Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
  • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
  • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
  • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms

Definitions

• the present invention relates to N-glycans comprising L-fucopyranosyl that is a-1 ,3-lin- ked to the distal /V-acetylglucosamine of the N-glycan core, preferably N-glycans comprising D-galacto-pyranosyl-a-1 ,2-L-fucopyranosyl that is a-1 ,3-linked to the distal /V-acetylglucosamine of the N-glycan core as well as glycoconjugates comprising and cells containing these N-glycans.
• the invention is directed to medical uses, pharmaceutical compositions, food and animal feed products comprising the N-glycans, N-glycan-conjugates and/or cells of the invention.
• Parasitic worms or helminths are eukaryotic parasites that live inside their host and feed off living cells. They are categorized into cestodes, trematodes and nematodes. Typical diseases mediated by helminths are ascariasis, dracunculiasis, elephantiasis, hookworm, lymphatic filariasis, onchocerciasis, schistosomiasis and trichuriasis, The "roundworms" or "nematodes” are the most diverse phylum of pseudocoelomates and one of the most diverse of all animals. Nematode species are difficult to distinguish; over 80,000 have been described, of which over 15,000 are parasitic.
• Nematodes are ubiquitous in freshwater, marine and terrestrial environments. Most of them are predators of bacteria and fungi. Parasitic forms include pathogens of plants, animals and also humans.
• Caenorhabditis elegans (C. elegans) is a model nematode and is unsegmented, vermiform, bilaterally symmetrical with a cuticle integument, four main epidermal cords and a fluid-filled pseudocoelomate cavity. In the wild it feeds on bacteria that develop on decaying vegetable matter. The glycobiology of the organism is intensely investigated (reviewed in Berninsone, Wormbook, 1 -22, 2006; Schachter, Curr. Opin. Struct. Biol., 14:607, 2004). In particular, the N-glycosylation pattern of C. elegans is well characterized and was recently reviewed in Paschinger et al.
• Characteristic nematode N-glycans are e.g. D-galactopyranosyl-3-1 ,4-L-fucopyra- nosyl-a-1 ,6-D-GlcNAc (Gal-Fuc) epitopes at the core of N-glycans from C. elegans (Hannemann et al. Glycobiology, 16:874, 2006) as well as the "plant epitope", the a-1 ,3- fucosylation on the Asn-linked GlcNAc-residue of N-glycan cores.
• WO 2011/047794 A2 teaches the use of glycans as a medicament for treating helminthic infections and immune diseases.
• the document discloses medical utility for galacto-pyranosyl- ⁇ -1 ,4-containing saccharides as well as for glycoconjugates thereof.
• the glycans are D-galacto-pyranosyl-3-1 ,4-L-fucopyranosyl-contai- ning saccharides and D-galacto-pyranosyl-3-1 ,4-L-fucopyranosyl-a-1 ,6-GlcNAc- containingsaccharides such as GnGnF 6 Gal- and MMF 6 Gal-containing saccharides.
• N-glycan which comprises L-fucopy- ranosyl that is a-1 ,3-linked to the distal /V-acetylglucosamine of the N-glycan core.
• glycan refers to mono-, oligo- or polysaccharides of homogenous or heterogenous composition with regard to linkage, substitution, modify- cation or identity of the monosaccharide building blocks.
• glycans are categorized into N-, O- or lipoglycans depending on the type of bond and conjugate component, e.g. a polypeptide or lipid.
• N-linked glycans are attached to a nitrogen of aspa- ragine or arginine side-chains.
• N-glycan is meant to indicate any glycan comprising the below-defined N-glycan core, preferably but not necessarily bound to a nitrogen, preferably an amine, more preferably an amine of an asparagine, glutamine or arginine, either isolated or forming part of a polypeptide.
• N-glycan also encompasses those glycans, wherein the proximal N- acetylglucosamine is unbound or bound to the nitrogen of a natural or non-natural compound such as a polymer, e.g. polyacrylamide, or polypeptides containing a biologically suitable glycosylation site such as Asn-X-Ser/Thr, wherein X is any amino acid except Pro.
