Plant-based production of a protective vaccine antigen against the bovine parasitic nematode Ostertagia ostertagi.

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.

Structural insights into a cooperative switch between one and two FimH bacterial adhesins binding pauci- and high-mannose type N-glycan receptors.

The FimH type-1 fimbrial adhesin allows pathogenic Escherichia coli to adhere to glycoproteins in the epithelial linings of human bladder and intestinal tract, by using multiple fimbriae simultaneously. Pauci- and high-mannose type N-glycans are natural FimH receptors on those glycoproteins. Oligomannose-3 and oligomannose-5 bind with the highest affinity to FimH by using the same Manα1,3Man branch. Oligomannose-6 is generated from oligomannose-5 in the next step of the biogenesis of high-mannose N-glycans, by the transfer of a mannose in α1,2-linkage onto this branch. Using serial crystallography and by measuring the kinetics of binding, we demonstrate that shielding the high-affinity epitope drives the binding of multiple FimH molecules. First, we profiled FimH glycan binding on a microarray containing paucimannosidic N-glycans and in a FimH LEctPROFILE assay. To make the transition to oligomannose-6, we measured the kinetics of FimH binding using paucimannosidic N-glycans, glycoproteins and all four α-dimannosides conjugated to bovine serum albumin. Equimolar mixed interfaces of the dimannosides present in oligomannose-6 and molecular dynamics simulations suggest a positive cooperativity in the bivalent binding of Manα1,3Manα1 and Manα1,6Manα1 dimannosides. The binding of core α1,6-fucosylated oligomannose-3 in cocrystals of FimH is monovalent but interestingly the GlcNAc1-Fuc moiety retains highly flexibility. In cocrystals with oligomannose-6, two FimH bacterial adhesins bind the Manα1,3Manα1 and Manα1,6Manα1 endings of the second trimannose core (A-4'-B). This cooperative switch towards bivalent binding appears sustainable beyond a molar excess of oligomannose-6. Our findings provide important novel structural insights for the design of multivalent FimH antagonists that bind with positive cooperativity.

Glyco-Engineering Plants to Produce Helminth Glycoproteins as Prospective Biopharmaceuticals: Recent Advances, Challenges and Future Prospects.

Glycoproteins are the dominant category among approved biopharmaceuticals, indicating their importance as therapeutic proteins. Glycoproteins are decorated with carbohydrate structures (or glycans) in a process called glycosylation. Glycosylation is a post-translational modification that is present in all kingdoms of life, albeit with differences in core modifications, terminal glycan structures, and incorporation of different sugar residues. Glycans play pivotal roles in many biological processes and can impact the efficacy of therapeutic glycoproteins. The majority of biopharmaceuticals are based on human glycoproteins, but non-human glycoproteins, originating from for instance parasitic worms (helminths), form an untapped pool of potential therapeutics for immune-related diseases and vaccine candidates. The production of sufficient quantities of correctly glycosylated putative therapeutic helminth proteins is often challenging and requires extensive engineering of the glycosylation pathway. Therefore, a flexible glycoprotein production system is required that allows straightforward introduction of heterologous glycosylation machinery composed of glycosyltransferases and glycosidases to obtain desired glycan structures. The glycome of plants creates an ideal starting point for N- and O-glyco-engineering of helminth glycans. Plants are also tolerant toward the introduction of heterologous glycosylation enzymes as well as the obtained glycans. Thus, a potent production platform emerges that enables the production of recombinant helminth proteins with unusual glycans. In this review, we discuss recent advances in plant glyco-engineering of potentially therapeutic helminth glycoproteins, challenges and their future prospects.

Contributions of the international plant science community to the fight against human infectious diseases - part 1: epidemic and pandemic diseases.

