Expression system for glycoengineered antigens

Abstract

The present invention relates to an expression system for the extracellular production of recombinant tri-fucosylated N-glycoproteins in insect cells. The invention also relates to an expression cassette comprising a glyco-module comprising polynucleotide sequences of a nematode galactosyltransferase, fucosyltransferase, and mannosidase; and a polynucleotide sequence encoding one or more tri-fucosylated N-glycoproteins derived from a nematode; a recombinant baculovirus comprising said expression cassette, a method for producing said tri-fucosylated N-glycoproteins in transfected insect cells, and a transfected insect cell. The invention also relates to an immunological composition comprising said tri-fucosylated N-glycoproteins, which can be administered to ruminants to induce protective anti-nematode immunity.

Description

[0001] VT016P

[0002] -1 -

[0003] EXPRESSION SYSTEM FOR GLYCOENGINEERED ANTIGENS

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to an expression system for the production of extracellular recombinant tri-fucosylated N-glycoproteins in insect cells.

[0006] The invention also relates to an expression cassette comprising a glyco-module comprising polynucleotide sequences of a nematode galactosyltransferase, fucosyltransferase, and mannosidase; a polynucleotide sequence encoding one or more tri-fucosylated N-glycoproteins derived from a nematode; a recombinant baculovirus comprising said expression cassette, a method for the production of said tri-fucosylated N-glycoproteins in transfected insect cells, and a transfected insect cell.

[0007] The invention further relates to an immunological composition comprising said tri- fucosylated N-glycoproteins, which can be administered to ruminants to induce protective anti-nematode immunity.

[0008] BACKGROUND OF THE INVENTION

[0009] Parasitic nematode infection is one of the biggest health problems for farmed ruminants worldwide. Parasitic worm infections are harmful to host animals for many reasons. For example, they deprive the host of feed, damage internal tissues and organs, cause anemia, weight loss, diarrhea, dehydration, and loss of appetite. Such parasitic infections cause costly production losses and if left untreated, animals may die causing further economic loss to farmers. Therefore, nematode infection severely compromises animal welfare.

[0010] Currently, prophylactic measures mainly rely on the use of anthelmintic agents (such as benzimidazoles, levamisole, morantel, monepantel, or ivermectin) to control parasitic nematodes, however resistance of parasites to one or more of these agents is now widespread. Besides this, most anthelmintic drugs are excreted via the feces of the animals and the majority of the active substances are toxic for the environment, especially for aquatic organisms and dung bugs.

[0011] Alternative methods of controlling on-farm parasite infections have been proposed and include altered grazing management, use of nematode trapping fungi, dietary supplements, selective breeding of animals for host resistance and vaccines.

[0012] Attempts to develop recombinant vaccines against parasitic nematodes have had limited success and to date there are no commercial recombinant vaccines available for VT016P

[0013] -2- any nematode parasites. However, the development of such a vaccine is viewed by the industry as a solution to the resistance problem.

[0014] Haemonchus contortus, the barber’s pole worm, is the most pathogenic bloodfeeding nematode in small ruminants (sheep and goats) and New World camelids (alpaca and lama). H. contortus infections lead to decreased weight gain in animals, decreased fertility rates, and loss of young stock, therefore threatening animal welfare and causing tremendous production losses for farmers worldwide. As a single worm ingests up to 50 pl of blood per day, even moderate infections can cause severe blood loss (more than 100 ml daily), leading to anaemia, hypoproteinaemia, haemonchosis (blood loss and hypoproteinemia) and even death of infected animals.

[0015] Parasite control primarily relies on the administration of anthelmintic drugs; however, disadvantages include ecotoxicological effects, long-lasting withdrawal period and a high prevalence of anthelminthic resistant worms. Animals infected with resistant H. contortus may die due to acute haemonchosis and due to a lack of treatment response. Moreover, when anthelmintic drugs, such as avermectins, are administered to livestock, between 80% and 98% of these drugs leave the body without being metabolized and can affect soil invertebrates.

[0016] Anti- H. contortus vaccines have been developed to induce protective immunity in sheep and goats. Among the protein-based vaccine candidates tested, the highest efficacy of inducing protective immunity was observed by using antigens such as aminopeptidase H11 and H-gal-GP, which are purified from the intestinal fraction of worm extract

[0020] . Such antigens have since been developed into the vaccine Barbervax®, which is the only commercially available vaccine against H. contortus

[0001] . However, to produce Barbervax®, donor sheep have to be infected with H. contortus and subsequently sacrificed, while the obtained vaccine yield is rather low and, as the antigen is extracted from worms derived from donor sheep, there may be a risk of crosscontamination with infectious agents such as viruses.

[0017] As an alternative, recombinant expression of digestive aminopeptidase H11 antigens has been attempted, however, the efficacy of recombinant antigens has shown to be lower than the native form. H11 antigens are naturally glycosylated and carry distinct N-glycan structures, which are crucial to the antigenicity of H11 [2], A crucial feature of nematode antigen glycosylation is the tri-fucosylation of N-glycoproteins, with the N-glycan chitobiose core being modified with up to three fucose residues [3]. As a result, H11 produced through recombinant expression in eukaryotic hosts, such as VT016P

[0018] -3-

[0019] Pichia and non-engineered insect cell lines, lacks such glycan modifications, whereas H11 expressed in C. elegans can carry tri-fucosylated structures [4], However, it was demonstrated that C. elegans cannot be used to produce H11 for an immunogenic H. contortus animal vaccine, since fucose residues of H11 expressed in C. elegans are often capped with galactose residues.

[0020] The Swiss company Malcisbo AG attempted recombinant H11 production in insect cells, however, only the Gal-Fuc glycan epitope on H11 glycoproteins could be introduced.

[0021] Fleurkens and colleagues described the recombinant production of mono- or di- fucosylated N-glycoproteins H11 , AC1 , and PEP1 in insect cells co-expressing GALT-1. Vaccination of lambs with a combination of the three glycoproteins showed reduction in egg counts and worm burden, however, efficacy was significantly less compared to vaccination with native H11 extract

[0019] .

[0022] WO2013182603A1 discloses the production of H11 of Haemonchus contortus or aspartic protease APR-1 of Ancylostoma caninum, whereas isolated or purified N-glycan comprising L-fucopyranosyl is alpha-1 , 3-linked to the distal N-acetylglucosamine of the N-glycan core and further discloses insect cells overexpressing nematode glycosyltransferases using e.g. a heterologous expression system for overexpression of GALT- 1 or FUT-1 from C. elegans. EP2490707A2 discloses the use of glycan-binding polypeptides which bind to one or more nematode glycans as a medicament. However, none of these documents disclose a recombinant H11 with tri-fucosylated N- glycoproteins.

[0023] Taken together, the production of recombinant tri-fucosylated N-glycoproteins in insect cells remains technically challenging. Therefore, there is still a strong and yet unmet need for a production system for recombinantly produced glycoengineered tri- fucosylated N-glycoproteins in insect cells for a vaccine protecting animals from nematode infections.

[0024] SUMMARY OF THE INVENTION

[0025] It is the objective of the present invention to provide soluble recombinant H. contortus antigens with nematode-type glycans which can induce protective immunity against H. contortus infection in livestock.

[0026] The problem is solved by the present invention. It has been shown by the inventors that an expression system described herein, specifically comprising AMAN-3, VT016P

[0027] -4-

[0028] GALT-1 and FUT-6 in combination with vaccine antigens can produce recombinant tri- fucosylated N-glycoproteins in insect cells which, when administered as a vaccine, induce protective immunity protecting ruminants from nematode infections.

[0029] According to the invention there is provided an expression cassette for the production of extracellular recombinant tri-fucosylated N-glycoproteins in insect cells comprising a polynucleotide sequence encoding one or more tri-fucosylated N- glycoproteins derived from a nematode and a glyco-module comprising polynucleotide sequences of a nematode galactosyltransferase, specifically GALT-1 , of a nematode fucosyltransferase, specifically FUT-6, and of a nematode mannosidase, specifically AMAN-3.

[0030] According to an embodiment of the invention, the expression cassette comprises a Caenorhabditis elegans (C. elegans) GALT-1 , a C. elegans FUT-6, and a C. elegans AMAN-3, and the polynucleotide sequence encoding one or more tri-fucosylated N- glycoproteins is derived from Haemonchus contortus.

[0031] In an embodiment of the invention, the expression cassette comprises polynucleotide sequences of a nematode galactosyltransferase which comprises the nucleotide sequence SEQ ID NO: 1 or SEQ ID NO: 2, a fucosyltransferase which comprises the nucleotide sequence SEQ ID NO: 3 or SEQ ID NO: 4, and / or a mannosidase which comprises the nucleotide sequence SEQ ID NO: 5.

[0032] According to a further embodiment, the glyco-module of the expression cassette comprises polynucleotide sequences from C. elegans, specifically GALT-1 , FUT-6, and / or AMAN-3; or from Haemonchus contortus (H. contortus), specifically GALT-1 and / or FUT-6.

[0033] According to a further embodiment, the tri-fucosylated N-glycoproteins are intestine proteins, specifically derived from any one of H. contortus, Haemonchus place!, Teladorsagia circumcincta, Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Ascaris suum, Oesophagostomum dentatum, or Ostertagia ostertagi.

[0034] In a further embodiment, the tri-fucosylated N-glycoproteins are derived from H. contortus glycoprotein H11 , encoded by SEQ ID NO: 6; H11-1 , encoded by SEQ ID NO: 7; H11-2, encoded by SEQ ID NO: 8; H11-4, encoded by SEQ ID NO: 9; H11-5a, encoded by SEQ ID NO: 10; H11-5b, encoded by SEQ ID NO: 11 ; H11-5c, encoded by SEQ ID NO: 12; and / or GA1 , encoded by SEQ ID NO: 13.

[0035] Provided in a further embodiment is a recombinant baculovirus comprising the expression cassette. Provided in a further embodiment is a method for production of VT016P

[0036] -5- recombinant tri-fucosylated N-glycoproteins derived from a nematode in insect cells comprising the sequential steps: a) transfecting insect cells with the expression cassette or with the baculovirus comprising the expression cassette, b) expressing the tri-fucosylated N-glycoproteins by the insect cells, c) harvesting and optionally purifying the tri-fucosylated N-glycoproteins from the insect cells.

[0037] In a further embodiment, the insect cells used for the production of recombinant tri-fucosylated N-glycoproteins are Lepidoptera- erived cells, more specifically Trichoplusia ni High Five (Hi5) cells, Spodoptera frugiperda Sf9, Spodoptera frugiperda Sf21 , or Trichoplusia ni Tnao38 cells.

[0038] According to a further embodiment, the polynucleotide sequences of the glycomodule and the polynucleotide sequences encoding the one or more tri-fucosylated N- glycoproteins are co-expressed.

[0039] Provided in a further embodiment is an immunological composition against a nematode, comprising at least one recombinant tri-fucosylated N-glycoprotein, wherein the recombinant tri-fucosylated N-glycoprotein comprises two mannose residues and up to one galactose residue, specifically a recombinant intestine tri-fucosylated N- glycoprotein, and optionally one or more adjuvants.

[0040] In a further embodiment, the tri-fucosylated N-glycoproteins produced by the aforementioned method or part of the immunological composition are derived from any one of Haemonchus contortus, Haemonchus placei, Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Ascaris suum, Oesophagostomum dentatum, or Ostertagia ostertagi.

[0041] According to a further embodiment, the tri-fucosylated N-glycoproteins produced by the method described herein or being part of the immunological composition are derived from H. contortus glycoprotein H11 comprising SEQ ID NO: 14; H11-1 comprising SEQ ID NO: 15; H11-2 comprising SEQ ID NO: 16; H11-4 comprising SEQ ID NO: 17; H11-5a comprising SEQ ID NO: 18; H11-5b comprising SEQ ID NO: 19; H11- 5c comprising SEQ ID NO: 20; and / or GA1 comprising SEQ ID NO: 21.

[0042] In a further embodiment, the intestine tri-fucosylated N-glycoprotein produced by the method described herein, or being part of the immunological composition is H. contortus glycoprotein lacking its trans-membrane domain. VT016P

[0043] -6-

[0044] In a further embodiment, the immunological composition is a lyophilized powder, a frozen liquid, or a liquid.

[0045] Provided in a further embodiment is an immunological composition for use in vaccinating an animal, specifically a ruminant, or a New World camelid.

[0046] According to an embodiment, the immunological composition is for use in reducing nematode parasitic worm burden in an animal, specifically in ruminants or New World camelids.

[0047] According to an embodiment, the immunological composition is for use in stimulating or boosting acquired immunity in an animal, specifically in a ruminant, or a New World camelid. According to an embodiment, the immunological composition for use in vaccinating an animal induces protective anti-nematode immunity in an animal, specifically in ruminants or New World camelids.

[0048] According to a further embodiment, the immunological composition for use in vaccinating an animal induces protective anti-nematode immunity for a nematode selected from the group consisting of Haemonchus contortus, Haemonchus placei, Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Ascaris suum, Oesophagostomum dentatum, and Ostertagia ostertagi.

