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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]

Summary: Mounting an effective immune response is crucial for the host to protect itself against invading pathogens. It is now well appreciated that reprogramming of core metabolic pathways in immune cells is a key requirement for their activation and function during infections. The role of several ancillary metabolic pathways in shaping immune cell function is less well understood. One such pathway, for which interest has recently been growing, is the hexosamine biosynthesis pathway (HBP) that generates uridine diphosphate N‐acetylglucosamine (UDP‐GlcNAc), the donor substrate for a specific form of glycosylation termed O‐GlcNAcylation. O‐GlcNAc is an intracellular post‐translational modification that alters the functional properties of the modified proteins, in particular transcription factors and epigenetic regulators. An increasing number of studies suggest a central role for the HBP and O‐GlcNAcylation in dictating immune cell function, including the response to different pathogens. We here discuss the most recent insights regarding O‐GlcNAcylation and immunity, and explore whether targeting of O‐GlcNAcylation could hold promise as a therapeutic approach to modulate immune responses to infections. [ABSTRACT FROM AUTHOR]

Glycoproteins and glycolipids of parasitic helminths play important roles in biology and host-parasite interaction. This review discusses recent helminth glycomics studies that have been expanding our insights into the glycan repertoire of helminths. Structural data are integrated with biological and immunological observations to highlight how glycomics advances our understanding of the critical roles that glycans and glycan motifs play in helminth infection biology. Prospects and challenges in helminth glycomics and glycobiology are discussed. [ABSTRACT FROM AUTHOR]

Abstract: 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: GalNAcβ1-4GlcNAc (LDN), GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F), Fucα1-3GalNAcβ1-4GlcNAc (F-LDN), Fucα1-3GalNAcβ1-4(Fucα1-3)GlcNAc (F-LDN-F), GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF), Fucα1-2Fucα1-3GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (DF-LDN-DF), Galβ1-4(Fucα1-3)GlcNAc (Lewis X) and the truncated trimannosyl N-glycan Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (TriMan). All but Lewis X were variously expressed by miracidia and sporocysts of S. mansoni. Most notably, α3-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, Fucα2Fucα3-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. [Copyright &y& Elsevier]

We here present a printed covalent glycan microarray for protein-binding studies, using low-femtomole quantities of glycans. Glycans, either natural glycans, which were released from glycoproteins and glycolipids from natural sources, or synthetic glycans, were labeled with common fluorescent labels (e.g., 2-aminobenzamide or 2-aminobenzoic acid) by reductive amination and purified by HPLC. The purified glycoconjugates were covalently immobilized on commercial epoxide-activated glass slides via the secondary amine group that links the glycan moiety with the fluorescent tag. This immobilization procedure is generally applicable to reductively aminated glycans with different established fluorescent labels and allows the spatial arrangement of oligosaccharides. The microarray comprised a variety of natural glycans from various biological sources and synthetic glycans and provided informative binding fingerprints for the leclin concanavalin A as well as 14 monoclonal antibodies. Recognized glycans were characterized by tandem mass spectrometry revealing binding motifs. This natural glycan array allowed the characterization of the specificity of carbohydrate-binding proteins for oligosaccharide ligands from sparse biological sources. Moreover, it was applied for the characterization of the microarray glycans by using known carbohydrate-binding proteins. [ABSTRACT FROM AUTHOR]

Abstract: Schistosome glycans and glycoconjugates play a prominent role in the parasite’s biology, in particular in the interaction with the human host. A large amount of structural data regarding glycosylation of different schistosome life stages and glycoconjugate subsets has been collected in the last decade, but many significant gaps in our knowledge of the schistosomal glycome remain. Here we will present a concise review of the already available data guided by a selection of recently generated stage-specific glycan profiles, and discuss implications and prospects of glycomics studies of schistosomes. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Schistosoma mansoni eggs trapped in the liver of an infected host cause the major pathological manifestations of schistosomiasis. Miracidia within the deposited eggs secrete soluble egg antigens (SEA) that induce periovular granuloma formation, which may lead to severe hepatic fibrosis. Several reports have highlighted the immunomodulatory capacities of carbohydrate determinants present in the glycoproteins of SEA. These glycans contain among others the immunogenic Galβ1-4(Fucα1-3)GlcNAc (LewisX) and GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF) elements. Due to cross-reactivity with schistosomal glycan antigens, keyhole limpet haemocyanin (KLH) has been used extensively for diagnostic and therapeutic studies on schistosomiasis. In the present study, a granulomatous response with numerous eosinophils towards SEA- and KLH-coated beads implanted in the liver by mesenteric injection was observed. Immunophenotyping of these experimentally induced granulomas for cellular recruitment, chemokines, adhesion and extracellular matrix proteins revealed very close resemblance with hepatic lesions evoked by native schistosome eggs, hence demonstrating the usefulness of the bead model, in general, as well as of KLH as a model antigen to study the immunopathological mechanisms of schistosome infections. While trypsin digestion of KLH did not alter its antigenic characteristics, beads coated with SEA or KLH treated with sodium periodate to destroy the immunological properties of their carbohydrate chains, yielded only a monolayer of macrophages similar to negative control beads. Up-regulation of ICAM-1, LFA-1 and fibronectin in SEA-induced granulomas and in native and trypsinised KLH-induced granulomas indicates a major role of the carbohydrate elements of SEA and KLH in the initiation and homeostasis of the inflammatory response. These data provide new insights in the complex and multifactorial carbohydrate-dependent host–parasite immunological interactions. [Copyright &y& Elsevier]

