Recombinant activation-related secretory proteins

Abstract

The present invention relates to recombinant activation-associated secretory protein (ASP) or a fragment thereof, wherein the ASP or fragment comprises an N-glycan containing core α1,3-fucose and / or core α1,6-fucose (Fuc). The present invention further relates to a pharmaceutical composition comprising such recombinant ASP or a fragment thereof. Furthermore, the present invention relates to recombinant ASP or a fragment thereof for use as a human or veterinary drug, particularly as a vaccine, more specifically for use against parasitic nematode infections.

Description

Technical Field

[0001] The present invention relates to a recombinant activation-related secreted protein (ASP) or a fragment thereof that can be obtained from, for example, Nicotiana spp., Pichia spp., or an insect cell line expression system. The present invention further relates to a pharmaceutical composition comprising such a recombinant ASP or a fragment thereof. Furthermore, the present invention relates to a recombinant ASP or a fragment thereof for use as a pharmaceutical for humans or animals, particularly as a vaccine, and more specifically for use against parasitic nematode infections.

Background Art

[0002] Control of the intestinal parasitic nematode Ostertagia ostertagi is of great economic importance to livestock farmers because the infection accounts for a large proportion of production losses. Treatment of these similar helminth infections in cattle relies almost entirely on the application of anthelmintics, but reports of future anthelmintic resistance in Cooperia oncophora are increasing, hindering the future outlook for these compounds. Also, anthelmintic resistance has been reported for O. ostertagi. To overcome this obstacle, alternative and sustainable control measures such as vaccination have been investigated in recent years.

[0003] Currently, an experimental vaccine against O. ostertagii exists, which is based on an endogenous activation-associated secretory protein (ASP) purified from excretory-secretory (ES) proteins released primarily by adult worms, and will be hereafter referred to as Oo-ASP. Although the exact role of ASP remains largely unknown, its component in ES proteins suggests its involvement in helminth survival, reproduction, host infection, and immune evasion. The fact that CAP (cysteine-rich secretory / antigen 5 / pathogenesis-related 1), the superfamily to which ASP belongs, can capture small hydrophobic ligands such as sterols further emphasizes the possibility that the host immune response is modulated to favor the parasite.

[0004] Under experimental conditions, intramuscular inoculation of these ASP antigens into cattle significantly reduces the amount of parasite eggs excreted and the amount of parasites. Further immunological studies have demonstrated that vaccination with these antigens results in significant proliferation of NK cells, secretion of IFN-γ, and a mixed IgG1 / IgG2 antibody response compared to the negative (adjuvant only) vaccine group.

[0005] Obtaining large quantities of endogenous vaccine antigens is time-consuming and uneconomical; therefore, recombinant expression is essential for its future prospects. Moreover, the economic use of these endogenous antigens is unethical, and in fact, obtaining Oo-ASP requires the euthanasia of calves. Despite the protective capacity of endogenous Oo-ASP, no significant reduction in fecal egg excretion and / or parasite count was observed after immunization of calves with recombinant Pichia pastoris (Non-Patent Literature 1). This study also noted that only animals vaccinated with endogenous Oo-ASP showed a significant secondary antibody response in the gastrointestinal tract, particularly with prominent IgG1 and IgG2 levels. In addition, the cellular memory response induced by endogenous Oo-ASP was remarkably stronger compared to that of recombinant P. pastoris. Furthermore, regarding C. oncophora, comparative analysis of immune responses induced by native and recombinant ASP-based vaccines demonstrated a significant level of protection after experimental challenge infection, whereas this was not demonstrated with the recombinant vaccine (Non-Patent Literature 2). These findings indicate that endogenous ASP activates the (bovine) immune system in a unique way, which has not been reproducible using recombinant ASP. This, in turn, suggests that recombinants have structural differences and / or substantially lack the antigenic epitopes present in endogenous ASP.

[0006] To better understand the immunogenic properties of ASPs, it is essential to determine which (primary and / or secondary) structural features, which subunits (or multiple subunits) of the protein, and / or which N-glycosylations thereof are part of the antibody epitope and consequently lead to the development of protective immunity.

[0007] In the research leading to the present invention, the inventors compared endogenous ASP with its recombinant counterpart and explored the biochemical differences that influence protection. In addition, they constructed peptide and glycan microarrays and studied the interaction between antigen-specific antibodies and highly specific regions or epitopes on these antigens. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Gonzalez-Hernandez et al., 2016 [Non-Patent Document 2] Gonzalez Hernandez et al. 2018 [Overview of the project] [Problems that the invention aims to solve]

[0009] The object of the present invention is to provide recombinant ASP that can induce an immune response against parasitic nematodes and can be produced on a large scale.

[0010] Surprisingly, it was found that the presence of core α1,3-fucose and / or core α1,6-fucose, particularly N-glycans containing core α1,3-fucose, is essential for the binding of native ASP-induced IgG to recombinant ASP. Therefore, recombinant ASP having N-glycans containing core α1,3-fucose, core α1,6-fucose, or a combination thereof can be used to induce an immune response. Furthermore, since recombinant ASP having N-glycans containing α1,3-fucose and / or α1,6-fucose can be produced on a large scale, it can be manufactured via various expression systems, such as Nicotiana benthamiana expression systems, Pichia pastris expression systems, or insect cell line expression systems. [Means for solving the problem]

[0011] In a first embodiment, the present invention provides a recombinant activation-associated secretory protein (ASP) or a fragment thereof, wherein the ASP or fragment thereof comprises core α1,3-fucose and / or core α1,6-fucose (Fuc), particularly an N-glycan containing core α1,3-fucose.

[0012] In certain embodiments of the present invention, the ASP or fragment thereof comprises an N-glycan further comprising a coreglycan structure constructed by a combination of two N-acetylglucosamine (GlcNAc) residues and either α-1,3-fucose or α-1,6-fucose linked to the GlcNAc residues linked to Asn. The mannose residues (located at positions X and / or Y in structure I) may each independently contain additional glycan branches constructed from N-acetylglucosamine (GlcNAc), galactose (gal), fucose (fuc), or mannose (man) residues (including combinations thereof). In another embodiment of the present invention, the recombinant ASP or fragment thereof comprises an N-glycan comprising the structure of structure (I) (Figure 17).

[0013] Preferably, the recombinant ASP or fragment thereof contains an N-glycan having a structure according to structure (I), where X and / or Y are substituted with GlcNac, particularly Gal-GlcNAc.

[0014] In certain embodiments of the present invention, recombinant ASP is produced by synthesis or obtained from an expression system containing a core-modified fucosyltransferase. Examples of core-modified fucosyltransferases include fucosyltransferase 8 derived from mouse or fruit fly in the case of core α-1,6-fucose, fucosyltransferase C derived from Schistosoma mansoni in the case of core α-1,3-fucose, or plant endogenous fucosyltransferase 11 or plant endogenous fucosyltransferase 12.

[0015] In another specific embodiment, recombinant ASP is obtained from a Nicotiana genus expression system (e.g., Nicotiana bentamiana or Nicotiana tabacum expression system), a Pichia genus expression system (e.g., Pichia pastris expression system), or an insect cell line expression system.

[0016] In further embodiments of the present invention, the amino acid sequences of recombinant ASP or fragments thereof have at least 90% sequence identity, preferably at least 95%, more preferably at least 99%, and most preferably 100% sequence identity with respect to the amino acid sequences of ASP of Ostertagia ostertagii, Cooperia oncophora, or Teladorsagia circumcincta, in particular the amino acid sequences of ASP of Ostertagia ostertagii, Cooperia oncophora, or Teladorsagia circumcincta, respectively, with Genbank accession numbers CAD23183.1 (especially amino acids 22-236; AA 1-21 include the signal sequence MQALIGIAALYLVLVTSNTEA (SEQ ID NO: 13)), Genbank accession numbers CCQ71722.1, and Genbank accession numbers CBJ15404.1 (especially amino acids 22-236; AA 1-21 are represented by the signal sequence MFTPIGIAVLYLALVTPHAKA (sequence number 14).

[0017] In another specific embodiment of the present invention, the recombinant ASP amino acid sequence of Ostertagia ostertagia of the present invention is represented by SEQ ID NO: 1, the ASP amino acid sequences of Cooperia oncophora are represented by SEQ ID NOs: 2 to 4, and the recombinant ASP amino acid sequence of Terradorsagia kirkumkinkuta is represented by SEQ ID NO: 5.

[0018] In a further embodiment, the present invention provides a pharmaceutical composition comprising a recombinant ASP or a fragment thereof and a pharmaceutically acceptable carrier and / or additive.

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

[0020] In another specific embodiment, the present invention relates to a pharmaceutical composition comprising the recombinant ASP of the present invention or a fragment thereof, further comprising an adjuvant.

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

[0022] In a more specific embodiment, the present invention provides a recombinant ASP of the present invention or a fragment thereof, or a pharmaceutical composition comprising the recombinant ASP of the present invention or a fragment thereof, for use in the treatment, prevention and / or alleviation of parasitic nematode infections in mammals.

[0023] In another specific embodiment, the present invention relates to a recombinant ASP of the present invention or a fragment thereof, or a pharmaceutical composition comprising recombinant ASP, for use in the treatment, prevention and / or alleviation of parasitic nematode infections in mammals, wherein the parasitic nematodes belong to the genus Ostertagia and / or Cooperia and / or Teladorsagia, and more specifically, the parasitic nematodes are Ostertagia ostertagi, Ostertagia leptospicularis, Cooperia onchophora, Cooperia punctata, Cooperia pectinata, Teladorsagia trifurcata, and / or Teladorsagia circumcincta, and even more particularly, the parasitic nematodes are Ostertagia ostertagi, Cooperia onchophora, or Teladorsagia circumcincta.

[0024] In a further embodiment, the present invention relates to a method for producing the recombinant ASP of the present invention or a fragment thereof, the method comprising a) Preparing an expression system containing fucosyltransferase, more particularly core-modifying fucosyltransferase; b) Introducing fucose sugars, particularly core α1,3-fucose and / or core α1,6-fucose, into the expression system; c) Expressing ASP using the expression system prepared in step a) to obtain the recombinant ASP of the present invention or a fragment thereof; and the method includes the above steps.

[0025] In a further embodiment, the present invention provides a method for treating, preventing and / or alleviating parasitic nematode infections in a subject who needs treatment, prevention and / or alleviation of parasitic nematode infections, the method comprising administering to the subject who needs treatment, prevention and / or alleviation of parasitic nematode infections the recombinant ASP of the present invention or a fragment thereof, or the pharmaceutical composition of the present invention.

[0026] It is emphasized that when specifically referring to the drawings, the details shown are illustrative and are only intended to exemplarily explain various embodiments of the present invention. Those details are presented to provide what is considered to be the most useful and easy explanation of the principles and conceptual aspects of the present invention. In this regard, it is not intended to show the details of the structure of the present invention in more detail than is necessary for a basic understanding of the present invention. It will be apparent to those skilled in the art how some forms of the present invention can be actually implemented through the description in conjunction with the drawings.