[0049] In a further embodiment, the vaccinated animals have reduced fecal egg count and worm burden in the gastrointestinal tract compared to unvaccinated animals infected with the nematode, wherein the nematode is specifically selected from the group consisting of Haemonchus contortus, Haemonchus placei, Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Ascaris suum, Oesophagostomum dentatum, and Ostertagia ostertagi.

[0050] According to a further embodiment, the immunological composition is administered as injection, specifically as subcutaneous injection, intramuscular injection, or intradermal injection.

[0051] In a further embodiment, the immunological composition is administered to sheep, goats, alpacas, llamas, or cattle.

[0052] Provided in a further embodiment is a recombinant H. contortus H11 or GA1 antigen having core tri-fucosylated N-glycans, wherein the N-glycans are nematode-type N-glycans comprising any one of amino acid sequences SEQ ID No: 14 to 21 , or amino acid sequences having at least 90%, 91 %, 92%, 93%, 94%, 95%, specifically at least 98%, more specifically 99% sequence identity to SEQ ID NOs: 14 to 21. VT016P

[0053] -7-

[0054] Provided in a further embodiment is an engineered insect cell comprising the expression cassette or the recombinant baculovirus comprising the expression cassette.

[0055] FIGURES

[0056] Figure 1. Overview of a H. contortus vaccine trial. Schematic overview of the conducted vaccine trial. Seven sheep were used in each experimental group, d, day; wk, week.

[0057] Figure 2. Construction of engineered H. contortus antigen sequences. Functional peptide fragments such as melittin signal peptide, HisFLAG duo-affinity tag and thrombin cleavage site were incorporated to the N-termini of predicted transmembrane domains (TMD)-truncated antigen sequences (A, SEQ ID NOs: 25 and 26). A phylogenetic tree of recombinant H11 and GA1 antigens constructed according to (A) is shown in (B). Amino acid sequences of H11 isoforms (KB strain) and GA1 (US strain) were aligned using the online server “multalin” (http: / / multalin.toulouse.inra.fr / multalin / ) and a phylogenetic tree was generated to indicate the homology of different antigens. A comparison of the generated mature recombinant products is shown in (C). Lengths (amino acids, aa), molecular weights (kDa) and predicted N-glycosites of mature recombinant products were compared using online servers Expasy Compute pl / Mw tool and NetNGIyc - 1.0. PAM, point accepted mutation; KB strain, an isolate of Katharinenhof in Bavaria (KB); US strain, a Beltsville strain maintained by the United States Department of Agriculture (US).

[0058] Figure 3. Comparison of typical N-glycan core modifications in different species. Mammalian, C. elegans, H. contortus, and insect glycoproteins are shown in (A). Major biosynthetic pathways of N-glycans in non-engineered and glycoengineered Hi5 insect cells are shown in (B). An overview of N-glycan structures detected in glycoengineered antigens (GEA) is given in (C). In contrast to the native insect enzymes (shown in regular font), the introduced C. elegans glyco-enzymes are highlighted in bold. A summary of the definition of the N-glycan core structure, and N-glycans identified in H. contortus and in GEA is given in (D). Glycan structures are drawn in symbol nomenclature for glycans (SNFG) and the annotations of monosaccharides as well as abbreviations are given [5].

[0059] Figure 4. Schematic workflow of the preparation of glycoengineered recombinant antigens. Molecular cloning of glyco-genes from wild type C. elegans and construction of baculovirus expression system (bacmid) DNAs for containing synthetic antigenencoding DNAs with (I) or without (II) the “glyco-module” (A). Preparation of high-titer VT016P

[0060] -8- recombinant baculovirus stocks using Sf9 insect cells (B). Recombinant expression and purification of soluble antigens of H. contortus (C).

[0061] Figure 5. Overview of generated DNA constructs carrying the glyco-module and different H. contortus antigen encoding genes. Verification was performed by Nanopore DNA sequencing.

[0062] Figure 6. Coomassie blue staining to determine purity and concentration of purified recombinant antigens.

[0063] Figure 7. MALDI-TOF MS spectra of 2-aminopyridine (PA)-labelled N-glycans. Matrix-assisted laser desorption / ionization (MALDI) time-of-flight (TOF) mass spectrometer (MS) spectra released from non-glycoengineered antigens (NEA) and glycoengineered antigens (GEA). Peaks indicating tri-fucosylated glycan compositions are annotated in bold italic. H, hexose; N, N-acetyl-hexosamine; F, fucose.

[0064] Figure 8. MALDI-TOF MS / MS spectra of N-glycans before and after hydrofluoric acid (HF) treatment. The glycan present on GEA (A) exhibits similar structural characteristics to a glycan purified from H. contortus (B), both losing two core a1 ,3- fucose residues upon HF treatment.

[0065] Figure 9. HPLC chromatograms of 2-aminopyridine (PA)-labelled N-glycans released from non-glycoengineered antigens (NEA) and glycoengineered antigens (GEA). Peaks are annotated with major glycans structures in SNFG format, which were detected by MALDI TOF MS / MS. HPLC, High-performance liquid chromatography.

[0066] Figure 10. Aminopeptidase activity of recombinant H11 antigens. Enzymatic assays were carried out at 37°C (pH 7.0) by incubating normalised recombinant H11 antigens (1 pg / reaction) with a pNP-leucine substrate. Absorptions at optical density (OD) 405nm were measured after 24-hour incubation. Reaction mixtures containing either no substrate (Neg.Ctl.1) or no recombinant antigen (Neg.CtL2) are set as negative controls. Barbervax® was used as a positive control (Pos.Ctl.Bvax). ge, glycoengineered.

[0067] Figure 11. In vitro enzymatic property of H. contortus FUT-6. MALDI-TOF MS spectra of H. contortus FUT-6 and C. elegans FUT-6 show that FUT-6 from H. contortus and C. elegans show similar enzymatic properties.

[0068] Figure 12. In vitro assay of biological activity of recombinant antigens. Recombinant antigens, either used alone or as a cocktail (GEA or NEA), vaccine adjuvant Quil-A® and a detergent-free Barbervax® were incubated with ovine PBMC cells isolated from a healthy blood sheep donor. Cytokines in cell culture supernatants VT016P

[0069] -9- were measured by Luminex assays using an ovine cytokine panel. Supernatant of nonstimulated PMBC was included as a negative control (Neg.Ctl.).

[0070] Figure 13. Time course analysis of vaccination-induced parasite-specific antibodies in sheep. Haemonchus-specific serum IgG (top) and IgE (bottom) produced in sheep are shown. Western blotting images demonstrate the binding patterns of BarbervaxO-induced serum IgG to larval and adult parasite proteins (Top right).

[0071] Figure 14. Vaccination-induced antigen-specific IgG antibodies in sheep serum. Serum samples were analysed using ELISA plates coated with the corresponding vaccine antigens. Experimental groups according to Figure 1 B.

[0072] Figure 15. Vaccination induced antigen-specific IgE antibodies in sheep serum. Serum samples were analysed using ELISA plates coated with the corresponding vaccine antigens. Experimental groups according to Figure 1 B.

[0073] Figure 16. Comparison of hemoglobin levels of sheep after vaccine challenge. Sheep in the GEA group showed significantly higher hemoglobin levels than the NEA group (p = p < 0.05).

[0074] Figure 17. Eggs per Gram of sheep feces (EpG) after vaccine challenge. Comparison of EpG of five experimental groups after challenge on day 63 (A). Sheep of the GEA group showed significantly lower EpG values than the NEA group (p < 0.05). Mean EpG is shown for G2 to G5 in (B). Significant differences between groups are highlighted with superscript letters. For instance, letter “a” highlights the significant difference between G2 (no vaccination / challenge) and G3 (Barbervax® I challenge). Letter “b” highlights the difference between G2 (no vaccination / challenge) and G4 (GEA / challenge) (a-e). The significance level was set at p < 0.05.

[0075] Figure 18. Total worm count in abomasum of sheep after vaccine challenge. Log transformed worm counts are shown for experimental groups G2 to G5 in (A). Mean worm counts of the experimental groups G2 to G5 are shown in (B). Significant differences between groups are highlighted with superscript letters (a-c). The significance level was set at p < 0.05.

[0076] Figure 19. Eggs per female worm. The number of eggs per female worm was calculated by dividing the cumulative egg shedding by the number of adult worms in the abomasum. An analysis of the total number of worms shed by one female worm is shown in (B). The value was calculated using the cumulative egg excretion divided by the number of adult worms.

[0077] Figure 20. Sequence information. VT016P

[0078] -10-

[0079] DETAILED DESCRIPTION

[0080] Successful glycoengineering of recombinant nematode antigens to obtain tri- fucosylated N-glycan glycosylation was surprisingly achieved by co-expression of the nematode glycoprotein with an expression cassette comprising genes encoding a mannosidase, a fucosyltransferase, and a galactosyltransferase.

[0081] Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (4th Ed.), Vols. 1 -3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., "Lewin's Genes XI", Jones & Bartlett Learning, (2017), and Murphy & Weaver, "Janeway's Immunobiology" (9th Ed., or more recent editions), Taylor & Francis Inc, 2017.

[0082] The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.

[0083] The term “about” as used herein refers to the same value or a value differing by + / - 5 % of the given value.

[0084] As used herein and in the claims, the singular form, for example “a”, “an” and “the” includes the plural, unless the context clearly dictates otherwise.

[0085] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1 , 1.1 , 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1 .5 to 5.5, and 3.1 to 4.7) and, therefore, all subranges of all ranges are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0086] The terms “expression cassette" or "expression vector" or “expression construct” refer to nucleotide sequences that are capable of effecting expression of a sequence or a product in host cells or host organisms compatible with such sequences, and wherein the coding sequence is operably linked to the appropriate expression control sequences, which at least comprises a suitable transcription regulatory sequence VT016P

[0087] -11- and optionally, 3' transcription termination signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements. The expression cassette will be introduced into a suitable host cell and be able to affect the expression of the coding sequence in an in vitro cell culture of the host cell.

[0088] As is known, expression of a gene product requires the presence of expression control and / or regulatory sequences such as one or more promoters and any other nucleic acid sequences, such as introns, necessary for expression of the selected nucleic acid sequence, all operably linked to the selected sequence, and may include an enhancer sequence.

[0089] As used herein, the term "promoter" or "transcription regulatory sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer or biological entity. The expression cassette can include multiple foreign genes, each regulated by a specific promoter. As a nonlimiting example, antigen-coding genes and AMAN-3 can be driven by a polyhedrin promoter, while FUT-6 and GALT-1 expression can be regulated by a P10 promoter (promoter of the late p10 gene).

[0090] The term "gene" means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region and a 3'-nontranslated sequence (3'-end) comprising a polyadenylation site. "Expression of a gene" refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which VT016P

[0091] -12- is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide.

[0092] An expression control sequence is "operably linked" to a nucleotide sequence when the expression control sequence controls and regulates the transcription and / or the translation of the nucleotide sequence. Thus, an expression control sequence can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, a start codon in front of a protein-encoding gene, splicing signal for introns, and stop codons.

[0093] The term "expression control sequence" is intended to include, at a minimum, a sequence whose presence is designed to influence expression, and can also include additional advantageous components. For example, leader sequences and fusion partner sequences are expression control sequences. The term can also include the design of the nucleic acid sequence such that undesirable, potential initiation codons in and out of frame, are removed from the sequence. It can also include the design of the nucleic acid sequence such that undesirable potential splice sites are removed. It includes sequences or polyadenylation sequences (pA) which direct the addition of a polyA tail, i.e., a string of adenine residues at the 3'-end of a mRNA, sequences referred to as polyA sequences. It also can be designed to enhance mRNA stability. Expression control sequences that affect the transcription and translation stability, e.g., promoters, as well as sequences that affect the translation, e.g., Kozak sequences, are known in insect cells. Expression control sequences can be of such nature as to modulate the nucleotide sequence to which it is operably linked such that lower expression levels or higher expression levels are achieved.

[0094] As used herein, the term “expression cassette” refers to a unit cassette that contains i) a glyco-module comprising polynucleotide sequences of a nematode a) galactosyltransferase, specifically GALT-1 , and b) fucosyltransferase, specifically a core alpha-1 ,3 fucosyltransferase, more specifically FUT-6, and c) mannosidase, specifically an alpha-mannosidase III, more specifically AMAN- 3, and ii) a polynucleotide sequence encoding one or more tri-fucosylated N-glycoproteins derived from a nematode, and is capable of expressing the gene product (glyco-enzymes) and leads to the biosynthesis of glycoengineered antigens, i.e. the tri-fucosylated N-glycoproteins. VT016P

[0095] -13-

[0096] In a specific embodiment, the expression cassette described herein comprises a C. elegans GALT-1 , a C. elegans FUT-6, and a C. elegans AMAN-3, and the polynucleotide sequence encoding one or more tri-fucosylated N-glycoproteins is derived from Haemonchus contortus.

[0097] Herein, the expression cassette or construct is an insect cell-compatible vector. An insect cell-compatible vector is understood to be a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell. Exemplary insect cell compatible vectors include plasmids, linear nucleic acid molecules, and recombinant viruses, such as baculoviruses. Any vector can be employed as long as it is insect cell compatible. The mammalian or insect cell-compatible vector may integrate into the cell’s genome but the presence of the vector in the cell needs not be permanent and transient episomal vectors are also included.