• N-glycan core is defined herein to mean a glycan comprising at least Man- ⁇ ,4-GlcNAc ⁇ 1 ,4-GlcNAc, i.e. the mannosylchitobiosyl core as indicated in Fig. 3A, preferably comprising (Man-a1 ,6-)(Man-a1 ,3-) ⁇ 8 ⁇ - ⁇ 1 ,4-GlcNAc ⁇ 1 ,4-GlcNAc as indicated in Fig. 3A, more preferably comprising (Man-a1 ,3-) ⁇ 8 ⁇ - ⁇ 1 ,4-GlcNAc ⁇ 1 ,4- GlcNAc.
• the distal /V-acetylglucosamine of the N-glycan core is the one bound to mannose and is located next to the proximal (also termed innermost) /V-acetylglucosamine, which is bound to asparagines or glutamine in nature.
• the N-glycan of the invention comprises D-galacto-pyra- nosyl-a-1 ,2-L-fucopyranosyl that is a-1 ,3-linked to the distal /V-acetylglucosamine of the N-glycan core.
• the N-glycan of the invention is a ga- lactoside-containing oligo/polysaccharide and/or glycoconjugate.
• the N-glycan core is selected from galacto-pyranosyl-3-1 ,4-containing oligo/polysaccharides and/or glycoconjugates, preferably D-galacto-pyranosyl- ⁇ -1 ,4-L-fucopyranosyl-containing oligo/polysaccharides and/or glycoconjugates, more preferably D-galacto-pyranosyl-3-1 ,4-L-fucopyranosyl-a- 1 ,6-GlcNAc-containing oligo/polysaccharides and/or glycoconjugates, most preferably GnGnF 6 Gal- and/or MMF 6 Gal-containing oligosaccharides and glycoconjugates.
• galacto-pyranosyl-3-1 ,4-containing oligo/polysaccharides and/or glycoconjugates preferably D-galacto-pyranosyl- ⁇ -1 ,4-L-fu
• glycoconjugate refers to conjugates of glycans and carrier molecules, for example, glycoconjugates produced enzymatically (in vitro or in vivo) or chemically, preferably glycans conjugated to carrier molecules selected from polypeptides, virus-like particles (VLP ' s), natural and non-natural polymers, lipids and any other type of molecule capable of being conjugated to a glycan of the invention.
• Typical embodiments of glycoconjugates are conjugates of glycans and carrier proteins, e.g. inactivated bacterial toxins or keyhole limpet hemocyanin (KLH).
• the N-glycan core of the N-glycan of the present invention is a fucoside-containing oligo/polysaccharide and/or glycoconjugate. More preferably, the N-glycan core is selected from L-fucopyranosyl-a-1 ,3-containing oligo/polysaccharides and/or glycoconjugates, more preferably L-fucopyranosyl-a-1 , 3-GlcNAc-containing oligo/- polysaccharides and/or glycoconjugates, most preferably GnGnF 3 - and/or MMF 3 -contai- ning oligosaccharides and glycoconjugates.
• N-glycan core is selected from
• glycoconjugates most preferably GnGnF 6 Gal- and/or MMF 6 Gal-containing oligosaccharides and glycoconjugates and/or
• the N-glycan of the present invention is either isolated or purified, preferably purified at least to the extent that it is no longer comprised in a complete cell that naturally produces the N-glycan.
• the term "purified N-glycan” as used herein indicates that the N-glycan forms part of a composition from which some or even most of the cellular components of the cells producing the N-glycan have been removed, e.g. by common protein purification techniques.
• at least 10, 20, 30, 40, 50, more preferably more than 50, most preferably more than 80 or 90 % by weight of the cellular components of the cells producing the N-glycan have been removed.
• isolated' indicates that the composition of the N-glycan comprises less than 10 %, preferably less than 5 %, more preferably less than 1 % by weight of the components of the cells producing the N-glycan.
• the present invention is also directed to N-glycans of the invention forming part of cells that do not naturally produce these (wild type cells), but which have been manipulated to produce these, e.g. by recombinant techniques.
• the present invention also relates to non-natural cells producing the N-glycans of the invention, preferably bacterial or insect cells, for example Escherichia coli, Salmonella enterica, Trichopulsia ni or Spodoptera frugiperda cells.
