Single domain activation-associated secreted protein and uses thereof

Abstract

The present invention relates to a single domain activation-associated secreted protein (ASP) or fragment thereof. The invention further relates to a composition comprising said ASP or fragment thereof and the use as a veterinary medicine, in particular as a vaccine; more in particular for use against parasitic nematode infection by Cooperia.

Description

[0001] Single domain activation-associated secreted protein and uses thereof

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a single domain activation-associated secreted protein (ASP) or fragment thereof. The invention further relates to a composition comprising said ASP or fragment, and the use as a veterinary medicine, in particular as a vaccine; more in particular for use against parasitic nematode infection by Cooperia.

[0004] BACKGROUND OF THE INVENTION

[0005] Gastrointestinal nematode infections in cattle present a considerable challenge to both animal health and farm productivity. The standard therapeutic approach for managing these infections involves periodic administration of anthelmintic drugs. However, there has been widespread documentation of resistance to anthelmintics, including benzimidazoles and macrocyclic lactones. Consequently, the scientific community is actively exploring sustainable methods of parasite control, among which vaccination is being considered as a promising approach. Progress in this field has been limited, resulting in only a small number of commercially available antiparasitic vaccines so far.

[0006] For Cooperia oncophora, one of the most common bovine intestinal parasitic nematodes found in temperate climate regions, anthelmintic resistance has been widely reported. To identify potential vaccine candidates, excretory-secretory (ES) material obtained from adult worms was previously examined (Borloo et al., 2013). In this study, this ES material was further fractionated into three fractions based on molecular weight: high-molecular weight (HMW), mid-molecular weight (MMW), and low-molecular weight (LMW).

[0007] Within the HMW fraction, a double domain activation-associated secreted protein (ASP), referred to as Co-dd-ASP, was the major constituent, and its relative purity made it suitable to further assess its efficacy in conferring protection against C. oncophora infections. Several bovine vaccination studies were conducted, and it was demonstrated that this antigen had the capability to reduce C. oncophora egg output up to 91 % (Gonzalez-Hernandez et al., 2018). Despite these prospects, recombinant expression of this protein has been challenging due to the complex N-glycosylation and protein folding of the native Co-dd-ASP. In contrast to HMW, the composition of the MMW and LMW fractions are more complex and the protective properties were never explored.

[0008] Despite strong efforts to develop antiparasitic vaccines, only 2 nematode vaccines are currently on the market, i.e. against Dictyocaulus viviparous in cattle and against Haemonchus contortus in sheep.

[0009] Hence, a need exists for vaccines against Cooperia infections. SUMMARY OF THE INVENTION

[0010] In a first aspect, the invention provides a single domain activation-associated secreted protein (ASP) or fragment thereof.

[0011] In a particular embodiment of the invention, the ASP or fragment thereof comprises a N- glycan.

[0012] In a particular embodiment of the invention, the ASP or fragment is synthetically produced or obtained from an expression system.

[0013] In another particular embodiment, the ASP is obtained from a plant expression system e.g. Nicotiana spp. expression system (e.g. a Nicotania benthamiana or Nicotania tabacum expression system), a yeast expression system e.g. Pichia spp. expression system, a bacterial, mammalian or insect cell line expression system.

[0014] In a further embodiment of the present invention the amino acid sequence of the ASP or fragment thereof of the invention has at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 99%, most preferably 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 1 .

[0015] In another particular embodiment of the present invention the single domain ASP amino acid sequence of Cooperia oncophora is represented by SEQ ID NO: 1 .

[0016] A further embodiment of the present invention provides a nucleic acid sequence encoding the ASP or fragment thereof of the invention, said nucleic acid sequence having at least 85%, in particular at least 90%, more in particular at least 95%, even more in particular at least 99% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 2.

[0017] The current invention also encompasses a vector comprising said nucleic acid and a host cell comprising said vector.

[0018] In a further aspect, the present invention provides a composition, in particular a pharmaceutical composition comprising the ASP or fragment thereof, the nucleic acid encoding the ASP or fragment, the vector or the host cell of the invention, and a pharmaceutically acceptable carrier and / or excipient.

[0019] In a further embodiment, the present invention relates to the pharmaceutical composition comprising the ASP or fragment thereof of the invention, wherein the composition is a vaccine.

[0020] In another particular embodiment, the invention relates the pharmaceutical composition comprising the ASP or fragment thereof of the invention additionally comprising an adjuvant.

[0021] In yet a further embodiment, the present invention provides a ASP or fragment thereof of the invention, or the pharmaceutical composition comprising the ASP or fragment thereof of the invention, for use as a human or veterinary medicine.

[0022] In a further particular embodiment, the present invention provides an ASP or fragment thereof of the invention, or the pharmaceutical composition comprising the ASP or fragment thereof of the invention, for use in the treatment, prevention and / or reduction of a parasitic nematode infection in a mammal.

[0023] In another particular embodiment, the present invention relates to the ASP or fragment thereof, or a pharmaceutical composition comprising the ASP or fragment of the invention, for use in the treatment, prevention and / or reduction of a parasitic nematode infection in a mammal, wherein the parasitic nematode belongs to the genus Cooperia, more in particular wherein the parasitic nematode is Cooperia oncophora, Cooperia punctate, or Cooperia pectinate.

[0024] In a further embodiment, the present invention relates to a method for producing a recombinant single domain ASP or fragment thereof of the invention, said method comprising the steps of: a) providing an expression system, b) optionally introducing in the expression system an enzyme, in particular an carbohydrate-processing enzyme, such as mannosidase I, c) expressing an ASP with the expression system provided in step a) to obtain a recombinant ASP or fragment thereof of the invention.

[0025] Preferably, the expression system of said method comprises a nucleic acid sequence encoding an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 99%, most preferably 100% sequence identity with the amino acid sequence represented by SEQ ID NO: 1 .

[0026] In yet a further embodiment, the present invention provides a method of treatment, prevention and / or reduction of a parasitic nematode infection in a subject in need thereof, comprising the administration to said subject in need thereof of a single domain ASP or fragment thereof of the invention or a pharmaceutical composition of the invention.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0029] Fig. 1 : Immunisation-challenge study in calves with the low-molecular weight (LMW) and mid-molecular weight (MMW) fraction of adult Cooperia oncophora excretory-secretory products. Calves were immunised three times with either the LMW fraction + QuilA (n=7), MMW fraction + QuilA (n=7) or QuilA alone (n=7) prior to a trickle infection with L3-stage Cooperia oncophora. Evaluated parasitological parameters include (Fig. 1 a) faecal egg output measured over time, (Fig. 1 b) cumulative faecal egg output and (Fig. 1 c) C. oncophora worm counts at the time of necropsy. The data is presented as mean values + / - standard error of the mean. P values for (b) and (c) were calculated using a Kruskal-Wallis test with Dunn’s test for multiple comparison. *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 versus QuilA-adjuvant control. P values for (Fig. 1 d) were calculated using a Two-way ANOVA with Dunnet’s test for multiple comparison. Degrees of freedom for the numerator: 9. Degrees of freedom for the denominator: 72. F distribution value: 14.37. The experiments for all figures were conducted once.

[0030] Fig. 2: Purification of native Co-sd-ASP. SDS-PAGE under non-reducing conditions before and after ConA-based affinity chromatography, showing the migration patterns for Precision Plus Protein™ standard (M), MMW fraction before purification (partially contains LMW fraction) (lane 1 ), flowthrough fractions (lane 2), wash fractions (lane 3) and eluted fraction with native Co-sd-ASP (lane 4). Molecular weight of 25, 37, 50 and 75 kDa protein bands are displayed on the left side of the figure.

[0031] Fig. 3: Immunisation-challenge study in calves with native Co-sd-ASP.

[0032] Calves were immunised three times with either native Co-sd-ASP + QuilA (n=7) or QuilA alone (n=8) prior to a trickle infection with L3-stage Cooperia oncophora. Evaluated parasitological parameters include (Fig. 3a) faecal egg output measured over time, (Fig. 3b) cumulative faecal egg output and (Fig. 3c) C. oncophora worm counts at the time of necropsy, (d) The systemic IgG 1 response to native Co-sd-ASP in an ELISA was measured prior to immunisation (imm), one week after each immunisation and at necropsy, displayed in optical density 405 nm with background correction at 492 nm. (Fig. 3e) The mucosal lgG1 response to native Co-sd-ASP was measured at time of necropsy, displayed in optical density 405 nm with background correction at 492 nm. Data are presented as mean values + / - standard error of the mean. P values for (b), (c) and (e) were calculated using a two-tailed Mann-Whitney test. P values for (d) were calculated using a Two-way ANOVA with Dunnet’s test for multiple comparison. Degrees of freedom for the numerator: 4. Degrees of freedom for the denominator: 52. F distribution value: 137.7. *P < 0.05, **P < 0.01 , ***P < 0.001 and ****p < 0.0001 versus QuilA-adjuvant control. The experiments for all figures were conducted once.