[0098] The nucleic acid or vectors of the invention may be delivered into the cell of the invention by any suitable methods, included but not limited to, chemical treatment of the cells, infection, transfection, transformation, transduction, nucleofection, electroporation, microinjection. For example, the expression cassette or nucleic acid of the invention may be comprised in a plasmid contained in bacteria. The expression cassette or nucleic acid of the invention may also be comprised in an insect production cell or in a baculovirus vector.

[0099] Specifically, the insect cell-compatible vector can be a recombinant baculovirus carrying the expression cassette described herein. The recombinant baculovirus can be harvested from insect cells, e.g. Sf9, or Sf21 cells, which were transfected with recombinant bacmid DNA carrying antigen encoding genes and genes encoding the galactosyltransferase, fucosyltransferase, and mannosidase described herein. The term “Bacmid” refers to baculovirus genomes that contain a bacterial origin of replication so that they can replicate in bacteria as a plasmid.

[0100] A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. Cells used herein to establish plasmid DNA can be E. coli cells e.g. NEB5alpha, TOP10 cells, or PIR1 cells.

[0101] Plasmids may be introduced into a cell via transformation, specifically, plasmid DNA carrying antigen genes and glyco-genes can be transformed to competent cells to VT016P

[0102] -14- establish Bacmid DNA. Cells which can be used for this purpose are e.g. E. coll strains engineered for uptake of plasmids, such as but not limited to DH EMBacY cells.

[0103] A “glyco-module” is part of a gene expression cassette. Specifically, a glycomodule comprises polynucleotide sequences of genes encoding enzymes for glycosylation. The glyco-module of the expression cassette comprises a polynucleotide sequence encoding a galactosyltransferase, such as, but not limited to C. elegans GALT-1 , specifically comprising the nucleotide sequence SEQ ID NO: 1 , or H. contortus GALT-1 , specifically comprising the nucleotide sequence SEQ ID NO: 2.

[0104] The glyco-module of the expression cassette further comprises a polynucleotide sequence encoding a fucosyltransferase, such as, but not limited to a core alpha-1 ,3 fucosyltransferase, specifically C. elegans FUT-6, specifically comprising the nucleotide sequence SEQ ID NO: 3 or H. contortus, specifically comprising the nucleotide sequence SEQ ID NO: 4. Alternatively, the fucosyltransferase may also be FUT-1 or FUT-8 from C. elegans, or FUT-1 or FUT-8 from H. contortus.

[0105] The glyco-module of the expression cassette further comprises a polynucleotide sequence encoding a mannosidase, specifically C. elegans AMAN-3 comprising the nucleotide sequence SEQ ID NO: 5.

[0106] Herein encompassed are also sequences having at least 90%, specifically 91 , 92, 93, 94, 95, 96 ,97, 98, 99, or 99.5 % sequence identity with any one of SEQ ID NOs: 1 to 5.

[0107] In a specific embodiment, the gene expression cassette of the invention comprises a glyco-module comprising a polynucleotide sequence of nematode genes for tri-fucosylated glycosylation comprising a) the galactosyltransferase GALT-1 , specifically comprising SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence having at least 98%, specifically at least 98,5%, 99%, or 99.9% identity to SEQ ID NO: 1 or SEQ ID NO: 2, or SEQ ID NO: 1 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 point mutations, or SEQ ID NO: 2 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27 point mutations. SEQ ID NO: 1 can also be referred to as Ce GALT-1, SEQ ID NO: 2 as He GALT- 1. b) the fucosyltransferase FUT-6, specifically comprising SEQ ID NO: 3 or SEQ ID NO: 4, or a nucleotide sequence having at least 98%, specifically at least 98,5%, VT016P

[0108] -15-

[0109] 99%, or 99.9% identity to SEQ ID NO: 3; or SEQ ID NO: 3 or SEQ ID NO: 4 having

[0110] 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 point mutations.

[0111] SEQ ID NO: 3 can also be referred to as Ce FUT-6, SEQ ID NO: 4 as He FUT-6. c) the mannosidase AMAN-3, specifically comprising SEQ ID NO: 5, or a nucleotide sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 5; or SEQ ID NO: 5 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27, up to 64 point mutations. SEQ ID NO: 5 can also be referred to as Ce AMANS.

[0112] Nematode genes of the glyco-module can be genes from H contortus, C. elegans or homologous genes of other nematodes.

[0113] “Glycoengineering” refers to the engineering of proteins with modified glycosylation patterns. The tri-fucosylated N-glycoproteins expressed according to the invention can be intestine proteins, derived from any one of e.g. Haemonchus contortus, Haemonchus placet, Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Ascaris suum, Oesophagostomum dentatum, Ostertagia ostertagi, Ostertagia (Teladorsagia) circumcincta, Cooperia curticei, Nematodirus spathiger, Trichostrongylus axi, Trichostrongylus vitrinus, Cooperia oncophera, Nematodirus brasiliensis, Dictyocaulus eckerti, genus Strongylida, specifically, Strongylus vulgaris, Nematodirus filicollis, Ashworthius sidemi, Mecistocirrus digitatus, Bunostomum trigonocephalum, Toxacara vitulorum, or Trichostrongylus colubriformis.

[0114] In a specific embodiment, the tri-fucosylated N-glycoproteins are intestine proteins derived from H. contortus, Haemonchus place!, T. circumcincta, Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Ascaris suum, Oesophagostomum dentatum, or Ostertagia ostertagi.

[0115] In a specific embodiment, the tri-fucosylated N-glycoproteins are derived from H. contortus.

[0116] In a further embodiment, the glycoprotein is a H. contortus microsomal peptidase H11 , specifically comprising SEQ ID NO: 6, or a nucleotide sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 6, or SEQ ID NO: 6 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27, up to 60 point mutations. SEQ ID NO: 6 can also be referred to as Hc / ?77.

[0117] In a further specific embodiment, the glycoprotein is a H. contortus microsomal peptidase H11-1 , specifically comprising SEQ ID NO: 7, or a nucleotide sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 7, or SEQ ID NO: 7 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20,

[0118] 21 , 22, 23, 24, 25, 26 or 27, up to 60 point mutations. SEQ ID NO: 7 can also be referred

[0119] In a further specific embodiment, the glycoprotein is a H. contortus microsomal peptidase H11-2, specifically comprising SEQ ID NO: 8, or a nucleotide sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 8, or SEQ ID NO: 8 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20,

[0120] 21 , 22, 23, 24, 25, 26 or 27, up to 59 point mutations. SEQ ID NO: 8 can also be referred to as He h11-2.

[0121] In a further specific embodiment, the glycoprotein is a H. contortus microsomal peptidase H11-4, specifically comprising SEQ ID NO: 9, or a nucleotide sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 9, or SEQ ID NO: 9 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20,

[0122] 21 , 22, 23, 24, 25, 26 or 27, up to 59 point mutations. SEQ ID NO: 9 can also be referred to as He h11-4.

[0123] In a further specific embodiment, the glycoprotein is a H. contortus microsomal peptidase H11-5a, specifically comprising SEQ ID NO: 10, or a nucleotide sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO:

[0124] 10, or SEQ ID NO: 10 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27, up to 61 point mutations. SEQ ID NO: 10 can also be referred to as He h11-5a.

[0125] In a further specific embodiment, the glycoprotein is a H. contortus microsomal peptidase H11-5b, specifically comprising SEQ ID NO: 11 , or a nucleotide sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO:

[0126] 11 , or SEQ ID NO: 11 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27, up to 58 point mutations. SEQ ID NO: 11 can also be referred to as He h11-5b.

[0127] In a further specific embodiment, the glycoprotein is a H. contortus microsomal peptidase H11-5c, specifically comprising SEQ ID NO: 12, or a nucleotide sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO:

[0128] 12, or SEQ ID NO: 12 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27, up to 58 point mutations. SEQ ID NO: 12 can also be referred to as He h11-5c. -17-

[0129] In a further specific embodiment, the glycoprotein is a H. contortus apical gut protein GA1 , specifically comprising SEQ ID NO: 13, or a nucleotide sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 13, or SEQ ID NO: 13 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27, up to 50 point mutations. SEQ ID NO: 13 can also be referred to as He gal.

[0130] An “immunological composition” as used herein refers to a composition comprising molecules, such as proteins or polypeptides, that are immunologically active in the sense that once administered to an animal, it can evoke an immune response of the humoral and / or cellular type directed against the molecule. The immunological composition described herein may comprise one or more adjuvants.

[0131] The immunological composition described herein may be a lyophilized powder, a frozen liquid, or a liquid.

[0132] In a specific embodiment, the immunological composition described herein comprises at least one recombinant tri-fucosylated N-glycoprotein, wherein the recombinant tri-fucosylated N-glycoprotein comprises two mannose residues and up to one galactose residue.

[0133] Specifically, the immunological composition described herein comprises at least one recombinant tri-fucosylated N-glycoprotein, wherein the recombinant tri-fucosylated N-glycoprotein is a recombinant intestine tri-fucosylated N-glycoprotein.

[0134] As described herein, the immunological composition comprises at least one recombinant glycoengineered H. contortus glycoprotein carrying tri-fucosylated N- glycans, taken from the group of microsomal peptidases e.g., H11 , H11-1 , H11-2, H11- 4, H11-5a, H11-5b, or H11-5c, or apical gut protein GA1 :

[0135] A recombinant product KB H11 , specifically comprising amino acid sequence SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, or 98%,, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 14, or SEQ ID NO: 14 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17,

[0136] 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,

[0137] 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63,

[0138] 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86,

[0139] 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, or 98 point mutations.

[0140] A recombinant product KB H11-1 , specifically comprising amino acid sequence SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91 %, 92%, 93%, 94%, VT016P

[0141] -18-

[0142] 95%, 96%, 97%, or 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 15, or SEQ ID NO: 15 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17,

[0143] 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,

[0144] 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63,

[0145] 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86,

[0146] 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, or 97 point mutations.

[0147] A recombinant product KB H11-2, specifically comprising amino acid sequence SEQ ID NO: 16, or an amino acid sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 16, or SEQ ID NO: 16 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17,

[0148] 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,

[0149] 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63,

[0150] 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86,

[0151] 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, or 98 point mutations.

[0152] A recombinant product KB H11-4, specifically comprising amino acid sequence SEQ ID NO: 17, or an amino acid sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 17, or SEQ ID NO: 17 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17,

[0153] 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,

[0154] 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63,

[0155] 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86,

[0156] 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, or 98 point mutations.

[0157] A recombinant product KB H11-5a, specifically comprising amino acid sequence SEQ ID NO: 18, or an amino acid sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 18, or SEQ ID NO: 18 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17,

[0158] 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,

[0159] 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63,

[0160] 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86,

[0161] 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 point mutations.

[0162] A recombinant product KB H11-5b, specifically comprising amino acid sequence SEQ ID NO: 19, or an amino acid sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 19, or SEQ ID NO: 19 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, VT016P

[0163] -19-

[0164] 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,

[0165] 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63,

[0166] 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86,

[0167] 87, 88, 89, 90, 91 , 92, 93, 94, 95, or 96 point mutations.

[0168] A recombinant product KB H11-5c, specifically comprising amino acid sequence SEQ ID NO: 20, or an amino acid sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 20, or SEQ ID NO: 20 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17,

[0169] 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,

[0170] 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63,

[0171] 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86,

[0172] 87, 88, 89, 90, 91 , 92, 93, 94, 95, or 96 point mutations.

[0173] A recombinant product GA1 , specifically comprising amino acid sequence SEQ ID NO: 21 , or an amino acid sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO:

[0174] 21 , or SEQ ID NO: 21 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18,

[0175] 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,

[0176] 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64,

[0177] 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, or 83 point mutations.

[0178] The parasitic nematodes treatable by the methods of this invention include, but are not limited to Haemonchus contortus, Haemonchus placei, Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Ascaris suum, Oesophagostomum dentatum, Ostertagia ostertagi, Ostertagia (Teladorsagia) circumcincta, Cooperia curticei, Nematodirus spathiger, Trichostrongylus axi, Trichostrongylus vitrinus, Cooperia oncophera, Nematodirus brasiliensis, Dictyocaulus eckerti, Strongylus vulgaris, Toxascaris vitolorum, Nematodirus filicollis, Ashworthius sidemi, Mecistocirrus digitatus, Bunostomum trigonocephalum, Toxacara vitulorum, Trichostrongylus colubriformis.

[0179] The immunological composition according to the invention may comprise of one or more adjuvants. The term “adjuvant” refers to a substance, or a combination of substances, that is used to increase the efficacy or potency of drugs, specifically, a substance that increases the immune response to the immunological composition described herein. Suitable adjuvants for the vaccination of farmed or wild ruminant animals include but are not limited to oil emulsions such as Freund's complete adjuvant, VT016P

[0180] -20-

[0181] Freund's incomplete adjuvant, squalane or squalene; mineral gels such as aluminum hydroxide, aluminum phosphate, calcium phosphate, calcium phosphate and alum; surfactants such as hexadecylamine, lysolecithin, and methoxyhexadecylglcerol; polyanions such as dextran sulphate and carbopol; peptides such as muramyl dipeptide and dimethylglycine; or other adjuvants including Quil A, lipopolysaccharide, montanide, lipovant, bacterial flagellin, adjuvant 65, imiquimod, gamma inulin, gardiquimod, or any combinations thereof. A preferred adjuvant is Montanide or Quil A.