Brief Description of the Drawings

[0027] [Figure 1]Amino acid sequences of ASP from Ostertagia ostertagii and Cooperia oncophora. (A) Shows the Oo-ASP-1 amino acid sequences of recombinant and European and North American isolates. Significant sequence diversity was found at a total of nine positions in the isolate sequences. Corresponding residues are shown in bold. Dotted lines indicate residues with approximately the same polymorphism ratio. (B) Nucleotide sequences encoding C. oncophora bidomain activation-associated secretory protein, and amino acid sequences of C. oncophora bidomain activation-associated secretory protein. [Figure 1-1] Same as above [Figure 1-2] Same as above [Figure 1-3] Same as above [Figure 2] Indirect ELISA: four peptides for three vaccine group pools; indirect ELISA: PUS 028 029 for three vaccine group individuals. [Figure 3] Overview of synthetic N-glycans present on array slide A. [Figure 3-1] B. N-glycan recognition by Oo-ASP-1 specific antibodies derived from native Oo-ASP-1 immunized cows, expressed as the mean fluorescence index (MFI) per glycan spot with the standard error of the mean based on four technical iterations. All core α1,3-fucosylated N-glycans are clearly recognized, while α1,6-fucosylation is not necessarily required for antibody recognition. [Figure 4] The ability of anti-ASP antibodies to inhibit the binding of native Oo-ASP to native Oo-ASP, N. benthamiana recombinant ASP, and P. pastris recombinant ASP was evaluated. A lower OD405-492 signal corresponds to less antibody binding to native Oo-ASP coated on a 96-well plate, and therefore to a higher level of inhibition. [Figure 5] Determination of cell proliferation upon (re)exposure to nASP. In the CD335+ NK cell population, cell proliferation was observed in most animals vaccinated with nASP. Proliferation was minimal in other cell types and / or vaccine groups. [Figure 6]Demonstration of systemic IgG1 response in the vaccine group. Clear (cross-reactive) IgG1 responses were demonstrated in both the nASP and rASP vaccine groups after the second immunization. The increase at autopsy in the QuilA group was a result of the trickle-infection period experienced by all animals. [Figure 6-1] Same as above [Figure 7] Determination of local IgG1, local IgG2, and local IgA reactions. Similar to systemic reactions, clear (cross-reactive) IgG1 and IgG2 reactions were observed in the mucus at autopsy. Similarly, these reactions were mainly observed in nASP and rASP. [Figure 7-1] Same as above [Figure 8] Daily fecal egg count (with SEM) for each vaccine group. Daily fecal egg count for each vaccine group, starting 21 days after the initial infection. [Figure 9] The average daily excretion of individual parasite eggs by vaccine group. Within each group, every animal is assigned a different symbol, indicating that animals with a high number of eggs in their feces are usually the same animal. Importantly, in the nASP and rASP vaccine groups, there are few or no animals excreting large numbers of parasite eggs. [Figure 9-1] Same as above [Figure 9-2] Same as above [Figure 10] Cumulative fecal egg counts shown in EPG. AUC per animal was calculated and shown as cumulative EPG. Compared to QuilA controls, a non-significant 57% reduction was observed in animals immunized with nASP+QuilA, while a similar non-significant 45% reduction was observed in animals immunized with rASP+QuilA. [Figure 11] The number of parasites in the control and vaccine groups. When comparing the QuilA control group with the nASP vaccine group and the rASP vaccine group, no clear differences were found in the total number of parasites, total number of adults, or total number of L4 parasites. [Figure 12]Determination of parasite length in the control and vaccine groups. No clear differences in parasite length were observed among the three vaccine groups. When the measured parasites were assigned to the vaccine group to which the animals belonged, rather than to individual animals, it was observed that the parasite length was significantly shorter in both male and female animals vaccinated with nASP. [Figure 13] Overview of synthetic N-glycans. An overview of the synthetic N-glycans present on this array slide, with reference names added to the data shown in Figures 11 and 12. [Figure 14] N-glycan recognition by Co-dd-ASP-specific antibodies derived from cows immunized with native Co-dd-ASP. N-glycan recognition by Co-dd-ASP-specific antibodies derived from cows immunized with native Co-dd-ASP, expressed as the mean fluorescence index (MFI) per glycan spot with the standard error of the mean based on four technical replicates. All core α1,3-fucosylated N-glycans are clearly recognized, while α1,6-fucosylation is not necessarily required for antibody recognition. [Figure 15] N-glycan recognition by Co-dd-ASP-specific antibodies derived from cows immunized with P. pastris ASP. N-glycan recognition by Co-dd-ASP-specific antibodies derived from cows immunized with P. pastris ASP is expressed as the mean fluorescence index (MFI) per glycan spot, with the mean standard error of the mean based on four technical replicates. N-glycan recognition is more limited compared to animals vaccinated with native ASP. [Figure 16] Differences in peptide recognition among pooled serum samples from the three vaccine groups. There appear to be no clear differences in peptide recognition among the pooled serum samples from the three vaccine groups. [Figure 17]A. Structure (I). B. a) Structure (I) + Gal-GlcNAc (position X or Y) - core α1,6 fucose. b) Structure (I) + Gal-GlcNAc + GlcNAc (position X or Y (only one shown)) - core α1,6 fucose. c) Structure (I) + Gal-GlcNAc + Gal-GlcNAc (positions X and Y (only one shown)) - core α1,6 fucose. d) Structure (I) + Gal-GlcNAc (position X or Y (only one shown)) - core α1,3 fucose. e) Structure (I) + Gal-GlcNAc + GlcNAc (position X or Y (only one shown)) - core α1,3 fucose. f) Structure (I) + Gal-GlcNAc + Gal-GlcNAc (positions X and Y (only one shown)) - core α1,3 fucose. [Figure 18] A: N. bentamiana Oo-ASP-1. Core α1,3-fucose, before terminal galactose enrichment. Estimated: Galactosylated N-glycan <5%. B: N. bentamiana Oo-ASP-1. Core α1,3-fucose, after terminal galactose enrichment. [Figure 19] A: N. bentamiana Oo-ASP-1. Core α1,6-fucose, before terminal galactose enrichment. Estimated: Galactosylated N-glycan <5%. B: N. bentamiana Oo-ASP-1. Core α1,6-fucose, after terminal galactose enrichment. [Modes for carrying out the invention]

[0028] The present invention will be described further below. Different aspects of the present invention will be defined in more detail in the following text. Each of the aspects defined in this manner may be combined with any other aspect or more of the invention unless explicitly stated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with any other feature or more of the features indicated as preferred or advantageous. When describing the compounds of the present invention, unless otherwise specified in the context, the terms used shall be interpreted according to the following definitions.

[0029] When used herein, the terms "about" or "approximately" refer to measurable values ​​such as parameters, quantities, and time intervals, and mean that they include variations of ±10%, preferably ±5%, more preferably ±1%, and even more preferably ±0.1% or less from a specified value, provided that such variations are appropriate for carrying out the disclosed invention. Naturally, the values ​​to which the modifiers "about" or "approximately" apply are themselves specifically and preferably disclosed.

[0030] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural subjects unless the context clearly indicates otherwise.

[0031] As already described in detail above in this specification, the present invention provides recombinant activation-associated secretory proteins (ASPs) or fragments thereof comprising α1,3-fucose (Fuc) and / or α1,6-fucose, more specifically core α1,3-fucose and / or core α1,6-fucose, and even more specifically an N-glycan containing core α1,3-fucose.

[0032] Therefore, the present invention preferably provides an N-glycan containing α1,3-fucose, and more particularly a recombinant activation-associated secretory protein (ASP) or fragment thereof containing an N-glycan containing core α1,3-fucose.

[0033] The ability of N-glycans to be recognized by ASP-specific antibodies was investigated using glycan microarrays. Among a diverse group of N-glycans, all recognized N-glycans contained core α1,3-fucose, optionally in combination with N-glycans containing core α1,6-fucose. Core α1,3-fucose, and / or to a lesser extent core α1,6-fucose, were considered essential for recombinant ASP to be recognized by native ASP-induced IgG.

[0034] Therefore, by recombinant ASPs containing N-glycans containing core α1,3-fucose or core α1,6-fucose, and in particular by combinations of recombinant ASPs containing N-glycans containing core α1,3-fucose and recombinant ASPs containing N-glycans containing core α1,6-fucose, a novel use and / or vaccine against intestinal parasitic nematodes, such as Ostertagia ostertagii, Ostertagia leptospicularis, Cooperia oncophora, Cooperia punctate, Cooperia pectinate, Telladorsagia triflucata, and / or Telladorsagia kilchumcincta, a novel use and / or vaccine can be provided.

[0035] Obtaining native ASP requires the euthanasia of calves, which hinders the large-scale production of the vaccine. The recombinant ASP of the present invention can be produced using various expression systems that enable the large-scale production of the above vaccine.

[0036] The importance of N-glycosylation in the immune response was further demonstrated by glycan microarray results, which confirmed the ability of antigen-specific antibodies to exclusively bind to N-glycans in the absence of proteins. Recognition of N-glycans containing core α1,3-fucose is surprisingly significant, both alone and in combination with core α1,6-fucose.

[0037] Therefore, in particular, the present invention further relates to recombinant activation-associated secretory protein (ASP) or fragment thereof, the ASP or fragment comprising a glycan, particularly an N-glycan, the glycan comprising α1,3-fucose attached to an Asn-bound GlcNAc residue, and / or α1,6-fucose (Fuc) attached to an Asn-bound GlcNAc residue. More specifically, the ASP or fragment thereof comprises a coreglycan structure constructed by a combination of two N-acetylglucosamine (GlcNAc) residues and two, particularly three mannose residues and either α-1,3-fucose or α-1,6-fucose attached to an Asn-bound GlcNAc residue. In certain embodiments, the (outer) mannose residues (at positions X and / or Y in structure I) each independently contain additional glycans constructed by or containing mannose, galactose, N-acetylgalactosamine, N-acetylglucosamine and / or fucose residues (including combinations thereof). More specifically, the N-glycan includes structures according to structure (I) (including Ia and Ib) presented herein. In the above structures, X and / or Y may be absent, or if present, they are independently selected from mannose, galactose, N-acetylgalactosamine, N-acetylglucosamine, and fucose (including combinations thereof), and in particular X and / or Y are selected from N-acetylglucosamine and galactose. In one embodiment, X is absent. In another embodiment, Y is absent. In a further embodiment, X comprises GlcNAc and Gal, and Y is absent. In yet another embodiment, Y comprises GlcNAc and Gal, and X is absent. In a further embodiment, X and Y comprise GlcNAc and Gal.

[0038] As referred to herein, N-linked glycosylation is a process called N-glycosylation in which an oligosaccharide (sometimes called a glycan), which is a carbohydrate consisting of several sugar molecules, is linked to a nitrogen atom (the amide nitrogen of the asparagine (Asn) residue in a protein).

[0039] The recombinant ASP or fragment of the present invention may also be referred to as isolated ASP or fragment.

[0040] In the context of this invention, the term "recombinant" refers to a protein produced by synthesis or expressed in an expression system different from its native expression system. Therefore, a recombinant protein or fragment thereof is interpreted as a protein expressed in an expression system different from the one in which it is expressed in its native form. The term "recombinant" also includes molecules formed by laboratory genetic engineering methods that create sequences not present in the natural genome by fusing genetic material of different origins.

[0041] Furthermore, in the context of the present invention, the term "recombinant ASP of the present invention" shall be interpreted as a recombinant activation-associated secretory protein or fragment thereof comprising core α1,3-fucose and / or core α1,6-fucose (Fuc), and more particularly core α1,3-fucose, at least one glycan, particularly an N-glycan. In one embodiment, the recombinant ASP or fragment comprises two, three, four or more N-glycans having core α1,3-fucose and / or core α1,6-fucose (Fuc), and more particularly core α1,3-fucose.

[0042] The term "isolated" is used to indicate that cells, peptides, or nucleic acids have been separated from their natural environment. Isolated proteins, peptides, and nucleic acids can be substantially pure, meaning they essentially contain no other substances to which they might bind in nature.

[0043] In one embodiment of the present invention, the amino acid sequence of the recombinant ASP or fragment thereof has at least 90% sequence identity (and therefore including at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity), preferably at least 95% sequence identity, more preferably at least 99%, and most preferably 100% sequence identity, with respect to the amino acid sequences of the ASP of Ostertagia ostertagia, Couperia oncophora, and Terradorsagia kirchumcinct, which are particularly as shown in Figure 1, or exist under Genbank accession numbers CAD23183.1 (excluding the signal sequence in particular), CCQ71722.1, or CBJ15404.1 (excluding the signal sequence in particular). In another embodiment, the nucleic acid sequences encoding recombinant ASP or fragments thereof provided herein have at least 90% sequence identity (and therefore include at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity), preferably at least 95%, more preferably at least 99%, and most preferably 100% sequence identity with the nucleic acid sequences of ASP of Ostertagia ostertagia, Cooperia oncophora, or Terradorsagia kilchumkinkuta, represented by accession number CAD23183.1 (excluding the signal sequence in particular), accession number CCQ71722.1, or accession number CBJ15404.1 (excluding the signal sequence in particular), respectively.