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. Author summary: Asthma is a chronic inflammatory disease, leading to cough, wheeze, and shortness of breath. The prevalence has increased drastically in Westernized societies and is now increasing in low- and middle-income countries. Chronic infection with the parasitic helminth, Schistosoma (S.) mansoni protects against allergic airway inflammation (AAI) in mice, and is associated with reduced skin prick test positivity to inhaled allergens in humans. Here we show that peritoneal administration of ω1, a single glycoprotein secreted by S. mansoni eggs, reduced OVA/alum-induced AAI. ω1 is taken up in the peritoneal cavity by dendritic cells (DCs) expressing the mannose receptor, reducing their expression of CCR7 and migratory capacity to the draining mediastinal lymph nodes. This results in accumulation of DCs in the peritoneal cavity of allergic mice and reduced numbers of DCs reaching the draining lymph nodes. The effects observed for ω1 is dependent on its RNAse activity, since the RNAse mutant form of ω1 was unable to reduce AAI nor affect DC migration. Our findings provide insights into how ω1 can modulated the immune response during allergic inflammation, and this may open new avenues for the development of novel therapeutic strategies for allergic asthma. [ABSTRACT FROM AUTHOR]

Presents a structural analysis of the sialylated nitrogen- and oxygen-linked carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. Proliferation and differentiation of erythroid progenitor cells; Modification of the carbohydrate chains by mutagenesis.

The N-linked carbohydrate chains of porcine zona pellucida glycoproteins were released by digestion with peptide-N4-(N-acetyl-β- glucosaminyl)asparagine amidase F and subsequently separated from the O- glycoprotein by gel-permeation chromatography on Bio-Gel P-100. The O- linked carbohydrate chains were released from the O-glycoprotein by alkaline borohydride treatment. Fractionation of the extremely heterogeneous mixture of O-linked oligosaccharide alditols was achieved by a combination of chromatographic techniques comprising gel- permeation chromatography on Bio-Gel P-4 and P-6, anion-exchange FPLC on Mono Q, and high-pH anion-exchange chromatography on CarboPac PA-1. The primary structures of 32 O-glycans were determined by one- and two- dimensional ¹H-NMR spectroscopy. The major part of the analyzed compounds contain a combination of the structural elements Gal β 1- 4GlcNAc β 1-3Gal β 1-3GalNAc-ol, Gal β 1-4(6SO4 )GlcNAc, and α 2-3-linked Neu5Gc or Neu5Ac. This series of compounds has the following structure, where n = 0 to > 6: [Neu5Gc/Ac α2-3]0-1[Gal β 1-4(6SO4-)GlcNAc β1-3]nGal β 1-4GlcNAc β 1-3Gal β 1- 3GalNAc-ol. In addition, smaller compounds were identified in which the Gal β 1-3GalNAc-ol core is substituted by Neu5Gc/Ac α 2-6-linked to GalNAc-ol and/or Neu5Gc/Ac α 2-3-linked to Gal. Furthermore, oligosaccharides were obtained in which the distribution of 6-O- sulfated GlcNAc residues differs from that in the above-mentioned general structure, and a small portion of the oligosaccharides has the GlcNAc β 1-3GalNAc-ol core structure. Analysis of the endo-β- galactosidase digests of pools of N- and O-glycans indicated that the two types of oligosaccharides contain qualitatively similar poly(N- acetyllactosamine) chains. [ABSTRACT FROM AUTHOR]

The development of effective recombinant vaccines against parasitic nematodes has been challenging and so far mostly unsuccessful. This has also been the case for Ostertagia ostertagi, an economically important abomasal nematode in cattle, applying recombinant versions of the protective native activation-associated secreted proteins (ASP). To gain insight in key elements required to trigger a protective immune response, the protein structure and N-glycosylation of the native ASP and a non-protective Pichia pastoris recombinant ASP were compared. Both antigens had a highly comparable protein structure, but different N-glycan composition. After mimicking the native ASP N-glycosylation via the expression in Nicotiana benthamiana plants, immunisation of calves with these plant-produced recombinants resulted in a significant reduction of 39% in parasite egg output, comparable to the protective efficacy of the native antigen. This study provides a valuable workflow for the development of recombinant vaccines against other parasitic nematodes. [ABSTRACT FROM AUTHOR]