[0182] The immunological composition or vaccine composition of the invention may also be a pharmaceutical preparation, further comprising one or more pharmaceutically or veterinary acceptable vehicles, diluents, excipients, and / or optionally, a carrier.

[0183] The carrier can be selected from, but is not limited to, solgel (a chitin based slow release compound), hollow mesoporous silicon nanoparticles (HMSNs), poly(d,l-lactide- co-glycolide) (PGC) nanoparticles, poly(d,l-lactic-coglycolic acid) (PGCA) nanoparticles, liposomes, virosomes, cochleate delivery vehicles, etc. The composition or vaccine composition may contain salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition as would be understood by a skilled worker.

[0184] The composition or vaccine composition of the present invention can be given to an animal before any infection is detected to act as a preventative or can be given as a treatment to infected animals.

[0185] The term “animal” as used herein refers to any farmed animal, wild animal, or animal kept in enclosures or in zoos which belong to the group of ruminant animals or New World camelids, such as, but not limited to sheep, cattle, goat, deer, buffalo, bison, camelids, lamas etc. Said animals can be specifically young animals, less than one year old, i.e. lambs, calves, kid goats etc. In one aspect, the animal is less than 6 months old. In a further aspect, the animal is at least 3 months old. In one aspect, the animal can also be older than 6 months old.

[0186] The composition or vaccine composition of the present invention is preferably in a form for administering to an animal via subcutaneous or intramuscular injection.

[0187] The composition or vaccine composition of the present invention will be formulated for subcutaneous or intramuscular administration as a parenterally injectable aqueous solution which is pyrogen-free and has a suitable pH, isotonicity, and stability. Preferably, the composition or vaccine composition is administered twice, with a primary immunization given followed by a booster 2-8 week later, preferably 3 weeks later. In VT016P

[0188] -21- some ruminants, a second booster may be required around 4-8 weeks after the first booster depending on antibody levels as would be understood by a skilled worker.

[0189] The composition or vaccine composition of the invention can be administered as a single or multiple doses in a therapeutically effective amount. The herein referred to “dose” refers to each administration of the immunological composition. The dosevolume of the herein described immunological composition for the target species, e.g. the dose volume of sheep compositions, is generally in the range of about 0.1 to about 5.0 ml, specifically of about 0.1 to 2.0 ml, specifically of 0.5 to 2.0 ml, or specifically of 1.0 to 2.0 ml.

[0190] The “dosage” describes the amount of antigen administered per dose. The dosage of the herein described immunological composition may be in the range of about 50 pg to about 200 pg, specifically, of about 70 to 150 pg, preferably, of about 100 pg per dose (per shot). Additional doses can be administered as required to treat or prevent infection as would be understood by a skilled worker.

[0191] The composition or vaccine composition of the invention will be administered to an animal in a therapeutically effective amount, i.e. an amount that results in an immunologic response such as the production of desirable antibodies.

[0192] The composition or vaccine composition of the invention can be administered with anthelmintic agents such as levamisole, morantel, oxfendozole, monepantel and / or ivermectin to increase the overall EpG reduction rates and worm burden of the treated animals at the time of vaccination.

[0193] The composition or vaccine composition of the invention can also be administered with other vaccine treatments commonly administered to ruminants and New World camelids such as clostridial diseases (including enterotoxaemia, pulpy kidney, tetanus, malignant oedema, black disease and black leg); bovine viral diarrhoea (BVD); footrot; leptospirosis; salmonella; scabby mouth, etc.

[0194] The term “antigen” refers to proteins, peptides, polysaccharides (chains of simple sugars), lipids, or nucleic acids, which can bind to a specific antibody and may trigger an immune response. Specifically, the term “vaccine antigen” refers to glycoproteins described herein, specifically, H. contortus tri-fucosylated N-glycoproteins H11 , H11-1 , H11-2, H11-4, H11-5a, H11-5b, H11-5c, or GA1. One type of a vaccine using said antigens is a subunit vaccine. Subunit-vaccine encompasses a vaccine that contains parts of the pathogen that are antigenic, or necessary to elicit a protective immune VT016P

[0195] -22- response. Subunit vaccine can be made from dissembled pathogen particles or recombinantly produced proteins.

[0196] An "immune response" to a composition or vaccine is the development of a cellular and / or antibody-mediated response to the composition described herein in the host, i.e. , the animal, specifically, in sheep, cattle, goats, deer, buffalo, bison, camelids, or llamas. Usually, an immune response includes, but is not limited to, one or more of the following effects: the production of antibodies, B cells, helper T cells, and / or cytotoxic T cells, directed specifically to the proteins included in the composition described herein.

[0197] In general, an immune response is generated to an antigen (also referred to as immunogen) through the interaction of the antigen with the cells of the immune system. Stimulation of an immune response can result from a direct or indirect response of a cell or component of the immune system to exposure to an immunogen. Immune responses may be broadly categorized into two categories: cell-mediated (Th1-type) and humoral (Th2-type, antibody-mediated) immune responses. Immune responses can be measured in many ways including stimulation of immunoglobulin (Ig) IgA, IgM, or IgG titer; activation, proliferation, or differentiation of cells of the immune system (e.g., B cells, T cells, dendritic cells, APCs, macrophages, NK cells, NKT cells, etc.); up- regulated or down-regulated expression of markers and cytokines; splenomegaly (including increased spleen cellularity); hyperplasia and mixed cellular infiltrates in various organs. Other responses, cells, and components of the immune system that can be assessed with respect to immune stimulation are known in the art.

[0198] Efficacy of the vaccine composition of the invention can be measured by an increase in expulsion of larvae and / or adult nematodes, larvae and / or adult nematodes in the gastrointestinal tract, e.g. the abomasum of sheep, and / or by a reduced eggs per gram of sheep feces (EpG), as well as by the presence of one or more protective antibodies targeted by the antigens present in the vaccine composition.

[0199] Preferably, the vaccinated animal will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and / or the clinical severity of the disease will be reduced. Such protection can be demonstrated by either a reduction or lack of symptoms and / or clinical disease signs normally displayed by an infected host, or a quicker recovery time of the infected host.

[0200] Due to vaccination with the composition described herein, the cumulative egg shedding and the worm burden of vaccinated sheep are decreased in comparison to the challenged non-vaccinated control group. Specifically, infected vaccinated sheep have VT016P

[0201] -23- at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% less cumulative egg shedding in comparison to the unvaccinated sheep. Specifically, the vaccinated sheep have at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% less worm burden compared to unvaccinated sheep.

[0202] The term “therapy” as referred to herein comprises administration of the immunological composition to an animal suffering from or going to suffer from a helminthic parasite infection. Therapy also refers to prophylaxis against helminthic parasite infection, specifically the composition described herein is used as vaccine for vaccination. Specifically, it refers to prophylactic or metaphylactic administration. The term “infectious” as used herein refers to a parasite, specifically H. contortus, that can invade and multiply in a host cell and causes a reaction of host tissue, specifically an immune response.

[0203] The vaccine or immunological composition can be a lyophilized powder, a frozen liquid, or a liquid. The term “lyophilized” refers to substances processed by lyophilization, a low temperature dehydration process that involves freezing the product and lowering pressure, thereby removing the ice by sublimation. As used herein “lyophilized powder” refers to an immunological composition processed by lyophilization. As used herein, a “lyophilized powder” can be dissolved in solvent, specifically water, buffer, or in an adjuvant.

[0204] A “protein” is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but nonetheless may be present.

[0205] The term “recombinant protein” refers to proteins which are formed by transfecting foreign genes into a host cell. Specifically, in the present invention, the host insect cell is selected from, but not limited to, Lep / doptera-derived cells, more specifically Trichoplusia ni High Five (Hi5) cells, Spodoptera frugiperda Sf9, Spodoptera frugiperda Sf21 , or Trichoplusia n / -derived Tnao38 cells.

[0206] Production of recombinant proteins can be in the expression system, specifically, expression of a recombinant protein in host cells, harvesting of host cells, purification of the recombinant protein, and mixing of recombinant proteins to obtain the immunological composition, a process well-known to the person skilled in the art. VT016P

[0207] -24-

[0208] Extracellular production of antigens can be enhanced or achieved by removal of the trans-membrane domain (TMD) and the introduction of one or more foreign signal peptide sequences. For this purpose, TMDs can be predicted by structure analysis and removed from the native protein sequence. Such prediction is well known to the skilled person and may be methods and services such as HMMTOP [6], PredictProtein server [7] or TMHMM [8,9],

[0209] In addition, further modifications may be incorporated to the N-terminus of the antigen sequence, specifically, the TMD-truncated antigen sequence, melittin signal peptides to enhance secretion of the tri-fucosylated glycoprotein described herein of the insect cell (e.g., MKFLVNVALVFMWYISYIYA, SEQ ID NO: 22), a HisFLAG duo-affinity tag for optimization if purification (e.g. HHHHHHDYKDDDDK, SEQ ID NO: 23) and / or a thrombin cleavage site (e.g. LVPRGS, SEQ ID NO: 24).

[0210] Described herein are glycoproteins lacking native trans-membrane domains and with a signal peptide inserted. This design enables extracellular secretion of recombinant glycoproteins, thereby increases expression yield, increases protein solubility, and ensures glycan modifications by ensuring glycan post-translational modifications (PTMs).

[0211] In a specific embodiment, the intestine tri-fucosylated N-glycoprotein described herein is a H. contortus glycoprotein lacking its trans-membrane domain.

[0212] One can also express the glycoprotein antigens with tri-fucosylation without removing their trans-membrane domains. In this case, the recombinant products may stay inside the host cells.

[0213] Transfection systems for the expression cassette described herein can be, but are not limited to, baculovirus systems. Recombinant baculoviruses have been proven to be efficient gene delivery vectors for cell systems and are well known to the person skilled in the art. Recombinant baculoviruses can e.g. act as transfection systems for expression cassettes. Described herein is a baculovirus system comprising the expression cassette to transfect insect cells.

[0214] Glycoproteins are proteins which contain oligosaccharide (sugar) chains covalently attached to amino acid side-chains. The carbohydrate is attached to the protein in a co-translational or posttranslational modification. This process is known as glycosylation. Secreted extracellular proteins are often glycosylated. In N-glycosylation, sugars are attached to nitrogen, typically on the amide side-chain of asparagine (Asn). VT016P

[0215] N-glycans are glycoproteins with glycosylation at Asn residues (Asn-x-Serine / Threonine motifs).

[0216] Below the structure of N-glycans is shown, comprising e.g. a core alpha-1 , 3- fucose and a core alpha-1 , 6-fucose. Glycan structures are drawn in symbol nomenclature for glycans (SNFG). In this example, the monosaccharide GIcNAc is annotated as a square. The core modifications e.g. the core alpha-1 , 3-fucose variant and a core alpha-1 , 6-fucose variant are indicated by a triangle, and a hexose by a filled

[0217] The expression cassette of the invention comprises polynucleotide sequences encoding nematode parasite proteins and the glycol-module which enables the presentation of nematode-like N-glycans, specifically core fucosylated N-glycans, more specifically tri-fucosylated N-glycans with or without a Gaipi ,4Fuca-R epitope.

[0218] Enzymes for N-glycosylation can be, but are not limited to, galactosyltransferase, specifically GALT-1 , fucosyltransferase, specifically FUT-6, or mannosidase, specifically

[0219] AMAN-3.

[0220] The term “amino acid” refers to one of the naturally occurring amino carboxylic acids of which proteins are comprised. Those amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, and valine. These 22 amino acids can be split into those that have neutral charges, positive charges, and negative charges:

[0221] The “neutral” amino acids are shown below along with their respective three-letter and single-letter code and polarity: alanine (Ala, A; nonpolar, neutral), asparagine (Asn, N; polar, neutral), cysteine (Cys, C; nonpolar, neutral), selenocysteine (Sec, U, nonpolar, VT016P

[0222] -26- neutral), glutamine (Gin, Q; polar, neutral), glycine (Gly, G; nonpolar, neutral), isoleucine (He, I; nonpolar, neutral), leucine (Leu, L; nonpolar, neutral), methionine (Met, M; nonpolar, neutral), phenylalanine (Phe, F; nonpolar, neutral), proline (Pro, P; nonpolar, neutral), serine (Ser, S; polar, neutral), threonine (Thr, T; polar, neutral), tryptophan (Trp, W; nonpolar, neutral), tyrosine (Tyr, Y; polar, neutral), valine (Vai, V; nonpolar, neutral), and histidine (His, H; polar, positive (10%) neutral (90%)). The “positively” charged amino acids are: arginine (Arg, R; polar, positive), lysine (Lys, K; polar, positive), and pyrrolysine (Pyl, O, positive). The “negatively” charged amino acids are: aspartic acid (Asp, D; polar, negative), and glutamic acid (Glu, E; polar, negative). Furthermore, the term “amino acid” includes both D- and L-amino acids (stereoisomers).