[0044] In a further embodiment of the present invention, the amino acid sequence of the recombinant ASP of the present invention for Ostertagia ostertagia is represented by SEQ ID NO: 1, the amino acid sequences of the recombinant ASP of the present invention for Cooperia oncophora are represented by SEQ ID NOs: 2 to 4, and the amino acid sequence of the recombinant ASP of the present invention for Terradoursagia kirkumkinkuta is represented by SEQ ID NO: 5. Any N-terminal and / or C-terminal sequence of C. oncophora is characterized by the amino acids LCSLDNGMT (SEQ ID NO: 6) and DEDCKCSSCRCSTQLSMCINPN (SEQ ID NO: 7), respectively.

[0045] The identity percentage of nucleic acid and polypeptide sequences can be calculated using commercially available algorithms that compare a reference sequence with a query sequence. The following programs (provided by the National Center for Biotechnology Information) can be used to determine homology / identity: BLAST, GapBLAST, BLASTN, and PSI BLAST (available with default parameters).

[0046] As used herein, the term “fragment” refers to a partial amino acid sequence (and the nucleic acid sequence encoding it) that shares at least one immunological or immunogenic property (i.e., the ability to produce antibodies in immunized animals) with the native molecule. Therefore, the full-length sequence of the ASP is not required, nor is 100% identity to the sequence numbers presented herein. This means that one or more amino acid modifications are possible. For example, a fragment may have up to 30 amino acids removed from the N-terminus or C-terminus of a protein, e.g., up to approximately 1, 5, 10, 15, 20, or 30 amino acids. As stated elsewhere in this disclosure, as long as the protein contains at least one N-glycan, including α1,3-fucose and / or α1,6-fucose (Fuc), as provided herein, it is highly likely that a sufficient level of antibody production will be obtained with 90% sequence identity. As used herein, the term “amino acid modification” refers to the addition, deletion, and / or substitution of amino acids compared to the reference sequence. Preferably, the above substitution of one or more amino acids is a "conservative" substitution of amino acids, i.e., the substitution of one amino acid with another amino acid of the same class, which is as follows: Examples of amino acid classes Non-polar Ala, Val, Leu, Pro, Met, Phe, Trp, Ile Non-charged electrodes: Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic Asp, Gly Basic Lys, Arg, His

[0047] Different amino acids may be conserved substitutions and / or located outside the immunodominant epitope(s) of the ASP fragment. As used herein, the term “immunogenic fragment” in relation to the ASP protein is a fragment of protein that is immunogenic, i.e., capable of specifically interacting with antigen-recognizing molecules of the immune system, such as immunoglobulins (antibodies) or T cell antigen receptors. Preferably, the immunogenic fragments of the present invention are immunodominant for the recognition of antibodies and / or T cell receptors. In certain embodiments, the immunogenic fragments referred to herein are fragments of the ASP provided herein that retain at least 50%, 60%, 70%, 80%, or 90% of the immunogenicity of the full-length protein. The fragments may range from small fragments of about eight amino acids to large fragments that are missing only one amino acid from the full-length protein provided herein. In certain embodiments, the fragments contain eight to 249 amino acid residues of the full-length protein. In other embodiments, the fragments contain or consist of 10 to 200, 10 to 150, 10 to 120, 10 to 100, or 10 to 50 amino acid residues. Such fragments contain at least one epitope (or antigenic determinant) of the native molecule. In one embodiment, they have a length of at least 8 amino acids, preferably at least 10, 11, 12, 13, 14, 15, or 20 amino acids.

[0048] A suitable non-limiting example of such a fragment of ASP of Ostertagia ostertagii is represented by a sequence comprising at least Asn9 and / or Asn37 (containing the N-glycan of the present invention), the fragment comprising or consisting of, for example, the amino acid sequence represented by SEQ ID NO: 8 (GFCCPADLNQTDEARKIFLDFHNQVRRDIAGASPLLNLTGAV).

[0049] In specific embodiments, the recombinant ASP or fragment of O. osteragi provided herein comprises or consists of the amino acid sequence represented by SEQ ID NO: 9 (GFCCPADLNQTDEARKIFLDFHN), and therefore comprises at least Asn9 (containing the N-glycan of the present invention).

[0050] A suitable non-limiting example of a suitable fragment of the C. oncophora dd-ASP is represented by a sequence containing at least one of Asn83, Asn277, or Asn340, according to the numbering in SEQ ID NOs. 2 to 4. In specific embodiments, the recombinant ASP or fragment of C. oncophora provided herein contains at least the amino acids Asn83 and Asn277, or Asn277 and Asn340, or Asn83, Asn277 and Asn340.

[0051] A suitable non-limiting example of a suitable fragment of Terradorsagia kirkumkinkuta ASP (Tc-ASP) is represented by a sequence containing at least Asn37 (containing the N-glycan of the present invention) according to the numbering in Sequence ID No. 5.

[0052] Fragments comprising two or more specific asparagine residues (Asn) selected from one or a combination of different species can be combined as a mixture or in a fusion protein. The term "fusion protein" refers to a polypeptide sequence translated from a nucleic acid transcript produced by combining a first nucleic acid sequence encoding a first peptide fragment provided herein with a second nucleic acid encoding at least a second peptide provided herein, and the fusion protein is not a spontaneously occurring protein. The nucleic acid construct may encode two, three, four or more peptides linked in the fusion protein and optionally separated by a linker sequence. Methods for producing fusion proteins are generally known to those skilled in the art.

[0053] As used herein, "glycan" generally refers to glycosidic monosaccharides, oligosaccharides, and polysaccharides. Therefore, the carbohydrate portion of complex carbohydrates such as glycoproteins, glycolipids, or proteoglycans is referred to herein as "glycan." Glycans may be homopolymers or heteropolymers of monosaccharide residues, and may be linear or branched. The terms "N-glycan" or, alternatively, "N-linked glycan" refer to glycans linked to the nitrogen atom of the asparagine (Asn) side chain. Such N-glycans are linked to eukaryotic proteins and play important roles in the structure and function of those proteins. Typical N-glycans can be selected from a list including N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, or other monosaccharides.

[0054] Another aspect of the present invention is a recombinant ASP or fragment thereof N-glycan comprising at least N-acetylglucosamine (GlcNAc), mannose (Man), and fucose (Fuc). In a broader sense, the present invention relates to formula: N-acetylglucosamine (X) and mannose Abbreviation: GlcNAc(X)Man The present invention provides an ASP or fragment thereof containing a carbohydrate compound, particularly an N-glycan, (wherein X is α1,3-fucose or α1,6-fucose) (wherein X is α1,3-fucose or α1,6-fucose).

[0055] More specifically, the carbohydrate compound comprises at least two GlcNAc residues and at least two, particularly at least three, Man residues. In a further embodiment, the carbohydrate compound further comprises galactose.

[0056] In specific embodiments, the ASP or fragment has two hybrid N-glycan structures comprising a composite 1,3-arm and an untreated 1,6-arm. The 1,3-arm is composed of an N-acetylglucosamine residue and a terminal galactose residue. Furthermore, the innermost N-acetylglucosamine residue of the N-bonded glycan has either an α1,6-fucose residue or an α1,3-fucose residue. The N-glycan according to the present invention more preferably comprises fucose, GlcNAc, Man, and Gal in the structure of structure (I) (Figure 17A).

[0057] An example of the recombinant ASP of the present invention is shown in Figure 17B.

[0058] In a further specific embodiment, the recombinant ASP of the present invention comprises or consists of an N-glycan structure as shown in Figure 17B, the N-glycan structure being structure (I) + Gal-GlcNAc (position X or Y) and core α1,6 fucose, or structure (I) + Gal-GlcNAc + GlcNAc (position X or Y) and core α1,6 fucose, or structure (I) + Gal-GlcNAc + Gal-GlcNAc (positions X and Y) and core α1,6 fucose, or structure (I) + Gal-GlcNAc (position X or Y) and core α1,3 fucose, or structure (I) + Gal-GlcNAc + Gal-GlcNAc (positions X and Y) and core α1,3 fucose.

[0059] In the case of recombinant Oo-ASP-1 (SEQ ID NO: 1), glycans are present in Asn9 and Asn37, which are identical to those found in endogenous Oo-ASP.

[0060] In the case of recombinant Co-dd-ASP (SEQ ID NOs. 2-4), the glycans are located at Asn83, Asn277, and Asn340.

[0061] In the case of recombinant Tc-ASP (SEQ ID NO: 5), the glycan is located at Asn39.

[0062] The chemical structure of the glycan related to the ASP antigen according to the present invention conforms to the general nomenclature in carbohydrate biochemistry.

[0063] Fucosylation is a type of glycosylation and can be defined as a process of adding fucose sugar units to a molecule. Therefore, an expression system having fucosylation ability can provide the recombinant ASP or fragment of the present invention. The enzyme that can perform such a fucosylation process is a fucosyltransferase. These enzymes may be endogenously present in the expression host or can be co-expressed with the ASP protein. In embodiments of the present invention, the recombinant ASP or fragment of the present invention is produced by synthesis or obtained from an expression system comprising a specific fucosyltransferase that transfers either α1,6-fucose or α1,3-fucose, particularly core α1,3-fucose, to the core of an N-glycan. In specific embodiments, the fucosyltransferase is a core-modifying fucosyltransferase. These can be fucosyltransferase 8 derived from mouse or fruit fly in the case of core α-1,6-fucose, or fucosyltransferase C derived from Schistosoma mansoni, or plant endogenous fucosyltransferase 11 or plant endogenous fucosyltransferase 12 in the case of core α-1,3-fucose.

[0064] According to further embodiments of the present invention, the ASP protein is produced by the expression of polynucleotides as described herein. Suitable vectors for protein expression include plasmids, bacteriophages, cosmids, viruses, minichromosomes, or stably integrated vectors. Generally, these vectors have autonomous replication properties, except for stably integrated vectors which are inserted into the genetic material of a host cell and replicate together with the host's genetic material. Host cells suitable for protein expression are not limited to, but may include bacteria such as Escherichia coli, yeasts such as Saccharomyces cerevisiae and Pichia pastris, mycoplasmas, algae, plant cells such as Arabidopsis thaliana and Nicotiana species (such as Nicotiana bentamiana or Nicotiana tabacum), vertebrate cells, or baculovirus / insect cells, and may be either prokaryotes or eukaryotes. Plant cells or animal cells may be cultured in vitro or may form part of an intact plant or animal.

[0065] According to certain embodiments, the (host) cells of the present invention are glyco-engineered cells. “Glyco-engineered cells” refer to cells that have been genetically modified to express proteins with altered N-glycan structures compared to unengineered cells or expression systems. Typically, this involves the use of specific enzymes involved in the glycosylation pathway. Generally, glycans in N-linked glycosylation can be classified into three types: high-mannose glycosylation (typically yeast), complex glycosylation (typically mammals), and hybrid glycosylation. All different types of N-glycosylation are well known to those skilled in the art and are defined in the literature. Significant efforts have been made to identify and optimize strategies for engineering eukaryotic cells that have a desired N-glycosylation pattern and / or O-glycosylation pattern and produce glycoproteins known in the art (van der Kaaij et al., 2022, Ma et al. 2020 (partially incorporated herein by reference)). Enzymes required for complex glycosylation include, but are not limited to, N-acetylglucosaminyltransferase I, N-acetylglucosaminyltransferase II, mannosidase II, galactosyltransferase, fucosyltransferase, and sialyltransferase, as well as enzymes involved in the synthesis or transport of donor sugar nucleotides. Further glycoengineered cells envisioned herein, particularly yeast cells, are characterized by the absence of expression of at least one enzyme involved in the production of high-mannose structures (high-mannose glycans). The enzyme involved in the production of high-mannose structures is typically mannosyltransferase. In particular, α-1,3-mannosyltransferases and β-1,2-mannosyltransferases of the α-1,6-mannosyltransferase Och1p, Alg3p, and Mnn1p families may not be expressed. Therefore, cells can be additionally or alternatively engineered to express one or more enzymes or enzymatic activities that enable the production of the specific N-glycan structures of the present invention in high yield.Such enzymes can be targeted to host intracellular organelles in which the enzyme has optimal activity, for example, by using signal peptides that are not typically associated with the enzyme. It will be apparent that the enzymes and their activities described herein are well known in the art.