• the non-natural cells, preferably bacterial and insect cells present the N-glycans of the invention on the cell surface.
• N-glycans of the present invention encompass N- glycans, wherein
• the glycans are displayed on the surface of cells, preferably on bacterial cells, more preferably on enterobacteria, more preferably Escherichia coli or Salmonella enterica, or on the surface of insect cells, preferably Trichopulsia ni or Spodoptera frugiperda;
• the glycans form part of a glycoproteinconjugate, wherein the protein is preferably selected from the group consisting of, horseradish peroxidase (HRP), Bromelain, Jack Bean mannosidase, Ulex europaeus agglutinin (UEA), Erythrina cristagalli lectin, honeybee phospholipase A2, keyhole limpet hemocyanin (KLH) and haemocyanins of other mollusks such as Limulus polyphemus, a functional fragment or derivative of any of these,
• HRP horseradish peroxidase
• Bromelain Bromelain
• Jack Bean mannosidase Ulex europaeus agglutinin
• Erythrina cristagalli lectin Erythrina cristagalli lectin
• honeybee phospholipase A2 keyhole limpet hemocyanin
• VLPs virus-like particles
• VLPs selected from the group consisting of bluetongue virus VLPs, ⁇ VLPs, porcine parvovirus VLPs, porcine circovirus VLPs, Newcastle disease virus VLPs, avian influenza virus VLPs, Rift Valley fever virus VLPs, African horse sickness virus VLPs,
• the glycans are displayed on the surface of polymers, preferably polymers selected from the group consisting of polyacrylamides, polyamines, preferably spermine and spermidine.
• a protein as used above is meant to indicate that the protein can by fragmented, e.g. enzymatically or chemically, and derivatized, e.g. by substitution, deletion, addition or modification of amino acids to the extent that the function, either biological function or carrier function is not abrogated.
• the present invention relates to a cell, preferably bacterial or insect cell comprising an N-glycan of the invention, preferably displaying said N-glycan on its surface, preferably bacterial or insect cells selected from the group consisting of Escherichia coli, Salmonella enterica, Trichopulsia ni or Spodoptera frugiperda cells.
• the N-glycan epitopes of the present invention confer an increased sensitivity for treating and/or preventing a helminthic, preferably a nematode infection or an immune disease when compared to other N-glycan epitopes, e.g. those taught in WO 201 1/047794 A2.
• the N-glycans of the present invention have medical utility for treating and/or preventing a helminthic, preferably a nematode infection or an immune disease.
• the present invention pertains to an N-glycan or cell of the invention for medical use, preferably for treating and/or preventing a helminthic, preferably a nematode infection or an immune disease.
• the invention relates to an N-glycan or cell of the invention for producing a medicament, preferably a medicament for treating and/or preventing a helminthic, preferably a nematode infection or an immune disease.
• glycans should be formulated to elicit glycan-specific antibody-based immune responses in the treated animal or person.
• glycan-specific antibody immunogenicity can be achieved by conjugation to or co-formulation of immune adjuvants.
• the glycans can be presented in or on inactivated or live cells presenting the glycans, preferably on the cell surface, or in the form of a homoge- nate thereof, or in mixture with immunity enhancing cells.
• the glycans are presented by, preferably displayed on the surface of bacterial cells, preferably enterobacteria, more preferably Escherichia col i or Salmonella typhimurium, or of insect cells, preferably Trichopulsia ni or Spodoptera frugiperda cells.
• the N-glycan or cell of the invention is used for treating and/or preventing a helminthic infection, preferably nematode infection resulting from a helminth selected from the family Trichostrongylidae, preferably Haemonchus contortus, Trichostrongylus colubriformis, Teladorsagia circumcincta, Cooperia oncophora, Nema- todirus battus, Ostertagia leptospiarias, Chabertia ovina, Oesophagostomum dentatum, and nematode species Trichinella spiralis, Trichuris trichuria, Angiostrongylus vasorum, Ancylostoma caninum, Ancylostoma duodenale, Ancylostoma ceylanicum, Necator ame- ricanus, Dictyocaulus spp., Ascaris lumbricoides, Ascaris su
• the N-glycan or cell of the invention is used for treating and/or preventing an immune disease selected from the group consisting of allergies, preferably allergies against plants and mites, and autoimmune diseases, preferably Crohn's disease.