[0223] The term “antibody” refers to the secreted form of a B cell receptor. The term immunoglobulin (Ig) can refer to either the membrane-bound form or the secreted form of the B cell receptor. The terms antibody and immunoglobulin are herein used interchangeably. One method to measure the presence of antibodies in liquid samples is the enzyme-linked immunosorbent assay (ELISA). The quantitative analysis of the ELISA is usually based on detection of intensity of transmitted light by spectrophotometry, which involves quantitation of transmission of some specific wavelength of light through the liquid, e.g., optical density (OD) measurements. As described in the examples herein, the ELISA was used to detect antibody levels in serum.

[0224] The present invention is also encompassed by the following embodiments.

[0225] 1. An expression cassette for the production of extracellular recombinant tri- fucosylated N-glycoproteins in insect cells comprising i) a glyco-module comprising polynucleotide sequences of a nematode a) galactosyltransferase, specifically GALT-1 , and b) fucosyltransferase, specifically FUT-6, and c) mannosidase, specifically AMAN-3, and ii) a polynucleotide sequence encoding one or more tri-fucosylated N- glycoproteins derived from a nematode.

[0226] 2. The expression cassette of embodiment 1 , wherein the galactosyltransferase comprises the nucleotide sequence SEQ ID NO: 1 or SEQ ID NO: 2, the VT016P

[0227] -27- fucosyltransferase comprises the nucleotide sequence SEQ ID NO: 3 or SEQ ID NO: 4, and / or the mannosidase comprises the nucleotide sequence SEQ ID NO: 5.

[0228] 3. The expression cassette of embodiment 1 or 2, wherein the glyco-module comprises polynucleotide sequences from Caenorhabditis elegans (C. e / egans), specifically GALT-1 , FUT-6, and / or AMAN-3; or from Haemonchus contortus (H. contortus), specifically GALT -1 and / or FUT-6.

[0229] 4. The expression cassette of any one of embodiments 1 to 3, wherein the tri- fucosylated N-glycoproteins are membrane-bound intestine proteins, specifically derived from any one of Haemonchus contortus, Haemonchus place!, Teladorsagia circumcincta (T. circumcincta) Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Ascaris suum, Oesophagostomum dentatum, or Ostertagia ostertagi.

[0230] 5. The expression cassette of any one of embodiments 1 to 4, wherein the tri- fucosylated N-glycoproteins are derived from H. contortus glycoprotein H11 , encoded by SEQ ID NO: 6; H11-1 , encoded by SEQ ID NO: 7; H11-2, encoded by SEQ ID NO: 8; H11-4, encoded by SEQ ID NO: 9; H11-5a, encoded by SEQ ID NO: 10; H11-5b, encoded by SEQ ID NO: 11 ; H11-5c, encoded by SEQ ID NO: 12; and / or GA1 , encoded by SEQ ID NO: 13.

[0231] 6. A recombinant baculovirus comprising the expression cassette of any one of embodiments 1 to 5.

[0232] 7. A method for production of recombinant tri-fucosylated N-glycoproteins derived from a nematode in insect cells comprising the sequential steps: a. Transfecting insect cells with the expression cassette of any one of embodiments 1 to 5, or with the baculovirus of embodiment 6; b. Expressing the tri-fucosylated N-glycoproteins by the insect cells; c. Harvesting and optionally purifying the tri-fucosylated N-glycoproteins from the insect cells.

[0233] 8. The method of embodiment 7, wherein the insect cells are Lepidoptera-derived cells, specifically Trichoplusia ni High Five (Hi5) cells, Spodoptera frugiperda Sf9, Spodoptera frugiperda Sf21 , or Trichoplusia ni Tnao38 cells.

[0234] 9. The method of embodiment 7 or 8, wherein the polynucleotide sequences of the glyco-module and the polynucleotide sequences encoding the one or more tri- fucosylated N-glycoproteins are co-expressed. VT016P

[0235] -28-

[0236] 10. An immunological composition against a nematode, comprising at least one recombinant tri-fucosylated N-glycoprotein, specifically a recombinant membrane-bound intestine tri-fucosylated N-glycoprotein, and optionally one or more adjuvants.

[0237] 11 . The method of any one of embodiments 7 to 9, or the immunological composition of embodiment 10, wherein the tri-fucosylated N-glycoproteins are derived from any one of H. contortus, Haemonchus placei, Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Ascaris suum, Oesophagostomum dentatum, or Ostertagia ostertagi.

[0238] 12. The method of any one of embodiments 7 to 9, or the immunological composition of embodiment 10, wherein the tri-fucosylated N-glycoproteins are derived from H. contortus glycoprotein H11 comprising SEQ ID NO: 14; H11-1 comprising SEQ ID NO: 15; H11-2 comprising SEQ ID NO: 16; H11-4 comprising SEQ ID NO: 17; H11-5a comprising SEQ ID NO: 18; H11-5b comprising SEQ ID NO: 19; H11-5c comprising SEQ ID NO: 20; and / or GA1 comprising SEQ ID NO: 21.

[0239] 13. The method of any one of embodiments 7 to 9, or the immunological composition of embodiment 10, wherein the membrane-bound intestine tri-fucosylated N- glycoprotein / s H. contortus glycoprotein lacking its trans-membrane domain.

[0240] 14. The composition of any one of embodiments 10 to 13, wherein the composition is a lyophilized powder, a frozen liquid, or a liquid.

[0241] 15. The composition of any one of embodiments 10 to 14 for use in vaccinating an animal, specifically a ruminant, or a New World camelid.

[0242] 16. The composition of any one of embodiments 10 to 14 for use in reducing nematode parasitic worm burden in an animal, specifically a ruminant, or a New World camelid.

[0243] 17. The composition of any one of embodiments 10 to 14 for use in stimulating or boosting acquired immunity in an animal, specifically a ruminant, or a New World camelid.

[0244] 18. The composition for use of embodiment 15 to induce protective anti-nematode immunity in an animal, specifically in a ruminant or a New World camelid.

[0245] 19. The composition for use of embodiment 16, wherein the nematode is selected from the group consisting of Haemonchus contortus, Haemonchus placei, Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Ascaris suum, Oesophagostomum dentatum, and Ostertagia ostertagi. VT016P

[0246] -29-

[0247] 20. The composition for use of any one of embodiments 15 to 17, wherein the vaccinated animals have reduced fecal egg count and worm burden in the gastrointestinal tract compared to unvaccinated animals infected with the nematode, wherein the nematode is specifically selected from the group consisting of Haemonchus contortus, Haemonchus placei, Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Ascaris suum, Oesophagostomum dentatum, and Ostertagia ostertagi.

[0248] 21. The composition for use of any one of embodiments 15 to 18, wherein the composition is administered as injection, specifically as subcutaneous injection, intramuscular injection, or intradermal injection.

[0249] 22. The composition for use of any one of embodiments 15 to 19, wherein the composition is administered to sheep, goats, alpacas, llamas, or cattle.

[0250] 23. A recombinant H. contortus H11 or GA1 antigen having core tri-fucosylated N- glycans, wherein the N-glycans are nematode-type N-glycans comprising any one of amino acid sequences SEQ ID No: 14 to 21 , or amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, specifically at least 98%, more specifically 99% sequence identity to SEQ ID NOs: 14 to 21.

[0251] 24. An engineered insect cell comprising the expression cassette of any one of embodiments 1 to 5, or the recombinant baculovirus of embodiment 6.

[0252] The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Different embodiments of the present invention have been described according to the present invention. Many modifications and variations may be made to the techniques described and illustrated herein without departing from the scope of the invention.

[0253] EXAMPLES

[0254] 1 . Construction of recombinant baculoviruses for the recombinant expression of H. contortus vaccine antigens

[0255] Amino acid sequences of H11 antigens were determined based on DNA sequencing results of PCR products, which were amplified using cDNAs of adult H. contortus (KB strain, an isolate of Katharinenhof in Bavaria) as a template. In total, seven H11 isoforms were determined, namely KB H11 , KB H11-1 , KB H11-2, KB H11-4, KB H11-5a, KB H11-5b and KB H11-5c. In addition, the sequence of an apical gut VT016P

[0256] -30- membrane polyprotein (US GA1) was obtained from GenBank (AAB01192.1). To achieve extracellular production of recombinant proteins, antigens were designed as follows: Step 1) predicted transmembrane domains (TMDs) were removed from native protein sequence and Step 2) functional peptide fragments, including a melittin signal peptide (MKFLVNVALVFMWYISYIYA (SEQ ID NO: 22), cleaved from mature proteins), a HisFLAG duo-affinity tag (HHHHHHDYKDDDDK, SEQ ID NO: 23) and a thrombin cleavage site (LVPRGS, SEQ ID NO: 24), were incorporated to the N-termini of TMD- truncated antigen sequences (Fig. 2). Amino acid sequences of desired recombinant H. contortus antigens are shown in Fig. 2A. Fig. 2B shows a phylogenetic tree to demonstrate the homology of different H11 isoforms and GA1. A comparison of antigens regarding size, molecular weight and predicted N-glycosylation site is summarized in Fig. 2C. Based on the amino acid sequences of designed antigens, codon-optimized synthetic DNA fragments were ordered from GenScript Biotech and subcloned into a pACEBad acceptor vector. Constructs were verified by PCR and DNA sequencing.

[0257] Based on the current knowledge of N-glycosylation of insect cells and nematodes [10,11], three nematode genes are required to introduce new N-glycan biosynthetic pathways in Hi5 cells that lead to the formation of nematode-type N-glycan structures on recombinant proteins, i.e. , tri-fucosylated chitobiose core and Gal|31 ,4Fuca-R epitope (Fig. 3). Fig. 3A shows a comparison of typical N-glycan core modifications of mammalian, C. elegans, H. contortus, and insect glycoproteins. Fig. 3B shows major biosynthetic pathways of N-glycans in non-engineered and glycoengineered Hi5 insect cells. Fig. 3C gives an overview of N-glycan structures as shown in Fig. 3A and 3B. A summary of the definition of the N-glycan core structure, and N-glycans identified in H. contortus and in GEA is given in Fig. 3D.

[0258] Glycosylation engineering of recombinant H. contortus antigens was achieved by co-expression of a parasite antigen with a gene cassette (glyco-module) consisting of three C. elegans genes, which encode a Golgi alpha-mannosidase III (AMAN-3, Genbank accession: NP 001361919.1 , SEQ ID NO: 5), a core cd , 3 fucosyltransferase (FUT-6, Genbank accession: NP_494823.2, SEQ ID NO: 3) and a p1 ,4- galactosyltransferase (GALT-1 , Genbank accession: NP 504545.2, SEQ ID NO: 1).

[0259] Using the MultiBac™ cloning system, coding sequences (CDSs) of the three corresponding C. elegans genes (SEQ ID NO: 1 , SEQ ID NO: 3 and SEQ ID NO: 5) were first subcloned into donor vectors (plDC, pIDK or pIDS) and then integrated together via Cre-LoxP recombination to form a big donor vector that hosts all three CDSs VT016P

[0260] -31- as the glyco-module. Subsequently, this construct is integrated with a pACEBad acceptor vector that hosts one CDS of desired antigen-coding genes, e.g., H11. Presence and organization patterns of genes in such constructs were verified by Nanopore DNA sequencing. Fig. 4 shows the schematic workflow of the preparation of glycoengineered recombinant antigens. Fig. 4A shows the molecular cloning of glycogenes from wild type C. elegans and construction of baculovirus expression system (bacmid) DNAs for containing synthetic antigen-encoding DNAs with (I) or without (II) the “glyco-module”. Fig. 4B shows the preparation of high-titer recombinant baculovirus stocks using Sf9 insect cells. Fig. 4C shows the recombinant expression and purification of soluble antigens of H. contortus.

[0261] Finally, all relevant genes were integrated into the baculovirus genome (DHWEMBacY) via Tn7 transposition and the obtained bacmid DNAs were PCR verified (Bacmid-GEA, Fig. 5 and Fig. 4 A(l)). In parallel, bacmids without the glyco-module were successfully prepared by integrating pACEBad constructs into DHWEMBacY (Bacmid- NEA, Fig. 4 A(ll)). Fig. 5 gives an overview of the generated DNA constructs carrying the glyco-module and different H. contortus antigen encoding genes based on results of nanopore DNA sequencing.

[0262] High-titer recombinant baculovirus stocks (V1) were obtained by transfecting Sf9 insect cells in 6-well cell culture plates using bacmid DNAs followed by the infection of Sf9 cells with VO baculoviruses in T-flasks. Infectivity of viruses were assessed by observing YFP fluorescence using a fluorescence microscope (Fig. 4B).

[0263] 2. Recombinant expression of H11 aminopeptidase isoforms and GA1 in insect cells

[0264] Recombinant expression of H11 and GA1 antigens was achieved by infecting suspension Hi5 cell culture using V1 baculovirus stocks. Three days post infection recombinant products were purified from cell culture supernatants using a AKTA protein purifier (Fig. 4C). Proteins were quantified by Bradford protein assays and their purities were assessed by Coomassie blue staining post SDS-PAGE (Fig. 6). Fig. 6 shows Coomassie blue staining to determine purity and concentration of purified recombinant antigens. All ten proteins used to formulate vaccine cocktails displayed as a single band on the gel with approximal molecular weights at 110 kDa (H11 isoforms) and 100 kDa (GA1).