Bacterial capsular polysaccharides are important vaccine immunogens. However, the study of polysaccharide-specific immune responses has been hindered by technical restrictions. Here, we developed and validated a high-throughput method to analyse antigen-specific B cells using combinatorial staining with fluorescently-labelled capsular polysaccharide multimers. Concurrent staining of 25 cellular markers further enables the in-depth characterization of polysaccharide-specific cells. We used this assay to simultaneously analyse 14 Streptococcus pneumoniae or 5 Streptococcus agalactiae serotype-specific B cell populations. The phenotype of polysaccharide-specific B cells was associated with serotype specificity, vaccination history and donor population. For example, we observed a link between non-class switched (IgM+) memory B cells and vaccine-inefficient S. pneumoniae serotypes 1 and 3. Moreover, B cells had increased activation in donors from South Africa, which has high-incidence of S. agalactiae invasive disease, compared to Dutch donors. This assay allows for the characterization of heterogeneity in B cell immunity that may underlie immunization efficacy. A high-throughput method is developed for simultaneous quantification and in-depth characterization of bacterial polysaccharide antigen-specific B cells. [ABSTRACT FROM AUTHOR]

There is currently no vaccine against schistosomiasis. With few Schistosoma vaccine candidates in clinical trials, unexplored antigens from the vulnerable schistosomulum should be considered as possible vaccine candidates. In addition, we suggest developing synthetic vesicles as a new delivery vehicle and adjuvant for immunoprophylactic schistosomula vaccine candidates. [ABSTRACT FROM AUTHOR]

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]

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross‐species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well‐recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth‐derived EVs in these processes and highlights EVs as an important participant in cross‐phylum communication. While the mammalian EV field is guided by a community‐agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan‐helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth‐derived EVs to complement the MISEV guidelines. We summarise community‐agreed standards for studying EVs derived from this broad set of non‐model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved. [ABSTRACT FROM AUTHOR]

Serum N-glycan profiling studies during the past decades have shown robust associations between N-glycan changes and various biological conditions, including infections, in humans. Similar studies are scarcer for other mammals, despite the tremendous potential of serum N-glycans as biomarkers for infectious diseases in animal models of human disease and in the veterinary context. To expand the knowledge of serum N-glycan profiles in important mammalian model systems, in this study, we combined MALDI-TOF-MS analysis and HILIC-UPLC profiling of released N-glycans together with glycosidase treatments to characterize the glycan structures present in rhesus macaque serum. We used this baseline to monitor changes in serum N-glycans during infection with Brugia malayi, a parasitic nematode of humans responsible for lymphatic filariasis, in a longitudinal cohort of infected rhesus macaques. Alterations of the HILIC-UPLC profile, notably of abundant structures, became evident as early as 5 weeks post-infection. Given its prominent role in the immune response, contribution of immunoglobulin G to serum N-glycans was investigated. Finally, comparison with similar N-glycan profiling performed during infection with the dog heartworm Dirofilaria immitis suggests that many changes observed in rhesus macaque serum N-glycans are specific for lymphatic filariasis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Abstract: 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 Fucα1-2Fucα1-3GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAcβ1- (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. [Copyright &y& Elsevier]

Abstract: Liquid chromatography (LC)–mass spectrometry (MS) has developed into an invaluable technology for the analysis of protein glycosylation. This review focuses on the recent developments in LC and combinations thereof with MS for this field of research. Recently introduced methods for the structural analysis of released glycans (native or derivatised) as well as glycopeptides, on normal phase, reverse phase and graphitized carbon LC columns with online MS(/MS) will be reviewed. Performed on nano-scale or capillary-scale, these LC–MS methods operate at femtomole sensitivity and support the further integration of glycosylation analysis in proteomics methodology. [Copyright &y& Elsevier]

The immunogenic O-glycan of circulating anodic antigen (CAA) is a high-molecular-mass polysaccharide with the unique →6)-[β-D-Glc pA-(1→3)]-β-D-Gal pNAc-(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 β-D-Gal pNAc for antibody binding was investigated using the following series of analogues: β-D-Glc pA-(1→3)-β-D-Glc pNAc-(1→O); β-D-Glc pNAc-(1→6)-[β-D-Glc pA-(1→3)]-β-D-Glc pNAc-(1→O); and β-D-Glc pA-(1→3)-β-D-Glc pNAc-(1→6)-[β-D-Glc pA-(1→3)]-β-D-Glc pNAc-(1→O). In the second series of analogues, β-D-Glc p6 S-(1→3)-β-D-Gal pNAc-(1→O), β-D-Gal pNAc-(1→6)-[β-D-Glc p6 S-(1→3)]-β-D-Gal pNAc-(1→O), and β-D-Glc p6 S-(1→3)-β-D-Gal- pNAc-(1→6)-[β-D-Glc p6 S-(1→3)]-β-D-Gal pNAc-(1→O), the native β-D-Glc pA moiety was replaced by β-D-Glc p6 S 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 (β-D-Gal pNAc replaced by β-D-Glc pNAc, and β-D-Glc pA replaced by β-D-Glc p6 S) completely eradicated the binding. [ABSTRACT FROM AUTHOR]

