WO2010130683A1

WO2010130683A1 - Glycan antigen

Info

Publication number: WO2010130683A1
Application number: PCT/EP2010/056353
Authority: WO
Inventors: Irma Marianne Van Die; Boris Tefsen
Original assignee: Stichting Voor De Technische Wetenschappen; Vereniging Voor Christelijk Hoger Onderwijs
Priority date: 2009-05-11
Filing date: 2010-05-10
Publication date: 2010-11-18
Also published as: UY32621A; AR076555A1

Abstract

The present invention relates to the field of veterinary parasite immunology. More specifically the invention relates to a carbohydrate structure useful for the immunoprotective treatment and prevention or reduction of parasitic nematode infections in ruminants. Particularly the invention pertains to an antigen comprising the carbohydrate structure, to an antibody specific for the carbohydrate structure, to a vaccine comprising the antigen or the antibody, to the use of the antigen, antibody or vaccine, and to methods of treatment and methods of manufacture.

Description

TITLE: Glycan antigen

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

The treatment of parasitic nematode (or nematode helminth) infections in ruminants forms a major need in livestock farming, and a challenge to the veterinary medicine community. The infections cause a range of side effects and diseases which result in suffering of the infected animals and economic losses to the farmers. For instance the parasites may be eating from the intestinal sidewalls or from the blood, leading to internal bleeding, anaemia and indigestion. Also the sheer number of the worms that may develop and migrate through the body may cause mechanical obstructions of lungs, intestines, or the vascular system. The general reduction in physical condition that results leaves the infected animal prone to secondary infections.

Highly relevant also to commercial animal keeping is the economic harm caused due to a lower yield or quality of, e.g., meat, milk, or offspring, and because of the increased expenses for feed consumption or veterinary care.

Of special interest are the parasitic ruminant nematodes that can be grouped under the denomination^ gastrointestinal nematodes (GI nematodes) of cattle and small ruminants^ the genera Haemonchus, Ostertagia, Teladorsagia, Trichostrongylus and Dictyocaulus. These parasites enter and leave the host via the digestive tract, as well as mature one or more of their developmental stages (larval stages 1 - 5, or adult stage) in the gastro -intestinal tract of a ruminant. Taxonomically they are grouped in the super-family Trichostrongyloidea, and in the sub-order of the Strongylida. In that sense these are essentially different from other well known parasitic nematodes such as the genera Ascaris, and Schistosoma! for these the clinical relevance is mostly limited to human and porcine species, which -being meat eaters and omnivores- have an essentially different gastro-intestinal biology and -environment than grazing ruminants.

Traditional antiparasitic (anthelminthic) drugs to a considerable extent have ceased to be effective, due to increasing resistance in nematode populations. For example no effective drugs exist anymore against a sheep gastric parasite, the nematode Haemonchus contortus, also known as red stomach worm, wire worm or Barber's pole worm. This is particularly problematic since H. contortus is a very common parasite and one of the most pathogenic ruminant nematodes. Thus, effective means to combat such nematodes are desired in order to improve ruminant's quality of life and to avoid the large economic losses caused.

As an improvement to drug therapy, particularly in view of the nematodes' drug-resistance, it is desired to be able to vaccinate ruminants against nematodes such as H. contortus. This, however, has yet been impossible to achieve.

The classical way of providing a vaccine, by growing the organism to be combated, and killing or attenuating it for use in an inactivated- or live- attenuated vaccine, is not normally available for parasites of the genera

Haemonchus, Ostertagia, Teladorsagia, Trichostrongylus and Dictyocaulus! it has proven to be difficult, if not impossible, to achieve full maturity in vitro of all the parasites' developmental stages of larvae and adults.

Another approach is that of providing a subunit vaccine, i.e. a vaccine based on a molecule or part thereof carrying an epitope, usually a protein subunit of the organism to be combated. An "epitope" is that part of an antigenic molecule that reacts with the antigen receptor of a T- and/or B- lymphocyte. An epitope will therefore induce and/or activate specific T- and/or B-lymphocytes such that these cells give rise to an immune reaction that interferes with the course of an infection or disease. Thus, through such epitopes, an antigen can induce an immune response via generation of antibodies and/or cellular immunity.

However, in the case of nematodes it has so far turned out to be virtually impossible to provide an antigen that was capable of inducing an effective immune response against the entire worm. This is to a large extent because nematodes are relatively large "micro"-organisms, displaying a bewildering array of candidate antigens of various biochemical shapes and sizes. In addition, nematodes may possess active mechanisms for immune- evasion, overcoming an immune-response that may be induced. For instance, parasitic nematodes may have masking carbohydrate structures in their outer coat which makes them less immunogenic to the infected hosts' immune system. One example is the presence of a blood group type antigen on parasite- surfaces^: LewisX (also known as CD15, or fucosylated lactosamine), with the structure^ galactose beta 1-4 (fucose alpha 1-3) N-acetylglucosamine, which is commonly also written as Galβl-4(Fucαl-3)GlcNAc.

Additionally the parasite's coat may change its appearance over time, depending on the parasites developmental stage, or environmental selection pressure.

In general, carbohydrate antigens (also: glycan antigens) have been investigated as candidate antigens for parasite vaccines. A brief review of this field was given by Nyame et al. in 2004 (Nyame, A.K., et al., 2004, Archives of

Biochem. and Biophys., vol. 426, p. 182-200).

Examples of carbohydrate structures of parasites that were investigated are: N-acetylgalactosamine beta 1-4 N-acetylglucosamine (GalNAcBl-4GlcNAc; a.k.a. LacDiNAc, or LDN) and: N-acetylgalactosamine beta 1-4 (fucose alpha 1-3) N-acetylglucosamine (GalNACBl-4(Fucal-3)GlcNAc; a.k.a. LacDiNAc- fucose, or LDNF), which were found on the human trematode parasite:

Schistosoma mansoni. These structures have also been described as antigen for diagnosis of human helminth infection, see for instance: WO 2000/017654. For H. contortus the carbohydrate structure LDNF has been described in the context of the parasite's excretory/secretory (ES) glycoproteins, which mixture was tested in vaccination of sheep (Vervelde, L. et ah, 2003,

Glycobiology, vol. 13, p. 795-804,): in lambs vaccinated with ES glycoproteins antibodies were found against the LDNF epitope.

However, in a later study, the LDNF carbohydrate epitope was found not to contribute to the immune response in, inter alia, the Vervelde 2003 study. A reference in this respect is Geldhof et ah, 2005 (Geldhof, P. et ah,

2005, Parasite Immunology, vol. 27, p. 55-60).

The structure: Galal-3GalNACBl-4(Fucal-3)(PC6)GlcNAcBl-3Man-R, with "PC" indicating: phosphorylcholine, "Man": mannose, and "R" a carrier group, has been described by Friedl et al. in 2003 (Friedl, CH. , et al, 2003,

Biochem. J., vol. 369, p. 89-102) as a structure on glycosphingolipids of the porcine nematode Ascaris suum.

Thus an undiminished desire exists to provide an antigen, suitable for use in vaccination for the immunoprotective treatment and prevention or reduction of parasitic nematode infections in ruminants. SUMMARY OF THE INVENTION

In order to better address one or more of the foregoing desires, the invention, in one aspect, is an antigen comprising a carbohydrate satisfying the structural formula: Galαl-3GalNAcβl-4(Fucαl-3)GlcNAc (hereinafter GaI-LDNF).

The invention, in another aspect, is an antigen comprising GaI-LDNF for use as a medicament, in particular for the immunoprotective treatment, prevention or reduction of parasitic nematode infections in a ruminant. Preferably the parasitic nematode is from the order Strongylida, more preferably from the sub-family Trichostrongyloidea, even more preferably selected from the group of genera consisting of Haemonchus, Ostertagia, Teladorsagia, Trichostrongylus and Dictyocaulus.

In yet another aspect, the invention is the use of an antigen comprising GaI- LDNF for the manufacture of a medicament, preferably the medicament is a vaccine, preferably for the immunoprotective treatment, prevention or reduction of parasitic nematode infections in a ruminant.