[0265] In addition, protein sequences of all recombinant products were verified by LC- MS / MS analysis, which indicated > 90% coverage of peptides. Equal amount of KB H11 , VT016P

[0266] -32-

[0267] KB H11-1 , KB H11-2, KB H11-4 and GA1 were mixed to form the non-glycoengineered antigen cocktail (NEA). Similarly, equal amount of KB geH11 , KB geH11-1 , KB geH11- 2, KB geH11-4 and US geGA1 were used to prepare the glycoengineered antigen cocktail (GEA).

[0268] Glycoengineered hidden antigens carry nematode-type glycan modifications

[0269] N-glycans were released from two antigen cocktails (NEA and GEA), labelled with 2-aminopyridine (PA) and measured by MALDI TOF MS. Apart from many shared glycan compositions, primarily being oligo- / pauci-mannosidic glycans (Hex2-9HexNAc2) and fucosylated glycans (Hex2-3HexNAc2-3Fuci-2), a major difference in glycoform between the two samples was the appearance of a new peak (m / z 1427.5) on the MS spectrum of GEA, indicative of a tri-fucosylated glycan structure (HexsHexNAc2Fuc3) as the result of the successful glycoengineering (Fig. 7). Fig. 7 shows MALDI TOF MS spectra of PA- labelled glycans.

[0270] Structural characterization of glycans present on GEA and on a glycan purified from H. contortus demonstrated similar characteristics (Fig. 8). Fig. 8 shows MALDI- TOF MS / MS spectra of N-glycans before and after hydrofluoric acid (HF) treatment. Structural characterisation of N-glycans is based on multiple analytical parameters, including elution pattern (HPLC retention time, typically expressed in glucose units, g.u.), sensitivity to specific glycosidase or chemical treatments (here, HF treatment), and changes in key MS / MS fragment ions before and after treatment. The glycan present on GEA (A) exhibits similar characteristics to a glycan purified from H. contortus (B), both losing two core a1 ,3-fucose residues upon HF treatment.

[0271] Separation of PA-glycans on RP-amide HPLC led to two distinct chromatograms (Fig. 9). Fig. 9 shows HPLC chromatograms of PA-labelled N-glycans released from NEA and GEA antigens. Further structural analysis of glycans in HPLC fractions by MALDI TOF MS / MS revealed that the glycoengineered antigens carry a series of nematode-type N-glycan structures, including a major tri-fucosylated glycan structure carrying a Gal-Fuc epitope.

[0272] Recombinant H. contortus antigens are enzymatically and biologically active

[0273] As H11 antigens are aminopeptidases belonging to the peptidase M1 family, in vitro enzymatic assays were carried out to assess activities of the recombinant proteins. Our data demonstrated that all eight recombinant H11s, in non-engineered (NEA) and VT016P

[0274] -33- glycoengineered (GEA) forms, were able to hydrolyse a pNP-Leucine substrate (Fig. 10). Fig. 10 shows the aminopeptidase activity of recombinant H11 antigens. Among the isoforms, H11-4 and geH11 -4 displayed the highest activity towards the given substrate.

[0275] It was further demonstrated that recombinant H. contortus FUT-6 exhibits similar in vitro enzymatic properties to C. elegans FUT-6 (Fig. 11). Fig. 11 shows that H. contortus FUT-6 fucosylates the antennae of two complex-type N-glycans (Dabsyl- GalGal and Dabsyl-PGn |3Gn) as well as the distal GIcNAc residue of the chitobiose core in a PA-labelled glycan lacking the upper a1 ,6-mannose arm.

[0276] In addition, the biological properties of recombinant antigens were assessed using ovine peripheral blood mononuclear cells (PBMCs). Recombinant antigens, either used as a cocktail (e.g., GEA and NEA) or a single antigen, were able to stimulate the secretion of cytokines from PBMCs. A set of fourteen different cytokine molecules were tested and it was demonstrated that six cytokines (IL-1 (3, IL-6, IL-8, INFy, TNFa and IP- 10) were significantly induced by recombinant antigens post overnight incubation with PBMCs (Fig. 12). Fig. 12 shows an in vitro assay of biological activity of recombinant antigens. In comparison to the native antigens of Barbervax® (Bvax), GEA was capable of inducing equivalent levels of cytokines.

[0277] These data confirmed that the insect cell-expressed glycoengineered recombinant proteins are correctly folded into enzymatically and biologically active antigens.

[0278] 3. Vaccine Trial

[0279] Vaccination with GEA and NEA induces parasite-specific and vaccine-specific antibodies in sheep

[0280] A vaccine trial was conducted using the glycoengineered H. contortus antigens (Fig. 1). Fig. 1 gives a schematic overview of the vaccine trial. Experimental groups were as follows: VT016P

[0281] -34-

[0282] The dosage (antigen per shot), the number of vaccinations, the time span between the vaccinations and the adjuvant was chosen following literature

[0012] , For this trial, a dosage of 100 pg per shot was used. For vaccine challenge, the sheep were infected with 5,000 L3 H. contortus. It is known that the average larval establishment rate is 0.24 (SE ± 0.02), which decreased as a function of the infection dose and host age.

[0283] To determine if vaccination with recombinant antigens could induce serum antibodies in animals, sheep sera were collected during the vaccine trial and examined by indirect ELISA assays. For assessing parasite-specific antibodies, combined serum samples of the same experimental group were incubated with ELISA plates coated with whole worm lysate of adult H. contortus (Fig. 13). Fig. 13 shows a time course analysis of vaccination-induced parasite-specific antibodies IgG and IgE in sheep sera. The data demonstrated clearly that IgG antibody response was significantly induced in vaccine groups (group 3, 4 and 5). Vaccination with GEA (group 4) and NEA (group 5) led to a rapid increase of IgG level, whereas vaccination with Barbervax® (Bvax, group 3) indicated a gradual increase of IgG. Animals which received Bvax maintained the highest IgG level of all vaccine groups after the third shot; and GEA performed better than NEA in inducing IgG antibodies. A very similar pattern was observed for parasite specific IgE antibody.

[0284] Furthermore, antigen-specific antibodies were measured using serum samples of individual animals. Fig. 14 shows vaccination-induced antigen-specific IgG antibodies in sheep. Fig. 15 shows vaccination-induced antigen-specific IgE antibodies in sheep. Serum samples of an experimental group were analyzed using ELISA plates coated with the corresponding vaccine antigens. For samples of the control groups 1 and 2, Bvax- coated ELISA plates were used. The results indicated that antigen-specific IgG (Fig. 14) and IgE (Fig. 15) antibodies were elevated in animals post vaccination (groups 3, 4 and 5). In contrast to animals in group 3 that received Bvax, animals in group 4 and 5 displayed rapid and high antibody responses. This suggested that the recombinant H. contortus antigens (GEA and NEA) are able to induce antibody response, specifically IgG and IgE antibodies, more efficiently than the native worm antigens in Bvax. VT016P

[0285] -35-

[0286] Sheep vaccinated with GEA and NEA showed higher blood hemoglobin concentrations than the non-vaccinated group

[0287] As shown in Fig. 16, group 1 (no vaccination / no challenge) and Group 3 (Barbervax® / challenge) showed no differences in hemoglobin concentrations after the vaccine challenge. Fig. 16 shows a comparison of hemoglobin levels of sheep after the vaccine challenge. In comparison to Group 2 (no vaccination / challenge) all groups showed significantly higher hemoglobin levels. Group 4 (GEA / challenge) showed significantly higher hemoglobin levels than Group 5 (NEA / challenge).

[0288] Sheep vaccinated with GEA showed on median 81.1 % less eggs per gram of feces than the non-vaccinated group

[0289] The sheep began to shed eggs 17 days after the infection. In total, the sheep shed eggs for 24 days (infection day until euthanasia). The median cumulative egg excretion in G1 was 0 EpG, in G2 36,930 (minimum = 10,005; maximum = 60,425), in G3 620 (min=340; max=3,630), in G4 6,985 (min=3,580, max=6,985) and in G5 24,845 (min=3,080, max=28,480). Using REML analysis, significant differences between Group 2 and Group 3 (p < 0.01), Group 2 and Group 4 (p < 0.01), Group 3 and Group 4 (p < 0.01), Group 3 and Group 5 (p < 0.01) and between Group 4 and Group 5 (p = 0.05) were detected. There was no difference in egg excretion between Group 2 and Group 5 (p = 0.08). The detailed results are shown in Fig. 17 A and Fig. 17 B. Fig. 17 shows eggs per gram of sheep feces after the vaccine challenge.

[0290] Sheep vaccinated with GEA showed on median 25.4% less worms in the abomasum than the non-vaccinated group

[0291] The median worm count was as follows: G2 = 2,587, G3 = 353, G4 = 1 ,931 and G5 = 2,232. There was a statistically significant difference between G2 and G3 (p < 0.01). There was no difference between G2 and G4 (p =1.0), G2 and G5 (p = 0.99) and G4 and G5 (p = 0.98). Results are shown in Fig. 18 A and Fig. 18 B. Fig. 18 shows the total worm count in the abomasum of sheep after the vaccine challenge.

[0292] Sheep vaccinated with GEA showed the same number of eggs per female worm as the sheep vaccinated with Barbervax®

[0293] The median female / male ratio was 1.28 (minimum = 0.78; maximum = 1.67) in G2, 0.58 (0.18; 1.02) in G3, 1.15 (0.82; 2.10) in G4 and 1.23 (0.94; 1.48) in G5. VT016P

[0294] -36-

[0295] Regarding the number of eggs per female worm G3, G4 and G5 were significantly lower than G2. No differences were seen between the groups G3, G4 and G5 (p > 0.05). The average establishment of the L3 (infection dose 5,000 L3) in the abomasum was 44.85% in G2, 10.27% in G3, 41.89% in G4 and 48.43% in G5 (Fig. 19 A and Fig. 19 B). Fig. 19 shows the eggs per female worm detected.

[0296] Summary vaccine trial

[0297] The described in vivo vaccine trial using glycoengineered H. contortus antigens revealed highly promising results. The cumulative egg shedding of vaccinated sheep decreased by 81.1 % and the worm burden decreased by 25.4% in comparison to the positive control group (Group 2 = no vaccination / challenge).

[0298] Additionally, the trial showed that glycoengineering is essential to achieve protection in terms of egg shedding and worm burden since the reduction of the cumulative egg shedding was only 32.7% in the non-glycoengineered group and there was no reduction in the worm burden in comparison to the positive control group. Moreover, sheep vaccinated with non-glycoengineered rH11 (G5) shed more eggs per female worm than the ones in the glycoengineered (G4) and Barbervax® (G3) group. Taken together, EpG and worm burden data showed that the glycoengineered antigens exhibited clearly a higher efficacy than the non-glycoengineered antigens.

[0299] Since H. contortus is a blood-feeding endoparasite, it is well known that packed cell volume and hemoglobin levels can decrease severely after infection since the sheep can lose up to 0.2 top 0.6 liters of blood per day, depending on the level of infection

[0013] . Therefore, blood hemoglobin concentration was used to compare the efficacy of the glycoengineered vaccine with the non-glycoengineered vaccine. The results showed that hemoglobin concentrations were higher in the glycoengineered (G4) than in the non- glycoengineered group (G5).

[0300] Currently, the only vaccine against H. contortus achieving high protection in terms of egg shedding and worm burden is Barbervax®. In the present vaccine trial Barbervax® showed a reduction of cumulative egg shedding of 98.3% and worm burden of 86.4%. The major disadvantage of this vaccine is that donor sheep, which need to be infected and slaughtered, are used to harvest the worms from their stomach. In addition to this animal welfare issue, the detailed structure of the antigens is not completely understood and biosecurity is difficult to guarantee. Moreover, the vaccination schedule states that the sheep need to be vaccinated at least every six weeks since otherwise the VT016P

[0301] -37- sheep are not protected any more. Therefore, lambs need to be vaccinated subcutaneously up to six times per year according to the developer, which also is a major disadvantage of the commercial vaccine [1].

[0302] Another study by Cachat et al. used insect cells as expression hosts for the recombinant production of H. contortus metalloendopeptidases to vaccinate sheep. The results showed only a very low reduction of egg shedding (2.5%) and worm burden (1.0%)

[0014] ,

[0303] Methods

[0304] Cultivation of E. coll and insect cells

[0305] Different E. coll strains were used for the preparation of DNA constructs. NEB5a was purchased from New England Biolabs, chemical competent cells One Shot™ PIR1 was purchased from Fisher Scientific and DH EMBacY was from Geneva Biotech. In brief, E. coll cells were cultivated in Lysogeny-Broth medium (Carl Roth) supplied with antibiotics of choice, including kanamycin, streptomycin, chloramphenicol and tetracycline purchased from Sigma-Aldrich and gentamycin from Fisher Scientific. Sf9 insect cell line was obtained from ATCC via LGC Standards and the High Five™ cell line (Hi5) was ordered from Fisher Scientific. Both cell lines were adapted to grow in HyClone™ SFM4lnsect cell culture medium, whereas for Sf9 cells 2% fetal bovine serum (Gibco) was supplied. Cells were maintained in T-flasks in a 27°C incubator without CO2 and passaged twice a week.