[0066] Recombinant polynucleotides may include, as inserts, complete polynucleotides encoding ASP or fragments thereof. Expression systems in bacterial, yeast, fungal, insect, plant, and vertebrate cells (e.g., CHO cells) are very frequently used systems. Such systems are well known in the art and are generally available.

[0067] In particular, the present invention relates to recombinant ASP obtained from an expression system containing a fucosyltransferase, such as a Nicotiana genus expression system (e.g., Nicotiana bentamiana or Nicotiana tabacum), a Pichia pastris expression system, or an insect cell line expression system.

[0068] In a further embodiment, the present invention provides a method for producing recombinant ASP or a fragment thereof, the method comprising the steps of introducing an expression vector containing a nucleotide sequence provided herein into a suitable expression host, and expressing and isolating the protein or fragment of the present invention. The term “suitable cell” refers to higher eukaryotic cells such as mammalian cells or plant cells, or lower eukaryotic cells such as filamentous fungal cells or yeast cells, as described above, and the cells may optionally be glycoengineered.

[0069] In particular, this specification envisions the production of recombinant ASPs or fragments provided herein, wherein the proteins or fragments are glycosylated and contain one or more glycans, in particular N-glycans.

[0070] For example, as presented herein, an ASP or fragment comprising an N-glycosylated mixture of GlcNAc, Gal, and lycans containing α1,3-fucose and / or α1,6-fucose can typically be obtained by expression in glycoengineered cells of higher or lower eukaryotes known to those skilled in the art.

[0071] In another embodiment, the present invention relates to a method for producing a recombinant ASP or fragment of the present invention, the method being a) A step of preparing an expression system containing fucosyltransferase, more particularly core-modified fucosyltransferase, b) A step of introducing fucose sugar, particularly core α1,3-fucose and / or core α1,6-fucose, more particularly core α1,3-fucose, into the expression system described above. c) A step of expressing ASP using the expression system prepared in step a) to obtain the recombinant ASP of the present invention, Includes.

[0072] In this method, the expression system containing fucosyltransferase may be a Nicotiana bentamiana expression system, a Pichia pastris expression system, or an insect cell line expression system. Fucosyltransferase is an enzyme that transfers L-fucose sugar from a GDP-fucose (guanosine diphosphate-fucose) donor substrate to an acceptor substrate. The acceptor substrate may be another sugar that transfers fucose to a core GlcNAc (N-acetylglucosamine) sugar, as in the case of N-linked glycosylation, or to a protein, as in the case of O-linked glycosylation produced by O-fucosyltransferase. In certain embodiments, the present invention includes recombinant ASP or fragments obtained by the above method. Furthermore, recombinant ASP of the present invention obtained by this method can be included in pharmaceutical compositions. The recombinant ASP of the present invention, and / or pharmaceutical compositions comprising the recombinant ASP of the present invention, can be used as pharmaceuticals, preferably as vaccines, for treating, preventing, or alleviating infections of the parasitic nematodes presented herein, particularly Ostertagia ostertagii, Cooperia oncophora, and / or Terradorsagia kirkumcincta.

[0073] In further embodiments, the present invention relates to a pharmaceutical composition comprising a recombinant ASP or fragment thereof according to the present invention, and a pharmaceutically acceptable carrier and / or additive. In certain embodiments, the pharmaceutical composition comprises a recombinant ASP or fragment thereof comprising an N-glycan containing (core) α1,3-fucose or (core) α1,6-fucose, and a pharmaceutically acceptable carrier and / or additive. In further embodiments, the pharmaceutical composition comprises a recombinant ASP or fragment thereof comprising an N-glycan containing (core) α1,3-fucose, a recombinant ASP or fragment thereof comprising an N-glycan containing (core) α1,6-fucose, and a pharmaceutically acceptable carrier and / or additive. In further specific embodiments, the composition comprises one or more ASPs or fragments of the present invention (structure I), and further comprises additional glycans present at (outer) mannose residues (positions X and / or Y of structure I), each of which is independently constructed of or comprises residues (may be more than one) of mannose, galactose, N-acetylgalactosamine, N-acetylglucosamine and / or fucose. More specifically, the N-glycan includes structures according to structure (I) (including Ia and Ib) presented herein. In the above structures, X and / or Y may be absent, or if present, independently selected from mannose, galactose, N-acetylglucosamine, and fucose (including combinations thereof), and in particular X and / or Y are selected from N-acetylglucosamine and galactose. In one embodiment, X is absent. In another embodiment, Y is absent. In a further embodiment, X comprises GlcNAc and Gal, and Y is absent, or alternatively, Y comprises GlcNAc and Gal, and X is absent. In yet another embodiment, X and Y comprise GlcNAc and Gal.

[0074] In specific embodiments, the composition includes one or more of the ASPs shown in Figure 17B, particularly two, three, four, five, six, seven, eight, nine, or all combinations thereof.

[0075] The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable additive" refer to solvents, dispersions, emulsifiers, disintegrants, isotonic agents, absorption retarders, etc., suitable for pharmacopoeia administration. Effective amounts of preservatives may also be included in the formulation. Suitable preservatives include, but are not limited to, benzalkonium chloride (0.003% w / v to 0.03% w / v), chlorobutanol (0.3% w / v to 0.9% w / v), parabens (0.01% w / v to 0.25% w / v), and thimerosal (0.004% w / v to 0.02% w / v). The use of such carriers and agents for pharmaceutically active substances, and the determination of effective amounts, are known in the art.

[0076] For these purposes, the pharmaceutical compositions of the present invention can be formulated by means known in the art, for example, in the form of tablets, pellets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, fine powders or aerosols or nebulizers for inhalation, and sterile aqueous or oily solutions or suspensions for parenteral use (including intravenous, intramuscular or injectable), or sterile emulsions.

[0077] In carrying out this method, the vaccine or composition of the present invention is preferably administered via an intramuscular or subcutaneous route, but other routes of administration such as oral, intranasal (e.g., aerosol or other needle-free administration), intra-lymph node, intradermal, intraperitoneal, rectal or vaginal administration, or a combination of routes may also be used. Formulations of the composition or vaccine can be prepared in various forms depending on the route of administration. For example, the composition may be prepared in the form of a sterile aqueous solution or dispersion suitable for injection, or in a lyophilized form using lyophilization technology. The lyophilized immunogenic composition is usually maintained at about 4°C and can be reconstituted in a stabilizing solution, such as physiological saline and / or HEPES, with or without adjuvants.

[0078] Additional immunotherapy may be required, and the medication plan can be adjusted to provide optimal immunity. The immunization protocol can be optimized using procedures known in the art. Animals may be administered a single dose, or alternatively, two, three, or more doses may be administered at intervals of 2 to 10 weeks. Depending on the age of the animals, the immunogenic composition or vaccine composition may be re-administered. For example, the present invention considers vaccinating healthy calves (3 to 12 months of age) 6 weeks and / or 3 weeks before the first grazing season, and re-vaccinating at the start of the first grazing season.

[0079] ASP or fragments thereof of the present invention, for use in the treatment, prevention and / or mitigation of nematode infections, particularly those caused by Ostertagia ostertagii, Cooperia oncophora and / or Terradorsagia kirkumkinkuta, are preferably administered in a therapeutically effective dose. The term “therapeutally effective dose” refers to an amount sufficient to induce an immune response and / or confer a protective effect in the animal to which it is administered. The immune response may include, but is not limited to, the induction of cellular immunity and / or humoral immunity. The amount of therapeutically effective vaccine may vary depending on the condition of the animal (e.g., ruminants or cattle) and / or the degree of infection, and may be determined by a veterinarian. As used herein, “protection” (and related terms such as “immunoprotection” and “immunoprotective”) means inducing an immune response to help prevent, improve, reduce susceptibility to, or treat a disease or disorder resulting from infection with a parasitic nematode. The term “mitigation” relates to reducing the incidence of nematode infection. In the context of the present invention and as demonstrated herein, this means a reduction in the number and / or size of adult and / or larval parasites, the number of eggs, or the intensity of clinical signs caused by parasitic nematode infection in the target subject. This may result from reduced parasitic colonization or a decrease in the parasitic infection rate, leading to a reduction in the number or severity of lesions, shedding, and effects caused by the parasite or the subject's response to the parasite.

[0080] Since the recombinant ASP of the present invention can induce an immune response against parasitic nematodes such as Ostertagia ostertagii, Cooperia oncophora, and / or Theradorsagia kilchumcincta, the pharmaceutical composition of the present invention, comprising this recombinant ASP, can be used as a vaccine against these nematodes, particularly against nematodes of the genera Ostertagia, Cooperia, and Theradorsagia, more specifically against Ostertagia ostertagii, Cooperia oncophora, and / or Theradorsagia kilchumcincta. In a preferred embodiment, the pharmaceutical composition of the present invention is therefore a vaccine.

[0081] To provide such immunity against the above-mentioned nematodes, particularly Ostertagia ostertagii, Cooperia oncophora, and / or Terradorsagia kirkum cinctula, the vaccine may optionally further contain an adjuvant. Therefore, in a further embodiment of the present invention, the pharmaceutical composition of the present invention comprises an adjuvant.

[0082] Adjuvants are known to act in many different ways to enhance the immune response. Generally, immunomodulatory adjuvants induce the general upregulation of certain cytokines and the simultaneous downregulation of other cytokines, resulting in cellular Th1 and / or humoral Th2 responses.

[0083] Suitable adjuvants include, but are not limited to, oil emulsions (water-in-oil, oil-in-water, water-in-oil-in-water, etc.), complete Freund's adjuvants, saponins such as QuilA, ISCOM (a complex of saponin, sterol, and phospholipid), aluminum compounds including aluminum phosphate and aluminum hydroxide, mycobacterial cell wall extracts, quaternary ammonium compounds such as MPL-A and dimethyldioctadecylammonium bromide (DDA), acrylic acid polymers such as carbomers containing CARBOPOL™, glycolipids such as BAY™ R1005, and oligonucleotides containing CpG motifs. Combinations of these compounds are also conceivable. The compositions described herein are preferably immunogenic compositions. "Immunogenicity" means the ability to induce an immune response to a pathogen / parasite in a subject. Accordingly, the present invention provides compositions used to induce an immune response that can be used as a vaccine against nematodes belonging to the genera Ostertagia and / or Couperia and / or Theradorsagia, more specifically against the nematodes Ostertagia ostertagii, Ostertagia leptospicularis, Couperia oncophora, Couperia punctate, Couperia pectinate, Theradorsagia triflucata, and / or Theradorsagia kilchumcincta, and even more specifically against the nematodes Ostertagia ostertagii, Couperia oncophora, and / or Theradorsagia kilchumcincta. The immune response may be a cellular immune response mediated mainly by NK cells and cytotoxic T cells, and / or a humoral immune response mediated mainly by helper T cells, which then activate B cells to produce antibodies. More specifically, "to induce or trigger an immune response" means that the antigen stimulates the synthesis and / or cellular proliferation of a particular IgG1 antibody, for example, by 3H thymidine uptake by NK cells, T cells, and B cells.