• the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising at least one N-glycan or cell of the present invention as described above.
• Said N-glycan most preferably comprises
• N-glycan core is selected from
• a further aspect of the invention relates to a food product or animal feed comprising N-glycans or cells according to the invention for providing the treatment and/or preventive medical effects described above, in particular anti-helminthic and immune- stimulating or suppressing effects.
• the invention relates to a food or feed for humans or animals, preferably livestock, comprising N-glycans and/or N-glycan-com- prising cells as defined above and a physiologically acceptable excipient and/or food stuff, preferably N-glycan-comprising bacteria, in particular enterobacteria, more preferably human or livestock enterobacteria, most preferably Escherichia col i or Salmonella typhimurium.
• enterobacteria more preferably human or livestock enterobacteria, most preferably Escherichia col i or Salmonella typhimurium.
• insect cells preferably Tri- chopulsia ni or Spodoptera frugiperda cells can be used for the food or feed of the invention.
• such a food or feed would greatly reduce helminth colonisation in humans or livestock, respectively, and/or stimulate or suppress the immune system against helminth-related antigens.
• the present invention relates to a method for treating and/or preventing helminthic, preferably nematode infections and/or immune diseases, comprising administration of an N-glycan or cell, pharmaceutical composition, food or feed of the present invention to a human or animal in need thereof in a physiologically active amount.
• compositions of the invention may be administered in any conventional dosage form in any conventional manner.
• Routes of administration include, but are not limited to, intravenously, intramuscularly, subcutaneously, intranasally, intrasynovially, by infusion, sublingually, transdermal ⁇ , orally (e.g. tablet, gavage), topically or by inhalation.
• the preferred modes of administration are oral, intravenous and intranasal, oral and intranasal being most preferred.
• N-glycans and cells comprising these N-glycans according to the invention may be administered alone or in combination with adjuvants that enhance stability and/or im- munogenicity of the medically effective compounds, facilitate administration of pharmaceutical compositions containing them, provide increased dissolution or dispersion, increase propagative activity - if cells are involved, e.g. cells producing the medically effective compounds, provide adjunct therapy, and the like, including other active ingredients.
• Recombinant or native digestive proteases of parasitic helminths preferably gut proteases of animal parasitic nematodes, more preferably aminopeptidase H1 1 of Hae- monchus contortus or aspartic protease APR-1 of Ancylostoma caninum, may be combined with the N-glycans or cells mentioned above.
• the digestive proteases are combined with N-glycans, more preferably with MMF 6 Gal or MMF 3 , for example by using a heterologous expression system, preferably insect cells overexpressing nematode glycosyl- transferases, more preferably in SF9 cells overexpressing GALT-1 or FUT-1 from C. elegans (the functional expression of GALT-1 in SF9 cells was demonstrated previously: PCT 50086).
• a composition combining said digestive proteases and N-glycan epitopes of the invention will provide an effective medicament, preferably a vaccine, in particular against parasitic helminths.
• the present invention is also directed (i) to compositions, preferably pharmaceutical compositions, food products and/or animal feed comprising N-glycans of the invention and/or cells comprising these together with recombinant or native digestive proteases, preferably recombinant digestive proteases, of parasitic helminths including functional fragments and functional derivatives thereof, i.e. fragments and derivatives still comprising at least some of the original protease activity, as well as directed (ii) to corresponding uses of these compositions, products and feed, preferably for treating and/or preventing helminthic infections.
• compositions preferably pharmaceutical compositions, food products and/or animal feed comprising N-glycans of the invention and/or cells comprising these together with recombinant or native digestive proteases, preferably recombinant digestive proteases, of parasitic helminths including functional fragments and functional derivatives thereof, i.e. fragments and derivatives still comprising at least some of the original protease activity,
• glycoproteins naturally display glycans that are involved in glycan-mediated nematotoxicity. At the same time these glycoproteins are known to be highly immunogenic. These proteins include keyhole limpet hemocyanin (KLH) whose N-glycans carry the Gal- ⁇ -1 ,4-Fuc-a-1 ,6 epitope on the core (Wuhrer et al, Bio- chem. J., 378:625, 2004) and the Fuc-a-1 ,3-GlcNAc epitope on the antenna (Geyer et al, J. Biol.
