed for many years, served as controls. Associations with responses to S. mansoni worm, egg (SEA), and cercarial (CERC) antigens were examined. In addition, we measured responses to keyhole limpet hemocyanin, a glycoprotein which carries glycan epitopes that are also expressed by schistosomes. Specific IgG1 responses were most pronounced against FLDN and LDN-DF and strongly associated with those previously measured to SEA and CERC. Similarly to previously published age profiles of IgG1 and IgG2 responses to SEA, levels of IgG1 against LDN-DF decreased with age. In contrast, specific IgM responses against the three schistosome-derived carbohydrate structures were most marked against LDNF. Our results indicate that, of the three glycan structures tested, the acute response against schistosome glycoconjugate antigens in young children is mainly directed against the LDN-DF epitope. The response to LDN-DF in older individuals and the responses to the two other epitopes were similar in the two cohorts, suggesting that these antigens are recognized in the early stages of infection and that the immune response persists. The biological significance of these observations needs further elucidation.

During chronic schistosome infections, a complex regulatory network is induced to regulate the host immune system, in which IL-10-producing regulatory B (Breg) cells play a significant role. Schistosoma mansoni soluble egg antigens (SEA) are bound and internalized by B cells and induce both human and mouse IL-10 producing Breg cells. To identify Breg-inducing proteins in SEA, we fractionated SEA by size exclusion chromatography and found 6 fractions able to induce IL-10 production by B cells (out of 18) in the high, medium and low molecular weight (MW) range. The high MW fractions were rich in heavily glycosylated molecules, including multi-fucosylated proteins. Using SEA glycoproteins purified by affinity chromatography and synthetic glycans coupled to gold nanoparticles, we investigated the role of these glycan structures in inducing IL-10 production by B cells. Then, we performed proteomics analysis on active low MW fractions and identified a number of proteins with putative immunomodulatory properties, notably thioredoxin (SmTrx1) and the fatty acid binding protein Sm14. Subsequent splenic murine B cell stimulations and hock immunizations with recombinant SmTrx1 and Sm14 showed their ability to dose-dependently induce IL-10 production by B cells both in vitro and in vivo. Identification of unique Breg cells-inducing molecules may pave the way to innovative therapeutic strategies for inflammatory and auto-immune diseases. Author summary: Worm parasites, such as schistosomes, are master regulators of the human immune system, manipulating the host response in order to prolong their survival in their host. As a bystander effect, they also reduce immune responses to allergens and/or auto antigens, thus protecting their host against inflammatory and auto-immune diseases. One of the immune cells involved in immune regulation is the IL-10-producing regulatory B (Breg) cells. Schistosome eggs release various molecules that induce the development of Breg cells, but the identity of these molecules is not yet fully known. In this study, the authors aimed to identify some of these molecules. They investigated both the involvement of glycans on high molecular weight schistosomal egg molecules, as well as dissected the role of proteins with a lower molecular weight coming from schistosomal eggs. Using proteomics, they targeted various interesting molecules, which they recombinantly expressed and confirmed the IL-10 inducing capacity in B cells in vitro and in vivo for 2 molecules. This new knowledge may explain the hyporesponsiveness found in chronic schistosome-infected people and may pave the way to new innovative therapies for inflammatory and auto-immune diseases. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis is a major global health problem caused by blood-dwelling parasitic worms, which is currently tackled primarily by mass administration of the drug praziquantel. Appropriate drug treatment strategies are informed by diagnostics that establish the prevalence and intensity of infection, which, in regions of low transmission, should be highly sensitive.Methods: To identify sensitive new serological markers of Schistosoma mansoni infections, we have compiled a recombinant protein library of parasite cell-surface and secreted proteins expressed in mammalian cells.Results: Together with a time series of sera samples from volunteers experimentally infected with a defined number of male parasites, we probed this protein library to identify several markers that can detect primary infections with as low as 10 parasites and as early as 5 weeks postinfection.Conclusions: These new markers could be further explored as valuable tools to detect ongoing and previous S mansoni infections, including in endemic regions where transmission is low. [ABSTRACT FROM AUTHOR]