[0306] Determination of Protein Sequences of H11 antigens in Haemonchus contortus (KB strain), molecular cloning and DNA sequencing

[0307] Isolation of mRNA molecules from wild type Caenorhabditis elegans and adult Haemonchus contortus (KB strain, an isolate of Katharinenhof in Bavaria, Germany) was performed using a QIAamp® RNA Blood Mini kit (QIAGEN). Conversion from mRNAs to cDNAs was done using a GoScript™ reverse transcriptase (Promega) following the user manual. Predicted H11-coding genes were PCR amplified, verified by agarose gel electrophoresis and subsequently cloned into a pCR4TOPO vector by TA cloning (Invitrogen™ TOPO™ TA Cloning™ Kit, Fisher Scientific). Sanger DNA sequencing of PCR products and DNA constructs was performed at LGC Genomics in Berlin. Assembly of contigs was carried out manually using a SnapGene software (GSL Biotech LLC, San Diego, US) and corresponding protein sequences of H11 antigens (KB strain) were deduced based on DNA sequences. Codon-optimization of coding sequences for protein VT016P

[0308] -38- expression in Trichoplusia ni (Hi5 cells) was performed using GenSmart™ 2.0 online server and synthetic DNAs of seven hi 1 isoforms (KB strain) and GA1 (US strain) were ordered from GenScript.

[0309] Construction of recombinant baculoviruses

[0310] Gibson assembly approach was employed for all molecular cloning work using a NEBuilder® HiFi DNA assembly cloning kit (New England Biolabs) and specific primers. A MultiBac™ cloning kit (Geneva Biotech) was used to prepare recombinant baculovirus following the user manual (version 8.3). Briefly, synthetic antigen-coding genes were subcloned to the pACEBad acceptor vector and transformed to competent NEB5a cells, whereas C. elegans glyco-genes (AMAN-3, FUT-6 and GALT-1 ) were subcloned to pIDC / K / S donor vectors and transformed to competent PIR1 cells. Post antibiotic selection and PCR screening, plasmid DNAs were prepared and sequenced. Construction of the glyco-module was achieved by combining plDC, pIDK and pIDS constructs via Cre-LoxP recombination. For expressing glycoengineered antigens, eight pACEBad constructs, each hosting one antigen-coding gene was combined with pIDC / K / S construct hosting the glyco-module. Resulted plasmids were verified by PCR and Nanopore sequencing (Microsynth). Subsequently, the resulted plasmids were transformed to DH EMBacY competent cells and positive bacmids were obtained post blue-white screening and were PCR verified.

[0311] Transfection of bacmid DNAs to Sf9 insect cells was done in 6-well cell culture plate using the FuGENE® HD transfection reagent (Promega). 1 pg of bacmid DNA was mixed with 10 pl of transfection reagent and added to Sf9 cells. Typically, recombinant baculoviruses (V0) were ready for harvesting 4 to 6 days post transfection, when YFP fluorescence could be detected in most of the cells. High-titre viruses (V1) were obtained by infecting Sf9 cells with V0 viruses either in T75 cell culture flasks (Sarstedt) or in 100 ml Erlenmeyer shake flasks (Fisher Scientific).

[0312] Expression of Recombinant Antigens in Insect cells

[0313] Suspension culture of Hi5 insect cells was carried out in 1 litre Erlenmeyer cell culture flask (Duran) at 27°C on a horizontal shaking platform (80 rpm). Typically, a 200 ml culture with a density of 1 *106cells / ml was infected with 4 ml V1 viral stock (1 :50) and harvested 3 days post infection (dpi). Cells and debris were removed by centrifugation and filtration (0.22 pm) prior to concentration and buffer-exchange steps using an ultrafiltration device (Amicon® Stirred Cell, Millipore) equipped 50 kDa MWCO disc. Concentrated cell culture supernatants in binding buffer (20 mM sodium phosphate, VT016P

[0314] -39-

[0315] 0.5 M NaCI, 20 mM imidazole, pH 7.4) were subject to affinity purification using an AKTA™ Start protein purifier equipped with a fraction collector (Cytiva) and a 1 ml prepacked HisTrap™ High Performance column (GE healthcare). A 20-minute linear gradient of elution buffer (20 mM sodium phosphate, 0.5 M NaCI, 500 mM imidazole, pH 7.4) was used to elute His-tagged proteins from the column. Purified recombinant antigens were desalted and buffered in PBS prior to quantification using a Pierce™ Bradford Plus protein assay kit (Thermo Fisher Scientific).

[0316] Analysis of N-glycoforms of recombinant antigens: 50 pg of GEA and NEA were dissolved in 8 M urea and trypsinised overnight at 37°C. Tryptic peptides were purified using hand-packed C18 cartridges prior to overnight deglycosylation by PNGase A (New England Biolabs). Released N-glycans were purified using two tandem cartridges, one filled with Dowex (50W*8) and C18, and another one with nPGC. Lyophilized native glycans were labeled with 2-aminopyridine (PA) to introduce a fluorescent tag at the reducing ends and the excess linker was removed by gel filtration (Sephadex G15, 0.5% acetic acid as solvent) as previously described

[0015] . PA-glycans were fractionated on a RP-amide column by a Shimadzu Nexera UPLC system equipped with a RF 20AXS fluorescence detector (excitation / emission: 320 nm / 400 nm). Pooled and HPLC- fractionated N-glycans were analysed by MALDI TOF MS / MS (Bruker rapifleX, Bremen) in positive ion mode using 6-aza-2-thiothymine as a matrix.

[0317] Aminopeptidase activity of recombinant antigens: 1 pg of recombinant H11 antigens were incubated in a 96-well plate with 0. 5 mM L-Leucine-p-nitroanilide (Sigma-Aldrich) in the presence of 2 mM ZnCI2 in Mcllveine buffer, pH 7. Reactions were carried out in triplicate at 37°C. Equivalent amount of native H11 antigen (Barbervax®, obtained from Merlin Vet UK) was included as a control. Post overnight incubation, reactions were stopped by adding 200 pl of 0.4 M glycine-NaOH buffer and OD405 was measured using a microplate reader (Molecular Devices).

[0318] Isolation of sheep PBMCs and cytokine assay (optional for patent application)

[0319] Ovine peripheral blood mononuclear cells (PBMCs) were obtained from 200 ml whole blood collected from a clinically healthy sheep. Briefly, PBMCs were isolated from blood using Lymphoprep™ density gradient medium (Stemcell), counted, resuspended in Gibco™ RPMI 1640 medium supplied with 10% FBS and 1% Pen / Strep solution and finally, seeded into 6-well cell culture plates. After 24-hour incubation at 37°C with 5% CO2, antigens were added to the cells to stimulate cytokine production for 24 hours. Cell culture supernatants were collected by centrifugation and kept at -20°C prior to Luminex VT016P

[0320] -40- cytokine assays. An ovine cytokine / chemokine panel (Merck) with a mixture of 14 probes was used to screen cytokine expression. Post incubation and washing steps, beads were measured by a Luminex Bio-Plex 200 instrument (Bio-Rad). Median fluorescent intensities (Fl) of the analytes were exported from the raw data to Excel. Data analysis was carried out in GraphPad Prism 10.

[0321] Indirect ELISA

[0322] Native and recombinant antigens were diluted to a concentration of 2 pg / ml in coating buffer (200 mM carbonate / bicarbonate buffer, pH 9.6). 96-well Nunc-lmmuno™ MicroWell™ ELISA plates (Sigma-Aldrich) were coated overnight at 4°C with 50 pl of antigens (100 ng / well). Post blocking with 2% BSA, sheep sera (1 :100 diluted) were added to the wells and incubated at 37°C for 1 hour. Serum antibodies were probed with anti-sheep IgG (clone GT-34, Sigma-Aldrich; 1 :10,000 diluted), IgM (rabbit polyclonal, Sigma-Aldrich; 1 :10,000 diluted), IgE (clone 1 E7, Bio-Rad; 1 :10,000 diluted), and IgA (rabbit polyclonal, Bio-Rad; 1 :1 ,000 diluted). HRP-conjugated secondary antibodies, anti-mouse IgG (Sigma-Aldrich; 1 :5,000 diluted) and anti-rabbit IgG (Sigma-Aldrich; 1 :10,000 diluted), were used to react with tetramethylbenzidine-hydrogen peroxide (TMB, Thermo Fisher Scientific). Post addition of a reaction stopping buffer (0.5 M H2SO4, Carl Roth GmbH, Karlsruhe, Germany), OD450 was measured using a microplate reader (Molecular Devices).

[0323] Vaccine Trial

[0324] The double-blinded randomized controlled trial has been carried out at the Clinic for Ruminants, University of Veterinary Medicine, Vienna. The sheep were housed in a separate building, divided into five groups of seven sheep, and multiple samples were collected during the study period (see Fig. 1). The detailed description of the five groups is shown in Table 1 .

[0325] Ethical considerations

[0326] Animal trial was approved by the Ethics and Welfare Committee of the University of Veterinary Medicine, Vienna in accordance with the University’s guidelines for Good Scientific Practice using the number GZ: 2023-0.734.950 and GZ: 2022-0.599.404 (Haemonchus L3 production).

[0327] Sample size calculation and randomization

[0328] The effectiveness of the vaccination was assessed by evaluating the number of eggs excreted (eggs per gram of feces) and the worm burden (N worms in the abomasum). In the publication by Gonzalez-Sanchez

[0016] , the standard deviation is VT016P

[0329] -41- approximately half of the mean of the total egg excretion, and the difference between the vaccinated and non-vaccinated groups is about 75%

[0016] . With a significance level of 5%, a power of 80%, and using a one-sided test procedure (vaccinated animals excrete fewer eggs per gram of feces), the calculated group size was seven animals per group. The sheep were allocated to each study group randomly (stratified randomization) depending on their body weight and breed using the “rand()” function in Microsoft Excel.

[0330] Sheep

[0331] The sheep were born between August and October 2023 and raised on one dairy sheep farm close to the town Amstetten, federal state Lower Austria. The farm is run as a commercial organic dairy sheep farm with ~200 dairy sheep. The ewes were thoroughbred Lacaune and the three rams were thoroughbred Lacaune or Jura. Therefore, the lambs were Lacaune x Lacaune or Lacaune x Jura breed. The lambs were born and stayed with their mothers for 14 days and afterwards they were separated from the mother and kept in a separate barn. The lambs did not have any access to pasture. The farmer separated 39 male lambs for our animal trial and housed them according to the national regulations. All lambs received a vaccine against clostridial disease within the regular herd health management procedure using Miloxan 50 ml (2 ml per sheep, subcutaneously). They received the first immunization on October 25th and the second on November 28th, 2023.

[0332] Deworming strategy before the trial

[0333] The first deworming was carried out on December 15th, 2023, where the sheep received Ivomec 10 mg / ml (Ivermectin, subcutaneously, dosage: 0.2 mg / kg). At the farm of origin no scale was available therefore the dosage for the heaviest sheep was used. At the day of arrival (January 2nd and 3rd, 2024) the sheep were dewormed using Panacur 250 mg (Fenbendazol, oral, dosage: 5mg / kg), Hapadex 50 mg / kg (Netobimin, oral, dosage: 7.5 mg / kg) and Baycox Muti 50 mg / ml (Toltrazuril, oral, dosage: 20 mg / kg). Challenge with Haemonchus contortus

[0334] The infective L3 larvae of H. contortus (KB strain) used for challenge were obtained by performing larval culture of parasite eggs, collected from experimentally infected donor sheep at the Institute of Parasitology. In the vaccine trial, four groups (G2, G3, G4, G5) were challenged using 5,000 L3. All sheep were challenged on the same day using a 2 ml disposable pipette.

[0335] Clinical assessment and blood samples VT016P

[0336] -42-

[0337] The sheep were assessed according to Baumgartner and Wittek

[0017] and blood samples (EDTA, serum, Tempus Tubes) were drawn on defined time point throughout the study period (for details see Fig. 1).

[0338] Efficacy assessment

[0339] Mini-FLOTAC method

[0340] Individual fecal samples were analyzed quantitatively by Mini-FLOTAC. The lower limit of detection of the method was five eggs per gram (EpG) of feces

[0018] , Samples were weighed (5 g feces) and mixed with 45 ml of a saturated saline solution (density 1.18 g / ml) using pistil and mortar to a homogeneous mixture and sieved (0 1.3 mm) into a plastic cylinder. By using a magnetic stirrer (IKA-COMBIMAG REO, Janke & Kunkel GmbH u. Co. KG, Germany), an equal distribution of the eggs was ensured. Samples were aspirated with a disposable 2 ml pipette and transferred to the Mini-FLOTAC system. After ten minutes of resting on a flat surface, the disks were rotated with the key. If air bubbles were visible in the analysis chambers the sample had to be discarded and the steps had to be repeated. Fecal samples were quantitatively evaluated under the light microscope (Eclipse Ci-S, 100 x magnification, Nikon, Vienna, Austria). The EpG is calculated with the following formula: EpG=egg counts in both chambers x(45+5) / (5*2).