In a further aspect, the invention is an antibody or part thereof that can bind specifically to an antigen according to the invention, preferably to GaI-LDNF.

In a further aspect the invention is a vaccine comprising GaI-LDNF. Preferably the vaccine is for the immunoprotective treatment, prevention or reduction of parasitic nematode infections in a ruminant. Preferably the parasitic nematode is from the order Strongylida, more preferably from the sub-family Trichostrongyloidea, even more preferably selected from the group of genera consisting of Haemonchus, Ostertagia, Teladorsagia, Trichostrongylus and Dictyocaulus.

In a further aspect, the invention is a method of treatment of parasitic nematode infection in a ruminant, wherein the ruminant is vaccinated with a vaccine comprising GaI-LDNF.

In one or more of the foregoing aspects, the GaI-LDNF is preferably comprised in a chemical structure of the formula GaI-LDNF-R, wherein R denotes an immunogenic carrier structure, preferably selected from the group consisting of carbohydrates, proteins, and lipids, or combinations thereof, to which the GIcNAc moiety of GaI-LDNF is attached. More preferably, the GaI-LDNF is conjugated to an immunogenic protein. DETAILED DESCRIPTION OF THE INVENTION

In a broad sense, the invention provides an antigen, comprising a carbohydrate compound satisfying the structural formula ^: galactose alpha 1-3 N-acetylgalactosamine beta 1-4 (fucose alpha 1-3) N- acetylglucosamine, which is in shortened notation:

Galαl-3GalNAcβl-4(Fucαl-3)GlcNAc, hereinafter: GaI-LDNF.

The description used herein of the chemical structure of the carbohydrate compound comprised in the antigen according to the invention, is in accordance with the common nomenclature in carbohydrate biochemistry. In this respect, the skilled person will immediately understand that the indication of a moiety between brackets (here fucose) denotes the binding of this fucose to the moiety following in reading direction (here N- acetylglucosamine) at a different position than that of the moiety (here N- acetylgalactosamine) preceding the moiety between brackets. Also the abbreviations used are the ones commonly used, thus: "Gal" stands for galactose, "GaINAc" stands for N-acetylgalactosamine, "Fuc" stands for fucose and "GIcNAc" stands for N-acetylglucosamine. Also, a common way is used to represent the stereochemical conformation of the molecular linkage between the carbohydrate moieties, such as alpha 1-3 and beta 1-4, abbreviated as αl-3, and βl-4 respectively.

As will be described herein, and detailed in the examples, the inventors of the present invention have surprisingly found, for the very first time, that the glycan GaI-LDNF can advantageously be used for conjugation to an immunogenic carrier and formulation into a vaccine, which vaccine can induce an effective immune response against a parasitic ruminant nematode.

Therefore, in general, the antigen according to the invention will comprise the GaI-LDNF in a form attached to a carrier, preferably via a spacer moiety. Thus, preferably, the antigen will comprise the structural moiety GaI- LDNF-R, wherein R generally represents an immunogenic carrier structure, which carrier preferably is a carbohydrate, a protein, or a lipid, or combinations thereof.

The use of an immunogenic carrier to activate or enhance the immune response to an epitope is well known in the art. This is because (as is known to the skilled person) in order to actually be antigenic, i.e. to induce an immune response, an antigen needs to be of a certain length; too small fragments will not be processed by antigen presenting cells to fragments that are able as such to associate with MHC molecules, which association is required for proper antigen presentation to lymphocytes, such as in MHC I or MHC II receptor binding. The relatively short carbohydrate structure according to the invention may thus not be antigenic as such, even though it presents several epitopes to the target's immune system! to function as effective antigen the glycan needs to be coupled to a carrier giving it volume and helping to stimulating the target's immune system. When coupled, the glycan antigen according to the invention will be able to induce an immune response that is within the scope of the invention.

The GaI-LDNF carbohydrate structure according to the invention is thus preferably coupled to a carrier -using techniques known in the art- to increase the immune response to the glycan epitopes. For a molecule to be suitable as immunogenic carrier for the invention, it is only relevant that it can induce an immune response and present the glycan antigen according to the invention effectively to the target's immune system. Preferably the immunogenic carrier is a carbohydrate, a protein or a lipid, or combinations thereof. Such carriers (or combinations) conjugated to a carbohydrate may be called by a variety of names: glycoprotein, proteoglycan, glycolipid, lipoglycan, lipopolysaccharide, lipoglycoprotein, etc.. All are within the scope of the invention.

Well known carriers are bacterial toxoids, such as Tetanus toxoid or Diphteria toxoid; alternatively KLH, BSA, or bacterial cell-wall components (derived from) lipid A, etc. may be used. Also polymers may be useful, or other particles or repeated structures such as virus like particles etc.

Preferably the immunogenic carrier is an immunogenic protein, more preferably Tetanus toxoid. As the skilled person will appreciate, the coupling of the carbohydrate structure according to the invention to the immunogenic carrier must be set up in such a way to preserve the biological properties of both. The skilled person is very well capable to select and optimise common methods and materials for efficient, effective and economical coupling, which will determine for instance how many carbohydrate moieties will be bound (on average) per molecule of the carrier.

The term "protein" is meant to incorporate a molecular chain of amino acids. A protein is not of a specific length, structure or shape and can, if required, be modified in vivo or in vitro, by, e.g. glycosylation, amidation, carboxylation, phosphorylation, or changes in spatial folding. Inter alia, peptides, oligopeptides and polypeptides are included within the definition of protein. A protein can be of biologic and/or of synthetic origin.

When referring to the chemical connection between the carbohydrate-, spacer-, and carrier groups described herein, the terms "couple", "attach", "bind" "conjugate" or "connect" have the same meaning. As the skilled person will understand, such couplings can be performed by chemical coupling, -conjugation or -cross-linking, through dehydration, esterification etc., of the carbohydrates moieties of the GaI-LDNF, or of the GaILDNF to the carrier either directly or through an intermediate structure.

Preferably the carbohydrate structure is coupled to the immunogenic carrier via one or more spacer groups. Such spacer groups are molecules that can be of a variety of shapes and sizes! only requirement is that they provide for the efficient and economic coupling, while allowing the biological properties of the carbohydrate- and immunogenic carrier groups to be displayed. Clearly unsuitable would be spacers that are of such a shape and/or size that they disturb the intended function of the glycan epitopes or of the immunogenic carrier in the present invention! for instance by stereochemical hindering of the display of the glycan epitopes or by creating so much distance between the glycan and the carrier that the target's immune system does not respond to the two together. The skilled person is perfectly capable to select and optimize spacer structures for the invention.

Therefore, the GaI-LDNF-R moiety preferably comprises the structure GaI- LDNF-R1-R2, wherein Rl is 61-4GIcNAc and wherein R2 has the same meaning as R.

More preferably, R2 is a spacer structure comprising a diaminopyridine (DAP) moiety. For the derivatisation of glycans with DAP, reference is made to Xia et al. (2005, Nature Meth., vol. 2, p. 845-850).

The DAP moiety can be connected directly or indirectly to the carrier. More preferably, the DAP moiety is linked to a squarate moiety, which can then be connected to the immunogenic carrier. For glycan to protein coupling via squarates, reference is made to Benaissa-Trouw et al. (2001, Inf. and Imm., vol. 69, p. 4698-4701), Hou et al. (2008, Carbohydrate Res., vol. 343, p. 196- 210), Lefeber et al. (2001, Chem. Eur. J., vol. 7, p. 4411-4421), and Kamth et al. (1996, Glycoconjugate J., vol. 13, p. 315-319).

The herein described antigens, antibody and vaccines according to the invention are suitable for use as a medicament, and particularly in the immunoprotective treatment, prevention or reduction of parasitic nematode infections in ruminants. The term "ruminants" includes many domesticated animals, or animals that otherwise are of agricultural, veterinary or economic importance (e.g. domestic herds), such as sheep, goats, cattle, bison, yaks, water buffalo, deer, camels, llamas, alpacas, as well as various wild animals. "Small ruminants" are understood to include sheep, goats, and deer.