• KLH keyhole limpet hemocyanin
• N-glycans comprise N-glycans and have medical use for immunization against parasitic helminths, more preferably for immunization of livestock against parasitic nematodes, most preferably for immunization of sheep against Haemonchus contortus.
• the present invention relates to (i) the use of KLH, HRP, Bromelain, Jack Bean mannosidase, Ulex europaeus agglutinin (UEA), honeybee phospholipase A2 and haemocyanines of mollusks such as Limulus polyphe- mus for producing glycoconjugates comprising N-glycans of the present invention, in particular for preparing a medicament, preferably for treating and/or preventing helminthic infections, preferably parasitic helminthic infection as well as (ii) corresponding compositions, preferably pharmaceutical compositions, food products and/or animal feed comprising these.
• Pharmaceutical dosage forms of the N-glycans and cells comprising N-glycans of the invention as described herein include pharmaceutically acceptable carriers and/or adjuvants known to those of ordinary skill in the art.
• These carriers and adjuvants include, for example, ion exchangers, alumina, aluminium stearate, lecithin, serum proteins, buffer substances, water, salts, electrolytes, cellulose-based substances, gelatine, water, pretrolatum, animal or vegetable oil, mineral or synthetic oil, saline, dextrose or other saccharide and glycol compounds such as ethylene glycol, propylene glycol or polyethylene glycol, antioxidants, lactate, etc.
• Preferred dosage forms include tablets, capsules, solutions, suspendsions, emulsions, reconstitutable powders and transdermal patches.
• Methods for preparing dosage forms are well known, see, for example, H. C. Ansel and N. G. Popovish, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5 th ed., Lea and Febiger (1990) and, in particular, Pastoret et al., Veterinary Vaccinology, Elsevier March 1999).
• Dosage levels and requirements are well-recognized in the art and may be selected by those of ordinary skill in the art from available methods and techniques suitable for a particular patient. As the skilled artisan will appreciate, lower or higher doses may be required depending on particular factors. For instance, specific doses and treatment regimens will depend on factors such as the patient's (human or animal) general health profile, the severity and course of the patient's disorder or disposition thereto, and the judgment of the treating physician or veterinarian.
• Fig. 1 shows the mass spectrometry and HPLC analysis data of N-glycans isolated from a C. elegans hex-2; hex-3 double mutant displaying the new glycan structure.
• N-glycans were released by PNGase F and analysed by MALDI-TOF MS as free oligosaccharides prior to fluorescent labelling.
• Hex3- 4 HexNAc 2 - 4 Fuc 0 -2 (labelled a-g in with the m/z 933, 1 136, 1282, 1339, 1428, 1444and 1590, respectively; mass of the native form), a corresponds to Hex3HexNAc 2 , b to Hex3HexNAc3, c to HexsHexNAcsFuc, d to Hex3HexNAc 4 , e to Hex3HexNAc3Fuc 2 , fto Hex 4 HexNAc3Fuc, g to Hex 4 HexNAc3Fuc2)
• Fig. 2 shows further structure determinations of the new Hex 4 HexNAc 3 Fuc 2 glycan epitope by enzymatic and chemical analyses.
• the 2D HPLC purified glycan (m/z 1646, Hex 4 HexNAc 3 Fuc 2 -PA, (A)) was subject to various exoglycosidase digestions followed by
• MALDI-TOF MS B-E.
• the glycan was insensitive to bovine kidney a-L-fucosidase (B), but sensitive to digestion by Aspergillus 31 ,4-galactosidase (C), which resulted in a reduced mass (m/z 1484) on MALDI-MS indicative of loss of terminal galactose.
• the loss of a deoxyhexose (146, fucose) only occurred with a combination of ⁇ -galactosidase and a-L-fucosidase(D).