Infectious diseases, also known as transmissible or communicable diseases, are caused by pathogens or parasites that spread in communities by direct contact with infected individuals or contaminated materials, through droplets and aerosols, or via vectors such as insects. Such diseases cause ˜17% of all human deaths and their management and control places an immense burden on healthcare systems worldwide. Traditional approaches for the prevention and control of infectious diseases include vaccination programmes, hygiene measures and drugs that suppress the pathogen, treat the disease symptoms or attenuate aggressive reactions of the host immune system. The provision of vaccines and biologic drugs such as antibodies is hampered by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, particularly in developing countries where infectious diseases are prevalent and poorly controlled. Molecular farming, which uses plants for protein expression, is a promising strategy to address the drawbacks of current manufacturing platforms. In this review article, we consider the potential of molecular farming to address healthcare demands for the most prevalent and important epidemic and pandemic diseases, focussing on recent outbreaks of high-mortality coronavirus infections and diseases that disproportionately affect the developing world.

Contributions of the international plant science community to the fight against infectious diseases in humans-part 2: Affordable drugs in edible plants for endemic and re-emerging diseases.

The fight against infectious diseases often focuses on epidemics and pandemics, which demand urgent resources and command attention from the health authorities and media. However, the vast majority of deaths caused by infectious diseases occur in endemic zones, particularly in developing countries, placing a disproportionate burden on underfunded health systems and often requiring international interventions. The provision of vaccines and other biologics is hampered not only by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, but also by challenges caused by distribution and storage, particularly in regions without a complete cold chain. In this review article, we consider the potential of molecular farming to address the challenges of endemic and re-emerging diseases, focusing on edible plants for the development of oral drugs. Key recent developments in this field include successful clinical trials based on orally delivered dried leaves of Artemisia annua against malarial parasite strains resistant to artemisinin combination therapy, the ability to produce clinical-grade protein drugs in leaves to treat infectious diseases and the long-term storage of protein drugs in dried leaves at ambient temperatures. Recent FDA approval of the first orally delivered protein drug encapsulated in plant cells to treat peanut allergy has opened the door for the development of affordable oral drugs that can be manufactured and distributed in remote areas without cold storage infrastructure and that eliminate the need for expensive purification steps and sterile delivery by injection.

ß-Hexosaminidases Along the Secretory Pathway of Nicotiana benthamiana Have Distinct Specificities Toward Engineered Helminth N-Glycans on Recombinant Glycoproteins.

Secretions of parasitic worms (helminths) contain a wide collection of immunomodulatory glycoproteins with the potential to treat inflammatory disorders, like autoimmune diseases. Yet, the identification of single molecules that can be developed into novel biopharmaceuticals is hampered by the limited availability of native parasite-derived proteins. Recently, pioneering work has shown that helminth glycoproteins can be produced transiently in Nicotiana benthamiana plants while simultaneously mimicking their native helminth N-glycan composition by co-expression of desired glycosyltransferases. However, efficient "helminthization" of N-glycans in plants by glyco-engineering seems to be hampered by the undesired truncation of complex N-glycans by ß-N-acetyl-hexosaminidases, in particular when aiming for the synthesis of N-glycans with antennary GalNAcß1-4GlcNAc (LacdiNAc or LDN). In this study, we cloned novel ß-hexosaminidase open reading frames from N. benthamiana and characterized the biochemical activity of these enzymes. We identified HEXO2 and HEXO3 as enzymes responsible for the cleavage of antennary GalNAc residues of N-glycans on the model helminth glycoprotein kappa-5. Furthermore, we reveal that each member of the HEXO family has a distinct specificity for N-glycan substrates, where HEXO2 has strict ß-galactosaminidase activity, whereas HEXO3 cleaves both GlcNAc and GalNAc. The identification of HEXO2 and HEXO3 as major targets for LDN cleavage will enable a targeted genome editing approach to reduce undesired processing of these N-glycans. Effective knockout of these enzymes could allow the production of therapeutically relevant glycoproteins with tailor-made helminth N-glycans in plants.

Minimal epitope for Mannitou IgM on paucimannose-carrying glycoproteins.