[0033] Fig. 4: N-glycosylation of native Co-sd-ASP. Mass-spectrometry (MS) analysis of the N- glycosylation profile of native Co-sd-ASP after PNGase-F release (upper panel) and PNGase-A release (lower panel). The X-axis displays the mass to charge ratio (m / z), whilst the Y-axis displays the relative intensity in arbitrary units (intens. [a.u.]).

[0034] Fig. 5: Impact of (partial) N-glycan removal on antibody recognition. Competition ELISA with a pooled serum sample from calves immunised with native Co-sd-ASP, pre-incubated with intact or enzymatically treated native Co-sd-ASP. A lower ODR corresponds with a higher degree of competition with intact native Co-sd-ASP for antibody binding.

[0035] Fig. 6. Production of recombinant Co-sd-ASP in N. benthamiana. (Fig. 6a) Schematic overview of the N-glycosylation pathway in different compartments of N. benthamiana resulting in paucimannose and oligomannose-type N-glycosylation without core fucose. The suppressed Xylosyltransferase (XYLT) and Fucosyltransferase 1 1 / 12 (FUT1 1 / 12) are displayed (Fig. 6b) SDS- PAGE under non-reducing conditions before and after ConA-based affinity chromatography, showing the migration patterns for Precision Plus Protein™ standard (M), N. benthamiana recombinant Co-sd-ASP (wild-type plant) (lane 1 ), N. benthamiana recombinant Co-sd-ASP (AXT / FT transgenic plant) (lane 2) and MMW fraction with native Co-sd-ASP antigens visible (partially contains LMW fraction) (lane 3). Molecular weight of 10, 15, 20, 25, 37, 50 and 75 kDa protein bands are displayed on the left side of the figure. (Fig. 6c) Mass-spectrometry (MS) analysis of the N- glycosylation profile of AXT / FT N. benthamiana recombinant Co-sd-ASP after PNGase-F release (upper panel) and PNGase-A release (lower panel). The X-axis displays the mass to charge ratio (m / z), whilst the Y-axis displays the relative intensity in arbitrary units (intens. [a.u.]). (Fig. 6d) Competition ELISA with a pooled serum sample from calves immunised with native Co-sd-ASP was pre-incubated with either native Co-sd-ASP or AXT / FT N. benthamiana recombinant. A lower ODR corresponds with a higher degree of competition with native Co-sd-ASP for antibody binding.

[0036] Fig. 7: Correlation between mucosal lgG1 and (Fig. 7a) cumulative faecal egg counts, (Fig. 7b) worm counts, (Fig. 7c) male worm length and (Fig. 7d) female worm length. The correlation between mucosal lgG1 and cumulative faecal egg counts, worm counts and worm measurements was evaluated for statistical significance via a nonparametric Spearman R correlation.

[0037] Fig. 8: Immunisation-challenge study in calves with recombinant Co-sd-ASP

[0038] Calves were immunised three times with the recombinant Co-sd-ASP + QuilA (n=8) or QuilA alone (n=8) prior to a trickle infection with L3-stage Cooperia oncophora. The graph shows the cumulative faecal egg output (Fig. 8a) and worm count (Fig. 8b) over a period of 5 weeks for both the control and the vaccinated group.

[0039] Fig. 9: Amino acid sequence and nucleic acid sequence of the single domain ASP of C. oncophora.

[0040] Fig. 10: Structure I

[0041] DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. When describing the compounds of the invention, the terms used are to be construed in accordance with the following definitions, unless a context dictates otherwise.

[0043] The term "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / - 10 % or less, preferably + / - 5 % or less, more preferably + / - 1 % or less, and still more preferably + / - 0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically, and preferably, disclosed.

[0044] As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0045] The present invention shows for the first time that immunisation of calves with a single-domain ASP protein resulted in a protective immune response against Cooperia infection. This was evidenced by a significant reduction in faecal egg excretion. The ability to induce an immune response with the single domain ASP thus provides a new use and / or vaccine against gastrointestinal nematodes, in particular of the genus Cooperia.

[0046] In one embodiment, the present invention provides a single domain activation-associated secreted protein (ASP) of Cooperia oncophora, or fragment thereof, and a pharmaceutical composition comprising it. Moreover, the present invention provides said ASP or composition for use in preventing, reducing and / or treating nematode infections, in particular infections with nematodes of the genus Cooperia. In a further embodiment, the ASP is a native ASP or a recombinant ASP, which can be isolated or produced by various expression systems enabling large scale production of said protein.

[0047] ASPs belong to the superfamily of cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP)18 and are commonly found in the ES products of various parasite species, including Ancylostoma caninum, Necator americanus, Ostertagia ostertagi and Haemonchus contortus. At least three different types of ASPs have been identified in nematodes: i) double-domain ASPs, encompassing two distinct but related CAP domains, ii) N-type single-domain ASPs, and iii) C-type single-domain ASPs, the second and the latter bearing the highest homology to the N- and C-terminus of the double domain ASPs, respectively. The specific roles of these antigens are yet to be fully elucidated. It is hypothesised that these antigens may be involved in activities related to the transition from free-living to parasitic phase, immune evasion, and hostpathogen interactions. In C. oncophora, both single and double domain ASP antigens were expressed throughout all stages of development, with the exception of the eggs. Thus, it is plausible that the functionality of these antigens may extend beyond solely the parasitic stage.

[0048] In a particular embodiment, the invention provides a single domain ASP comprising or consisting of an amino acid sequence with at least 90% sequence identity (thus including at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity), preferably at least 95% sequence identity, more preferably at least 99%, most preferably 100% sequence identity with the amino acid sequence of the single domain ASP of Cooperia oncophora, represented by SEQ ID NO: 1 , or as provided in Fig. 9. The nucleic acid sequence encoding the single domain ASP or fragment thereof as provided comprises or consists of at least 85% sequence identity (thus including at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity), at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 99%, most preferably 100% sequence identity with the nucleic acid sequence of the single domain ASP of Cooperia oncophora, provided by SEQ ID NO: 2, or as represented by accession number HAAM01000002.1 (in particular excluding the signal sequence).

[0049] In a further aspect of the invention, the amino acid sequence of the single domain ASP of the invention of Cooperia oncophora is represented by SEQ ID NO:1 .

[0050] The term “sequence identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base or the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, “sequence identity” will be understood to mean the “match percentage” calculated by an appropriate method. The percentage identity of nucleic acid and polypeptide sequences can be calculated using commercially available algorithms, which compare a reference sequence with a query sequence. The following programs (provided by the National Center for Biotechnology Information) may be used to determine homologies / identities: BLAST, gapped BLAST, BLASTN and PSI BLAST, which may be used with default parameters.

[0051] In one embodiment, the nucleic acid sequence encoding the ASP is codon optimized. As known to the skilled person, codon optimization is an approach / tool designed to improve the codon composition of a recombinant gene based on various criteria without altering the amino acid sequence. This is possible because most amino acids are encoded by more than one codon. In heterologous expression systems, to maximize protein expression from the DNA sequence of the original species in the host, codon optimization improves the translation efficiency of a target gene by converting the DNA sequence of nucleotides of one species to that of another, such as e.g. converting human sequences to bacterial or yeast sequences, plant sequences to mammal sequences, and fungal sequences to yeast sequences.