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_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_089290's female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_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_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_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. Author summary: Schistosomiasis is a parasitic disease caused by infection with blood flukes, which leads to acute and chronic pathology in millions of infected individuals located in deprived tropical and subtropical regions. Elucidating the function of schistosome genes has provided a clearer view on their roles in various molecular pathways, which are critical to successful parasitism. This information is invaluable when progressing novel drug and vaccine candidates. Here, we add to the existing knowledge of the Schistosoma mansoni parasitic glycan processing and modification machinery by functionally characterising a glycosyl hydrolase (S. mansoni α-N-acetylgalactosaminidase, SmNAGAL). We demonstrate that this protein is enzymatically active and important in coordinating parasite movement in adult male and female schistosomes. Additionally, we provide evidence that this protein regulates pathways associated with egg production in female schistosomes, which is responsible for inducing pathological reactions. Developing drugs that inhibit SmNAGAL enzymatic activity could provide a novel approach for controlling schistosomiasis. [ABSTRACT FROM AUTHOR]

Conventional normal-phase high performance liquid chromatography of fluorescently labeled oligosaccharides with or without on-line mass spectrometry is an established tool for the structure characterization of protein and lipid derived glycans. Here we describe the miniaturization of such a system to the nano-scale using a 75 μm internal diameter normal-phase amide column on-line with electrospray ionization ion-trap mass spectrometry. 2-Aminobenzamide-labeled oligosaccharides have a predictable retention on this normal-phase column that can be expressed as glucose units by comparison to the retention of a standard 2-aminobenzamide glucose polymer mixture. Isobaric compounds are separated on the basis of their structural differences, and by on-line electrospray ionization ion-trap mass spectrometry, the sequence of the monosaccharides can be deduced. The major improvement of the on-line nano-LC-MS system in comparison to conventional systems is the gain in sensitivity with detection of low femtomole amounts of glycans. This implies that LC-MS of 2-aminobenzamide-labeled oligosaccharides can now be performed at higher sensitivity than their analysis with fluorescence detection. [Copyright &y& Elsevier]

Examines the crystallization of the anti-LewisX Fab fragment of the monoclonal antibody 291-2G3-A. X-ray crystallography; Crystal growth; Crystal space group; Unit-cell parameters.

Type 2 immunity plays an essential role in the maintenance of metabolic homeostasis and its disruption during obesity promotes meta‐inflammation and insulin resistance. Infection with the helminth parasite Schistosoma mansoni and treatment with its soluble egg antigens (SEA) induce a type 2 immune response in metabolic organs and improve insulin sensitivity and glucose tolerance in obese mice, yet, a causal relationship remains unproven. Here, we investigated the effects and underlying mechanisms of the T2 ribonuclease omega‐1 (ω1), one of the major S mansoni immunomodulatory glycoproteins, on metabolic homeostasis. We show that treatment of obese mice with plant‐produced recombinant ω1, harboring similar glycan motifs as present on the native molecule, decreased body fat mass, and improved systemic insulin sensitivity and glucose tolerance in a time‐ and dose‐dependent manner. This effect was associated with an increase in white adipose tissue (WAT) type 2 T helper cells, eosinophils, and alternatively activated macrophages, without affecting type 2 innate lymphoid cells. In contrast to SEA, the metabolic effects of ω1 were still observed in obese STAT6‐deficient mice with impaired type 2 immunity, indicating that its metabolic effects are independent of the type 2 immune response. Instead, we found that ω1 inhibited food intake, without affecting locomotor activity, WAT thermogenic capacity or whole‐body energy expenditure, an effect also occurring in leptin receptor‐deficient obese and hyperphagic db/db mice. Altogether, we demonstrate that while the helminth glycoprotein ω1 can induce type 2 immunity, it improves whole‐body metabolic homeostasis in obese mice by inhibiting food intake via a STAT6‐independent mechanism. [ABSTRACT FROM AUTHOR]