Preferred application is to parasitic nematodes that are gastro-intestinal ruminant nematodes, and more preferred to nematodes of the genera Haemonchus, Ostertagia, Teladorsagia, Trichostrongylus, and Dictyocaulus. With respect to the current taxonomic classification of these genera, the skilled person will realise this may change over time as new insights may lead to reclassification into new or other taxonomic groups. However, as this does not change the antigenic repertoire of the organism involved, only its classification, such re-classified organisms are considered to be within the scope of the invention.

More preferably, the antigens, antibody or vaccines according to the invention are used in the immunoprotective treatment of cattle and sheep against Heamonchus contortus infections, or the prevention or reduction of these infections therein.

The advantageous utility of the carbohydrate structure of the invention in the immuneprotection against a number of parasitic nematode genera results from a discovery by the inventors^: parasites of different taxonomic groups, when surviving in the environment of the gastro-intestinal tract of a ruminant, exhibit the same or similar carbohydrate structures to the environment.

Consequently, a glycan antigen effective in immuneprotection against one of these parasites, is also useful for other parasites in the same environmental conditions.

Therefore, the present invention is generally applicable to the group of parasitic nematodes which enter and leave the host via the digestive tract, as well as mature one or more of their developmental stages (larval stages 1 - 5, or adult stage) in the gastro-intestinal tract of a ruminant.

"Protection" (and related terms such as "immunoprotection" and "immunoprotective") as used for the present invention, means to induce an immune response for aiding in preventing, ameliorating, reducing sensitivity for, or treatment of a disease or disorder resulting from infection with a micro^¬ organism, such as for instance a parasitic nematode. The protection is achieved as a result of administering (a composition containing) one ore more antigens derived from that micro-organism in an appropriate form and dose. The term "reduction" relates to reducing susceptibility for microbial infection. In the sense of the present invention this means causing the target ruminant to show a reduction in the number, or the intensity of clinical signs caused by the parasitic nematode infection. This may be the result of a reduced colonization or of a reduced infection rate by the parasite, leading to a reduction in the number or the severity of lesions and effects that are caused by the parasite or by the target's response thereto.

The aforementioned treatment preferably involves vaccination. In this respect, the invention pertains to a vaccine comprising GaI-LDNF, and also to combination vaccines thereof, comprising in addition at least one other vaccine component. The vaccine particularly is a conjugate vaccine comprising GaI- LDNF conjugated with an immunogenic carrier.

The vaccine according to the invention is particularly suitable for use in the immunoprotective treatment, prevention or reduction of parasitic nematode infections, and particularly gastro- intestinal nematode infections, in a ruminant.

In a preferred embodiment the vaccine of the invention serves for use in the treatment, the prevention, or the reduction of infection selected from the group consisting of nematodes of the genera Haemonchus, Ostertagia, Teladorsagia, Trichostrongylus, and Dictyocaulus.

In particular, the vaccine according to the invention serves for use in the immunoprotective treatment of cattle, goats or sheep against Heamonchus contortus infections, or the prevention or reduction of these infections therein.

The vaccination serves to reduce, in the host (i.e. the target ruminant), one or more of the following characteristics of the nematodes infecting that host: the number, the length, and the gender ratio, and/or spread into the environment (such as stable, or meadow). All these reflecting some form of interference by the host's immune system with the health and reproductive capacity of the infecting nematode, thereby reducing the level of damage afflicted to the host and others in its surroundings.

Amongst many others, known mechanisms of an anti-parasite immune response are: so-called rapid expulsion, hypersecretion, and/or the induction of IgE type antibodies.

The vaccine, or the vaccine with additional immunoactive component(s) according to the invention may be applied by all possible routes into or on the body of the ruminant host, such as by injection (into or through the skin, e.g.^: intramuscular, intravenous, intraperitoneal, intradermal, or submucosal, or subcutaneous), by topical application, by spray, or in combination with the feed or drinking water.

The vaccine according to the invention can thus be in several forms, e.g.^: a liquid, a gel, an ointment, a powder, a tablet, or a capsule, depending on the desired method of application to the target. Preferably the vaccine is in the form of an injectable liquid.

The vaccine preferably comprises an adjuvant. An adjuvant in general comprises a substance that boosts the immune response of the host in a non- specific manner. A wide variety of different adjuvants are known in the art.

Examples of adjuvants are Freund's complete and -incomplete adjuvant, vitamin E, non-ionic block polymers and polyamines (e.g. dextransulphate, Carbopol™, Syntex adjuvant or pyran), carbohydrates (glucans and glycans), lipopolysaccharides, saponins, ISCOMs, peptides (e.g. muramyldipeptides, dimethylglycine, or tuftsin), mineral oils (e.g. Bayol™ or Markol™) or biological oils (e.g. squalene, coconut oil, montanide, etc.) or emulsions thereof, Diluvac Forte™, immune stimulatory nucleic acid motifs (e.g. CpG), etc.

Vaccines based upon the antigens according to the invention are also very suitable as marker vaccines. A marker vaccine is a vaccine that allows the discrimination between vaccinated and field-infected subjects e.g. on the basis of a characteristic antibody panel, different from the antibody panel induced by infection with the wild type infectious agent. A different antibody panel is induced e.g. when an immunogenic glycoprotein present on a wild type nematode is not present in a vaccine^: the host will then not make antibodies against that glycoprotein after vaccination.

For instance a simple ELISA test, having wells comprising e.g. the glycan antigen of the invention and wells comprising glycans from the wild type nematode would suffice to test antiserum from vaccinated subjects and tell if these were either vaccinated with the immunogenic composition according to the invention, or suffered from a field infection.

Therefore, an even more preferred form of this embodiment relates to marker vaccines based on the antigens or vaccines according to the invention.

Instead of using the vaccine or the vaccine with additional immunoactive component(s) according to the invention to induce an immune response in the target by active immunization, a further possible utility is so called "passive immunization", also considered to fall under the scope of the present invention. This method of vaccination, passive vaccination, is advantageous when an animal is already infected, and there is no time to allow the natural immune response to be triggered, which may take some weeks. It is also the preferred method for vaccinating immune-compromised animals, such as animals that are young, old, or sick.

Therefore the invention relates to an antibody (or a part thereof) that can bind specifically to GaI-LDNF.

The capacity of an antibody to recognise or "bind" to the epitope for which it is specific is well known in the art. In that sense "specificity" is reflected in the molecular structure of the antigen-binding site of the (variable domain of the) immunoglobulin (antibody) recognising a particular epitope. In practice specificity can be measured or detected by a variety of serological assays such as Elisa, immuno-fluorescence, Ouchterlony (gel- diffusion) etc., all well known in the art. Commonly specific binding of an antibody to its specific ligand is distinguished from aspecific- or background binding by applying a dilution range of the antibody and/or the ligand.

An antibody according to the invention can be produced in a variety of ways, all well known to the skilled person. One method may be by applying a vaccine according to invention to an appropriate animal species, harvesting serum containing these antibodies, purifying them, and using them to quickly but temporarily cause high antibody levels in a subject of the same or another species.

Alternatively monoclonal antibodies may be produced against GaI- LDNF, by applying well known methods to immortalise B-lymphocytes that produce a desired anti-Gal-LDNF antibody. Also such antibodies or parts thereof may be produced by well known recombinant DNA techniques and an appropriate expression system.

A "fragment" of an antibody according to the invention is meant to indicate that besides complete immunoglobulin molecules, also parts thereof are considered to be within the scope of the invention. Fragments of antibodies according to the invention are protein molecules that can still bind to GaI- LDNF. Examples are FAB, scFv, Fv, dAb, or Fd fragments, all well known in the art.

Such fragments may be obtained from intact antibodies by e.g. chemical or enzymatic digestion. Preferably such fragments are obtained from a recombinant expression system, for example a phage- display system.