• Fig. 3A shows a scheme of the eukaryotic N-glycan core to explain the concept of distal and proximal GlcNAc.
• the distal GlcNAc is the GlcNAc linked ⁇ 1 ,4 to the reducing-end GlcNAc which is linked to the asparagine side chain in polypeptides.
• Fig. 4 shows the RP-amide HPLC analysis of Ascaris and Oesophagostomum N- glycans.
• glycans hex-2;hex-3 double mutant The putatively phosphorylcholine-modified glycan (m/z 1503) in O. dendatum fraction 22 was also HF sensitive (f ⁇ k). Furthermore, glycans with m/z 1281 , 1297, 1443, 1485 and 1646 (marked with an asterisk) were sensitive to Aspergillus- ⁇ ,4-galactosidase.
• Fig. 5 shows the detailed MS/MS spectra of HPLC-fractionated Ascaris and Oesophagostomum N-glycans.
• Glycan species found in fractions 17 and 22 from A. suum and O. dendatum (see Fig. 5) were subjected to MS/MS to reveal the occurrence of the m/z 608 fragment present in the m/z 1646 glycan from the C. elegans double hexosaminidase mutant.
• Fig. 6 shows a bar chart of the lectin sensitivity of C. elegans wild-type and the hex-2;- hex-3 mutant. Toxicity assays comparing the development of the hex-2;hex-3 mutant (H2H3, black bars) to the development of wildtype (N2, white bars) nematodes when fed on increasing percentages of E.coli expressing four different nematotoxic lectins (CGL2, CCL2, TAP1 , and XCL) are shown. The error bars represent standard deviation from the mean.
• Fig. 7 shows a bar chart of the dose dependency of the toxicity induced by TAP1 and XCL.
• Toxicity assays comparing developmental profiles of hex-2;hex-3 mutant (H2H3) to wild-type (N2) nematodes when fed on different percentages of E. coli expressing either TAP1 or XCL lectin are given. Error bars represent standard deviation from the mean. The data indicates a far higher degree and earlier onset of developmental arrest by TAP1 as compared to XCL, when more than 1 % of the E. coli fed to the nematodes expressed the lectin.
• Fig. 8 shows the analysis of pig sera from a healthy animal and 2 animals infested with either A. suum (labelled Ascaris) or O. dendatum (labelled Osi). These sera were tested for reactivity against different C. elegans strains expressing the new glycan epitope D- galacto-pyranosyl-a-1 ,2-L-fucopyranosyl-a-1 ,3-linked to the distal /V-acetylglucosamine of the N-glycan core.
• Figure 8A is an SDS-PAGE stained with Coomassie blue to show equal protein loading amounts.
• Figure 8B shows the control membrane probed with secondary antibody only.
• Figure 8C shows Ponceau-Red-stained membranes to confirm transfer or equal protein amounts.
• Figure 8D are immunoblots developed using serum of a healthy pig as well as sera of animals infested with either A. suum or O. dendatum against several worm extracts.
• Fig. 9 shows the quantitative analysis of the reaction of pig sera from a healthy animal and 2 animals infested with either A. suum or O. dendatum by ELISA. Equal amounts of protein extracts were spotted on a 96 well plate and binding of IgG or IgM from different pigs was detected by colour development at 405 nm after 10 min.
• Figure 9A the reaction against the 2 different C. elegans strains N2 and hex2hex3 is shown while Figure 9B depicts the reaction against extracts of A. suum and O. dendatum.
• the glycan structures of a C. elegans mutant in the hexosaminidase genes hex-2 and hex-3 were evaluated as follows: N-glycans were released from worm peptic peptides using peptide:/V-glycosidase F (capable of removing most eukaryotic N-glycans other than those carrying a1 ,3-fucose on the reducing terminal GlcNAc), followed by peptide:/V-glycosidase A (which is capable of removing core a1 ,3-fucosylated N-glycans), according to the procedures described previously in Paschinger et al., Glycobiology 2012; 22:300-13.
• the /V-glycome of the mutant was profiled by MALDI-TOF MS (Ultraflex I, BrukerDaltonics, Germany) in positive mode.