Background: In high‐income, temperate countries, IgE to allergen extracts is a risk factor for, and mediator of, allergy‐related diseases (ARDs). In the tropics, positive IgE tests are also prevalent, but rarely associated with ARD. Instead, IgE responses to ubiquitous cross‐reactive carbohydrate determinants (CCDs) on plant, insect and parasite glycoproteins, rather than to established major allergens, are dominant. Because anti‐CCD IgE has limited clinical relevance, it may impact ARD phenotyping and assessment of contribution of atopy to ARD. Methods: Using an allergen extract‐based test, a glycan and an allergen (glyco)protein microarray, we mapped IgE fine specificity among Ugandan rural Schistosoma mansoni (Sm)‐endemic communities, proximate urban communities, and importantly in asthmatic and nonasthmatic schoolchildren. Results: Overall, IgE sensitization to extracts was highly prevalent (43%‐73%) but allergen arrays indicated that this was not attributable to established major allergenic components of the extracts (0%‐36%); instead, over 40% of all participants recognized CCD‐bearing components. Using glycan arrays, we dissected IgE responses to specific glycan moieties and found that reactivity to classical CCD epitopes (core β‐1,2‐xylose, α‐1,3‐fucose) was positively associated with sensitization to extracts, rural environment and Sm infection, but not with skin reactivity to extracts or sensitization to their major allergenic components. Interestingly, we discovered that reactivity to only a subset of core α‐1,3‐fucose‐carrying N‐glycans was inversely associated with asthma. Conclusions: CCD reactivity is not just an epiphenomenon of parasite exposure hampering specificity of allergy diagnostics; mechanistic studies should investigate whether specific CCD moieties identified here are implicated in the protective effect of certain environmental exposures against asthma. [ABSTRACT FROM AUTHOR]

Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host immunity to enable infection. Extracellular vesicles (EVs) are among these E/S products, yet molecular mechanisms and functionality of S. mansoni EV interaction with host immune cells is unknown. Here we demonstrate that EVs released by S. mansoni schistosomula are internalised by human monocyte-derived dendritic cells (moDCs). Importantly, we show that this uptake was mainly mediated via DC-SIGN (CD209). Blocking DC-SIGN almost completely abrogated EV uptake, while blocking mannose receptor (MR, CD206) or dendritic cell immunoreceptor (DCIR, CLEC4A) had no effect on EV uptake. Mass spectrometric analysis of EV glycans revealed the presence of surface N-glycans with terminal Galβ1-4(Fucα1-3)GlcNAc (LewisX) motifs, and a wide array of fucosylated lipid-linked glycans, including LewisX, a known ligand for DC-SIGN. Stimulation of moDCs with schistosomula EVs led to increased expression of costimulatory molecules CD86 and CD80 and regulatory surface marker PD-L1. Furthermore, schistosomula EVs increased expression of IL-12 and IL-10 by moDCs, which was partly dependent on the interaction with DC-SIGN. These results provide the first evidence that glycosylation of S. mansoni EVs facilitates the interaction with host immune cells and reveals a role for DC-SIGN and EV-associated glycoconjugates in parasite-induced immune modulation. [ABSTRACT FROM AUTHOR]

Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host immunity to enable infection. Extracellular vesicles (EVs) are among these E/S products, yet molecular mechanisms and functionality of S. mansoni EV interaction with host immune cells is unknown. Here we demonstrate that EVs released by S. mansoni schistosomula are internalised by human monocyte‐derived dendritic cells (moDCs). Importantly, we show that this uptake was mainly mediated via DC‐SIGN (CD209). Blocking DC‐SIGN almost completely abrogated EV uptake, while blocking mannose receptor (MR, CD206) or dendritic cell immunoreceptor (DCIR, CLEC4A) had no effect on EV uptake. Mass spectrometric analysis of EV glycans revealed the presence of surface N‐glycans with terminal Galβ1‐4(Fucα1‐3)GlcNAc (LewisX) motifs, and a wide array of fucosylated lipid‐linked glycans, including LewisX, a known ligand for DC‐SIGN. Stimulation of moDCs with schistosomula EVs led to increased expression of costimulatory molecules CD86 and CD80 and regulatory surface marker PD‐L1. Furthermore, schistosomula EVs increased expression of IL‐12 and IL‐10 by moDCs, which was partly dependent on the interaction with DC‐SIGN. These results provide the first evidence that glycosylation of S. mansoni EVs facilitates the interaction with host immune cells and reveals a role for DC‐SIGN and EV‐associated glycoconjugates in parasite‐induced immune modulation. [ABSTRACT FROM AUTHOR]

• The Schistosoma mansoni venom allergen-like (SmVAL) family relationships were investigated. • Transcription patterns of SmVALs associate with phylogenetic relationships. • There was clear antibody cross-reactivity between related native SmVAL proteins. • SmVAL4, 10, 18 and 19 all localized via WISH to pre-acetabular glands of cercariae. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract Image 1 Highlights • Comprehensive Lipidomics study of Schistosoma mansoni and its life stages. • Description of a Schistosoma mansoni lipid database. • Identification of several important immunomodulatory lipids, leads for further research. [ABSTRACT FROM AUTHOR]

α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall–associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-d-galactopyranoside substrate (Km = 0.17 mm) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management. [ABSTRACT FROM AUTHOR]