Paucimannosidic glycans are restricted to the core structure [Man1-3GlcNAc2Fuc0-1] of N-glycans and are rarely found in mammalian tissues. Yet, especially [Man2-3GlcNAc2Fuc1] have been found significantly upregulated in tumors, including in colorectal and liver cancer. Mannitou IgM is a murine monoclonal antibody that was previously shown to recognize Man3GlcNAc2 with an almost exclusive selectivity. Here, we have sought the definition of the minimal glycan epitope of Mannitou IgM, initiated by screening on a newly designed paucimannosidic glycan microarray; among the best binders were Man3GlcNAc2 and its α1,6 core-fucosylated variant, Man3GlcNAc2Fuc1. Unexpectedly and in contrast to earlier findings, Man5GlcNAc2-type structures bind equally well and a large tolerance was observed for substitutions on the α1,6 arm. It was confirmed that any substitution on the single α1,3-linked mannose completely abolishes binding. Surface plasmon resonance for kinetic measurements of Mannitou IgM binding, either directly on the glycans or as presented on omega-1 and kappa-5 soluble egg antigens from the helminth parasite Schistosoma mansoni, showed submicromolar affinities. To characterize the epitope in greater and atomic detail, saturation transfer difference nuclear magnetic resonance spectroscopy was performed with the Mannitou antigen-binding fragment. The STD-NMR data demonstrated the strongest interactions with the aliphatic protons H1 and H2 of the α1-3-linked mannose and weaker imprints on its H3, H4 and H5 protons. In conclusion, Mannitou IgM binding requires a nonsubstituted α1,3-linked mannose branch of paucimannose also on proteins, making it a highly specific tool for the distinction of concurrent human tumor-associated carbohydrate antigens.

Tumor Necrosis Factor and Schistosoma mansoni egg antigen omega-1 shape distinct aspects of the early egg-induced granulomatous response.

Infections by schistosomes result in granulomatous lesions around parasite eggs entrapped within the host tissues. The host and parasite determinants of the Schistosoma mansoni egg-induced granulomatous response are areas of active investigation. Some studies in mice implicate Tumor Necrosis Factor (TNF) produced in response to the infection whereas others fail to find a role for it. In addition, in the mouse model, the S. mansoni secreted egg antigen omega-1 is found to induce granulomas but the underlying mechanism remains unknown. We have recently developed the zebrafish larva as a model to study macrophage recruitment and granuloma formation in response to Schistosoma mansoni eggs. Here we use this model to investigate the mechanisms by which TNF and omega-1 shape the early granulomatous response. We find that TNF, specifically signaling through TNF receptor 1, is not required for macrophage recruitment to the egg and granuloma initiation but does mediate granuloma enlargement. In contrast, omega-1 mediates initial macrophage recruitment, with this chemotactic activity being dependent on its RNase activity. Our findings further the understanding of the role of these host- and parasite-derived factors and show that they impact distinct facets of the granulomatous response to the schistosome egg.

The helminth glycoprotein omega-1 improves metabolic homeostasis in obese mice through type 2 immunity-independent inhibition of food intake.

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.

Helminth Glycans at the Host-Parasite Interface and Their Potential for Developing Novel Therapeutics.

Helminths are parasitic worms that have successfully co-evolved with their host immune system to sustain long-term infections. Their successful parasitism is mainly facilitated by modulation of the host immune system via the release of excretory-secretory (ES) products covered with glycan motifs such as Lewis X, fucosylated LDN, phosphorylcholine and tyvelose. Evidence is accumulating that these glycans play key roles in different aspects of helminth infection including interactions with immune cells for recognition and evasion of host defences. Moreover, antigenic properties of glycans can be exploited for improving the efficacy of anti-helminthic vaccines. Here, we illustrate that glycans have the potential to open new avenues for the development of novel biopharmaceuticals and effective vaccines based on helminth glycoproteins.

Functional characterization of Schistosoma mansoni fucosyltransferases in Nicotiana benthamiana plants.

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.

Secreted venom allergen-like proteins of helminths: Conserved modulators of host responses in animals and plants.