[0052] The term "fragment" as used herein refers to partial amino acid sequences (and nucleic acid sequences coding therefore) having at least one immunologic or immunogenic property in common with the native molecule, and hence e.g. the ability to generate antibodies in an immunized animal. Thus, the full-length sequence of the ASP is not necessary, and neither is the 100% identity to SEQ ID Nos provided herein, meaning that one or more amino acid modifications are possible (e.g. 1 , 2, 3, 4, 5, etc). For example, the fragment may have up to 30 amino acids removed from the N- or C- terminal ends of the protein, e.g. up to about 1 , 5, 10, 15, 20, or 30 amino acids removed. As noted elsewhere in the present disclosure, 90% sequence identity is likely to be sufficient to provide suitable level of antibody production. As used herein, the term "amino acid modification" refers to an amino acid addition, amino acid deletion, and / or to an amino acid substitution as compared to the reference sequence. Preferably, said one or more amino acid substitution is a ‘conservative’ amino acid substitution, i.e. the substitution of an amino acid by another amino acid of the same class, in which the classes are as follows:

[0053] Class Amino acid examples

[0054] Nonpolar Ala, Vai, Leu, Pro, Met, Phe, Trp, lie

[0055] Uncharged polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin

[0056] Acidic Asp, Glu

[0057] Basic Lys, Arg, His

[0058] The differing amino acids can be conservative substitutions, and / or are located outside of immunodominant epitope(s) of the ASP fragment. As used herein the term "immunogenic fragment" in regard to the ASP protein is a fragment of that protein that is immunogenic, i.e., capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. Preferably, an immunogenic fragment of the present invention is immunodominant for antibody and / or T cell receptor recognition. In a particular embodiment, an immunogenic fragment as referred to herein is a fragment of the ASP provided herein that retains at least 50%, 60%, 70%, 80%, or 90% of the immunogenicity of the full-length protein. Fragments can be as small as 8 amino acids or at the other extreme, be large fragments that are missing as little as a single amino acid from the full-length protein as provided herein. In a particular embodiment the fragment comprises 8 to all minus one of the amino acid residues of the full-length protein. In other embodiments, the fragment comprises or consist of 10 to 288, 10 to 250, 10 to 200, 10 to 150, 10 to 100 or 10 to 50 amino acid residues. Such fragments will include at least one epitope (or antigenic determinant) of the native molecule. In one embodiment, they have a length of at least 8 amino acids, preferably at least 9, 10, 1 1 , 12, 13, 14, 15, 20 or 50 amino acids.

[0059] A suitable non-limiting example of a suitable fragment for Cooperia oncophora sd-ASP is represented by a sequence comprising at least a Asparagine (symbol Asn or N), such as Asn61 according to the numbering in SEQ ID NO: 1 . In one embodiment, said Asn is glycosylated as provided herein.

[0060] In particular, the invention further relates to a single domain activation-associated secreted protein (ASP) or fragment thereof, said ASP or fragment comprising a glycan, in particular an N- glycan. More specific, the ASP or fragment comprises a core glycan structure built up by 2 N- acetylglucosamine (GIcNAc) residues in combination with 2, in particular 3, mannose residues, and optionally a core-fucose. Additional glycans may be present on the (outer) mannose residues (on positions X and / or Y in Structure I; Fig. 10a), each independently built up by or comprising a mannose (Man), galactose (Gal), N-acetylgalactosamine (GalNac), N-acetylglucosamine (GIcNac) and / or fucose (Fuc) residue(s), including combinations thereof. More particular, the N-glycan comprises the structure according to Structure (I) as provided herein. In said structure X and / or Y can be absent or when present are independently selected from mannose, galactose, N-acetylgalactosamine, N- acetylglucosamine and fucose, including combinations thereof, in particular X and / or Y are selected from N-acetylglucosamine, mannose and galactose. In one embodiment X is absent. In another embodiment Y is absent. In a further embodiment, X comprises GIcNAc and Gal and Y is absent. In a preferred embodiment, X is absent or is Man and Y is Man, in particular Man2 or Man3.

[0061] In particular, the invention provides a single domain activation-associated secreted protein (ASP) or fragment thereof, said ASP or fragment comprising a glycan, in particular an N-glycan, having a structure selected from the list consisting of:

[0062] - Man3-GlcNAc2

[0063] - Man3-GlcNac3

[0064] - Man3-GlcNac4

[0065] - Man3-GlcNAc-(Fuc-)GlcNAc

[0066] - Man5-GlcNAc2

[0067] - Man6-GlcNAc2

[0068] - Man7-GlcNAc2,

[0069] - GalMan3GlcNAc3Fuc,

[0070] - GalMan3GlcNAc4Fuc,

[0071] - Gal(Fuc)GalNacMan3GlcNAc2,

[0072] - Gal(Fuc)GalNAc2Man3GlcNAc2, and

[0073] - GalNAc-(Fuc-)Gal-GlcNAc-Man3-GlcNAc-(Fuc-)GlcNAc.

[0074] The invention furthermore provides combinations and mixtures of one or more (i.e. 2, 3, 4, 5, 6, 7, 8, 9,10 or all) of the ASPs identified herein, such as the ASPs comprising an N-glycan having structure I or as given in Fig. 10a or 10b.

[0075] As referred to herein, N-linked glycosylation is the attachment of an oligosaccharide, a carbohydrate consisting of several sugar molecules, sometimes also referred to as glycan, to a nitrogen atom (the amide nitrogen of an asparagine (Asn) residue of a protein), in a process called N-glycosylation.

[0076] The ASP or fragment of the invention may also be referred to as isolated ASP or fragment. The term “isolated” is used to indicate that a cell, protein or nucleic acid is separated from its native environment. Isolated proteins and nucleic acids may be substantially pure, i.e. essentially free of other substances with which they may bound in nature.

[0077] In the context of the present invention the term “recombinant” refers to being synthetically produced or expressed in an expression system which is not the native expression system. A recombinant protein or fragment thereof should thus be interpreted as a protein expressed in an expression system which is different from the expression system it is natively expressed in. The term “recombinant” also includes molecules formed by laboratory methods of genetic recombination, which bring together genetic material from different sources, thereby creating sequences which would not otherwise be found in the native genome. Also, in the context of the present invention, the term “recombinant ASP of the invention” should be interpreted as a recombinant single domain activation-associated secreted protein or fragment thereof comprising at least one glycan, in particular a N-glycan.

[0078] A “glycan” as used herein generally refers to glycosidically linked monosaccharides, oligosaccharides and polysaccharides. Hence, carbohydrate portions of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan are referred to herein as a “glycan”. Glycans can be homo- or heteropolymers of monosaccharide residues, and can be linear or branched. The term “N- glycan” or alternatively ‘N-linked glycan’ relates to a glycan attached to the nitrogen atom of an asparagine (Asn) side chain. Such N-glycans are attached to eukaryotic proteins and play a major role in the structure and function of said proteins. Typical N-glycans may be selected from the list comprising N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, or other monosaccharides.

[0079] The N-glycan according to the invention preferably comprises GIcNAc, GalNac, Man, Fuc, and Gal in the conformation of structure (I) (Fig. 10a and 10b). For the Co-sd-ASP (SEQ ID NO: 1 ), glycans are present on Asn61 .

[0080] The chemical structure of the glycans associated with the ASP antigen according to the invention, is in accordance with the common nomenclature in carbohydrate biochemistry.

[0081] Glycosylation is the reaction in which a carbohydrate (or 'glycan'), i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate. Expression systems having the ability to glycosylate are therefore able to provide the recombinant ASP or fragments of the present invention. Enzymes able to perform such glycosylation processes are transferases, glucosidases and / or mannosidases. These enzymes can be endogenously present in the expression host or can be co-expressed with the ASP protein.

[0082] According to a further embodiment of the present invention the ASP protein is produced synthetically or by the expression of a nucleic acid as described herein in a suitable expression system. Typically an expression system comprises a vector and a host cell. Suitable vectors for expression of proteins are plasmids, bacteriophages, cosmids, viruses, minichromosomes or stably integrating vectors. Generally, these vectors have the property of autonomous replication except for the stably integrating vectors which insert themselves in the genetic material of the host cell and replicate with host's genetic material. Suitable host cells for the expression of proteins may either be prokaryotic or eukaryotic, such as but not limited to bacteria such as Escherichia coli, yeasts such as Saccharomyces cerevisiae and Pichia pastoris, mycoplasma's, algae, plant cells such as Arabidopsis thaliana and Nicotiana spp., such as Nicotania benthamiana or Nicotania tabacum, vertebrate cells, or baculovirus / insect cells; the plant or animals cells may be cultivated in vitro or may form part of an intact plant or animal, respectively.