In an embodiment of the passive vaccination that is within the scope of the invention, pregnant ruminant animals may be vaccinated before delivery, with an intention to protect the offspring passively against disease or infection. Such offspring will then become protected by the maternal antibodies, acquired either by trans-placental passage when unborn, or by drinking milk from the mother after birth; the first milk, the colostrum, then contains high levels of passively protective antibodies.

Vaccines according to the invention, can be administered in amounts containing between 0.1 and 1000 μg of an antigen according to the invention per ruminant target. Smaller or larger doses can in principle be used, and can be optimised to the level of protection required for each type of ruminant by known methods, taking due note of the economy of the costs involved. Preferably a dose of between 1 and 200 μg of the antigen is used. The scheme of the application of the vaccine according to the invention to the target ruminant can be in single or multiple doses, which may be given at the same time or sequentially, in a manner compatible with the dosage and formulation, and in such an amount as will be immunologically effective. The vaccines of the invention are advantageously applied in a single yearly dose.

The vaccine according to the invention comprises a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can be e.g. sterile water or a sterile physiological salt solution. In a more complex form the carrier can e.g. be a buffer. Pharmaceutically acceptable indicates the carrier does not adversely affect the health of the animal to be vaccinated, or at least not to the extent that the adverse effect is worse than the effects seen when the animal would not be protected by vaccination.

Often, a vaccine is mixed with stabilizers, e.g. to protect degradation-prone components from being degraded, to enhance the shelf- life of the vaccine, or to improve freeze-drying efficiency. Useful stabilizers are i.a. SPGA (Bovarnik et ah, 1950, J. Bacteriology, vol. 59, p. 509), skimmed milk, gelatine, bovine serum albumin, carbohydrates e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein or degradation products thereof, and buffers, such as alkali metal phosphates.

The vaccine according to the invention may additionally comprise a so-called "vehicle". A vehicle is a compound to which the compositions according to the invention adhere, without being covalently bound to it. Such vehicles are i.a. bio-microcapsules, micro-alginates, liposomes, macrosols, aluminium- hydroxide, -phosphate, -sulphate or -oxide, silica, Kaolin®, and Bentonite®, all known in the art.

An example is a vehicle in which the antigen is partially embedded in an immune- stimulating complex, the so-called ISCOM® (EP 109.942, EP 180.564, and EP 242.380). In addition, the vaccine according to the invention may comprise one or more suitable surface-active compounds or emulsifiers, e.g. Span® or Tween®. It goes without saying that other ways of adjuvating, adding vehicle compounds or diluents, emulsifying or stabilizing a vaccine are also within the scope of the invention. Such additions are for instance described in well-known handbooks such as: "Remington: the science and practice of pharmacy" (2000, Lippincot, USA, ISBN: 683306472), and: "Veterinary vaccinology" (P. Pastoret et al. ed., 1997, Elsevier, Amsterdam, ISBN 0444819681).

The vaccine according to the invention can equally be used as prophylactic and as therapeutic treatment, and interferes with the establishment and/or with the progression of an infection or its clinical symptoms of disease.

The vaccine according to the invention can advantageously be combined with another antigen. Therefore, in a more preferred embodiment the vaccine according to the invention is characterised in that it is a combination vaccine, comprising an additional immunoactive component.

The additional immunoactive component(s) may be an antigen, an immune enhancing substance, and/or a vaccine! either of these may comprise an adjuvant. The additional immunoactive component(s) may be in the form of an immune enhancing substance e.g. a chemokine, or an immunostimulatory nucleic acid, e.g. a CpG motif.

The additional immunoactive component(s) when in the form of an antigen may consist of any antigenic component of human or veterinary importance. It may for instance comprise a biological or synthetic molecule such as a protein, a carbohydrate, a lipopolysaccharide, a nucleic acid encoding a proteinacious antigen, or a recombinant nucleic acid molecule containing such a nucleic acid operably linked to a transcriptional regulatory sequence.

Also a host cell comprising such a nucleic acid, a recombinant nucleic acid molecule, or a live recombinant carrier containing such a nucleic acid, may be a way to deliver the nucleic acid or the additional immunoactive component.

Alternatively it may comprise a fractionated or killed micro-organism such as a parasite, bacterium or virus.

In an even more preferred embodiment, the vaccine according to the invention is characterised in that said additional immunoactive component or nucleic acid encoding said additional immunoactive component is obtained from an organism infective to ruminants, particularly sheep or cattle.

An antigen of human origin in this respect is advantageously used e.g. in the context of vaccinations against zoonotic diseases! diseases that can transfer from ruminant to human. Examples are^: the parasite Trichinella spiralis or the tuberculosis bacterium. Alternatively, the antigen, antibody or vaccine according to the invention, may themselves be added to another vaccine. For instance a vaccine according to the invention can be combined with a preparation of a parasitic subunit vaccine antigen, not being an antigen according to the invention, to form a combination subunit vaccine against parasitic infection or associated clinical signs of disease.

Alternatively, the vaccine according to the invention can advantageously be combined with a pharmaceutical component such as an antibiotic, a hormone, or an anti-inflammatory drug.

For reasons of e.g. stability or economy the antigen, antibody, or vaccine according to the invention may be freeze- dried. In general this will enable prolonged storage at temperatures above zero ° C, e.g. at 4°C. Procedures for freeze- drying are known to persons skilled in the art; equipment for freeze- drying at different scales is available commercially. Therefore, in a most preferred embodiment, the vaccines according to the invention are characterised in that said vaccines are in a freeze-dried form. To reconstitute a freeze-dried vaccine, it may be suspended in a physiologically acceptable diluent. Such a diluent can e.g. be as simple as sterile water, or a physiological salt solution. In a more complex form it may be suspended in an emulsion as outlined in PCT/EP99/10178.

Still another aspect of the invention relates to a method for the preparation of a vaccine according to the invention, said method comprising the admixing of an antigen according to the invention, and a pharmaceutically acceptable carrier.

The GaI-LDNF glycan structure can be synthesized by methods generally available to the person skilled in carbohydrate chemistry which will appreciate the importance of attaining the correct conformation, involving the right α and β type linkages.

Advantageously, the antigen according to the invention is prepared by a combination of chemical and biochemical steps. Such biochemical steps for instance involve the use of enzymes, which may be highly specific for the starting compound and the resulting end product; a similar specificity by purely chemical synthesis may be very hard to achieve. Therefore the invention also pertains to a method of making the aforementioned GaI-LDNF by a chemo-enzymatic method of synthesis. For the invention a number of known glycosyltransferases have been used in a certain order! the skilled person is perfectly capable to select and use other glycosyltransferases with similar catalytic properties that can catalyze the synthesis of the same structure. Also the optimisation of the order of the steps required, and the efficiency of the reactions is within the capability of the routine worker.

The invention also pertains to a method of treatment, prevention or reduction of a parasitic nematode infection in a ruminant, comprising the administration to said mammal of an antigen comprising GaI-LDNF. Herein the various embodiments of the antigen, the antibody, and vaccines and components thereof, as well as the target animals and nematode infections treated are as substantially described hereinbefore.

The invention will now be further described with reference to the following, non-limiting, examples, and the accompanying figures.

EXAMPLES

Example 1 : Synthesis of Gal-LDNF-PAP

GaI-LDNF-DAP and its synthesis are depicted in Figures 1 and 2. The various enumerated arrows in Figure 1 indicate the following reaction steps^: l) Commercial Chitobiose was derivatised with DAP (chemical details presented in Figure 2 A, part a.).

2) Addition of βl-4-GalNAc by the C. elegans enzyme: 61-4GaINAc Transferase (Ce6l-4-GalNAcT) (see methods below) (Figure l);

3) Addition of αl-3-Gal by commercial bovine αl-3-GalT . 4) Addition of αl-3-Fuc by human FUT6 (see methods below).

Details of the non-commercial glycosyltransferases used and their production are described in Example 2.

