Recombinant production of proteins with xylosylated N-glycans

Abstract

Provided are non-plant polypeptides or proteins comprising xylosylated N-linked glycans containing β1,2-xylose, wherein at least 25% of the individual protein species comprise xylosylated N-glycans, compositions comprising the non-plant proteins, cells engineered to produce the polypeptides or proteins, and methods for their production and use.

Description

[Technical Field]

[0001] The present invention relates to the field of active immunization. In particular, the present invention provides immunization antigens that have been glycoengineered to contain non-native glycosylation patterns with a view to enhancing their antigenic properties for use in areas such as vaccination and antibody production. The present invention relates to methods and means for producing glycomodified antigens, as well as methods and uses of glycomodified antigens. [Background technology]

[0002] Protein therapeutics, such as monoclonal antibodies (mAbs), peptides, and recombinant proteins, represent a large group of products under development in the biopharmaceutical industry. Among the FDA-approved biological products, the leading are recombinant glycoproteins, which are used to treat a variety of diseases, including metabolic, autoimmune, and cancer. These products are manufactured in a wide range of platforms, including mammalian expression systems, including CHO and human cell lines, and non-mammalian expression systems, such as bacteria, yeast, plants, and insects.

[0003] The most suitable expression system for a particular therapeutic protein depends on the specific protein being expressed and its intended use. In the past, proteins with therapeutic potential were derived directly from sources such as humans or pigs. Examples of this are insulin derived from pancreatic tissue and albumin derived from plasma. However, ensuring reproducibility, purity, and safety became difficult with the advent of genetic engineering technology, which led to the development of recombinant expression systems for protein production.

[0004] While different expression systems have different advantages, certain therapeutic drugs have glycosylation requirements, which means that currently CHO and other mammalian systems are primarily used for their production, and other expression systems have advantages such as speed and the ability to produce different proteins.

[0005] When producing vaccine antigens, it is important to consider glycosylation in general, and N-linked glycans in particular, as they affect glycoprotein half-life, direct migration, ensure protein stability, and mediate cell signaling.

[0006] The most common expression systems and their glycosylation characteristics are described below.

[0007] Bacteria as expression systems In 1982, the first recombinant biologic was approved: insulin (Humulin D, manufactured by Eli Lilly & Co.). (登録商標) ) and was produced in Escherichia coli. Since then, E. coli has been used for the commercial production of approved non-glycosylated therapeutic proteins, such as cytokines, monoclonal antibodies, and enzymes. Bacteria generally do not glycosylate proteins because they lack the glycosylation machinery. This inability to add N-glycans to proteins limits bacteria compared to more complex hosts in the production of proteins that require post-translational modifications (PTMs). However, the bacterium Campylobacter jejuni has demonstrated glycosylation machinery, which could be transferred into E. coli. While this is highly relevant for recombinant protein production, further optimization is still required to establish a cost-effective method.

[0008] Yeast as an expression system Yeast-based systems have been widely used for recombinant protein expression. Yeast and filamentous fungi offer numerous advantages as recombinant protein expression systems compared to mammalian cell cultures, including high recombinant protein titers, short incubation times, and the ability to grow in chemically defined media. Saccharomyces cerevisiae is the expression system for approximately 20% of all biopharmaceuticals, including insulin, hepatitis vaccines, and human serum albumin. Yeast can be grown on an industrial scale, exhibiting very robust expression, folding proteins, and secreting them into the culture medium. Furthermore, they exhibit well-characterized N-glycosylation, often hypermannosylation. Much effort has been devoted to human-type N-glycosylation in yeast to produce better pharmaceuticals. Then, in 2006, Hamilton et al. were able to construct a Pichia pastoris cell line that added 90.5% doubly sialylated N-glycan structures to purified erythropoietin (EPO).

[0009] Plant cells as expression systems Plant cells can be grown in basic culture media and are easily scaled up. Unlike E. coli, plant cells do not contain endotoxins and do not present the same drawbacks associated with producing recombinant proteins in whole plants. Plant cells exhibit greater similarity to human N-glycans than yeast. However, plant cells are also known to express α1,3-fucose and β1,2-xylose, both of which are thought to be immunogenic to the human immune system. In 2012, the first plant-based therapeutic was approved by the FDA. Eleyso (ProTalix BioTherapeutics), produced in carrot cells and containing α1,3-fucose and β1,2-xylose, was targeted at patients with a lysosomal storage disorder known as Gaucher disease. These individuals lack the glucocerebrosidase enzyme, and previous treatments for this condition involved administering recombinant glucocerebrosidase produced in mammalian cells. Because this product in mammalian cells was relatively expensive, efforts focused on producing glucocerebrosidase in a less expensive system. Notably, plant-produced glucocerebrosidase does not appear to elicit adverse immune responses in humans.