[0083] According to a particular embodiment, the (host) cell of the present invention is a glycoengineered cell. A “glyco-engineered cell” refers to a cell that has been genetically modified so that it expresses proteins with an altered N-glycan structure as compared to a non-engineered cell or expression system. Typically, this includes the use of specific enzymes involved in the glycosylation pathway. In general, sugar chains in N-linked glycosylation may be divided in three types: high- mannose (typically yeast), complex (typically mammalian) and hybrid type glycosylation. The different types of N-glycosylation are all well known to the skilled person and defined in the literature. Considerable effort has been directed towards the identification and optimization of strategies for the engineering of eukaryotic cells that produce glycoproteins having a desired N-and / or O-glycosylation pattern and are known in the art (van der Kaaij et al., 2022; Ma et al. 2020 - incorporated herein by reference). Enzymes needed for complex glycosylation include, but are not limited to: N- acetylglucosaminyl transferase I, N-acetylglucosaminyl transferase II, mannosidase II, galactosyltransferase, fucosyltransferase and sialyltransferase, and enzymes that are involved in donor sugar nucleotide synthesis or transport. Still other glyco-engineered cells, in particular yeast cells, that are envisaged here are characterized in that at least one enzyme involved in the production of high mannose structures (high mannose-type glycans) is not expressed. Enzymes involved in the production of high mannose structures typically are mannosyltransferases. In particular, alpha-1 ,6- mannosyltransferases Ochlp, Alg3p, alpha-1 ,3-mannosyltransferase of the Mnnlp family, beta-1 ,2- mannosyltransferases may not be expressed. Thus, a cell can additionally or alternatively be engineered to express one or more enzymes or enzyme activities, which enable the production of the particular N-glycan structures of the invention at a high yield. Such an enzyme can be targeted to a host subcellular organelle in which the enzyme will have optimal activity, for example, by means of signal peptide not normally associated with the enzyme. It should be clear that the enzymes described herein and their activities are well-known in the art. A signal peptide (also referred to as signal sequence) is a short peptide (usually 15-30 amino acids long) present at the N-terminus (or occasionally nonclassically at the C-terminus or internally) of the synthesized protein that is destined toward the secretory pathway. For this, the recombinant gene for the recombinant protein to be produced is linked with a signal sequence (“in-frame fusion”), which results in the production of a signal peptide-protein fusion product. The signal peptide encoded by the signal sequence mediates the secretion of the recombinant protein across the cytoplasmic membrane into the periplasm by means of the expression system. In this, the signal peptide is cleaved off at the cleavage site between signal peptide and the recombinant protein, and the desired recombinant protein is obtained in the periplasm. The recombinant protein can then be purified from the periplasm. Various signal sequences and the corresponding signal peptides are known and described in the art. An appropriate signal peptide will be selected by the skilled person based on the expression system. Hence in one embodiment, the invention provides a fusion protein comprising an amino acid sequence of the single domain ASP as defined herein and a signal peptide. The invention further encompasses a nucleic acid sequence encoding such a signal peptide and / or fusion protein.

[0084] The recombinant polynucleotide may contain as an insert a complete polynucleotide coding for the ASP or a fragment thereof. Bacterial, yeast, fungal, insect, plant and vertebrate cell expression systems (e.g. CHO cells) are very frequently used systems. Such systems are well known in the art and generally available. Accordingly, the present invention provides to a recombinant polynucleotide comprising the herein described nucleic acid sequence, in particular further including a functionally linked promoter and / or signal sequence. The invention furthermore provides a vector comprising the herein described nucleic acid sequence or recombinant polynucleotide, said vector in particular being a plasmid, bacteriophage, cosmid, virus or minichromosome; and a host cell comprising the herein described nucleic acid sequence, recombinant polynucleotide or vector, said host cell in particular being an animal cell, bacterial cell, yeast cell, insect cell or plant cell.

[0085] In particular, the invention relates to the recombinant ASP of the invention which is obtained from the expression systems such as a plant expression system, in particular Nicotiana spp. expression system (e.g. Nicotania benthamiana or Nicotania tabacum), a yeast expression system, in particular Pichia pastoris expression system, a mammalian expression system, or an insect cell line expression system. In a particular embodiment, the expression system comprises an enzyme capable of producing Man6, such as a mannosidase I (alpha mannosidase). In the alternative the expression system is transformed to comprise an enzyme capable of producing Man6, such as a mannosidase I.

[0086] In a further aspect, the invention provides a method to produce a recombinant ASP or fragment thereof, said method comprising the steps of introducing an expression vector comprising a nucleotide sequence provided herein in a suitable expression host, expressing and isolating said protein or fragment of the invention. By the term “a suitable cell” a higher eukaryotic cell, such as a mammalian cell, insect cell or a plant cell, a lower eukaryotic cell, such as a filamentous fungus cell or a yeast cell, or a prokaryotic cell such as a bacterial cell, said cell being optionally glycoengineered, is envisaged as explained above.

[0087] Particularly envisaged herein is the production of a recombinant ASP or fragment provided herein, wherein said protein or fragment is glycosylated and comprises one or more glycans, in particular N-glycans.

[0088] In another embodiment, the invention relates to a method for producing a recombinant ASP or fragment of the invention, said method comprising the steps of: a) providing an expression system comprising a nucleic acid sequence encoding an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 99%, most preferably 100% sequence identity with the amino acid sequence represented by SEQ ID NO: 1 ; b) optionally introducing in the expression system an enzyme, in particular a mannosidase I, c) expressing an ASP with the expression system provided in step a) to obtain a recombinant ASP of the invention.

[0089] In this method, the expression system can be a mammal, yeast, bacterial, plant or insect expression system, such as Nicotiana benthamiana expression system, Pichia pastoris expression system, or an insect cell line expression system. In a particular embodiment, the invention comprises a recombinant ASP or fragment obtained by said method. Furthermore, the recombinant ASP of the invention as obtained from this method can be comprised in a pharmaceutical composition. The recombinant ASP of the invention and / or the pharmaceutical composition comprising the recombinant ASP of the invention can be used as a medicine, preferably as a vaccine to treat, prevent or reduce infection of parasitic nematodes provided herein, in particular a Cooperia infection, more in particular an infection with Cooperia oncophora.

[0090] In a further embodiment, the invention relates to a pharmaceutical composition comprising an ASP or fragment thereof according to invention, a nucleic acid, a vector or host cell as provided herein, and a pharmaceutically acceptable carrier and / or excipient. More specific, the composition can comprise one or more of the glycosylated ASPs as provided herein. The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” means solvents, dispersion media, emulsifying agents, disintegrants, isotonic agents, absorption delaying agents, stabilizers, buffers (e.g. PBS), and the like, that are compatible with pharmaceutical administration. Effective amounts of preservatives can also be included into the formulations. Suitable preservatives include, without limitations, benzalkonium chloride (0.003-0.03% w / v); chlorobutanol (0.3-0.9% w / v); parabens (0.01 - 0.25% w / v) and thimerosal (0.004-0.02% w / v). The use of such media and agents for pharmaceutically active substances, and the determination of an effective amount, is well known in the art. In one embodiment, the pharmaceutical composition is a vaccine, such as a preparation used to stimulate the body's immune response against a specific disease.

[0091] For these purposes, the pharmaceutical composition of the present invention may be formulated by means known in the art into the form of, for example, tablets, pellets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely divided powders or aerosols or nebulizers for inhalation, and for parenteral use (including intravenous, intramuscular or infusion) sterile aqueous or oily solutions or suspensions or sterile emulsions.

[0092] In practicing the present methods, a vaccine or composition of the present invention is administered preferably via intramuscular or subcutaneous routes, although other routes of administration can be used as well, such as e.g. by oral, intranasal (e.g. aerosol or other needleless administration), intra-lymph node, intradermal, intraperitoneal, rectal or vaginal administration, or by a combination of routes. The formulation of the composition or the vaccine can be made in various forms depending upon the route of administration. For example, the compositions can be made in the form of sterile aqueous solutions or dispersions suitable for injectable use, or made in lyophilized forms using freeze-drying techniques. Lyophilized immunogenic compositions are typically maintained at about 4°C, and can be reconstituted in a stabilizing solution, e.g. saline or and HEPES, with or without adjuvant. In one embodiment, a single dose of the vaccine reduces nematode infestation in a previously unvaccinated animal. In the alternative, boosting regimens (e.g. a second, third, fourth, etc. dose) may be required and the dosage regimen can be adjusted to provide optimal immunization. Immunization protocols can be optimized using procedures well known in the art. A single dose can be administered to animals, or, alternatively, two, three or more inoculations can take place with intervals of one to ten weeks. Depending on the age of the animal, the immunogenic or vaccine composition can be re-administered. For example, the present invention contemplates the vaccination of healthy calves (3-12 months of age) about 6 and / or about 3 weeks prior to their first grazing season and revaccination at the beginning of the first grazing season.