[0084] In one embodiment, administration of the composition or vaccine of the present invention induces an immune response that reduces the mean cumulative number of fecal eggs in animals by at least about 30%, 40%, 45%, or 50% compared to unvaccinated (e.g., adjuvant only) control animals. Preferably, the reduction is about 55%, more preferably about 60%, and most preferably about 70% or more. In specific embodiments, the reduction in the mean cumulative number of fecal eggs lasts for at least 4 weeks, particularly at least 5, 6, 7, or 8 weeks, after the first administration of recombinant ASP or fragment. Thus, the immune response provides the subject with some beneficial protective effect against subsequent exposure to the infectious agent. More preferably, the immune response prevents or improves the onset of at least one symptom of a disease associated with the infectious agent, or reduces the severity of at least one symptom of a disease associated with the infectious agent upon subsequent exposure.

[0085] In consideration of the beneficial medical properties of the compositions of the present invention, according to another embodiment, the present invention relates to recombinant ASP or pharmaceutical compositions of the present invention for use as pharmaceuticals for humans or veterinary use.

[0086] The immune response induced by the recombinant ASP or fragments or pharmaceutical compositions of the present invention can treat, prevent or mitigate parasitic nematode infections, particularly those of parasitic nematodes belonging to the genera Ostertagia and / or Cooperia and / or Theradorsagia, more specifically Ostertagia ostertagii, Ostertagia leptospicularis, Cooperia oncophora, Cooperia punctate, Cooperia pectinate, Theradorsagia triflucata, and / or Theradorsagia kirchumcincta, and even more particularly Ostertagia ostertagii, Cooperia oncophora, and / or Theradorsagia kirchumcincta. Therefore, a further aspect of the present invention is that the recombinant ASP or fragments or pharmaceutical compositions of the present invention can be used for the treatment, prevention and / or mitigation of parasitic nematode infections in subjects, particularly mammals.

[0087] "Subject" or "host" means any mammal / animal that is infected with or susceptible to nematode infections, particularly infections caused by nematodes belonging to the genera Ostertagia and / or Cooperia and / or Theradorsagia, more specifically infections caused by Ostertagia ostertagii, Ostertagia leptospicularis, Cooperia oncophora, Cooperia punctate, Cooperia pectinate, Theradorsagia trifulcata, and / or Theradorsagia kirchumcinct, and more specifically any mammal / animal susceptible to infections caused by Ostertagia ostertagii, Cooperia oncophora, and / or Theradorsagia kirchumcinct, such as humans or non-human animals, especially ruminants, more specifically cattle. The term "ruminant" includes many domesticated animals, or other animals of agricultural, veterinary, or economic importance (e.g., herds of livestock), such as sheep, goats, cattle, bison, yaks, buffalo, deer, camels, llamas, alpacas, and various wild animals. "Small ruminants" is understood to include sheep, goats, and deer. The term "cattle" is not limited to but refers to animals of the Bovidae family, including castrated bulls, bulls, cows, and calves.

[0088] In preferred embodiments, the recombinant ASP or fragments or pharmaceutical compositions of the present invention can be used for the treatment, prevention and / or reduction of nematode infections, particularly infections caused by nematodes belonging to the genera Ostertagia and / or Cooperia and / or Theradorsagia, more specifically infections caused by Ostertagia ostertagii, Ostertagia leptospicularis, Cooperia oncophora, Cooperia punctate, Cooperia pectinate, Theradorsagia triflucata, and / or Theradorsagia kirchumcinct, and even more particularly infections caused by Ostertagia ostertagii, Cooperia oncophora, or Theradorsagia kirchumcinct.

[0089] In further embodiments, the present invention relates to a method for treating, preventing and / or reducing parasitic nematode infections in subjects requiring treatment, prevention and / or reduction of such infections, the method comprising administering a recombinant ASP or fragment or pharmaceutical composition of the present invention to such subjects requiring treatment, prevention and / or reduction of such infections.

[0090] The present invention will be explained below with reference to the following examples, but these examples do not limit the scope of the present invention in any way. [Examples]

[0091] Example 1 As a first step in investigating the reasons (and possibly multiple reasons) why Pichia-produced Oo-ASP-1 fails to induce an adequate immune response and protection after vaccination of calves (Non-Patent Literature 1), we examined the amino acid sequences (i.e., primary structures) of both the endogenous and recombinant molecules. The structural characteristics of two forms of Pichia-produced recombinant Oo-ASP-1 and its endogenous counterpart (purified from O. ostertagii excretions / secretions) were investigated by gel filtration (GF-Oo-ASP-1) and also by the thiol-sepharose protocol (TS-Oo-ASP-1).

[0092] Regarding the primary structure of endogenous Oo-ASP-1, nine hotspots exhibiting significant amino acid substitution frequencies were identified, but these were found not to be part of the predicted B cell epitopes. Antibody binding experiments confirmed that these substitutions do not play a significant role in antibody recognition. This was confirmed after fabricating a recombinant Oo-ASP-1 version (referred to as Oo-ASP-1-sub) containing all nine of the above substitutions (i.e., G1D, K16Q, Q24E, A65K, A69E, N100T, D121N, D179Q, and M185E) and incorporating it into an inhibitory ELISA mechanism.

[0093] Following the analysis of its effects on sequence analysis and antibody recognition, we attempted to investigate the possibility that any abnormal secondary structural features were introduced during the production of recombinant Oo-ASP-1. Therefore, circular dichroism (CD) experiments were performed on endogenous TS-Oo-ASP-1 and recombinant Oo-ASP-1. In addition, the LC-MS approach was applied to both ASP versions.

[0094] While the content of α-helices and β-sheets, as well as dimerization ability, were demonstrated to be identical between endogenous Oo-ASP-1 and recombinant Oo-ASP-1, it was found that recombinant ASP possessed all the disulfide bonds characteristic of CAP proteins, whereas endogenous ASP did not. This was thought to be due to the thiol-sepharose-based purification protocol, so endogenous ASP purified by gel filtration was included in this study, and it showed the same structural characteristics as the recombinant version. Since the disulfide bond pattern of recombinant Oo-ASP is almost identical to that of its endogenous counterpart, the root cause of the lack of protection must be sought in other tertiary and / or quaternary structural characteristics such as N-glycosylation.

[0095] Example 2 Acquisition of endogenous ASP and recombinant ASP As described in the previous study by Geldhof et al. (2000), ES material was obtained from adult O. ostertagii worms collected from the abomasum of infected calves. To purify Oo-ASP, the ES fraction was applied to a Superdex 200 16 / 70 column (GE Healthcare Bio-Sciences AB (Uppsala, Sweden)) at a flow rate of 1 ml / min and gel filtration chromatography was performed (Borloo et al., 2013a). A recombinant version of Oo-ASP was expressed in P. pastris and purified as described in Borloo et al. (2013b).

[0096] Peptide microarrays To gain insights into protein recognition by protective IgG antibodies, peptide microarrays were created using PepscanPresto BV. An array of linear peptide epitopes was generated on a solid support called a "minicard" by computer-based splitting of the Oo-ASP amino acid sequence into duplicate fragments. Furthermore, structural epitopes were constructed using CLIPS (Chemically Linked Peptides on Scaffolds) technology (Timmerman et al., 2007), and also as duplicate fragments. This allows for the reproduction of secondary structural elements such as loops, α-helices, and β-strands. Constructs forming incomplete parts of the antibody epitope may be recognized by the antibody, albeit with low affinity, while constructs not part of this epitope are not recognized. An overlapping battery of ASP peptides was synthesized using solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) synthesis, and amino-functionalized polypropylene supports were obtained by grafting with a proprietary hydrophilic polymer formulation. Next, dicyclohexylcarbodiimide (DCC) was reacted with N-hydroxybenzotriazole (HOBt) to t-butyloxycarbonylhexamethylenediamine (BocHMDA), and the Boc group was subsequently cleaved by the addition of trifluoroacetic acid (TFA). The peptide was synthesized on an amino-functionalized solid support using standard Fmoc peptide synthesis methods.

[0097] The binding of serum antibodies to each peptide was evaluated by pepscan-based ELISA. This was performed using serum samples from bovine animals vaccinated with either native Oo-ASP (n=5), P. pastris Oo-ASP (n=5), or QuilA adjuvant alone as a control (n=5). These samples were collected one week after the third immunization. To prevent variability in antibody binding, all pre-coat conditions (antibody concentration and relative amount of competing protein in ELISA buffer) were equal across the various serum samples. First, the array card and peptide array were incubated overnight at 4°C with serum antibodies in PBS containing 5% (v / v) horse serum, 5% (w / v) ovalbumin, and 1% (v / v) Tween 80. After washing with PBS / Tween 80, the array was incubated with HRP-labeled goat anti-bovine IgG (1 / 1000 dilution) at 25°C for 1 hour. After further washing, 2,2'-azino-di-3-ethylbenzthiazoline sulfonic acid (ABTS) and 20 μl / ml of 3% H2O2 were added to the array. One hour after incubation, the color development was measured and quantified using a charge-coupled device (CCD) camera and image processing system, as described by Slootstra et al. (1996).

[0098] The raw data consisted of optical values ​​obtained by a CCD camera, which was used to capture images of the cards before and after coloring with peroxidase. The value for each specific peptide was then obtained by calculating the difference between the two images. Values ​​for wells containing air bubbles were evaluated as zero, as this could lead to false positives. To perform quality validation of the synthetic peptides, separate sets of positive and negative control peptides were synthesized in parallel. These peptides were screened using commercial antibodies 3C9 and 57.9 (Posthumuse et al., 1990).

[0099] Further data analysis was performed by creating box plots, linear intensity profiles, and heatmaps. The TukeyHSD test was performed on the raw dataset to classify specific peptides as potential defense sites. Potential defense sites were selected only if peptide recognition by serum from the native vaccine group was significantly higher than that of both the P. pastris vaccine group and the negative control group.

[0100] Indirect ELISA was used to re-evaluate the recognition of potential defense sites and classified peptides using pooled and individual serum samples from animals vaccinated with either native Oo-ASP, P. pastris Oo-ASP, or QuilA adjuvant. To maintain linearity of the test results, serum samples for ELISA were collected from the same animals used for peptide microarray analysis. 96-well ELISA plates (MaxiSorp, NUNC) were coated with a 0.25 μg / ml peptide solution in 100 μl coating buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6) at 4°C for 20 hours. Subsequently, the plates were blocked at room temperature for 1 hour with 200 μl blocking buffer (2% BSA in 150 mM PBS / Tween 20), during which washing was performed with 300 μl washing buffer (150 mM PBS / Tween 20). Next, the plates were incubated with pooled bovine serum samples or individual bovine serum samples (diluted 1 / 200 in PBS) at room temperature for 1 hour. After further washing, the plates were incubated with HRP-labeled sheep anti-bovine IgG (diluted 1 / 1000 in blocking buffer) at room temperature for 1 hour. After the final washing, ABTS was added to the plates and incubated at room temperature for 10 minutes. The color development due to oxidation of ABTS appeared as OD405-492 and was quantified using an Infinite F50 absorbance microplate reader (Tecan Trading AG, Mönnendorf, Switzerland).

[0101] In addition, competitive inhibition ELISA was performed to evaluate whether the peptide could inhibit the binding of native Oo-ASP to native Oo-ASP by serum antibodies derived from animals vaccinated with native Oo-ASP. For this purpose, 96-well ELISA plates were coated with 1 μg / ml Oo-ASP in 100 μl of coating buffer at 4°C for 20 hours. Subsequently, the plates were blocked at room temperature for 1 hour with 150 μl of blocking buffer, during which washing was performed with 200 μl of washing buffer. Simultaneously, serum from vaccinated animals was pre-incubated at room temperature for 1 hour with native Oo-ASP in a concentration range of 0 pmol / ml to 500 pmol / ml, or with either of the peptides, before being transferred to the coated ELISA plates and incubated for another hour. Finally, HRP-labeled sheep anti-bovine IgG1 (diluted 1 / 1000 in blocking buffer) was added to the plates, incubated for 1 hour, and then ABTS was added. OD405-492 was determined using an Infinite F50 absorbance microplate reader.