1.1 Coupling of DAP to chitobiose

To derivatise N,N'-Diacetylchitobiose (chitobiose, Sigma, D1523) with the multifunctional spacer DAP (2,6 diaminopyridine, 98%, Sigma, D24404) the following method was used:

These solutions were made fresh before each derivatisation:

A. 1722 μl DMSO (Fluka, 41647) with 778 μl Acetic acid (glacial 99,99%, Sigma, 338826); total volume was 2500 μl; B. 82.5 mg DAP was resuspended in 1250 μl of mixture A;

C. 137.4 mg of sodium cyanoborohydride (NaCNBH3, Fluka, 71435) was resuspended in 1250 μl of mixture A, and heated at 65°C until completely dissolved; D. a mixture of B and C.

Four eppendorf tubes containing 1 μmol of dried chitobiose pellet per tube, dissolved in 10 μl DMSO, were each dissolved in 500 μl of mixture D and incubated for 18 hours at 65°C in a heat block.

Directly after incubation, 2 vials containing chitobiose-DAP and free DAP were purified on one 12 cc Sep-Pak C 18 column.

Both eluates were pooled in one 15 ml tube and dried in a speedvac (Thermo Fisher Scientific Speedvac®); the resulting pellet (containing approximately 4 μmol chitobiose-DAP and excess free DAP) was taken up in 500 μl Milli Q and stored at 4°C

1.2 Separation of chitobiose-DAP from excess DAP by HPLC

The 4 μmol chitobiose-DAP was separated from excess DAP by HPLC on a normal phase column (Zorbax, NH2 prep HT 21.2 x 250 mm, 7-micron, Agilent Technologies) in an Akta explorer (Amersham Pharmacia Biotech) at a flow rate of 10 ml/min. Detection of DAP could be done conveniently by UV detection at 235 nm.

For HPLC the following buffers were used: buffer A : 50 niM ammoniumformate pH 4.4 (made by dissolving 5 ml 10 M Ammoniumformate solution (Fluka, 78314) and 200 μl Formic acid (Fluka, 56302) in 1 1. Milli Q water) buffer B: 100% acetonitrile (Riedel de Haen, 34967);

The following gradient was conducted manually: 1. 81% buffer B / 19% buffer A for 1 column volume (around 40 ml); 2. sample injection;

3. 81% buffer B / 19% buffer A in 0 - 17 minutes to 30% buffer B / 70% buffer

A;

4. ratio brought back to 81% buffer B / 19% buffer A and started at l) again.

All flow trough of the column was fractionated by a fraction collector (Frac- 950, Amersham Pharmacia Biotech) in fractions of 3 ml. Fractions were dried in the speedvac, and pellets resuspended in 500 μl Milli Q water. To remove all salts, chitobiose-DAP, and similarly other oligosaccharides coupled to DAP (OS-DAPs) were purified on a 6 cc reverse- phase Sep-Pak C18 column.

The elutions were dried in the speedvac and resuspended in 100 μl of Milli Q water. The concentration of chitobiose-DAP was determined by measuring the optical density of a sample (1:100 dilution in Milli Q) at a wavelength of 345 with a spectrophotometer (Ultrospec 3100pro®, Amersham Biosciences).

The eluted chitobiose-DAP was dried and dissolved in Milli Q water (to a concentration of 2-10 niM) and stored at -20 °C (in the dark) until use for enzymatic synthesis.

1.3 Addition of a Bl-4-GalNAc residue to chitobiose-DAP

The following components were mixed in total volume of 100 μl: 1 μmol dried chitobiose-DAP (final concentration was 10 niM) 4 μl 1 M MnCb (final concentration was 40 mM) - 2.5 μl 4 M sodium cacodylate pH 7.0 (final concentration was 100 mM) - 20 μl 100 mM UDP-GalNAc (final concentration was 20 mM)

73.5 μl medium, containing recombinant Ce- βl, 4GaINAcT (enzyme activity 326 nmol/ml/hour on GlcNAc-S-pNp); the enzyme expression is described in Example 2.

The mixture was incubated at room temperature (20"24°C) until the acceptor had been completely converted to the desired end-product: LDN-DAP. The formation of LDN-DAP was monitored in time by HPLC: 0.5 μl of the 100 μl reaction mixture, containing 100-500 pmol oligosaccharide, was dissolved in 100 μl 80% Acetonitrile from which 25 μl was tested on a Surveyor® (Thermo) HPLC, using a LudgerSep Nl Amide column (250 x 4.6 mm, Ludger). A 30- minute gradient starting with 70% acetonitrile and 30% 50 mM ammonium- formate, pH 4.4 and ending in a 50^;50 ratio was run at 22°C to separate the formed product from the starting oligosaccharide. The column was subsequently equilibrated with 100% ammonium formate for 10 minutes, followed by 10 minutes of 70% acetonitrile and 30% 50 mM ammonium formate. Detection by fluorescence (Waters fluorimeter 470) was done with excitation at 345 nm and measuring the emission at 400 nm.

The chitobiose-DAP peak eluted in this system around minute 17,8, whereas the LDN-DAP eluted around minute 19,8. After complete conversion, LDN- DAP was purified from the fractions using a 6 cc Sep-Pak column, dried in the speedvac at room temperature and stored dry at -20°C.

1.4 Addition of an αl-3-Gal residue to LDN-DAP

The following components were mixed in a total volume of 140 μl^: 1 μmol LDN-DAP (final concentration was 7.1 mM)

- 14 μl 1 M MnCb (final concentration was 100 mM) - 7 μl 1 M Trizma (Sigma, in kit 74188, final concentration was 50 mM)

- 7 μl 10 x AP (Alkaline phosphatase, Sigma, in kit 74188)

- 14 μl 0.5% BSA (Sigma, in kit 74188, final concentration was 0.05%)

- 45 μl 100 mM UDP-GaI (final concentration was 10 mM)

50 μl αl,3-galactosyltransferase (αl, 3- GaIT) bovine enzyme, as recombinant expression product from E. coli, from Sigma kit 74188 (78 nmol/ml/hour on

LDN-DAP)

The mixture was incubated at 37°C until the desired end-product:

Galαl-3-LDN-DAP was formed in optimal amounts (approximately 72 hrs). The product formation was followed in time as described above by analytical

HPLC. The αl-3Gal-LDN-DAP eluted around minute 22,8 and LDN-DAP around minute 19,8.

After conversion, the products were purified with a 6 cc Sep-Pak column, dried in the speedvac and stored at — 20°C.

1.5 Addition of a αl-3Fuc residue to LDN-DAP, or αl -3GaI-LDN-DAP

* For synthesis of LDNF-D AP-' The following components were mixed in a total volume of 200 μl^: 1 μmol LDN-DAP (final concentration was 5 mM)

- 1 μl 1 M MnCl₂

2.5 μl 4 M sodium cacodylate pH 7.0 (final concentration was 50 mM) 40 μl 50 mM GDP-fucose (final concentration was 10 mM) - 156.5 μl medium containing FUT6-ProtA recombinant enzyme (175 nmol/ml/hour on LDN-O-(CH₂)₈COOCH₃)

The mixture was incubated at 37°C until acceptor had been completely converted to the desired end-product: LDNF-DAP (approximately 16 hrs). The conversion was monitored in time by analytical HPLC as described above. LDN-DAP eluted around minute 19,8 and LDNF-DAP around minute 22,6. After complete conversion, the LDNF-DAP was purified with a 6 cc Sep-Pak column, dried in the speedvac and stored at — 20°C. * For synthesis of Galal -3-LDNF-DAP (herein: GaI-LDNF-DAP): The following components were mixed in a total volume of 200 μl^: - 1 μmol of a mixture of Galαl-3-LDN-DAP and LDN-DAP (final concentration was 5 niM)

1 μl 1 M MnCb (final concentration was 5 niM)

2.5 μl 4 M sodium cacodylate pH 7.0 (final concentration was 50 niM) 40 μl 50 mM GDP-fucose (final concentration was 10 mM) 156.5 μl medium containing FUT6-ProtA recombinant enzyme. The mixture was incubated at 37°C until the GaI-LDN-DAP had been converted completely to the desired end-product: GaI-LDNF-DAP. Simultaneously, the LDN-DAP was completely converted to LDNF-DAP. The product formation was followed in time (sample 1 μl) by analytical HPLC.