[0093] The ASP or fragment thereof of the present invention for use in the treatment, prevention and / or reduction of a nematode infection, in particular with parasite of the genus Cooperia, is preferably dosed in a therapeutically effective amount. The term "therapeutically effective amount" refers to an amount sufficient to elicit an immune response and / or to confer protection in the animal to which it is administered. The immune response may comprise, without limitation, induction of cellular and / or humoral immunity. The amount of a vaccine that is therapeutically effective may vary depending on the condition of the animal (e.g. ruminant or cattle) and / or the degree of infection, and can be determined by a veterinary physician. "Protection" (and related terms such as "immunoprotection" and "immunoprotective") as used herein, 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 parasitic nematode. The term "reduction" relates to reducing susceptibility for nematode infection. In the context of the present invention and as demonstrated herein, this means causing the target subject to show a reduction in the number and / or size of adult and / or juvenile parasites, the number of eggs 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, shedding and effects that are caused by the parasite or by the subject’s response thereto.

[0094] The ability of the single domain ASP of the invention to induce an immune response to parasitic nematodes such as Cooperia oncophora, allows that the pharmaceutical composition of the invention comprising this ASP can be used as a vaccine against these nematodes, in particular against nematodes of the genera Cooperia, more in particular against Cooperia oncophora. In a preferred embodiment, the pharmaceutical composition of the invention is thus a vaccine.

[0095] To provide such immunity against said nematodes, the vaccine may optionally further comprise an adjuvant. Therefore, in a further aspect of the invention, the pharmaceutical composition of the invention comprises an adjuvant.

[0096] Adjuvants are known to act in a number of different ways to enhance the immune response. In general, immunomodulatory adjuvants cause a general up-regulation of certain cytokines and a concomitant down regulation of others leading to a cellular Th1 and / or a humoral Th2 response.

[0097] Suitable adjuvants include, without limitations, oil emulsions (such as water-in-oil, oil-in- water, watier-in-oi-in-water), Complete Freund’s adjuvants, saponins such as for example Quil A, ISCOMs (complexes of saponins, sterols and phospholipids), Aluminum compounds including Aluminum phosphate and Aluminum hydroxide, mycobacterial cell wall extracts, MPL-A, quaternary ammonym compounds such as dimethyl dioctadecyl ammonium bromide (DDA), acrylic acid based polymers such as carbomers including CARBOPOI®, glycolipids such as BAY®R1005 and oligonucleotides containing CpG motifs. Combinations of these compounds are also envisioned. Preferably, the composition as described herein is an immunogenic composition. By "immunogenic" is meant the capacity to provoke an immune response in a subject against the pathogen / parasite. The present invention accordingly provides compositions for use in eliciting an immune response which may be utilized as a vaccine, in particular against nematodes belonging to the genus Cooperia, more in particular against the nematodes Cooperia oncophora, Cooperia punctate, or Cooperia pectinate. The immune response can be a cellular immune response mediated primarily by NK cells, cytotoxic T-cells, and / or a humoral immune response mediated primarily by helper T-cells, which in turn activates B-cells leading to antibody production. More specific, by "eliciting or inducing an immune response" is meant that an antigen stimulates synthesis of specific IgG 1 antibodies and / or cellular proliferation as measured by, for example, 3H thymidine incorporation of NK cells, T-cells and B-cells. The importance if a IgG 1 response was demonstrated herein, more specific the mucosal IgG 1 showed a significant negative correlation with cumulative faecal egg output and worm length.

[0098] In one embodiment, administering the composition or vaccine of the invention elicits an immune response that results in a reduction in worm count of at least about 30%, 40%, 45% or 50% in an animal in relation to a non-vaccinated (e.g. adjuvant alone) control animal. In another embodiment, administering the composition or vaccine of the invention elicits an immune response that results in a reduction in mean cumulative fecal egg count of at least about 30%, 40%, 45% or 50% in an animal in relation to a non-vaccinated (e.g. adjuvant alone) control animal. Preferably, the level of the decrease is about 55%, more preferably about 60% and most preferably, about 70% or greater. In a specific embodiment, the reduction in mean cumulative fecal egg count is present for at least 4 weeks after the first administration of the single domain ASP or fragment, in particular for at least 5 weeks, 6 weeks, 7 weeks or 8 weeks. Hence the immune response confers some beneficial, protective effect to the subject against a subsequent challenge with the infectious agent. More preferably, the immune response prevents the onset of or ameliorates at least one symptom of a disease associated with the infectious agent, or reduces the severity of at least one symptom of a disease associated with the infectious agent upon subsequent challenge.

[0099] Given the beneficial medical properties of the composition of the invention, the present invention relates, according to another aspect, to the single domain ASP or the pharmaceutical composition of the invention, for use as a medicament, in particular a human or veterinary medicine.

[0100] The immune response induced by the ASP or fragment, or pharmaceutical composition of the invention can treat, prevent or reduce infection of parasitic nematodes, such as nematodes belonging to the genus Cooperia, more in particular the parasitic nematodes Cooperia oncophora, Cooperia punctate, or Cooperia pectinate. A further aspect of the invention is therefore that the ASP or fragment or the pharmaceutical composition of the invention can be used in the treatment, prevention and / or reduction of a parasitic nematode infection in a subject, in particular a mammal.

[0101] By “subject” or “host” is meant any mammal / animal that has or is susceptible to a nematode infection, in particular susceptible to infection with nematodes belonging to the genus Cooperia as provided herein, such as humans or non-human animals, in particular ruminants, more in particular cattle 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. The term "cattle" refers to bovine animals including but not limited to steer, bulls, cows, and calves.

[0102] In a preferred embodiment, the recombinant ASP or fragment or the pharmaceutical composition of the invention can be used in the treatment, prevention and / or reduction of nematode infections, in particular gastrointestinal nematode infections, more in particular infections with nematodes belonging to the genus Cooperia, such as Cooperia oncophora, Cooperia punctate, or Cooperia pectinate.

[0103] The invention further encompasses the use of the isolated ASP or fragment disclosed herein, the nucleic acid sequence endoding the ASP or fragment, or the vector or host cell containing the nucleic acid sequence, for the manufacture of a medicament for treating a nematode infection, in particular a parasitic nematode infection, more in particular a gastrointestinal parasitic infection, such as an infection with nematodes belonging to the genus Cooperia.

[0104] In a further embodiment, the invention relates to a method of treatment, prevention and / or reduction of a nematode infection in a subject in need thereof, comprising administering to said subject in need thereof an isolated single domain ASP or fragment, a nucleic acid sequence endoding the ASP or fragment, a vector or host cell containing the nucleic acid sequence, or a pharmaceutical composition, as provided herein. More specific, the nematode infection is a parasitic nematode infection, even more specific a gastrointestinal parasitic infection, such as an infection with nematodes belonging to the genus Cooperia.

[0105] The invention will now be illustrated by means of the following examples, which do not limit the scope of the invention in any way.

[0106] EXAMPLES

[0107] Materials and methods

[0108] Purification of MMW and native Co-sd-ASP from adult worm ES. Adult worm ES was obtained from C. oncophora infected calves as previously described (Borloo et al. 2013). The MMW fraction was purified from the ES fraction by applying the ES material to a HiLoad 16 / 70 Superdex 200 pg column (Cytiva) as previously described (Borloo et al. 2013).

[0109] From the MMW fraction, native Co-sd-ASP was isolated via affinity chromatography. For this, a HiTrap Con A 4B column (GE Healthcare 28-9520-85) was employed. Binding buffer (20 mM Tris- HCI, 0.5 M NaCI, 1 mM MnCIz, 1 mM CaCIz; pH 7.4) was added to the MMW fraction prior to loading onto the column. The bound fraction was eluted with an elution buffer composed of 0.5 M methyl-a- D-glucopyranoside (methyl-a-D-glucoside), 20 mM Tris-HCI, 0.5 M NaCI at pH 7.4. For binding and eluting, a flow rate of 0.5 ml / min was used. After elution, samples were put on PBS and evaluated on SDS-PAGE followed by staining with a Pierce™ Silver Stain Kit (ThermoFisher) according to the manufacturer’s instructions.

[0110] Production and purification of N. benthamiana Co-ASP-1.

[0111] The AXT / FT N. benthamiana plants for recombinant expression are a laboratory accession derived from RA-4, and were provided by Dr. Richard Strasser (Strasser et al. 2008). All handling and use of N. benthamiana and associated samples in this study was conducted in compliance with institutional, national, and international guidelines and legislation.