[0102] Glycan microarrays Prior to glycan microarray analysis, ASP-specific antibodies were isolated to prevent false positives and high background fluorescence caused by glycan recognition by non-ASP-specific antibodies. Bovine serum samples were collected one week after the third vaccination from animals vaccinated with either endogenous Oo-ASP or P. pastris Oo-ASP. In addition, serum samples from bovines vaccinated with QuilA adjuvant alone were collected to serve as a negative control in the in vitro test described later. To obtain ASP-specific antibodies, lipoproteins and lipids that could potentially clog the purification column were first removed from the serum. This was done by adding 10% dextran sulfate and 1 M calcium chloride solution, followed by centrifugation at 10000 × g. Due to the small sample volume, IgG antibodies were first purified using a Protein G HP SpinTrap column (GE Life Sciences) according to the manufacturer's protocol. Subsequently, IgG was placed on an NHS HP SpinTrap column (GE Life Sciences) coated with endogenous Oo-ASP antigen, also according to the manufacturer's protocol. The buffers were always adjusted to meet the requirements of both SpinTrap columns. Therefore, only IgG binding to the endogenous antigen was purified from the various serum samples. In other words, antibodies induced by P. pastris Oo-ASP, which did not show any level of cross-reactivity to the endogenous counterpart, were not captured. After purification, all samples were repeatedly dialyzed on a Slide-A-Lyzer MINI dialysis machine (ThermoFischer) to obtain PBS buffer solution. Subsequently, all samples were concentrated using an Amicon Ultra-15 centrifugal filter unit (Merck Millipore), and antibody activity was evaluated by Western blotting (data not shown).

[0103] After thawing the synthetic glycan microarray slides at room temperature, they were covered with a silicone gasket, and wells were formed on each of the synthetically printed N-glycan arrays. First, the arrays were incubated with 300 μl of ASP-specific antibody (1 / 500 dilution in PBS-0.01% Tween20 and 1% BSA) derived from animals vaccinated with either native Oo-ASP or P. pastris Oo-ASP. Serum from animals vaccinated with QuilA (similarly purified for ASP specificity) was used as a negative control. In addition, one well was used as a technical negative control and was left without antibody. After incubation at room temperature for 1 hour with shaking, the slides were washed with PBS-0.05% Tween20 and PBS. To incubate with secondary antibody, the wells were filled with a solution containing Cy3-labeled anti-bovine IgG (1 / 1000 dilution in PBS-0.01% Tween20 and 1% BSA) and stored in the dark at room temperature for 30 minutes with shaking. Finally, the slides were sequentially washed with PBS-0.05% Tween 20, PBS, and deionized water, dried, and stored in the dark until the next use. Subsequently, the binding of N-glycans by ASP-specific antibodies was measured using a G2565BA scanner (Agilent Technologies, California, USA) equipped with a 532 nm laser. Fluorescently labeled bovine serum albumin (BSA) was incorporated as an array printing control, always constituting the first four spots on each glycan microarray.

[0104] Data from the G2565BA scanner was analyzed using GenePixPro 7.0 software (Molecular Devices, California, USA) by implementing a spot detection algorithm used for aligning and resizing fluorescence spots in microarray images. The median fluorescence intensity (MFI) for each of the four spots was then obtained and exported to Microsoft Excel software. The background MFI was then subtracted for each spot. For further analysis, four technical iterations were exported to GraphPad Prism (version 6.0c, Faye Avenue, La Jolla, California, USA) for statistical analysis and data visualization. Since each array contained 135 structures in quadruple nesting, a p-value of 0.001 was adopted to minimize the possibility of false rejection of H0.

[0105] result 1. Peptide microarrays Analysis using PEPSCAN Presto BV showed no significant differences in peptide recognition among the three different vaccine groups. One of five serum samples from bovine vaccined with native Oo-ASP was able to recognize a peptide that was not recognized in the P. pastris Oo-ASP and control vaccine groups. In addition, TukeyHSD analysis was performed on the raw dataset, and only samples showing p<0.05 in both the native vs. recombinant group and the native vs. control group were selected as potential protective sites. Based on this analysis, four peptides (underlined in Figure 1) were selected for further evaluation by immunological in vitro assays.

[0106] 2. ELISA Indirect ELISA using four selected peptides demonstrated that PUS028 029, one of the four peptides, was strongly recognized in both pooled and individual serum samples from animals vaccinated with native Oo-ASP, while this recognition was substantially lower in the P. pastris Oo-ASP and QuilA control vaccine group (Figure 2).

[0107] Inhibitory ELISA demonstrated that none of the selected peptides could inhibit the binding of the native Oo-ASP-inducing antibody to the native Oo-ASP coated on the ELISA plate. Furthermore, a mixture of the four peptides showed no inhibitory activity whatsoever.

[0108] 3. Glycan microarrays The results of the glycan microarray projected in Figures 3A and 3B demonstrate that native Oo-ASP-specific IgG recognizes a diverse group of N-glycans. With few exceptions, all recognized N-glycans contained core α1,3-fucose, and in some cases were combined with N-glycans containing α1,6-fucose. No core α1,3-fucose-containing N-glycans were not recognized by these antigen-specific antibodies. N-glycans on this array were largely unrecognized by P. pastris-induced native Oo-ASP-specific IgG, with the exception of the trimannoside structure G99 (data not shown).

[0109] Example 3 Recombinant expression of fucosylated ASP antigen Expression of recombinant ASP antigen in Nicotiana bentamiana is achieved by infiltration of Agrobacterium tumefaciens, a natural plant pathogen capable of introducing DNA fragments into the plant genome. An engineered Agrobacterium clone (MOG101 strain) harbors a pHYG expression plasmid containing a uniquely codon-optimized gene encoding the mature ASP antigen. Expression from these plasmids is promoted by a dual 35S promoter, and co-infiltration with a viral silencing suppressor further enhances protein yield. To perform glycoengineering of recombinant ASP antigen, Agrobacterium clones containing constructs that promote the expression of core-modified fucosyltransferase can be added to the infiltration mix. The fucosyltransferases used to successfully modify the N-glycan core were mouse or fruit fly-derived fucosyltransferase 8 for core α-1,6-fucose, and manson's schistosomiasis-derived fucosyltransferase C, or plant endogenous fucosyltransferase 11 or plant endogenous fucosyltransferase 12 for core α-1,3-fucose.

[0110] Typically, multiple Agrobacterium clones are infiltrated into the leaves of 5-6 week old ΔXT / FT plants (RNAi knockdown plants lacking endogenous core α-1,3-fucose and β-1,2-xylose), and transient protein production and simultaneous glycoengineering of ASP antigens are promoted over 5-6 days. At this stage, the infiltrated leaves are harvested, and vacuum infiltration is performed using extraction buffer (50 mM sodium acetate buffer, pH 4.4) to isolate recombinant ASP antigens from the extracellular space (apoplast). Subsequently, the recombinant ASP antigens are purified from the collected apoplast solution using HS Poros 50 cation exchange chromatography (pH 4.4).

[0111] Competitive ELISA was performed to evaluate the ability of core α1,6 fucosylated recombinant N. bentamiana ASP antigen and core α1,3 fucosylated recombinant N. bentamiana ASP antigen to inhibit the binding of anti-ASP antibodies to native ASP. For this purpose, wells of an ELISA-maxisorp plate were coated with 100 μl of 0.5 μg / ml native Oo-ASP in 0.05 M carbonate coating buffer (pH 9.6) and incubated overnight at 4°C. Three 150 mM PBST solutions were added between each step. 0.05 After washing, rinse all wells with 150 mM PBST. 0.05 The mixture was blocked with 200 μl of 2% bovine serum albumin for 1 hour at room temperature. During blocking, pooled serum from animals vaccinated with native Oo-ASP was pre-incubated in a separate low-binding 96-well plate with various recombinant antigens added at different concentrations ranging from 0 pmol / ml to 1000 pmol / ml. After blocking, the contents of the pre-incubation plate were added to an ELISA-maxisorp plate to allow native and recombinant antigens to compete for binding to the anti-ASP antibody. After further incubation at room temperature for 1 hour, 100 μl of goat anti-bovine IgG1-HRP was added at a 1 / 500 dilution and incubated at room temperature for 1 hour. Subsequently, ELISA development was performed using ABTS (Roche), and the optical density at 405 nm was measured for each well, corrected for the background optical density at 492 nm.

[0112] result The results in Figure 4 demonstrate that recombinant N. bentamiana ASP can compete with native Oo-ASP for anti-ASP antibody binding, while recombinant P. pastris ASP exhibits significantly lower competition. These results indicate higher antibody affinity for recombinant N. bentamiana compared to P. pastris. These findings can be explained by the presence of specific antibody epitopes present in both native Oo-ASP and recombinant N. bentamiana ASP but not in P. pastris. These high-affinity antibody epitopes likely involve (partially) N-glycosylation, which is the main difference between recombinant N. bentamiana and recombinant P. pastris, particularly with respect to core fucosylation. At low concentrations, a comparison of N. bentamiana recombinants containing either core α-1,3-fucose or core α-1,6-fucose shows that the recombinant containing core α-1,3-fucose is slightly more effective at inhibiting antibody binding to the native antigen than its core α-1,6-fucose counterpart. This may indicate the presence of some highly specific antibody epitopes shared with the native antigen.

[0113] Example 4 To evaluate recombinant N. bentamiana ASP in cattle, 24 female cows were randomly divided into three vaccine groups of eight each. The study included a group receiving rASP + QuilA, a native ASP + QuilA group, and a negative control group receiving QuilA only. Negative control animals received 750 μg of QuilA per vaccination, while the recombinant and native vaccine groups received 30 μg of each antigen in combination with 750 μg of QuilA. The recombinant vaccine groups were administered N-glycan containing either core α-1,3-fucose or core α-1,6-fucose as antigens in a 50 / 50 ratio. Prior to the start of the study, all animals were confirmed to be negative for recent O. ostertagii infection by fecal egg counting and ELISA. The cows received three immunizations at three-week intervals, and for 25 days starting from the third immunization, they were exposed to L3 trickle infection at a rate of 1,000 per day.

[0114] To evaluate both humoral and cellular responses, blood samples were collected one week after each immunization and at autopsy. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by Lymphoprep gradient centrifugation. In addition, mononuclear cells (MNCs) were collected from the abomasal lymph nodes, the inflow region, by Lymphoprep gradient centrifugation after mechanically disrupting the tissue through a 70 μm cell strainer. These cells were labeled with PKH26 and divided into 200 μl portions of RPMI complete medium, either alone or in medium containing 5 μg / ml nASP, at a dose of 2.5 × 10⁶ cells per 200 μl. 5 Cells were cultured for 5 days. To obtain the initial PKH26 intensity, small amounts of PKH26-labeled cells were used and stained with the following antibody mixes: CD3-IgG1, CD21-IgM, CD335-IgG2b, IgG1-V450, IgM-APC-Cy7, IgG2b-FITC, and a viability dye. After 5 days of culture, cells were harvested and stained with the aforementioned antibody mixes to obtain the final PKH26 intensity for each cell type. Subsequently, the proliferation index of each cell population was calculated using ModFit LT software. The stimulation index was obtained by dividing the number of nASP-stimulated cells by the number of culture-stimulated cells and essentially represents a multiplicative change. Enzyme-linked immunosorbent assay (ELISA) was performed to examine the systemic and local (abomasum) (cross-reactive) IgG1, IgG2, and IgA responses in the three vaccine groups. For this purpose, nASP was coated overnight in 1 μg / ml of carbonate buffer (pH 9.6) on a 96-well Maxisorp plate. The wells were then PBST 0.05 The 2% BSA inside blocks the washing process, and the PBST process is performed. 0.05The procedure was performed three times. After blocking, the wells were incubated with a 1 / 200 dilution of bovine serum sample for 1 hour. HRP-conjugated anti-bovine IgG1, IgG2, and IgA were added to the wells and incubated at room temperature for 1 hour, after which ELISA was developed by adding 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) substrate. Optical density was measured at 405 nm. Parasitological parameters were evaluated as previously described in Van Meulder et al. (2015) and Vlaminck et al. (2015). Briefly, fecal egg counting was performed three times a week from day 21 after initial infection until necropsy, using the McMaster method with a sensitivity of 25 eggs per gram of feces (EPG). Adult worms for counting and measuring body length were collected by washing the abomasum after necropsy. On the other hand, L4 was collected after digesting the abomasal mucosa with hydrochloric acid-pepsin.