The GaI-LDN-DAP eluted around minute 22,8, the GaI-LDNF-DAP around minute 25,8; the LDN-DAP around minute 19,8 and LDNF-DAP around minute 22,6.

After conversion, the mixture of GaI-LDNF-DAP and LDNF-DAP was purified with a 6 cc Sep-Pak column, dried in the speedvac and stored at — 20°C.

Example 2- Construction of neo-glycoconjugates

The coupling of DAP-derivatised oligosaccharides (OS) is depicted in Figure 2. Herein the various linking steps are as follows^:

1) coupling of 2,6 diaminopyridine to the reducing end of the oligosaccharide!

2) coupling of diethylsquarate to the DAP moiety of DAP-OS (by one of the ethyl groups of diethylsquarate; the other ethyl group then being available for coupling to the carrier);

3) coupling of squarate-DAP-oligosaccharide to free NH2 groups of Tetanus toxoid;

2.1 Coupling of diethylsquarate to DAP-derivatised oligosaccharides (OS- DAPs)

4 μmol OS-DAP was dissolved completely in 1 ml of 0.1 M phosphate buffer pH 6.95 (made by mixing 6.3 ml 0.2 M NaH₂PO₄x2H₂O with 8.7 ml 0.2 M Na₂HPO₄ and 15 ml Milli Q water) and transferred to a glass coned vial (Wheaton). A freshly made mixture of 1 ml ethanol (via Brunschwig Chemie from Nedalco) with 20 μl 3,4-Diethoxy-3-cyclobutene-l,2-dione, (98%, Fw = 170.16, Aldrich, 310778"5G) was added and mixed. The sample was incubated at 22°C for 16 hr. The addition of squarate to the OS-DAP was checked by ESI-MS (add

0.5 μl of the 2 ml reaction mixture to 50 μl 50% Acetonitrile). Successful coupling resulted in a product with a mass of 124 Dalton added to the original OS-DAP mass.

30 ml of Milli Q water was added to the reaction mixture which was subsequently applied to a 6 cc Sep-Pak column for purification.

2.2 Coupling squarate -derivatized OS-DAP to tetanus toxoid (TT)

400 nmol squarate- derivatized OS-DAP was dissolved in 320 μl Conjugation buffer.

Conjugation buffer was made by dissolving 1.545 g boric acid (Gibco, 15705-023) and 1.305 g KCl (Fluka, 60130) in 30 ml Milli Q water; the pH was adjusted to 9 with 421 mg KOH (Merck, 5012)

To this mixture, 8 nmol tetanus toxoid (TT) (80 μl of a 15 μg/μl sample in 0.1 M NaHCO3, which equals about 1.2 mg) was added to a final volume of 400 μl. The reaction mixture was incubated at 22°C for 23 hr.

The addition of glycans to TT was defined by ELISA; 0.5 μl of reaction mix was dissolved in 300 μl coating buffer (50 mM Na₂CO₃ pH=9,6 (Merck, 1063985000)) and the ELISA plate was coated with 50 μl per well (= 5 μg/ml coat) overnight at 4°C. ELISA was performed with an anti-LDNF monoclonal antibody (named SMLDNFl) in accordance with procedures generally known in the art. The antibody SMLDNFl has been described in Nyame, A. K., et al. (2000, Exp. Parasitol., vol. 96, p. 202-212). To remove salts, 1.1 ml Milli Q water was added to the reaction mixture and the sample was injected in a Slide-A-Lyzer® Dialysis Cassette (10.000 MWCO, 0.5-3 ml capacity, Thermo Scientific, #66380) and dialyzed against 5 litre demi-water for at least 16 hr at 4°C.

The neoglycoconjugates were dried in the speedvac and stored at — 20°C.

2.3 The Ce-B4GalNAcT coding sequence

The Caenorhabditis elegans 64GaINAcT is a betal-4-N-acetyl- galactosaminyltransferase. Its encoding mRNA sequence was published in GenBank under accession number AY130767, as an 1152 bp sequence.

The sequence as used herein starts at nt 87 from start codon of the GenBank sequence, with Lys30 as the first amino acid, which marks the start of the catalytic domain! this was cloned into a plasmid for expression in HEK293T cells. The DNA sequence used is presented as SEQ ID NO: 1, and the encoded protein in SEQ ID NO: 2.

2.4 Production of recombinant Caenorhabditis elegans Bl,4-N-acetylgalactosaminyl-transferase (Ce-β4GalNAcT)

For synthesis of the GaINAcBl- 4GIcNAc (LDN) epitope, recombinant βl,4-N- acetylgalactosaminyltransferase from Caenorhabditis elegans (Ce- B4GalNAcT) was used. Ce-β4GalNAcT was produced as a secreted fusion protein, by expression of the plasmid pCMV"SH-CeB4GalNAcT in HEK293T cells. The cloning of Ce-β4GalNAcT and construction of the plasmid has been described by Kawar et al. (2002, J. Biol. Chem., vol. 277, p. 34924-34932).

2.5 Plasmid _PCMV-SH-CeB4GalNAcT

In the plasmid pCMV"SH-CeB4GalNAcT, the catalytic domain of the Ce- B4GalNAcT is present as insert in the EcoRV site of the pcDNA 3.l(+)-TH vector. This insert was a Psil (partial)/PvuII DNA fragment starting at nt 87 of the Ce-β4GalNAcT open reading frame as deposited in GenBank under accession number AY130767, and extending beyond the stop codon. The resulting plasmid (pCMV"SH-CeB4GalNAcT) encodes a fusion protein, designated SH-CeB4GalNAcT, which consists of a signal peptide at the N- terminus followed by an HPC4 epitope, and then the catalytic domain of the CeB4GalNAcT (beginning at Lys30, the first amino acid after the transmembrane domain). SH-CeB4GalNAcT is under the transcriptional control of the cytomegalovirus promoter, which is present in the vector. See SEQ ID NO's: 1 and 2.

2.6 Transfection of pCMV-SH-CeB4GalNAcT plasmid to HEK293T cells and characterization of the enzyme produced

HEK293T cells were cultured adherent in T75 flasks (Greiner, 658175) until the bottom was completely covered by a monolayer of cells (about 6xlO^A6 cells). Incubation was at 37°C/5% CO2 in an incubator (Hereas, Hera cell 150). Transfection with plasmid pCMV-SH-Ce64GalNAcT was done in accordance with methods customary in the art. After transfection, medium samples were harvested at several days (2, 3, 6, 7 days) and assayed for 61,4-GaINAcT activity as follows. 2.7 Bl-4GalNAcTransferase assay

The following components were mixed per reaction in total volume of 25 μl^: 5 μl of acceptor 10 mM GlcNAc-S-pNp (final concentration 1 niM) 1 μl 1 M MnCb (final concentration was 40 mM) - 2.5 μl 1 M sodium cacodylate pH 7.0 (final concentration was 100 mM)

- 2.5 μl 1 mM UDP-3H-GalNAc (specific activity = lCi/mol, final concentration was 0.5 mM)

Medium was added from Ce64GalNAcT recombinant enzyme until the end volume was 25 μl. This was incubated for 2 hr at RT. Acceptors (with and without GaINAc build-in) were purified on a 6 cc reverse-phase Sep-Pak C 18 column and the elutions were dried in an 100°C oven until all methanol was evaporated. 500 μl demi-water was added, the sample was vortexed, and 8 ml of counting fluid (Ultima Gold®, Packard Bioscience, 6013329) was added and vortexed again. The build-in radioactivity was counted on a liquid scintillation counter (Liquid Scintillation Counter 1900 TR®, Packard).

The highest activity on GlcNAc-S-pNp was measured at day 2 after transfection, at an activity of 326 nmol/ml/hour. The enzyme was stored at - 20°C.