[0341] Worm burden and worm sexing

[0342] The worm burden was evaluated after euthanizing the sheep. First, the abdominal cavity was opened, and the duodenum was ligated with a string and the omasum was cut to keep the content within the abomasum. Subsequently, the abomasum was removed from the body. The greater curvature was opened, and the content was transferred into a sieve with a mesh width of 150 pm and washed thoroughly. Subsequently, the abomasal content was transferred into a bucket for storage. The abomasal mucosa as well as the abomasal content was inspected for adult worms and all worms were gathered and counted. The abomasal content (number of worms and gender) was evaluated within three days.

[0343] Statistics

[0344] Efficacy evaluation

[0345] Descriptive and explorative statistical analysis was performed using Microsoft Excel 2010 (Microsoft®, Washington, USA) and IMB® SPSS® Statistics Version 29 (IBM®, New York, USA). The cumulative egg excretion was calculated by summarizing the egg excretions over time. The EpG values per worm was calculated by dividing the VT016P

[0346] -43- cumulative EpG values by the number of female worms. The female / male ratio was calculated by dividing female by male worms. The EpG values, worm counts and blood values were log transformed using the Ig 10 function in SPSS. All measures (EpG values, worm counts, blood values) were analyzed using a linear mixed model followed by a post hoc test (Sidak test) between the experimental groups. Group identification and sampling date were included as fixed values. Sampling date was included as repeated measure and sheep identification as subject. All missing values were excluded in the analysis. The Akaike’s information criterion (AIC) revealed 215.24 for the egg excretion. Comparison between groups without repeated measures was carried out using a 1-way ANOVA. For all tests the significance level was set at p 0.05.

[0347] VT016P

[0348] -44-

[0349] REFERENCES

[0350] 1. Smith D. Development of a commercial vaccine for Haemonchus contortus, the Barber’s Pole Worm; Meat and Livestock Australia, 2014, ISBN 9781740362832.

[0351] 2. Wang, C.; Liu, L.; Wang, T.; Liu, X.; Peng, W.; Srivastav, R.K.; Zhu, X.-Q.; Gupta, N.; Gasser, R.B.; Hu, M. H11-induced immunoprotection is predominantly linked to N-glycan moieties during Haemonchus contortus infection. Front. Immunol. 2022, 13, 1034820, doi: 10.3389 / fimmu.2022.1034820.

[0352] 3. Haslam, S.M.; Coles, G.C.; Munn, E.A.; Smith, T.S.; Smith, H.F.; Morris, H.R.; Dell, A. Haemonchus contortus glycoproteins contain N-linked oligosaccharides with novel highly fucosylated core structures. J. Biol. Chem. 1996, 271 , 30561-30570, doi: 10.1074 / jbc.271 .48.30561 .

[0353] 4. Roberts, B.; Antonopoulos, A.; Haslam, S.M.; Dicker, A.J.; McNeilly, T.N.; Johnston, S.L.; Dell, A.; Knox, D.P.; Britton, C. Novel expression of Haemonchus contortus vaccine candidate aminopeptidase H11 using the free-living nematode Caenorhabditis elegans. Vet. Res. 2013, 44, 111 , doi:10.1186 / 1297-9716-44-111.

[0354] 5. Varki, A.; Cummings, R.D.; Aebi, M.; Packer, N.H.; Seeberger, P.H.; Esko, J.D.; Stanley, P.; Hart, G.; Darvill, A.; Kinoshita, T.; et al. Symbol Nomenclature for Graphical Representations of Glycans. Glycobiology 2015, 25, 1323-1324, doi: 10.1093 / glycob / cwv091 .

[0355] 6. Tusnady, G.E.; Simon, I. Principles governing amino acid composition of integral membrane proteins: application to topology prediction. J. Mol. Biol. 1998, 283, 489-506, doi: 10.1006 / jmbi.1998.2107.

[0356] 7. Bernhofer, M.; Dallago, C.; Karl, T.; Satagopam, V.; Heinzinger, M.; Littmann, M.; Olenyi, T.; Qiu, J.; Schutze, K.; Yachdav, G.; et al. PredictProtein - Predicting Protein Structure and Function for 29 Years. Nucleic Acids Res. 2021 , 49, W535-W540, doi: 10.1093 / nar / gkab354.

[0357] 8. Krogh, A.; Larsson, B.; Heijne, G. von; Sonnhammer, E.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 2001 , 305, 567-580, doi:10.1006 / jmbi.2000.4315.

[0358] 9. Sonnhammer, E.L.; Heijne, G. von; Krogh, A. A hidden Markov model for predicting transmembrane helices in protein sequences. Proc. Int. Conf. Intell. Syst. Mol. Biol. 1998, 6, 175-182.

[0359] 10. Paschinger, K.; Yan, S.; Wilson, LB.H. N-glycomic Complexity in Anatomical Simplicity: Caenorhabditis elegans as a Non-model Nematode? Front. Mol. Biosci. 2019, 6, 9, doi: 10.3389 / fmolb.2019.00009. VT016P

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[0361] 11. Shi, X.; Jarvis, D.L. Protein N-glycosylation in the baculovirus-insect cell system. Curr. Drug Targets 2007, 8, 1116-1125, doi: 10.2174 / 138945007782151360.

[0362] 12. Adduci, I.; Sajovitz, F.; Hinney, B.; Lichtmannsperger, K.; Joachim, A.; Wittek, T.; Yan, S. Haemonchosis in Sheep and Goats, Control Strategies and Development of Vaccines against Haemonchus contortus. Animals (Basel) 2022, 12, doi: 10.3390 / ani12182339.

[0363] 13. Flay, K.J.; Hill, F.L; Muguiro, D.H. A Review: Haemonchus contortus Infection in Pasture-Based Sheep Production Systems, with a Focus on the Pathogenesis of Anaemia and Changes in Haematological Parameters. Animals (Basel) 2022, 12, doi:10.3390 / ani12101238.

[0364] 14. Cachat, E.; Newlands, G.F.J.; Ekoja, S.E.; McAllister, H.; Smith, W.D. Attempts to immunize sheep against Haemonchus contortus using a cocktail of recombinant proteases derived from the protective antigen, H-gal-GP. Parasite Immunol. 2010, 32, 414-419, doi:10.1111 / j.1365-3024.2010.01208.x.

[0365] 15. Yan, S.; Vanbeselaere, J.; Jin, C.; Blaukopf, M.; Wbls, F.; Wilson, I.B.H.; Paschinger, K. Core Richness of N-Glycans of Caenorhabditis elegans: A Case Study on Chemical and Enzymatic Release. Anal. Chem. 2018, 90, 928-935.

[0366] 16. Gonzalez-Sanchez, M.E.; Cuquerella, M.; Alunda, J.M. Vaccination of lambs against Haemonchus contortus with the recombinant rHc23. Effect of adjuvant and antigen dose. PLoS One 2018, 13, e0193118.

[0367] 17. Walter Baumgartner, T.W. Klinische Propadeutik der Haus- und Heimtiere, 9., aktualisierte und erweiterte Auflage; Stuttgart: Enke Verlag, 2018, ISBN 9783132402744.

[0368] 18. Cringoli, G.; Rinaldi, L.; Maurelli, M.P.; Utzinger, J. FLOTAC: new multivalent techniques for qualitative and quantitative copromicroscopic diagnosis of parasites in animals and humans. Nat. Protoc. 2010, 5, 503-515.

[0369] 19. Fleurkens S.M.; Carbohydrate-based vaccines against the sheep parasite Haemonchus contortus, 2017, PhD thesis, DISS. ETH NO. 24498, XP093274053.

[0370] 20. Smith, T. S., Munn, E. A., Graham, M., Tavernor, A. S., & Greenwood, C. A. (1993). Purification and evaluation of the integral membrane protein H11 as a protective antigen against Haemonchus contortus. International journal for parasitology, 23(2), 271-280.

Claims

VT016P-46-CLAIMS1 . An expression cassette for the production of extracellular recombinant tri-fucosylated N-glycoproteins in insect cells comprising i) a glyco-module comprising polynucleotide sequences of a nematode a) galactosyltransferase, specifically GALT-1 , and b) fucosyltransferase, specifically FUT-6, and c) mannosidase, specifically AMAN-3, and ii) a polynucleotide sequence encoding one or more tri-fucosylated N- glycoproteins derived from a nematode.

2. The expression cassette of claim 1 comprising a Caenorhabditis elegans (C. elegans) GALT-1 , a C. elegans FUT-6, and a C. elegans AMAN-3, and the polynucleotide sequence encoding one or more tri-fucosylated N-glycoproteins is derived from Haemonchus contortus.

3. The expression cassette of claim 1 , wherein the galactosyltransferase comprises the nucleotide sequence SEQ ID NO: 1 or SEQ ID NO: 2, the fucosyltransferase comprises the nucleotide sequence SEQ ID NO: 3 or SEQ ID NO: 4, and / or the mannosidase comprises the nucleotide sequence SEQ ID NO: 5.

4. The expression cassette of claim 1 , wherein the glyco-module comprises polynucleotide sequences from C. elegans, specifically GALT-1 , FUT-6, and / or AMAN- 3; or from Haemonchus contortus (H. contortus), specifically GALT-1 and / or FUT-6.

5. The expression cassette of any one of claims 1 or 3 to 4, wherein the tri-fucosylated N-glycoproteins are intestine proteins, specifically derived from any one of Haemonchus contortus, Haemonchus place!, Teladorsagia circumcincta, Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Ascaris suum, Oesophagostomum dentatum, or Ostertagia ostertagi.

6. The expression cassette of any one of claims 1 to 5, wherein the tri-fucosylated N-glycoproteins are derived from H. contortus glycoprotein H11 , encoded by SEQ ID NO: 6; H11-1 , encoded by SEQ ID NO: 7; H11-2, encoded by SEQ ID NO: 8; H11-4, encoded by SEQ ID NO: 9; H11-5a, encoded by SEQ ID NO: 10; H11 -5b, encoded byVT016P-47-SEQ ID NO: 11 ; H11-5c, encoded by SEQ ID NO: 12; and / or GA1 , encoded by SEQ ID NO: 13.

7. A recombinant baculovirus comprising the expression cassette of any one of claims 1 to 6.

8. A method for production of recombinant tri-fucosylated N-glycoproteins derived from a nematode in insect cells comprising the sequential steps: a. T ransfecting the insect cells with the expression cassette of any one of claims 1 to 6, or with the baculovirus of claim 7; b. Expressing the tri-fucosylated N-glycoproteins by the insect cells; c. Harvesting and optionally purifying the tri-fucosylated N-glycoproteins from the insect cells, specifically, the insect cells are Lep / doptera-derived cells, specifically Trichoplusia ni High Five (Hi5) cells, Spodoptera frugiperda Sf9, Spodoptera frugiperda Sf21 , or Trichoplusia ni Tnao38 cells.

9. An immunological composition against a nematode, comprising at least one recombinant tri-fucosylated N-glycoprotein, wherein the recombinant tri-fucosylated N- glycoprotein comprises two mannose residues and up to one galactose residue, specifically a recombinant intestine tri-fucosylated N-glycoprotein, and optionally one or more adjuvants, specifically, wherein the composition is a lyophilized powder, a frozen liquid, or a liquid.

10. The method of claim 8, or the immunological composition of claim 9, wherein the tri-fucosylated N-glycoproteins are derived from H. contortus glycoprotein H11 comprising SEQ ID NO: 14; H11-1 comprising SEQ ID NO: 15; H11-2 comprising SEQ ID NO: 16; H11-4 comprising SEQ ID NO: 17; H11-5a comprising SEQ ID NO: 18; H11- 5b comprising SEQ ID NO: 19; H11-5c comprising SEQ ID NO: 20; and / or GA1 comprising SEQ ID NO: 21.

11. The method of any one of claims 8 or 10, or the immunological composition of claims 9 or 10, wherein the intestine tri-fucosylated N-glycoprotein is a H. contortus glycoprotein lacking its trans-membrane domain.VT016P-48-12. The composition of any one of claims 9 to 11 for use in vaccinating an animal, specifically a ruminant, or a New World camelid, more specifically sheep, goats, alpacas, llamas, or cattle.

13. The composition of any one of claims 9 to 12 for use in reducing nematode parasitic worm burden, in stimulating or boosting acquired immunity, or to induce protective anti-nematode immunity in an animal, specifically a ruminant, or a New World camelid, more specifically sheep, goats, alpacas, llamas, or cattle.

14. The method of any one of claims 8 to 11 , the composition of any one of claims 9 to 12, or the composition for use of claim 13, wherein the vaccinated animals have reduced fecal egg count and worm burden in the gastrointestinal tract compared to unvaccinated animals infected with the nematode, specifically wherein the nematode is selected from the group consisting of Haemonchus contortus, Haemonchus placei, Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Ascaris suum, Oesophagostomum dentatum, and Ostertagia ostertagi.

15. A recombinant H. contortus H11 or GA1 antigen having core tri-fucosylated N- glycans, wherein the N-glycans are nematode-type N-glycans comprising any one of amino acid sequences SEQ ID No: 14 to 21 , or amino acid sequences having at least 90%, 91 %, 92%, 93%, 94%, 95%, specifically at least 98%, more specifically 99% sequence identity to SEQ ID NOs: 14 to 21 .

16. An engineered insect cell comprising the expression cassette of any one of claims 1 to 6, or the recombinant baculovirus of claim 7.

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