Despite causing considerable damage to host tissue at the onset of parasitism, invasive helminths establish remarkably persistent infections in both animals and plants. Secretions released by these obligate parasites during host invasion are thought to be crucial for their persistence in infection. Helminth secretions are complex mixtures of molecules, most of which have unknown molecular targets and functions in host cells or tissues. Although the habitats of animal- and plant-parasitic helminths are very distinct, their secretions share the presence of a structurally conserved group of proteins called venom allergen-like proteins (VALs). Helminths abundantly secrete VALs during several stages of parasitism while inflicting extensive damage to host tissue. The tight association between the secretion of VALs and the onset of parasitism has triggered a particular interest in this group of proteins, as improved knowledge on their biological functions may assist in designing novel protection strategies against parasites in humans, livestock, and important food crops.

Nicotiana benthamiana α-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease.

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.

Heligmosomoides polygyrus Venom Allergen-like Protein-4 (HpVAL-4) is a sterol binding protein.

Heligmosomoides polygyrus bakeri is a model parasitic hookworm used to study animal and human helminth diseases. During infection, the parasite releases excretory/secretory products that modulate the immune system of the host. The most abundant protein family in excretory/secretory products comprises the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. There are >30 secreted Heligmosomoides polygyrus VAL proteins (HpVALs) and these proteins are characterised by having either one or two 15 kDa CAP (cysteine-rich secretory protein (CRISP)/antigen 5/pathogenesis related-1) domains. The first known HpVAL structure, HpVAL-4, refined to 1.9 Šis reported. HpVAL-4 was produced as a homogeneously glycosylated protein in leaves of Nicotiana benthamiana infiltrated with recombinant plasmids, making this plant expression platform amenable for the production of biological products. The overall topology of HpVAL-4 is a three layered αßα sandwich between a short N-terminal loop and a C-terminal cysteine rich extension. The C-terminal cysteine rich extension has two strands stabilized by two disulfide bonds and superposes well with the previously reported extension from the human hookworm Necator americanus Ancylostoma secreted protein-2 (Na-ASP-2). The N-terminal loop is connected to alpha helix 2 via a disulfide bond previously observed in Na-ASP-2. HpVAL-4 has a central cavity that is more similar to the N-terminal CAP domain of the two CAP Na-ASP-1 from Necator americanus. Unlike Na-ASP-2, mammalian CRISP, and the C-terminal CAP domain of Na-ASP-1, the large central cavity of HpVAL-4 lacks the two histidines required to coordinate divalent cations. HpVAL-4 has both palmitate-binding and sterol-binding cavities and is able to complement the in vivo sterol export phenotype of yeast mutants lacking their endogenous CAP proteins. More studies are required to determine endogenous binding partners of HpVAL-4 and unravel the possible impact of sterol binding on immune-modulatory functions.

Crystal structure of Brugia malayi venom allergen-like protein-1 (BmVAL-1), a vaccine candidate for lymphatic filariasis.

Brugia malayi is a causative agent of lymphatic filariasis, a major tropical disease. The infective L3 parasite stage releases immunomodulatory proteins including the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (Sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. BmVAL-1 is a major target of host immunity with >90% of infected B. malayi microfilaraemic cases being seropositive for antibodies to BmVAL-1. This study is part of ongoing efforts to characterize the structures and functions of important B. malayi proteins. Recombinant BmVAL-1 was produced using a plant expression system, crystallized and the structure was solved by molecular replacement and refined to 2.1 Å, revealing the characteristic alpha/beta/alpha sandwich topology of eukaryotic SCP/TAPS proteins. The protein has more than 45% loop regions and these flexible loops connect the helices and strands, which are longer than predicted based on other parasite SCP/TAPS protein structures. The large central cavity of BmVAL-1 is a prototypical CRISP cavity with two histidines required to bind divalent cations. The caveolin-binding motif (CBM) that mediates sterol binding in SCP/TAPS proteins is large and open in BmVAL-1 and is N-glycosylated. N-glycosylation of the CBM does not affect the ability of BmVAL-1 to bind sterol in vitro. BmVAL-1 complements the in vivo sterol export phenotype of yeast mutants lacking their endogenous SCP/TAPS proteins. The in vitro sterol-binding affinity of BmVAL-1 is comparable with Pry1, a yeast sterol transporting SCP/TAPS protein. Sterol binding of BmVAL-1 is dependent on divalent cations. BmVAL-1 also has a large open palmitate-binding cavity, which binds palmitate comparably to tablysin-15, a lipid-binding SCP/TAPS protein. The central cavity, CBM and palmitate-binding cavity of BmVAL-1 are interconnected within the monomer with channels that can serve as pathways for water molecules, cations and small molecules.