2.8 The FUT6 coding sequence

The human fucosyltransferase 6 (FUT6) is an alphal-3 fucosyltransferase. Its encoding mRNA was published in GenBank under accession number: NM001040701, as a 3009 bp fragment, of a transcript variant 2

The sequence of the FUT6 enzyme as used herein for the expression of FUT6 in C0S7 cells, is taken from the catalytic domain, and starts with the 40^th amino acid from the starting methionine, at Asp40. The coding DNA fragment used is presented in SEQ ID NO: 3, the encoded protein in SEQ ID NO: 4.

2.9 Production of recombinant human αl-3-fucosyltransferase

For fucosylation of the oligosaccharides, the human αl-3-fucosyltransferase FUT6 was used. FUT6 was produced as a secreted fusion protein, containing a signal peptide part of Protein A and the catalytic domain of FUT6, see SEQ ID NO: 3 and 4. The enzyme was encoded by plasmid FUT6-pPROTA, and produced by expression of this plasmid in HEK293T cells. The cloning of human FUT6 has been described by Weston et al. (1992, J. Biol. Chem., vol. 267, p. 24575-24584). The construction of the FUT6-pPROTA plasmid has been described by Jost et al. (2005, Glycobiology, vol. 15, p. 165-175). The pPROTA plasmid is also depicted in Figure 3.

The FUT6-pPROTA plasmid contains the total catalytic domain of FUT6, fused to protein A. The FUT6 sequence (starting at amino acid 40) is inserted between the Xmal site and Xbal site of the pPROTA plasmid.

2.10 Transfection of FUT6-pPROTA plasmid into COS7 cells, and characterization of the enzyme produced

COS7 cells were grown adherent in T75 flasks (Greiner, 658175) until the bottom was completely covered by a monolayer of cells (about 6xlO^A6 cells).

Incubation was at 37°C/5% CO2 in an incubator (Hereas, Hera cell 150).

Transfection with plasmid pPROTA- FT6 was done in accordance with methods customary in the art. After transfection, medium samples were harvested 2 days after transfection and assayed for αl-3-FucT activity on the acceptors LN- pNp (from Sigma) and LDN-O-(CH₂)₈COOCH₃ (LDN-Lemieux).

2.11 Fucosyltransf erase assay

The following components were mixed per reaction in a total volume of 25 μl^: 5 μl of acceptor 5 mM LN-pNp (final concentration 1 niM) or 2.5 μl acceptor 10 mM LDN-Lemieux (final concentration 1 mM)

- 0.625 μl 200 mM MnCb (final concentration was 5 mM) - 1.25 μl 1 M sodium cacodylate pH 7.0 (final concentration was 50 mM)

- 2.5 μl 100 mM GDP-14C-fucose (specific activity = 1 Ci/mol, final concentration was 1 mM)

Medium with FUT6-ProtA recombinant enzyme was added until the end volume was 25 μl. This was incubated for 1,5 hr at 37°C. Acceptors (with and without fucose build-in) were purified on a 6 cc reverse-phase Sep-Pak C18 column and the elutions were dried in an 100°C oven until all methanol was evaporated. 500 μl demi-water was added, vortexed, and 8 ml counting fluid was added and vortexed again. The build-in radioactivity was counted on a liquid scintillation counter. The activity on LN-pNp was 61 nmol/ml/hour and 175 nmol/ml/hour on

LDN-Lemieux. The enzyme was stored at -8O⁰C. Example 3: Vaccination-challenge trial in sheep

3.1 Study design:

Three groups of seven sheep were used in the study: two vaccinated groups and one control group (adjuvant control). After priming and two booster vaccinations with neoglycoconjugate vaccines, the sheep were challenged with H. contortus larvae for three consecutive days. Humoral immune responses were monitored by ELISA.

3.2 Materials and methods^:

Test articles Vaccine preparations:

A. GaI-LDNF-TT + LDNF-TT

Composition: 100 μg of a mixture of GaI-LDNF (20%) and LDNF (80%), each coupled to TT.

B. LDNF-TT Composition: 100 μg LDNF coupled to TT.

C. TT (mock vaccine) Composition: 100 μg TT.

All antigens were emulgated at 100 μg in a proprietary water-in-oil emulsion. Transport and storage conditions: 4-10 °C.

Test system

Animals

Clinically healthy sheep, males and females, between five and seven months old, raised indoors (worm free), were included in the study! faecal samples were taken upon arrival for parasitological investigation, and sheep that had a patent ('established') infection with nematodes other then Strongyloides spp. were excluded from the study. Twenty one sheep were individually marked by an eartag with a unique life number. Sheep were allowed to acclimatise for three to seven days before vaccination.

The animals were housed in stables with hard flooring: contact with soil was prevented in order to prevent infection with nematode worms. Sheep were fed a standard ration with hay/silage and concentrates. Drinking water was provided ad libitum.

Assignment of animals to treatment groups

The animals were assigned to the treatment groups as they came to hand. Treatment, dosage and administration

Table l:

3.3 Experimental procedures

Bloodsampling

Blood samples for serology were taken at weekly intervals for the duration of the study. Blood was collected in serum-gel vacutainer® tubes (BD), serum was separated by centrifugation, aliquoted into 1 or 3 ml tubes and stored at - 20°C until use. At end of the experiment 100 ml of blood was collected and processed for serum collection by standard methods.

Serology

Serum antibody titres against the carbohydrate components of the candidate vaccines were determined by ELISA as follows^ 96-well ELISA plates were coated with either of the following antigens^:

- TT (2 μg/ml),

- BSA (20 μg/ml)

H. contortus adult worm extract (described below) (10 μg/ml). All were diluted to the indicated concentration, and coated in 50 mM NaH CO3, pH=9.6 after incubated overnight at 4°C and 50 μl/well.

All reagents were added at 50 μl/well and incubated for 1 hour at room temperature unless stated otherwise. All washes were performed with TSM buffer with 0.1% Tween-20. TSM buffer = 20 mM Tris-HCl pH 7,4; 150 mM NaCl; 2 mM CaCl₂; 2 mM MgCl₂.

Plates were washed, and subsequently blocked with TSM/1% BSA. After washing, 2-fold dilutions of sheep serum in TSM/1%BSA (1=25 to 1:800) were added to the plates.

After incubation (as described above) plates were washed and the second antibody was added: Rabbit anti-Sheep IgG (AdB serotec, 5184-2104) 1:5000 in TSM/1%BSA.

After washing, the conjugate was added: Goat anti rabbit (DAKO pO448) 1:5000 in TSM/ 0.1% Tween-20 and incubated at 37°C.

After the last wash, the substrate solution (TMB/ IHb(VNaAc) was added at 100 μl/well and incubated until the colour had developed sufficiently (5-30 minutes). TMB = peroxidase substrate: 3,3',5,5'-tetramethyl-benzidine salt.

The reaction was stopped by adding 0,8 M H2SO4 50 μl/well. The plates were read in an ELISA reader for optical density (OD) at 450nm.

Preparation of H. contortus adult worm extract:

Whole parasite extracts were prepared from adult H. contortus. Frozen worms were thawed, washed three times in PBS, pH 7.4 (6.7 mM KH₂PO₄, 150 mM NaCl) and suspended in 50 mM PBS, pH 7.0. The parasite suspension was homogenized on ice with a polytron® homogenizer using three pulses of 30 sec each. The homogenate was subsequently subjected to sonication (five pulses of 1 min each) and centrifuged at 10.000 x g, at 4°C for 30 min., and the supernatant fraction was collected. Next, the proteins of the homogenate were precipitated by adding four volumes of -20°C acetone, and incubation for 1 hour at — 20°C. The protein pellet was collected by centrifugation for 10 min at 13,000 x g, and resuspended in PBS. Protein concentration of the extract was determined by the BCA protein assay procedure (Pierce Co., Rockford, IL). The proteins were resuspended in coating buffer (50 mM Na₂COs pH=9.6 (Merck, 1063985000)) at a final concentration of 10 μg/ml, and the ELISA plates were coated with 50 μl per well overnight at 4°C.

Challenge infection

4.500 L3 larvae of an anthelmintic- sensitive strain of if. contortus, suspended in water, were administered orally. The challenge was divided into three doses of 1.500 larvae in 10 ml of water, administered on three consecutive days around 10 days after the last vaccination. Before use the bottle was shaken to stir up the larvae. Larvae had been stored at 2-8°C after production, until use.