Abstract: Teichoic acids (TAs) are key components of the Gram‐positive bacterial cell wall that are composed of alditol phosphate repeating units, decorated with alanine or carbohydrate appendages. Because of their microhetereogeneity, pure well‐defined TAs for biological or immunological evaluation cannot be obtained from natural sources. We present here a streamlined automated solid‐phase synthesis approach for the rapid generation of well‐defined glycosylated, glycerol‐based TA oligomers. Building on the use of a “universal” linker system and fluorous tag purification strategy, a library of glycerolphosphate pentadecamers, decorated with various carbohydrate appendages, is generated. These are used to create a structurally diverse TA‐microarray, which is used to reveal, for the first time, the binding preferences of anti‐LTA (lipoteichoic acids) antibodies at the molecular level. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Human immunity to Schistosoma infection requires many years of exposure, and multiple infections and treatments to develop. Unlike humans, rhesus macaques clear an established schistosome infection naturally at the same time acquiring immunity towards re-infection. In macaques, schistosome egg production decreases after 8 weeks post-infection and by week 22, physiological impairment of the worm caused by unclarified antibody-mediated processes is observed. Since strong antibody responses have been observed against schistosome glycan antigens in human and animal infections, we here investigate if anti-glycan antibodies are associated with immunity against schistosome infections in macaques. Methods: We used a microarray containing a large repertoire of glycoprotein- and glycolipid-derived glycans from different schistosome life stages to analyse anti-glycan serum IgG and IgM from S. japonicum-infected macaques during the course of infection and self-cure. We also used an in vitro schistosomula assay to investigate whether macaque sera containing anti-glycan antibodies can kill schistosomula. Conclusions/significance: Antibody responses towards schistosome glycans at week 4 post-infection were dominated by IgM while IgG was high at week 8. The profound increase in IgG was observed mainly for antibodies towards a large subset of glycans that contain (multi-)fucosylated terminal GalNAcβ1-4GlcNAc (LDN), and Galβ1-4(Fucα1–3)GlcNAc (LeX) motifs. In general, glycans with a higher degree of fucosylation gave rise to stronger antibody responses than non-fucosylated glycans. Interestingly, even though many IgG and IgM responses had declined by week 22 post-infection, IgG towards O-glycans with highly fucosylated LDN motifs remained. When incubating macaque serum with schistosomula in vitro, schistosomula death was positively correlated with the duration of infection of macaques; macaque serum taken 22 weeks post-infection caused most schistosomula to die, suggesting the presence of potentially protective antibodies. We hypothesize that IgGs against highly fucosylated LDN motifs that remain when the worms deteriorate are associated with infection clearance and the resistance to re-infection in macaques. [ABSTRACT FROM AUTHOR]

Background: The pig parasite Ascaris suum plays and important role in veterinary medicine and represents a suitable model for A. lumbricoides, which infects over 800 million people. In pigs, continued exposure to Ascaris induces immunity at the level of the gut, protecting the host against migrating larvae. The objective of this study was to identify and characterize parasite antigens targeted by this local immune response that may be crucial for parasite invasion and establishment and to evaluate their protective and diagnostic potential. Methodology/Principal Findings: Pigs were immunized by trickle infection for 30 weeks, challenged with 2,000 eggs at week 32 and euthanized two weeks after challenge. At necropsy, there was a 100% reduction in worms recovered from the intestine and a 97.2% reduction in liver white spots in comparison with challenged non-immune control animals. Antibodies purified from the intestinal mucus or from the supernatant of cultured antibody secreting cells from mesenteric lymph nodes of immune pigs were used to probe L3 extracts to identify antibody targets. This resulted in the recognition of a 12kDa antigen (As12) that is actively shed from infective Ascaris L3. As12 was characterized as a phosphorylcholine-containing glycolipid-like antigen that is highly resistant to different enzymatic and chemical treatments. Vaccinating pigs with an As12 fraction did not induce protective immunity to challenge infection. However, serological analysis using sera or plasma from experimentally infected pigs or naturally infected humans demonstrated that the As12 ELISA was able to detect long-term exposure to Ascaris with a high diagnostic sensitivity (98.4% and 92%, respectively) and specificity (95.5% and 90.0%) in pigs and humans, respectively. Conclusions/Significance: These findings show the presence of a highly stage specific, glycolipid-like component (As12) that is actively secreted by infectious Ascaris larvae and which acts as a major antibody target in infected humans and pigs. [ABSTRACT FROM AUTHOR]

Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F. hepatica NEJ glycosylation and the first report of N-glycosylation of F. hepatica cathepsins. The significance of these findings for immunological studies and vaccine development is discussed. [ABSTRACT FROM AUTHOR]

Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. Like other helminths, F. hepatica employs mechanisms of immune suppression in order to evade its host immune system. In this study the N-glycosylation of F. hepatica’s tegumental coat (FhTeg) and its carbohydrate-dependent interactions with bone marrow derived dendritic cells (BMDCs) were investigated. Mass spectrometric analysis demonstrated that FhTeg N-glycans comprised mainly of oligomannose and to a lesser extent truncated and complex type glycans, including a phosphorylated subset. The interaction of FhTeg with the mannose receptor (MR) was investigated. Binding of FhTeg to MR-transfected CHO cells and BMDCs was blocked when pre-incubated with mannan. We further elucidated the role played by MR in the immunomodulatory mechanism of FhTeg and demonstrated that while FhTeg’s binding was significantly reduced in BMDCs generated from MR knockout mice, the absence of MR did not alter FhTeg’s ability to induce SOCS3 or suppress cytokine secretion from LPS activated BMDCs. A panel of negatively charged monosaccharides (i.e. GlcNAc-4P, Man-6P and GalNAc-4S) were used in an attempt to inhibit the immunoregulatory properties of phosphorylated oligosaccharides. Notably, GalNAc-4S, a known inhibitor of the Cys-domain of MR, efficiently suppressed FhTeg binding to BMDCs and inhibited the expression of suppressor of cytokine signalling (SOCS) 3, a negative regulator the TLR and STAT3 pathway. We conclude that F. hepatica contains high levels of mannose residues and phosphorylated glycoproteins that are crucial in modulating its host’s immune system, however the role played by MR appears to be limited to the initial binding event suggesting that other C-type lectin receptors are involved in the immunomodulatory mechanism of FhTeg. [ABSTRACT FROM AUTHOR]

Background: Conventional diagnostic methods for human ascariasis are based on the detection of Ascaris lumbricoides eggs in stool samples. However, studies of ascariasis in pigs have shown that the prevalence and the number of eggs detected in the stool do not correlate well with exposure of the herd to the parasite. On the other hand, an ELISA test measuring antibodies to Ascaris suum haemoglobin (AsHb) has been shown to be useful for estimating transmission intensity on pig farms. In this study, we further characterized the AsHb antigen and screened samples from a population-based study conducted in an area that is endemic for Ascaris lumbricoides in Indonesia to assess changes in AsHb antibody rates and levels in humans following mass drug administration (MDA). Methodology/Principal findings: We developed and evaluated an ELISA to detect human IgG4 antibodies to AsHb. We tested 1066 plasma samples collected at different times from 599 subjects who lived in a village in rural Indonesia that was highly endemic for ascariasis. The community received 6 rounds of MDA for lymphatic filariasis with albendazole plus diethylcarbamazine between 2002 and 2007. While the AsHb antibody assay was not sensitive for detecting all individuals with Ascaris eggs in their stools, the percentage of seropositive individuals decreased rapidly following MDA. Reductions in antibody rates reflected decreased mean egg output per person both at the community level and in different age groups. Two years after the last round of MDA the community egg output and antibody prevalence rate were reduced by 81.6% and 78.9% respectively compared to baseline levels. Conclusion/Significance: IgG4 antibody levels to AsHb appear to reflect recent exposure to Ascaris. The antibody prevalence rate may be a useful indicator for Ascaris transmission intensity in communities that can be used to assess the impact of control measures on the force of transmission. [ABSTRACT FROM AUTHOR]

Glycan molecules from helminth parasites have been associated with diverse biological functions ranging from interactions with neighbouring host cell populations to down-modulation of specific host immunity. Glycoproteins secreted by the intestinal nematode Heligmosomoides polygyrus are of particular interest as the excretory–secretory products (termed HES) of this parasite contain both heat-labile and heat-stable components with immunomodulatory effects. We used MALDI-TOF-MS and LC–MS/MS to analyse the repertoire of N - and O -linked glycans released from Heligmosomoides polygyrus excretory–secretory products by PNGase A and F, β-elimination and hydrazinolysis revealing a broad range of structures including novel methylhexose- and methylfucose-containing glycans. Monoclonal antibodies to two immunodominant glycans of H. polygyrus , previously designated Glycans A and B, were found to react by glycan array analysis to a methyl-hexose-rich fraction and to a sulphated LacDiNAc (LDN; GalNAcβ1–4GlcNAc) structure, respectively. We also analysed the glycan repertoire of a major glycoprotein in Heligmosomoides polygyrus excretory–secretory products, VAL-2, which contains many glycan structures present in Heligmosomoides polygyrus excretory–secretory products including Glycan A. However, it was found that this set of glycans is not responsible for the heat-stable immunomodulatory properties of Heligmosomoides polygyrus excretory–secretory products, as revealed by the inability of VAL-2 to inhibit allergic lung inflammation. Taken together, these studies reveal that H. polygyrus secretes a diverse range of antigenic glycoconjugates, and provides a framework to explore the biological and immunomodulatory roles they may play within the mammalian host. [ABSTRACT FROM AUTHOR]