• glycans were pyridyl-ami- nated and subjected to NP-HPLC and RP-HPLC either singly or in succession. Free glycans were labelled with 2-aminopyridine prior to fractionation by normal phase HPLC (NP-HPLC) and reversed-phase HPLC (RP-HPLC). All the HPLC peaks were collected and examined by MALDI-TOF MS using either 2, 5-dihydrobenzoic acid (DHB) or 6-aza- 2-thiothymine (ATT) as matrices: Predicted glycan species were subjected to fragmentation by MS/MS (post-source decay) and the spectra were analyzed manually.
• NP-HPLC normal phase HPLC
• RP-HPLC reversed-phase HPLC
• the location of the putatively a-linked galactose and second fucose was initially less clear.
• the PA-glycan was subjected to GC-MS linkage analysis which indicated the presence of terminal galactose, terminal and di-substituted GlcNAc, 2- and 3-substituted mannose as well as 2- and 4-substituted fucose.
• Example 2 The new glycan epitope in parasites A. suum and O. dendatum
• suum consists of three sub-fractions, the most dominant of which co-elutes with the purified glycan from the C. elegans mutant (Fig. 4A).
• Fractions 17 and 22 contained a number of species and some of these glycans were sensitive to ⁇ 1 ,4-galactosidase and/or HF (Fig. 4B) indicating the presence of galactose, a1 ,3-linked fucose and phosphorylcholine modifications. This is also confirmed by the MS/MS data.
• the fragment of m/z 369.2 (labelled a in Fig 5A) upon MS/MS of the HF-sensitive O.
• dendatum glycan of m/z 1503 (labelled in Fig 4B) was consistent with a terminal PC-HexNAc (Fig. 5A).
• MS/MS data of a number of other glycans resulted in the appearance of fragments of m/z 607.7-608.2 (Figs 5 B-F, peaks labelled f, /, k, m, n).
• the galactosidase-sensitive O. dendatum glycan with a composition of Hex 4 HexNAc 2 Fuc i (m/z 1297; labelled *c in Fig 4B and unlabelled in 5B) is predicted to possess a GalFuc modification of the reducing-terminal GlcNAc residue.
• Another example is the HF- and galactosidase-sensitive O. dendatum glycan of m/z 1443 (Hex 4 Hex-
• GlcNAc galactosylated fucose on the proximal (reducing terminal) GlcNAc as well as unsubstituted or substituted a1 ,3-fucose attached to the distal core GlcNAc.
• Example 3 The new glycan epitope leads to hypersensitivity towards certain lectins
• a C. elegans liquid toxicity assay was performed on the wild-type N2 strain and on the hex-2;hex-3 double mutant with the following fungal lectins: CCL2, CGL2, TAP1 and XCL.
• E. coli were mixed at different percentages with vector control transformed E. coli. The percentage of individuals reaching each developmental stage was quantified after 48 h of incubation at 20°C; each treatment was performed in quintuplicate.
• the binding specificity of the lectins were the following: CCL2 and CGL2 from Coprinopsis cinerea target core a1 ,3-fucose and GalFuc epitopes, respectively; TAP1 from Sordaria macrospora binds to T-antigen (Gal31 ,3GalNAc); and XCL from Xeroco- mus chrysenteron binds both T-antigen and terminal GlcNAc.
• L1 larve of wild-type and double mutant C. elegans were fed with mixed populations of E. coli with a certain percentage of the bacteria expressing recombinant forms of the lectins. The percentage of larvae reaching L4 was determined.
• Fig. 6 shows that the sensitivity to CGL2 and XCL was significantly increased in the hex-2;hex-3 double mutant, whereas sensitivity to CCL2 was abolished due to the lack of core a1 ,3-fucose. Only small differences in the nematotoxicity were observed with TAP1. An closer examination of the developmental profile of the arrested worm population indicated no development to adults of either wild- type or mutant worms in the presence of a concentration of >5% of TAP1 -expressing E. coli, whereas wild-type worms showed a lower rate of developmental arrest across the concentration range (5-50%; see Fig. 7).
• Example 4 The new glycan epitope is immunogenic
• A. suum, O. dendatum (male and female), C. elegans N2, C. elegans hex2hex3 (labelled

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• 2013
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