Necropsy

At the end of the experiment all animals were necropsied, and final samples were taken. Blood was collected for serum preparation, and the abomasum was removed; its content was collected and the abomasum was washed with saline. By counting the worm burden in the abomasal content and -wash, it was established that a good 'take' of the challenge infection had occurred

3.4 Results:

The results of the vaccination-challenge experiment clearly indicated the significant immunogenic effect of the GaI-LDNF-TT glycan-conjugate vaccine over the marginal effects of the LDNF-TT vaccine or the background effect of the control TT vaccine. Results as measured by Elisa responses to various antigens coated in plates are indicated in Figures 4 A - D, wherein Figures 4 A - C depict the Elisa scores (measured in OD 450 nm) of duplex serum samples and their standard deviation, of the pooled sera (n=7) of the sheep in the three different treatment groups. Figure 4 D compares the difference in the induction of antibodies recognising the H. contortus extract in those three groups.

Serum- samples were taken at several time points during the experiment, these are indicated along the horizontal axis. Vaccinations are indicated by super-imposed black narrow arrows, and the time point of the challenge is indicated by an open block arrow. Coated test antigens in the Elisa plates were: H. contortus adult worm extract, to test for induction of a sero-response recognising worm antigens! TT-antigen, to illustrate the effect of the immunogenic carrier group! and BSA, as negative control antigen.

In all figures 4 A - C some level of induction of specific antibodies against the coated TT and H. contortus extract antigens can be seen, whereas only a background response to the BSA antigen was detected.

In all three groups, antibodies reactive with the coated TT antigen achieved plateau phase at 21 days after the first vaccination, and did not vary significantly after the 2^nd or 3^rd vaccination or after the challenge, as was expected. As also expected, no antibodies reactive with the coated BSA antigen were induced or measured at any time.

Most interesting was the detection of the induction of antibodies binding to the coated H. contortus extract, and the unexpected differences in that response between the different vaccinated groups.

The TT mock vaccine applied to the animals in group 3 (Figure 4 C), did not of itself induce specific antibodies which bound the coated H. contortus extract, as only after the animals were challenged (and thus came into contact with worm antigens) an increase in Elisa response can be seen.

The LDNF-TT vaccinated animals of group 2 (Figure 4 B) presented a slight increase in the level of antibodies that are reactive with the coated worm extract after the first and second vaccination. No further increase could be seen after the third vaccination or after the challenge. Similarly, the glycairconjugate mixture of GaI-LDNF-TT and LDNF-TT in the vaccine given to the animals in group 1 (Figure 4 A) induced antibodies that could bind to the coated H. contortus extract, after the first and second vaccinations, and no further increase was detected after the 3^rd vaccination, or after the challenge.

However, what was most surprising was the strong and steady increase in the amount of specific antibodies induced by the GaI-LDNF-TT + LDNF-TT vaccine, which resulted in antibody levels significantly higher than those induced by the LDNF-TT vaccine alone (see Figure 4 D). It was noted that the specific antibodies were induced before the animals came in contact with the worms upon challenge, and thus must have resulted from the vaccination with the glycan-conjugate.

The high level of response obtained was especially unexpected as only 20 % of the mixture consisted of GaI-LDNF-TT.

The inventors realised that a vaccine comprising the glycan GaI-LDNF could induce specific antibodies that recognised antigens from H. contortus.

Example 4'- Coupling of GaI-LDNF to BSA as carrier:

Coupling of GaI-LDNF to BSA as carrier kan be performed as described by Tefsen et al., 2009, "Chemoenzymatic synthesis of multivalent neoglyco- conjugates carrying the helminth glycan antigen LDNF", Carbohydr. Res, vol.

344, p. 1501-1507.

In short: GaI-LDNF-DAP was linked to squarate as described above.

Next, a solution of squarate-derivatized GaI-LNDF-DAP can be added to BSA and conjugation buffer (boric acid and KCl in MiIIiQ water, pH adjusted to 9 with KOH), and incubated at 22 °C for 23 h. To remove salts, Milli Q water can be added, and the mixture can be dialyzed against deionized water overnight at 4 °C. The neoglycoconjugates can be dried in a Speedvac and stored at

-20 °C. Synthesized oligosaccharide products can be characterized by ESI MS, for instance in an ion-trap mass spectrometer with a nano-ES ionization source.

In addition, to estimate the degree of coupling of the oligosaccharides to

BSA, the neoglycoconjugates can be subjected to MALDI-TO FMS. LEGEND TO THE FIGURES:

Figure l:

Presents a graphic depiction of the steps of a way of synthesis of GaI-LDNF coupled to DAP via a linearised GIcNAc group.

Figure 2, A, B:

Present details of the chemical reactions for the synthesis of a glycan conjugate according to the invention:

Figure 2 A, in part a., presents the coupling of Gal-LDNF-linked to a linearised GIcNAc group, to DAP; this part corresponds to Figure 1 where Chitobiose is the "oligosaccharide". Details are presented in Example 1.

Next , in Figure 2 A, part b., the subsequent coupling of the DAP group to squarate is depicted (see Example 2).

Figure 2 B, part c, presents the subsequent coupling of a number of derivatized glycans to the immunogenic carrier, here: Tetanus toxoid, (see Example 2).

Figure 3:

Represents the plasmid vector construct used for the expression of the recombinant human αl-3 fucosyltransferase 6 (FUT6) in COS7 cells (details in Example 2).

Figure 4, A - D:

Represent the results of the animal vaccination-challenge trials, as measured by Elisa. Full details are described in the results of Example 3, section 3.4.

Claims

1. An antigen comprising a carbohydrate satisfying the structural formula ^: Galαl-3GalNAcβl-4(Fucαl-3)GlcNAc (hereinafter: GaI-LDNF).

2. The antigen according to claim 1, comprising the GaI-LDNF in a chemical structure of the formula^: GaI-LDNF-R, wherein R denotes an immunogenic structure, to which the GIcNAc unit of GaI-LDNF is attached.

3. The antigen according to claim 2, wherein the immunogenic structure R is selected from the group consisting of carbohydrates, proteins, and lipids.

4. The antigen according to claim 3, wherein the GaI-LDNF-R structure comprises Gal-LDNF-Rl-R2, wherein Rl is 61-4GIcNAc and wherein R2 has the same meaning as R.

5. The antigen according to claim 4, wherein R2 additionally comprises a diaminopyridine moiety which attaches R2 to Rl.

6. The antigen according to claim 5, wherein R2 additionally comprises a squarate moiety attached to the diaminopyridine moiety, the squarate being further attached to the immunogenic structure.

7. An antibody or a part thereof that can bind specifically to GaI-LDNF.

8. Use of an antigen according to any one of the claims 1-6, or of an antibody according to claim 7, for the manufacture of a medicament for the immunoprotective treatment, prevention or reduction of parasitic nematode infection in a ruminant.

9. A vaccine comprising an antigen according to any one of the claims 1-6, or an antibody according to claim 7, and a pharmaceutically acceptable carrier.

10. The vaccine according to claim 9, additionally comprising an adjuvant.

11. An antigen according to any one of the claims 1-6, an antibody according to claim 7, or a vaccine according to any one of claims 9 or 10, for use in the immunoprotective treatment, prevention or reduction of parasitic nematode infection in a ruminant.

12. A method for the manufacture of a vaccine for ruminants, wherein an antigen according to any one of the claims 1-6, or an antibody according to claim 7, is admixed with a pharmaceutically acceptable carrier.

13. A method of treatment, prevention or reduction of a parasitic nematode infection in a ruminant, comprising the administration to said ruminant of an antigen according to any one of the claims 1-6, of an antibody according to claim 7, or of a vaccine according to any one of the claims 9 or 10.

14. A method according to claim 13, for the immunoprotective treatment of cattle and sheep against Heamonchus contortus infection, or the prevention or reduction of such an infection therein.