Human interleukin-22 ( IL-22) is a member of the IL-10 cytokine family that has recently been shown to have major therapeutic potential. IL-22 is an unusual cytokine as it does not act directly on immune cells. Instead, IL-22 controls the differentiation, proliferation and antimicrobial protein expression of epithelial cells, thereby maintaining epithelial barrier function. In this study, we transiently expressed human IL-22 in Nicotiana benthamiana plants and investigated the role of N-glycosylation on protein folding and biological activity. Expression levels of IL-22 were up to 5.4 μg/mg TSP, and N-glycan analysis revealed the presence of the atypical Lewis A structure. Surprisingly, upon engineering of human-like N-glycans on IL-22 by co-expressing mouse FUT8 in Δ XT/ FT plants a strong reduction in Lewis A was observed. Also, core α1,6-fucoylation did not improve the biological activity of IL-22. The combination of site-directed mutagenesis of Asn54 and in vivo deglycosylation with PNGase F also revealed that N-glycosylation at this position is not required for proper protein folding. However, we do show that the presence of a N-glycan on Asn54 contributes to the atypical N-glycan composition of plant-produced IL-22 and influences the N-glycan composition of N-glycans on other positions. Altogether, our data demonstrate that plants offer an excellent tool to investigate the role of N-glycosylation on folding and activity of recombinant glycoproteins, such as IL-22. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Author Summary: Schistosomiasis is one of the major parasitic diseases in developing countries and still causes 200,000 deaths per year. Mass drug administration programs with praziquantel, the drug of choice against schistosomiasis, are currently undertaken in Sub-Saharan Africa. Praziquantel, although efficient against adult worms, fails to cure early infection. The complex developmental stages of schistosomes and migration through varying host compartments with different local drug concentration are a challenge for drug treatment. After infecting their mammalian host, schistosomula traverse through skin and the vasculature of lung, liver and intestines. During their migration, they develop from larvae to paired adults in approximately 4 to 5 weeks. So far, drug effects have been analyzed on adult worms or freshly transformed schistosomula only. Information about the effects on the larval stages is lacking. We were able to transfer the larval development of the first three weeks into the culture dish. This tool can be used for the analysis of drug effects against schistosomula and for investigation of the accessibility, expression and localization of antigens. Rendering the parasite's larvae vulnerable to the host's immune system by increasing antigen presentation is an important aspect of drug activity. We demonstrate on in vitro-cultured Schistosoma mansoni larvae, that SmCD59a and SmKK7, as examples for hidden antigens, become accessible to antibodies following drug treatment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Penetration of skin, migration through tissues and establishment of long-lived intravascular partners require Schistosoma parasites to successfully manipulate definitive host defences. While previous studies of larval schistosomula have postulated a function for excreted/secreted (E/S) products in initiating these host-modulatory events, the role of extracellular vesicles (EVs) has yet to be considered. Here, using preparatory ultracentrifugation as well as methodologies to globally analyse both proteins and small non-coding RNAs (sncRNAs), we conducted the first characterization of Schistosoma mansoni schistosomula EVs and their potential host-regulatory cargos. Transmission electron microscopy analysis of EVs isolated from schistosomula in vitro cultures revealed the presence of numerous, 30–100 nm sized exosome-like vesicles. Proteomic analysis of these vesicles revealed a core set of 109 proteins, including homologs to those previously found enriched in other eukaryotic EVs, as well as hypothetical proteins of high abundance and currently unknown function. Characterization of E/S sncRNAs found within and outside of schistosomula EVs additionally identified the presence of potential gene-regulatory miRNAs (35 known and 170 potentially novel miRNAs) and tRNA-derived small RNAs (tsRNAs; nineteen 5′ tsRNAs and fourteen 3′ tsRNAs). The identification of S. mansoni EVs and the combinatorial protein/sncRNA characterization of their cargo signifies that an important new participant in the complex biology underpinning schistosome/host interactions has now been discovered. Further work defining the role of these schistosomula EVs and the function/stability of intra- and extra-vesicular sncRNA components presents tremendous opportunities for developing novel schistosomiasis diagnostics or interventions. [ABSTRACT FROM AUTHOR]

The unique features of Ig A, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by Ig G and Ig E, make it a promising antibody isotype for several therapeutic applications. However, in contrast to Ig G, reports on plant production of Ig A are scarce. We produced Ig A1κ and Ig G1κ versions of three therapeutic antibodies directed against pro-inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF-α, and Ustekinumab, directed against the interleukin-12p40 subunit. We evaluated antibody yield, quality and N-glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell-based assay. However, Ig A1κ-based Adalimumab and Ustekinumab were poorly secreted compared to their Ig G counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both Ig A1κ- and Ig G1κ-based Infliximab were enriched in oligomannose-type N-glycan structures. For Ig G1κ-based Ustekinumab and Adalimumab, the major N-glycan type was the typical plant complex N-glycan, biantennary with terminal N-acetylglucosamine, β1,2-xylose and core α1,3-fucose. In contrast, the major N-glycan on the Ig A-based antibodies was xylosylated, but lacked core α1,3-fucose and one terminal N-acetylglucosamine. This type of N-glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant-produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N-glycan an antibody receives. [ABSTRACT FROM AUTHOR]