Re-evaluation of IL-10 signaling reveals novel insights on the contribution of the intracellular domain of the IL-10R2 chain.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that plays a key role in maintaining immune homeostasis. IL-10-mediated responses are triggered upon binding to a heterodimeric receptor complex consisting of IL-10 receptor (IL-10R)1 and IL-10R2. Engagement of the IL-10R complex activates the intracellular kinases Jak1 and Tyk2, but the exact roles of IL-10R2 and IL-10R2-associated signaling via Tyk2 remain unclear. To elucidate the contribution of IL-10R2 and its signaling to IL-10 activity, we re-evaluated IL-10-mediated responses on bone marrow-derived dendritic cells, macrophages and mast cells. By using bone marrow from IL-10R-/- mice it was revealed that IL-10-mediated responses depend on both IL-10R1 and IL-10R2 in all three cell types. On the contrary, bone marrow-derived cells from Tyk2-/- mice showed similar responses to IL-10 as wild-type cells, indicating that signaling via this IL-10R2-associated kinase only plays a limited role. Tyk2 was shown to control the amplitude of STAT3 activation and the up-regulation of downstream SOCS3 expression. SOCS3 up-regulation was found to be cell-type dependent and correlated with the lack of early suppression of LPS-induced TNF-α in dendritic cells. Further investigation of the IL-10R complex revealed that both the extracellular and intracellular domains of IL-10R2 influence the conformation of IL-10R1 and that both domains were required for transducing IL-10 signals. This observation highlights a novel role for the intracellular domain of IL-10R2 in the molecular mechanisms of IL-10R activation.

Schistosome egg antigens, including the glycoprotein IPSE/alpha-1, trigger the development of regulatory B cells.

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.

Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants.

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.

Feeding preference as a main determinant of microscale patchiness among terrestrial nematodes.

Soil biota are responsible for essential ecosystem services such as carbon storage, nutrient cycling and water retention. However, assessment of the condition of soil biota is hampered by an overwhelming level of diversity. With representatives in all trophic levels of the food web, nematode communities can be used as bioindicators. Accurate assessment of nematode assemblages requires insight into the distribution of specimens with distinct food preferences. With the availability of taxon-specific quantitative PCR assays, distribution patterns of multiple nematode groups can be investigated simultaneously. Here, microscale patchiness of 45 nematode taxa was studied on 12 sampling sites (each with four adjacent microplots) located on arable fields or semi-natural grasslands ('system'), and on marine, river clay or sandy soils ('soil type'). From each microplot, five composite samples were collected. Contrary to our expectations, an increase in the number of cores per composite sample did not result in more accurate measurements, and apparently the levels of microscale patchiness of the taxa are low compared to what has been reported for oligophagous plant-parasites. System and soil type did not affect microscale distribution. To investigate the level of patchiness in more detail, detection probability (DP) and variability of abundances were calculated. Common and widespread bacterivorous and fungivorous taxa had DP ≥ 90%, confirming low level of microscale patchiness. With DPs of 40%-70%, predators and most omnivores showed degrees of local clustering. An overview of mean variabilities of abundances is presented that offers insight into how feeding preferences impact the microscale distribution both between and within trophic groups.