[0115] result Proliferation of PBMC cells upon (re)exposure to nASP antigens was mainly observed in the nASP vaccine group, particularly in CD335+ NK cells (Figure 5). Such proliferation was not observed or was barely observed in QuilA-controlled and rASP-immunized cattle. Proliferation of MNC cells upon (re)exposure to the antigens was limited and unconfirmed (data not shown).

[0116] ELISA results evaluating systemic antibody responses primarily demonstrated an IgG1 response accompanied by (cross-reactive) antibodies recognizing nASP (Figure 6). In the two vaccine groups containing either nASP or rASP antigen, an IgG1 response was observed one week or more after the second immunization. A more limited IgG2 response was observed, following a similar pattern to that described for IgG1 (data not shown). Notably, clear local IgG1 and local IgG2 responses were observed in both the nASP and rASP groups (Figure 7). In contrast, such responses were either absent or negligible in the control animals. Furthermore, such responses were not observed with recombinant antigens previously produced in P. pastris.

[0117] Figure 8 shows that the number of fecal eggs was generally reduced in both the nASP and rASP vaccine groups compared to the QuilA control. Animals vaccinated with nASP or rASP still excreted O. ostertagii eggs, but egg excretion appeared to be delayed. Importantly, both antigens prevented animals from excreting large numbers of eggs, as demonstrated in Figure 9. Finally, the cumulative number of eggs was obtained by calculating the area under the curve (AUC) per animal (Figure 10 Panel A). Compared to the QuilA control, a significant 57% reduction was observed in animals immunized with nASP + QuilA, while a similar non-significant 45% reduction was observed in animals immunized with rASP + QuilA. Based on these results, a second vaccination trial using rASP was conducted. Compared to the QuilA control, animals immunized with rASP + QuilA showed a significant 39% reduction in the cumulative number of eggs (Figure 10 Panel B).

[0118] Regarding the number of parasites, in the initial trial, no clear differences were observed in the total number of parasites, total number of adults, or total number of L4s when comparing the QuilA control with the nASP and rASP vaccine groups (Figure 11). However, when comparing the ratio of total L4s to total parasites per vaccine group, an increase was observed in the nASP vaccine group compared to the other groups, but this was only true for 2 out of 7 animals. In Trial 1, no clear differences in parasite length were observed among the three vaccine groups (Figure 12).

[0119] Example 5 Acquisition of endogenous ASP and recombinant ASP Adult C. oncophora were collected, excretions / secretions were prepared, and the dd-ASP protein fraction was purified as previously described (Borloo et al., 2013). Recombinant Cooperia ASP was expressed in P. pastris and purified as described in Non-Patent Literature 2.

[0120] Glycan microarrays Prior to the use of the Cooperia Oncophora glycan microarray, ASP-specific antibodies were isolated to prevent false positives and high background fluorescence caused by glycan recognition by non-ASP-specific antibodies. Bovine serum samples were collected one week after the third vaccination from animals vaccinated with either endogenous Co-dd-ASP or P. pastris Co-dd-ASP. Serum samples from bovines vaccinated with QuilA adjuvant alone were collected to serve as negative controls in the in vitro tests described later. To obtain ASP-specific antibodies, lipoproteins and lipids that could potentially clog the purification column were first removed from the serum. This was done by adding 10% dextran sulfate and 1 M calcium chloride solution, followed by centrifugation at 10000 × g. Due to the small sample volume, immunoglobulins (Ig) were first purified using a Protein G HP SpinTrap column (GE Life Sciences) according to the manufacturer's protocol. Next, following the manufacturer's protocol, immunoglobulins were placed in NHS HP SpinTrap columns (GE Life Sciences) coated with endogenous Co-dd-ASP antigen. Buffers were always adjusted to the requirements of both SpinTrap columns. From the various serum samples, only antibodies binding to the endogenous antigen were purified. Therefore, antibodies induced by P. pastris Co-dd-ASP, which showed no level of cross-reactivity to the endogenous counterpart, were not captured. After purification, all samples were repeatedly dialyzed on a Slide-A-Lyzer MINI dialysis machine (ThermoFischer) to obtain PBS buffer solution. Subsequently, all samples were concentrated using an Amicon Ultra-15 centrifugal filter unit (Merck Millipore), and antibody activity was evaluated by Western blotting (data not shown).

[0121] Synthetic microarray slides (Figure 13) were thawed at room temperature, covered with a silicone gasket, and wells were formed on each array of synthetically printed N-glycans. First, the arrays were incubated with 300 μl of ASP-specific antibody (1 / 500 dilution in PBS-0.01% Tween20 and 1% BSA) derived from animals vaccinated with either native Co-dd-ASP or P. pastris Co-dd-ASP. Serum from animals vaccinated with QuilA (similarly purified for ASP specificity) was used as a negative control. In addition, one well was used as a technical negative control and was left without antibody. After incubation at room temperature for 1 hour with shaking, the slides were washed with PBS-0.05% Tween20 and PBS. To incubate with secondary antibodies, the wells were filled with a solution containing Cy3-labeled anti-bovine IgG (1 / 1000 dilution in PBS-0.01% Tween20 and 1% BSA) and stored in the dark at room temperature for 30 minutes with shaking. Finally, the slides were sequentially washed with PBS-0.05% Tween 20, PBS, and deionized water, dried, and stored in the dark until the next use. Subsequently, N-glycan binding by ASP-specific antibodies was measured using a G2565BA scanner (Agilent Technologies, California, USA) equipped with a 532 nm laser. Fluorescently labeled bovine serum albumin (BSA) was incorporated as an array printing control.

[0122] Data from the G2565BA scanner was analyzed using GenePix Pro 7.0 software (Molecular Devices, California, USA) by implementing a spot detection algorithm used for aligning and resizing fluorescence spots in microarray images. The median fluorescence intensity (MFI) for each of the four spots was then obtained and exported to Microsoft Excel software. The background MFI was then subtracted for each spot. For further analysis, four technical iterations were exported to GraphPad Prism (version 6.0c, La Jolla, California, USA) for statistical analysis and data visualization. Since each array contained 135 structures in quadruple nesting, a p-value of 0.001 was adopted to minimize the possibility of false rejection of H0.

[0123] result Results from the glycan microarray projected in Figure 14 demonstrate that native Co-dd-ASP-specific antibodies recognize a diverse group of N-glycans. With few exceptions, all recognized N-glycans contained either core α1,3-fucose, α1,6-fucose, or a combination thereof. No N-glycans containing core α1,3-fucose were not recognized by these antibodies. Recognition of N-glycans on this array by native ASP-specific antibodies derived from animals vaccinated with P. pastris ASP was substantially lower compared to antibodies derived from animals vaccinated with native Co-dd-ASP (Figure 15).

[0124] Example 6 To gain insights into the recognition of Cooperia oncophora protein epitopes by protective IgG antibodies, peptide microarrays were performed using Pepscan Presto BV (Netherlands). An array of linear peptide epitopes was generated on a solid support called a "minicard" by computer-based splitting of the Co-dd-ASP amino acid sequence into duplicate fragments. Furthermore, structural epitopes were constructed using CLIPS (Chemically Linked Peptides on Scaffolds) technology (Timmerman et al., 2007) and also constructed as duplicate fragments. This allows for the reproduction of secondary structural elements such as loops, α-helices, and β-strands. Constructs forming incomplete parts of the antibody epitope may be recognized by the antibody, albeit with low affinity, while constructs not part of this epitope are not recognized. The duplicated group of ASP peptides was synthesized using solid-phase 9-fluorenyl methoxycarbonyl (Fmoc) synthesis, and amino-functionalized polypropylene supports were obtained by grafting with a proprietary hydrophilic polymer formulation. Next, dicyclohexylcarbodiimide (DCC) was reacted with N-hydroxybenzotriazole (HOBt) to t-butyloxycarbonylhexamethylenediamine (BocHMDA), and the Boc group was subsequently cleaved by the addition of trifluoroacetic acid (TFA). The peptide was synthesized on an amino-functionalized solid support using standard Fmoc peptide synthesis methods.

[0125] The binding of serum antibodies to each peptide was evaluated by pepscan-based ELISA. This was performed using serum samples from bovine animals vaccinated with either native Co-dd-ASP (n=5), P. pastris Co-dd-ASP (n=5), or QuilA adjuvant alone as a control (n=5). These samples were collected one week after the third immunization. To prevent variability in antibody binding, all pre-coat conditions (antibody concentration and relative amount of competing protein in ELISA buffer) were equal across the various serum samples. First, the array card and peptide array were incubated overnight at 4°C with serum antibodies in PBS containing 5% (v / v) horse serum, 5% (w / v) ovalbumin, and 1% (v / v) Tween 80. After washing with PBS / Tween 80, the array was incubated with HRP-labeled goat anti-bovine IgG (1 / 1000 dilution) at 25°C for 1 hour. After further washing, 2,2'-azino-di-3-ethylbenzthiazoline sulfonic acid (ABTS) and 20 μl / ml of 3% H2O2 were added to the array. One hour after incubation, the color development was measured and quantified using a charge-coupled device (CCD) camera and image processing system, as described by Slootstra et al. (1996).

[0126] The raw data consisted of optical values ​​obtained by a CCD camera, which was used to capture images of the cards before and after staining with peroxidase. The value for each specific peptide was then obtained by calculating the difference between the two images. Values ​​for wells containing air bubbles were evaluated as zero, as this could lead to false positives. To perform quality validation of the synthetic peptides, separate sets of positive and negative control peptides were synthesized in parallel. These peptides were screened using commercial antibodies 3C9 and 57.9 (Posthumuset al., 1990 J. Virol. 64).

[0127] Further data analysis was performed by creating box plots, linear intensity profiles, and heatmaps. The TukeyHSD test was performed on the raw dataset to classify specific peptides as "protective" antibody epitopes. Potential "protective" antibody epitopes were selected only if peptide recognition by serum from the native vaccine group was significantly higher than that of both the P. pastris vaccine group and the negative control group.

[0128] Using indirect ELISA, the recognition of peptides classified as potential "protective" epitopes was re-evaluated using pooled samples from animals vaccinated with either native Co-dd-ASP, P. pastris Co-dd-ASP, or QuilA adjuvant. To maintain linearity of the test results, serum samples for ELISA were collected from the same animals used for peptide microarray analysis. 96-well ELISA plates (MaxiSorp, NUNC) were coated with a 0.25 μg / ml peptide solution in 100 μl coating buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6) at 4°C for 20 hours. Subsequently, the plates were blocked at room temperature for 1 hour with 200 μl blocking buffer (2% BSA in 150 mM PBS / Tween 20), during which washing was performed with 300 μl washing buffer (150 mM PBS / Tween 20). Next, the plates were incubated with pooled bovine serum samples or individual bovine serum samples (diluted 1 / 200 in PBS) at room temperature for 1 hour. After further washing, the plates were incubated with HRP-labeled sheep anti-bovine IgG (diluted 1 / 1000 in blocking buffer) at room temperature for 1 hour. After the final washing, ABTS was added to the plates and incubated at room temperature for 10 minutes. The color development due to oxidation of ABTS appeared as OD405-492 and was quantified using an Infinite F50 absorbance microplate reader (Tecan Trading AG, Mennedorf, Switzerland).