[0112] The nucleic acid encoding the Co-sd-ASP protein sequence was subjected to in-house codon optimization by Wageningen University and Research, and its synthetic construction was performed at GeneArt. The gene fragment was flanked with Nhel / Kpnl restriction site for subcloning behind the Arabidopsis thaliana chitinase gene (cSP) in the plant expression vector pHYG (Westerhof et al. 2012).

[0113] Culturing of A. tumefaciens (strain MOG101 ), infiltration of AXT / FT transgenic N. benthamiana leaves with these bacteria and extraction of the apoplast fluid was done as described previously (Wilbers et al, 2017; Strasser et al. 2008). Apoplast fluid was desalted via Sephadex-G25 columns (Cytiva), buffer-exchanged to sodium acetate (pH 4.4) before purified using the HS POROS® 50 strong cation exchange resin (ThermoFisher) on the AKTA Prime Liquid Chromatography System (GE Healthcare). Bound Co-sd-ASP was eluted from the column using a gradient with cationexchange buffer supplemented with 1 M NaCI at 2 ml / min, followed by dialysis against phosphate- buffered saline (PBS). Protein concentration was determined using a BCA assay (Pierce, ThermoFisher). Successful expression and purification were confirmed on a 12% Bis-Tris SDS- PAGE gel (ThermoFisher) stained with Coomassie Brilliant Blue.

[0114] Protein evaluation on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

[0115] All antigens were evaluated on SDS-PAGE under non-reducing conditions, visualised via Coomassie blue staining as described previously (Borloo et al., 2013b). In addition, the protein bands associated with native Co-sd-ASP and the N. benthamiana recombinant were cut out and sequenced by the Vlaams Instituut voor Biotechnologie (VIB) for the presence of Co-sd-ASP (Genbank accession number: HAAM01000002.1 ).

[0116] Enzymatic treatment of native Co-sd-ASP

[0117] Native Co-sd-ASP was subjected to treatment with either PNGase F (New England Biolabs), EndoH (New England Biolabs) or a-mannosidase (New England Biolabs) according to the manufacturer’s instructions.

[0118] N-glycan profiling via MS

[0119] The N-glycosylation of native Co-sd-ASP and the recombinant produced in N. benthamiana was evaluated via MS after PNGase F and / or A release as described in a previous study (Wilbers et al, 2017).

[0120] Indirect and competition enzyme-linked immunosorbent assay.

[0121] Indirect ELISAs were performed as described previously (Gonzalez-Hernandez et al., 2018). Slight modifications include: 96-well ELISA plates (MaxiSorp, NUNC) were coated with 1 pg / ml native Co- sd-ASP in 100 pl carbonate buffer (pH 9.6) overnight at 4°C. Serum samples and HRP-conjugated antibodies were used at following concentrations: bovine serum at 1 / 200 in PBS, sheep anti-bovine lgG1 -HRP (AAI21 P; Bio-Rad) at 1 / 4000 in blocking buffer (2% bovine serum albumin in PBS-0.05% Tween20), sheep anti-bovine lgG2-HRP (AAI22P; Bio-Rad) at 1 / 1000 in blocking buffer, and 1 / 1000 anti-bovine IgA-HRP (AAI49P; Bio-Rad) at 1 / 1000 in blocking buffer. For competition ELISAs, 96- well Maxisorp plates (MaxiSorp, NUNC) were coated with 1 pg / ml native Co-sd-ASP and dilution series of test antigens ranging from 0 to 500 pmol / ml were generated as described previously (Gonzalez-Hernandez et al. 2018). A 1 / 4000 sheep anti-bovine lgG1 -HRP (AAI21 P; Bio-Rad) was used to monitor antibody binding against the lgG1 isotype. 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) (Roche) was used as substrate and the colour development, expressed as OD405-492, was quantified by using an Infinite F50 Absorbance Microplate Reader (Tecan Trading AG).

[0122] Bovine immunisation experiment

[0123] All animal experiments were conducted in accordance with the E.U. Animal Welfare Directives and VICH Guidelines for Good Clinical Practice, and ethical approval to conduct the studies were obtained from the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University (EC201 1 / 147, EC2022 / 047).

[0124] For the immunisation study to evaluate LMW and MMW, twenty-one helminth naive male Holstein calves of 4-6 months old were randomised over three groups of seven animals: LMW + QuilA, MMW + QuilA or QuilA alone. The study was conducted as described previously (Gonzalez-Hernandez et al. 2018; Geldhof et al. 2004; Gonzalez-Hernandez et al. 2016). In short, all animals received 30 |ag of antigen and / or 750 pg of QuilA percutaneous in the neck muscle, three times with a three-week interval. Starting on the day of the third immunisation, all calves were challenged via a 5-week long trickle infection of 25,000 O. ostertagi L3 (1000 L3 I day; 5 days / week; for 5 weeks). Faecal egg counts were determined three times per week with McMaster (Rossanigo et al. 1991 ) over the course of the study, starting 21 days after the first infection. The calves were euthanised three weeks after the last infection to determine worm counts and worm length measurements.

[0125] For the bovine immunisation-challenge study to evaluate native Co-sd-ASP, sixteen helminth naive male Holstein calves of 4-6 months old were randomised over two groups of eight animals: native Co-sd-ASP + QuilA or QuilA alone. The study was conducted as described above. Instead of McMaster, faecal egg counts were performed with MiniFLOTAC (Cringoli et al. 2017). Serum samples were collected before the first immunisation and one week after each immunisation. Mucus samples were collected at time of necropsy and processed as described in previous studies (Gonzalez-Hernandez et al. 2016).

[0126] Statistical analyses

[0127] The statistical analyses were performed using GraphPad Prism 9. For the bovine immunisationchallenge study involving the LMW and MMW fractions, cumulative faecal egg output and worm counts were evaluated for statistical significance via Kruskal-Wallis tests. The systemic lgG1 and lgG2 response was evaluated via a two-way ANOVA with Dunnet’s test for multiple comparison. For the bovine immunisation-challenge study with native Co-sd-ASP versus QuilA controls, cumulative faecal egg output, worm counts and worm measurements were evaluated for statistical significance via one-tailed Mann-Whitney tests. The systemic IgG 1 and lgG2 response was evaluated via a two- way ANOVA with Dunnet’s test for multiple comparison. The mucosal IgG 1 and lgG2 response was evaluated via one-tailed Mann-Whitney tests.

[0128] The correlation between mucosal lgG1 and cumulative faecal egg counts, worm counts and worm measurements was evaluated for statistical significance via a nonparametric Spearman R correlation. All data were presented as mean ± standard error of the mean (SEM). P < 0.05 was considered significant.

[0129] Results

[0130] Example 1

[0131] Calves immunised with the mid-molecular weight fraction of C. oncophora excretory / secretory products demonstrated protection against infection.

[0132] The LMW and MMW fractions were purified from adult worm ES products using size-exclusion chromatography, employing a previously described approach (Borloo et al., 2013). To investigate the protective capacity of these fractions against a C. oncophora infection, a bovine immunisation- challenge study was conducted. Twenty-one calves were randomly assigned to three groups (n=7) and each group received three immunisations with either the LMW fraction + QuilA, MMW fraction + QuilA or QuilA alone. Subsequently, calves underwent a trickle infection with infectious L3 stage C. oncophora, and the faecal egg excretion was measured over the course of the study (Fig. 1 a). The group immunised with the MMW fraction demonstrated a significant reduction in cumulative faecal egg excretion by 83% (p < 0.05), whereas the group immunised with the LMW fraction showed a non-significant 34% reduction (p > 0.05), both compared to the QuilA control group (Fig. 1 b; Table 1 ). The total worm burden was reduced by 48% for the MMW group compared to controls (Fig. 1 c; Table 1 ), but was not statistically significant (p > 0.05). No clear impact on worm burden was found for the LMW group. Calves immunised with MMW demonstrated a significantly increased MMW- reactive systemic IgG 1 response after the third immunisation (p < 0.05) and at necropsy (p < 0.001 ), compared to the control group (Fig. 1 d). For LMW immunised calves, the LMW-reactive systemic lgG1 response was significantly increased only at necropsy (p < 0.01 ), compared to the control group. For both vaccine groups, the systemic lgG2 response was not significantly increased compared to controls.