[0129] In addition, competitive inhibition ELISA was performed to evaluate whether the peptide could inhibit the binding of native Co-dd-ASP to native Co-dd-ASP by serum antibodies derived from animals vaccinated with native Co-dd-ASP. For this purpose, 96-well ELISA plates were coated with 1 μg / ml Co-dd-ASP in 100 μl of coating buffer at 4°C for 20 hours. Subsequently, the plates were blocked with 150 μl of blocking buffer at room temperature for 1 hour, during which washing was performed with 200 μl of washing buffer. Simultaneously, serum from vaccinated animals was pre-incubated at room temperature for 1 hour with native Co-dd-ASP in a concentration range of 0 pmol / ml to 500 pmol / ml, or one of the peptides, before being transferred to the coated ELISA plates and incubated for another hour. Finally, HRP-labeled sheep anti-bovine IgG1 (diluted 1 / 1000 in blocking buffer) was added to the plates, incubated for 1 hour, and then ABTS was added. OD405-492 was determined using an Infinite F50 absorbance microplate reader.

[0130] result TukeyHSD analysis was performed on the raw dataset, and only peptides showing p<0.05 in both the native Co-dd-ASP group vs. recombinant Co-dd-ASP group and the native Co-dd-ASP group vs. control group were selected as potential "protective" antibody epitopes. Based on this analysis, four peptides were selected for further evaluation by immunological in vitro assays. Indirect ELISA using the selected peptides demonstrated no clear difference in peptide recognition between pooled serum samples from animals vaccinated with native Co-dd-ASP, P. pastris Co-dd-ASP, or QuilA (Figure 16). In addition, inhibitory ELISA demonstrated that none of the selected peptides, or mixtures thereof, could inhibit the binding of native Co-dd-ASP-inducing antibodies to native Co-dd-ASP coated on the ELISA plate.

[0131] Example 7 To increase the amount of terminal galactose, each glycoform of N. bentamiana Oo-ASP-1 (either core α1,3-fucose or core α1,6-fucose) was subjected to affinity chromatography using agarose-bound Ricinus communis aglutinin-I (VectorLaboratories), primarily following the manufacturer's instructions. Modifications: A Pierce Spin column (Thermofisher) was packed with 0.5 ml of RCA I slurry along with five times the column volume of binding / wash buffer, and the eluate was removed after each packing by centrifugation at 75 × g for 1 minute. The N. bentamiana recombinant was applied to the column at a concentration of 1 mg / ml in 0.5 ml of binding / wash buffer and incubated at 4°C for 1 hour with continuous inversion mixing. Elution was performed by applying 0.5 ml of glycoprotein elution solution (VectorLaboratories) in two separate applications, followed by centrifugation at 100 × g for 1 minute. The results are shown in Figures 18 and 19.

[0132] result: Figure 18 demonstrates a clear increase in terminally galactosylated N-glycans. Since glycoproteins have two N-glycans, there are also N-glycans with terminal galactose. If only one of these N-glycans is galactosylated, the whole is purified.

[0133] Figure 19 also demonstrates a clear increase in terminally galactosylated N-glycans. Since glycoproteins have two N-glycans, there are also N-glycans with terminal galactose. If only one of these N-glycans is galactosylated, the whole molecule is purified.

[0134] References Borloo, J. et al. In-Depth Proteomic and GlycomicAnalysis of the Adult-Stage Cooperia oncophora Excretome / Secretome. J. ProteomeRes. 12(9), 3900-11 (2013a). Borloo, J. et al. Structure of Ostertagia ostertagiASP-1: insights into disulfide-mediated cyclization and dimerization. ActaCrystallogr. Sect. D Biol. Crystallogr.69, 1-11 (2013b). Gonzalez-hernandez, A. et al. Host protectiveASP-based vaccine against the parasitic nematode Ostertagia ostertagi triggersNK cell activation and mixed IgG1-IgG2 response. Scientific Reports 6, 29496(2016). Gonzalez-Hernandez et al. (2017) Comparative analysisof the immune responses induced by native and recombinant versions of theASP-based vaccine against the bovine intestinal parasite Cooperia oncophora.International Journal for Parasitology 2018 Jan;48(1):41-49 Ma at al. Protein Glycoengineering: An Approach forImproving Protein Properties, Front. Chem., 23 July 2020, 8, 1-14. Posthumus WP, Lenstra JA, Schaaper WM, van NieuwstadtAP, Enjuanes L, Meloen RH.Analysis and simulation of a neutralizing epitope oftransmissible gastroenteritis virus. J Virol. 1990 Jul;64(7):3304-9. Slootstra, JW, Puijk, WC, Ligtvoet, GJ,Langeveld, JPM andMeloen, RH (1996). Structural aspects ofantibody-antigen interactionrevealed through small random peptide libraries.Mol. Div. 1 , 87 – 96 Timmerman, P., Puijk, WC, Meloen, RH, 2007.Functional reconstruction and synthetic mimicry of a conformational epitopeusing CLIPS technology. Journal of molecular recognition 20, 283-299. Van der Kaaij et al., Glyco-Engineering Plants toProduce Helminth Glycoproteins as Prospective Biopharmaceuticals: RecentAdvances, Challenges and Future Prospects, Front. Plant Sci., 2022, 13, 1-12. Van Meulder et al. (2015) Analysis of the protective immune response following intramuscular vaccination of calves against the intestinal parasite Cooperia oncophora. International Journal for Parasitology2015 45:637-646 Vlaminck, J., Borloo, J., Vercruysse, J., Geldhof, P.,Claerebout, E., 2015. Vaccination of calves against Cooperia oncophora with adouble-domain activation-associated secreted protein reduces parasite eggoutput and pasture contamination. Int. J. Parasitol. 45, 209-213.

[0135] Drawing translation Figure 1 A. Ostertagia ostertagi - ASP (SEQ ID NO: 1) (SEQ ID NO: 1) B. Cooperia oncophora - Double-domain ASP variant A (SEQ ID NO: 2) (SEQ ID NO: 2) (SEQ ID NO: 10) (SEQ ID NO: 10) C. Cooperia oncophora - Double-domain ASP variant B (SEQ ID NO: 3) (SEQ ID NO: 3) (SEQ ID NO: 11) (SEQ ID NO: 11) D. Cooperia oncophora - Double-domain ASP variant C (SEQ ID NO: 4) (SEQ ID NO: 4) (SEQ ID NO: 12) (SEQ ID NO: 12) E. Teladorsagia circumcincta ASP (SEQ ID NO: 5) (SEQ ID NO: 5) Figure 2 Native Oo-ASP serum Pichia Oo-ASP serum QuilA serum QuilA serum Serum recognition Serum recognition Figure 3A Mannose Fucose N-acetylglucosamine Galactose Glucose N-acetylgalactosamine Xylose Sialic acid Figure 3B Native Oo-ASP specific IgG Mean fluorescence index Core alpha-1,6-fucose Core alpha-1,3-fucose Figure 4 P. pastoris N. benthamiana a1,6-fucose N. benthamiana a1,3-fucose Native Oo-ASP Concentration (pmol) Figure 5 Stimulation index Cell types Figure 6 Pre imm. Before immunization 1st immunization. 2nd immunization. 3rd immunization. Necropsy Figure 7 Mucus IgG1 Mucus IgG1 Mucus IgG2 Mucus IgG2 Mucus IgA Figure 8 Eggs per gram feces (EPG): Number of parasite eggs per gram of feces (EPG) Days after first infection Figure 9 Days after first infection Figure 10 Cumulative EPG Cumulative EPG Native ASP Nicotiana ASP Cum EPG Cumulative EPG Control Vaccinated Figure 11 Total number of worms Worm counts Adult worms L4-stage worms Figure 12 Average Male Length (μm) Average Female Figure 13 Mannose Fucose N-acetylglucosamine Galactose Glucose N-acetylgalactosamine Xylose Sialic acid Figure 14 Native Co-dd-ASP specific IgG Mean fluorescence index Core alpha-1,6-fucose Core alpha-1,3-fucose Figure 15 P. pastoris Co-dd-ASP specific IgG Mean fluorescence index Core alpha-1,6-fucose Core alpha-1,3-fucose Figure 16 Native Co-dd-ASP serum Pichia Co-dd-ASP serum Pichia Co-dd-ASP serum QuilA serum QuilA serum Peptide mix Figure 17A Core alpha-1,3-fucose variant Core alpha-1,6-fucose variant Mannose Fucose Structure (I) Figure 18 pre post after Smoothed, Baseline Subtracted Intens [au] Intensity [arbitrary unit] Figure 19 pre post after Smoothed, Baseline Subtracted Intens [au] Intensity [arbitrary unit]

Claims

1. Recombinant activation-associated secretory protein (ASP) or fragment thereof, wherein the ASP or fragment thereof is a structural (Ia) core α1,3-fucose mutant or a structural (Ib) core α1,6-fucose mutant: 【Chemistry 1】 In its structure, it contains core α1,3-fucose and / or core α1,6-fucose, GlcNAc, and an N-glycan containing Man. X and / or Y are absent or independently substituted with N-acetylglucosamine (GlcNAc), galactose (gal), fucose (fuc), or mannose (man), or combinations thereof. Recombinant ASP or fragment thereof.

2. The recombinant ASP or fragment thereof according to claim 1, wherein X and / or Y are replaced with Gal-GlcNAc.

3. The recombinant ASP or a fragment thereof according to claim 1 or 2, wherein the recombinant ASP is produced by synthesis or obtained from an expression system containing fucosyltransferase.

4. The recombinant ASP or fragment thereof according to claim 3, wherein the recombinant ASP or fragment thereof is obtained from a Nicotiana tabacum expression system, a Pichia pastris expression system, or an insect cell line expression system.

5. The recombinant ASP or fragment thereof according to any one of claims 1 to 4, wherein the amino acid sequence of the recombinant ASP or fragment thereof has at least 90% sequence identity, preferably at least 95%, more preferably at least 99%, and most preferably 100% sequence identity with the amino acid sequence of the ASP of Ostertagia ostertagii, Cooperia oncophora, or Terradorsagia kirkum kinkuta.

6. The recombinant ASP or fragment thereof according to claim 5, wherein the ASP amino acid sequence of Ostertagia ostertagii is represented by SEQ ID NO: 1, the ASP amino acid sequences of Cooperia oncophora are represented by SEQ ID NOs: 2 to 4, and the ASP amino acid sequence of Terradorsagia kirkum kinkuta is represented by SEQ ID NO:

5.

7. A pharmaceutical composition comprising a recombinant ASP or a fragment thereof according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier and / or additive.

8. A pharmaceutical composition according to claim 7, which is a vaccine.

9. The pharmaceutical composition according to claim 8, further comprising an adjuvant.

10. A recombinant ASP or a fragment thereof according to any one of claims 1 to 6, or a pharmaceutical composition according to any one of claims 7 to 9, for use as a pharmaceutical for human or veterinary use.

11. A recombinant ASP or a fragment thereof according to any one of claims 1 to 6, or a pharmaceutical composition according to any one of claims 7 to 9, for use in the treatment, prevention and / or reduction of parasitic nematode infections in mammals.

12. A recombinant ASP or fragment thereof, wherein the parasitic nematode belongs to the genera Ostertagia and / or Cooperia and / or Theradorsagia, and in particular the parasitic nematode is Ostertagia ostertagii, Ostertagia leptospicularis, Cooperia oncophora, Cooperia punctate, Cooperia pectinate, Theradorsagia triflucata, and / or Theradorsagia kirchumcincta, and more particularly Ostertagia ostertagii, Cooperia oncophora, or Theradorsagia kirchumcincta, or the pharmaceutical composition according to claim 10 or 11.

13. A method for producing a recombinant ASP or fragment according to any one of claims 1 to 6, a) A step of preparing an expression system containing fucosyltransferase, b) A step of introducing fucose sugars, particularly core α1,3-fucose and / or core α1,6-fucose, into the expression system, c) A step of expressing an ASP or fragment using the expression system prepared in step a) to obtain a recombinant ASP or fragment according to any one of claims 1 to 6, Methods that include...

14. A method for treating, preventing and / or alleviating a parasitic nematode infection in a subject requiring treatment, prevention and / or alleviation of such an infection, comprising administering to the subject requiring treatment, prevention and / or alleviation of a parasitic nematode infection a recombinant ASP or fragment according to any one of claims 1 to 6, or a pharmaceutical composition according to any one of claims 7 to 9.

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