[0133] Table 1 : Overview of parasitological parameters obtained in the immunisation-challenge study with the low-molecular weight (LMW) and mid-molecular weight (MMW) fractions. n, number of animals; EPG, mean cumulative eggs per gram faeces; % L4, percentage of L4 worms observed in post-necropsy worm counting; Male and female worm length in pm. All values represent arithmetic means (+ experimentally observed range). * p < 0.05

[0134] Example 2

[0135] Immunisation with the native single-domain ASP resulted in a significant reduction in faecal egg output and worm burden.

[0136] The reduction in faecal egg excretion observed after immunisation highlighted the considerable potential of the MMW fraction for further vaccine development, prompting the evaluation of its constituent antigens. Co-sd-ASP is one of the antigens identified in this fraction and accounts for approximately 50% of its contents. To purify the native Co-sd-ASP from the MMW fraction, a lectin- based approach was adopted, involving a Concanavalin A (ConA) Sepharose column. This approach was based on previous evidence indicating that Co-sd-ASP is primarily glycosylated with oligomannose-type N-glycans (Borloo et al., 2013). The purification process successfully yielded the single domain ASP, although the purity was not absolute as one of the LMW-component remained detectable on SDS-PAGE (Fig. 2).

[0137] To examine the involvement of native Co-sd-ASP in the observed 83% decrease in C. oncophora egg output after immunisation with the MMW fraction, a bovine immunisation-challenge study was conducted with the purified antigen. Calves were divided into two groups: one group was immunised with native Co-sd-ASP + QuilA (n=8), while the control group received only the QuilA adjuvant (n=8). The calves received three intramuscular immunisations followed by a trickle infection of infectious L3-stage C. oncophora larvae. Immunisation with native Co-sd-ASP resulted in an 86% reduction in cumulative faecal egg output, compared to the adjuvans control group (p < 0.001 ) (Fig. 3a, b; Table 2). In addition, immunisation with this native antigen also resulted in a significant (p < 0.001 ) reduction of the adult worm burden by 63% compared to the control group (Fig. 3c), and both male and female worm length was significantly reduced by 6% (p < 0.01 ) and 16% (p < 0.001 ), respectively (Table 2). Furthermore, calves immunised with native Co-sd-ASP demonstrated a clear systemic lgG1 response after the second immunisation (Fig. 3d), whereas the systemic lgG2 response became apparent after the third immunisation. At necropsy, mucosal lgG1 was significantly increased (p < 0.001 ) in Co-sd-ASP immunised animals (Fig. 3e) compared to QuilA controls, whereas no clear differences for mucosal lgG2 and IgA were observed between the two groups. Within the native Co-sd-ASP group, mucosal lgG1 showed a significant negative correlation with cumulative faecal egg output (p < 0.01 ; R2= 0.5812), male worm length (p < 0.05; R2= 0.5761 ) and female worm length (p < 0.05; R2= 0.6224) (Fig. 7). A negative correlation between mucosal IgG 1 and worm counts was not considered significant (p > 0.05; R2= 0.5222) .

[0138] Table 2: Overview of parasitological parameters obtained in the immunisation-infection study with the native Co-sd-ASP antigen. n, number of animals; EPG, mean cumulative eggs per gram faeces; % L4, percentage of L4 worms observed in post-necropsy worm counting; Male and female worm length in pm. All values represent arithmetic means (+ experimentally observed range). * p < 0.05; ** p < 0.01 ; *** p < 0.005

[0139] Example 3

[0140] Native Co-sd-ASP predominantly contains oligomannose-type N-glycans. Since immunisation with native Co-sd-ASP resulted in a protective immune response against C. oncophora, the subsequent phase involved the production and evaluation of a recombinant version of this antigen.

[0141] First, the N-glycosylation profile of purified native Co-sd-ASP was verified by mass spectrometric analysis. The analysis indicated the predominant presence of Mane-GlcNAc2 glycans (Fig. 4).

[0142] To get more insight on the role of these N-glycans on antibody recognition, it was attempted to enzymatically remove the antigen’s N-glycans. It was noted that treatment with PNGase F did not result in a complete deglycosylation if the protein remained intact. Nonetheless, Co-sd-ASP was treated with PNGase F as its N-glycans may be more accessible to this endoglycosidase compared to Oo-ASP-1 and Co-dd-ASP. In addition, native Co-sd-ASP was subjected to endoglycosidase H (EndoH) and a-mannosidase separately, as an alternative to PNGase F. EndoH cleaves between the two proximal GIcNAc residues of high mannose and some hybrid-type N-glycans, whereas a- mannosidase removes a-linked mannose residues.

[0143] In total, four different samples including an untreated control sample were evaluated on SDS-PAGE to evaluate the effectiveness of these glycosidases in removing or down-trimming the N- glycosylation of native Co-sd-ASP. Treating native Co-sd-ASP with PNGase F resulted in the emergence of a faint band with a lower molecular weight when compared to the intact Co-sd-ASP. Compared to the intact antigen, this lower molecular weight band displayed an increased intensity in the sample treated with EndoH, indicating a more effective removal of N-glycans. Last, the application of a-mannosidase caused a subtle shift in molecular weight, implying the removal of a- linked mannoses, theoretically resulting in a remaining Mani-GlcNAc-(Fuc-)GlcNAc N-glycan.

[0144] To conduct a more in-depth analysis of the remaining N-glycans after enzymatic treatment, all Co- sd-ASP samples were subjected to MS analyses. In terms of the N-glycan composition, no differences compared to intact Co-sd-ASP were found after PNGase F treatment. In contrast, EndoH treatment of the antigen resulted in the removal of oligomannose-type N-glycans. The remaining N- glycans were identified as small quantities of the core fucosylated, paucimannose-type, and hybridtype. Furthermore, no remaining N-glycan structures were identified following a-mannosidase treatment. This indicates that all N-glycans were down-trimmed to either Mani-(Fuc-)GlcNAc2 or Mani-GlcNAc2, which were outside the detection range during this analysis.

[0145] To evaluate the impact of (partial) N-glycan removal or down-trimming on antibody recognition of this antigen, a competition enzyme-linked immunosorbent assay (ELISA) was conducted. In this assay, serum from calves immunised with native Co-sd-ASP was pre-incubated with different concentrations of either intact native Co-sd-ASP, PNGase F treated native Co-sd-ASP, EndoH treated native Co-sd-ASP or a-mannosidase treated native Co-sd-ASP. The formed antibodyantigen complexes were transferred onto native Co-sd-ASP coated ELISA plates allowing the antibodies to either maintain their binding to the pre-incubation antigen or dissociate and bind to the antigen coated on the plate. Antibody binding to the coated antigen was measured and expressed in optical density ratio (ODR). As displayed in Fig. 5, partial removal or down-trimming of the antigen’s N-glycans had no impact on the antigen’s ability to compete with intact Co-sd-ASP for antibody binding. This indicates that the removed N-glycans do not play a role as part of the antibody epitope.

[0146] Instead, these oligomannose-type N-glycans are known to interact with specific soluble and cell-surface lectin receptors, such as mannose-binding lectin (MBL) and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN). Through this mechanism, glycans can facilitate antigen internalisation and modulate the adaptive immune response via lectin- mediated signalling pathways. For instance, a study demonstrated that mice lacking MBL-A exhibited reduced clearance of an experimental Brugia malayi infection. Similarly, in the context of human infections, specific polymorphisms in the MBL gene leading to low MBL expression were associated with significantly increased susceptibility to filarial infections caused by Wuchereria bancrofti.

[0147] Example 4

[0148] Nicotiana benthamiana recombinant of single-domain Co-ASP competes with the native antigen for antibody binding.

[0149] Recombinant versions of Co-sd-ASP were subsequently generated in wild-type and (31 ,2- xylosyltransferase and a1 ,3-fucosyltransferase down-regulated (AXT / FT) Nicotiana benthamiana plants, as illustrated in Fig. 6a. In a first step, the recombinants were produced without co-expression of external glycosyltransferases using the plant signal peptide MFTLCILLLSLCGLCTG (SEQ ID NO:3). The plant’s leaves were infiltrated with Agrobacterium tumefaciens containing transfer DNA for the Co-sd-ASP antigen. The expressed antigens were secreted in the plant’s apoplast fluid and harvested six days after infiltration via vacuum infiltration of an extraction buffer, followed by centrifugation. SDS-PAGE with Coomassie staining revealed two distinct protein bands for the recombinants with a mass of approximately 25 kDa (Fig. 6b), matching that of the native antigen. An LC-MS / MS in-gel protein sequence analysis confirmed that the upper and lower band contained Co- sd-ASP at a relative abundancy of 97 and 99%, respectively. The analysis also confirmed that the ASP antigens present in both bands possess an N-glycosylation site at Asparagine (Asn) residue 61 . Further evaluation was focused on the AXT / FT N. benthamiana recombinant. The N- glycosylation of this recombinant Co-sd-ASP was analysed by mass spectrometry, showing an N- glycosylation pattern that closely resembled that of the native Co-sd-ASP (Fig. 6c). Mane-GlcNAc2 was identified as the most prevalent N-glycan on the recombinant antigen. In addition, smaller quantities of Mans-GlcNAc2, Mans-GIcNAcs, Man5-GlcNAc2, Mans-GlcNAc4, and Gal-GIcNAc-Mans- GlcNAc-(Fuc-)GlcNAc were detected. A competition ELISA was carried out to assess the recognition of the recombinant antigen by serum from calves immunised with native Co-sd-ASP and to investigate the potential competition between the recombinant version and native Co-sd-ASP for antibody binding. In this assay, serum from calves immunised with native Co-sd-ASP was pre-incubated with different concentrations of either native Co-sd-ASP or the AXT / FT N. benthamiana recombinant. The formed antibody-antigen complexes were transferred onto native Co-sd-ASP coated ELISA plates allowing the antibodies to either maintain their binding to the pre-incubation antigen or dissociate and bind to the antigen coated on the plate. Antibody binding to the coated antigen was measured and expressed in optical density (OD) values. As displayed in Figure 4d, the AXT / FT N. benthamiana recombinant was able to partially inhibit the binding between the native antigen and native antigen-induced antibodies, suggesting a strong homology between the native and this recombinant antigen.

[0150] Example 5

[0151] Immunisation with the recombinant single-domain ASP resulted in a significant reduction in faecal egg output.

[0152] To investigate the protective capacity of the recombinant single-domain ASP against a C. oncophora infection, a bovine immunisation-challenge study was conducted. Sixteen calves were randomly assigned to 2 groups (n=8) and received three immunisations with either the recombinant singledomain ASP + QuilA or QuilA alone. Subsequently, calves underwent a trickle infection with 1000 infectious 3rd stage C. oncophora larvae for 25 consecutive days, and the faecal egg excretion was measured for a period of 5 weeks starting three post first infection. The group immunised with the recombinant single-domain ASP demonstrated a significant reduction in cumulative faecal egg excretion by 53 % (p < 0.05) as well as a significant reduction in worm count by 45% (p<0.05) compared to the QuilA control group (Fig. 8).

[0153] REFERENCES

[0154] Borloo, J. et al. In-Depth Proteomic and Glycomic Analysis of the Adult-Stage Cooperia oncophora Excretome / Secretome. J. Proteome Res. 12, 3900-3911 (2013).

[0155] Gonzalez-Hernandez, A. et al. Comparative analysis of the immune responses induced by native versus recombinant versions of the ASP-based vaccine against the bovine intestinal parasite Cooperia oncophora. International Journal for Parasitology 48, 41 -49 (2018).

[0156] Wilbers, R. H. P. et al. Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants. Sci. Rep. 10, 45910 (2017).

[0157] Strasser, R. et al. Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnol. J. 6, 392-402 (2008).

[0158] Borloo, J. et al. Structure of Ostertagia ostertagi ASP-1 : insights into disulfide-mediated cyclization and dimerization. Acta Crystallogr. Sect. D Biol. Crystallogr. 69, 1 -11 (2013b).

[0159] Geldhof, P. et al. Validation of the protective Ostertagia ostertagi ES-thiol antigens with different adjuvantia. Parasite Immunol. 26, 37-43 (2004).

[0160] Gonzalez-Hernandez, A. et al. Host protective ASP-based vaccine against the parasitic nematode Ostertagia ostertagi triggers NK cell activation and mixed lgG1 -lgG2 response. Sci. Rep. 6, 29496 (2016).

[0161] Rossanigo, C. E. & Gruner, L. Accuracy of two methods for counting eggs of sheep nematode parasites. Veterinary parasitology 39, 115-121 (1991 ).

[0162] Cringoli, G. etal. The Mini-FLOTAC technique for the diagnosis of helminth and protozoan infections in humans and animals. Nat. Protoc. 12, 1723-1732 (2017).

[0163] Van der Kaaij et al., Glyco-Engineering Plants to Produce Helminth Glycoproteins as Prospective Biopharmaceuticals: Recent Advances, Challenges and Future Prospects, Front. Plant Sci., 2022, 13, 1 -12.

[0164] Ma at al. Protein Glycoengineering: An Approach for Improving Protein Properties, Front. Chem., 23 July 2020, 8, 1 -14.

[0165] Westerhof, L. B. et al. 3D Domain Swapping Causes Extensive Multimerisation of Human I nter leukin- 10 When Expressed In Planta. Pios One 7, e46460 (2012)

Claims

CLAIMS1 . An isolated single domain activation-associated secreted protein (ASP), or a fragment or at least 15 amino acids thereof, wherein the amino acid sequence of the ASP or fragment thereof has at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 99%, most preferably 100% sequence identity with the amino acid sequence represented by SEQ ID NO: 1 .

2. The isolated ASP or fragment thereof according to claim 1 , comprising an N-glycan, in particular an N-glycan selected from the list consisting of:- Man3-GlcNAc2- Man3-GlcNac3- Man3-GlcNac4- Man3-GlcNAc-(Fuc-)GlcNAc- Man5-GlcNAc2- Man6-GlcNAc2- Man7-GlcNAc2,- GalMan3GlcNAc3Fuc,- GalMan3GlcNAc4Fuc,- Gal(Fuc)GalNacMan3GlcNAc2,- Gal(Fuc)GalNAc2Man3GlcNAc2, and- GalNAc-(Fuc-)Gal-GlcNAc-Man3-GlcNAc-(Fuc-)GlcNAc.

3. A nucleic acid sequence encoding the ASP, or the fragment thereof, according to claim 1 or 2, in particular wherein the nucleic acid sequence has at least 85% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 2.

4. A vector comprising the nucleic acid sequence according to claim 3.

5. A host cell comprising the nucleic acid sequence of claim 3 or the vector according to claim 4.

6. A pharmaceutical composition comprising a single domain activation-associated secreted protein (ASP) of Cooperia oncophora, or fragment thereof, a pharmaceutically acceptable excipient, and optionally an adjuvant.

7. The pharmaceutical composition according to claim 6 being a vaccine.

8. The isolated ASP or fragment thereof according to claim 1 or 2, the nucleic acid sequence according to claim 3, the vector according to claim 4, the host cell according to claim 5, or thepharmaceutical composition according to claim 6 or 7, for use as a medicament, preferably a human medicine or a veterinary medicine.

9. The isolated ASP or fragment thereof according to claim 1 or 2, the nucleic acid sequence according to claim 3, the vector according to claim 4, the host cell according to claim 5, or the pharmaceutical composition according to claim 6 or 7, for use in the treatment, prevention and / or reduction of a parasitic nematode infection, in particular in a mammal.

10. The isolated ASP or fragment thereof for the use of claim 9, wherein the parasitic nematode infection is gastrointestinal.11 . The isolated ASP or fragment thereof according to claim 1 or 2, the nucleic acid sequence according to claim 4, the vector according to claim 5, the host cell according to claim 6, or the pharmaceutical composition according to claim 7 or 8, for the use according to claims 9 or 10, wherein the parasitic nematode infection is a Cooperia infection, particularly a Cooperia oncophora infection, a Cooperia punctate infection, and / or a Cooperia pectinate infection.

12. A method for producing a recombinant ASP or fragment thereof, comprising the steps of: a) providing an expression system comprising a nucleic acid sequence encoding an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 99% sequence identity, most preferably 100% sequence identity with the amino acid sequence represented by SEQ ID NO: 1 ; b) optionally introducing into the expression system an enzyme, in particular a mannosidase I, and c) expressing ASP with the expression system provided in step a), or step b), to obtain the recombinant ASP or fragment thereof.

13. The method according to claim 12, wherein the expression system comprises a host cell selected from the group consisting of bacteria, yeasts, algae, plant cells, vertebrate cells, and insect cells.

14. The method according to claim 13, wherein the host cell is Nicotiana tabacum, Pichia pastoris, or an insect cell.