[0010] Another example is Medicago's SARS-CoV-2 vaccine candidate. The vaccine was produced in Nicotiana benthamiana and completed Phase III clinical trials, with no adverse reactions reported in subjects (C. Dubé et al., “Broad neutralization against SARS-CoV-2 variants induced by ancestral and B.1.351 AS03-Adjuvanted recombinant plant-derived virus-like particle vaccines,” Vaccine, vol. 40, no. 30, pp. 4017-4025, 2022, doi: 10.1016 / j.vaccine.2022.05.046 and KJ Hager et al., “Efficacy and Safety of a Recombinant Plant-Based Adjuvanted Covid-19 Vaccine,” N. Engl. J. Med., vol. 386, no. 22, pp. 2084-2096, 2022). 2022, doi: 10.1056 / nejmoa2201300).

[0011] While Medicago did not provide information on the glycosylation of their vaccine, Balieu et al. produced the spike protein of SARS-CoV-2 in N. benthamiana and analyzed its glycosylation; the predominant form observed was the F(3)A2Xyl-rich N-glycan seen in the bottom row of example plant glycans in Figure 1.

[0012] Mammalian cells as expression systems Over 50% of commercially available therapeutic proteins are produced using mammalian cells. In general, mammalian expression systems are suitable for producing biopharmaceuticals, which are large, complex proteins that require post-translational modifications (PTMs, most notably glycosylation), because they are often relatively similar to proteins produced by humans. Furthermore, mammalian cell lines, and animal cell lines in general, allow most proteins to be secreted directly into the culture medium. This is an advantage compared to bacterial / prokaryotic systems, which require cell lysis for protein extraction and potentially subsequent protein refolding. The most common mammalian (non-human) cell lines used for therapeutic protein production include mouse myeloma cells (NS0 and Sp2 / 0), Chinese hamster ovary (CHO) cells, and baby hamster kidney (BHK21) cells. However, these non-human mammalian cell lines also have drawbacks. They produce glycosylation that is not expressed in humans, more specifically, galactose-α1,3-galactose (α-Gal) and N-glycolylneuraminic acid (Neu5Gc). Antibodies to both of these N-glycans are not found in the human circulation, and therefore therapeutics are screened during cell line development and manufacturing for acceptable glycan profiles. Glycan profiles are considered an important quality parameter for therapeutic proteins.

[0013] Insect cells as an expression system Insect cells are easily cultured and can secrete correctly folded and post-translationally modified proteins into the culture medium. N-glycans in insect cells, like plant N-glycans, correspond to human structures but are generally simpler. Most proteins produced in insect cell lines also contain some high mannose structure, such as M3 or F(6)M3. The baculovirus expression system (BEVS) is the most common insect expression system and is used for many recombinant expression purposes. This insect cell based expression platform has been successfully used for the production of vaccine antigens and virus-like particles. To date, Cervarix (登録商標) (GlaxoSmithKline) and FluBlok(登録商標) (Protein Sciences) has been approved by the FDA as a vaccine. Regulators have approved Provenge, a drug containing proteins produced by the Sf21 cell line, as a component of an autologous prostate cancer treatment product. (登録商標) (Dendreon) also approved the use of N-glycans. The predominant N-glycan structure found in commonly used insect cells is a short, low-mannose structure (trimannosyl) with or without a core α1,6-fucose. The Spodoptera frugiperda 9 (Sf9) insect cell line has been glycoengineered to produce more complex N-glycosylation. However, relatively high levels of F(6)M3 and intermediate glycan structures still remain.

[0014] High Five from Trichoplusia ni (商標) YaTni PRO (商標) Some insect cells, such as Sf9 and Drosophila Schneider 2 (S2) cells, have glycosylation similar to the M3 structure, but with the immunogenic α1,3-linked fucose rather than the α1,6-linked fucose found in Sf9 and S2 cells. (商標) , and Tni PRO (商標) Efforts have been made to remove fucosylation from proteins expressed in Escherichia coli. These attempts did not directly target the core genes responsible for α1,3- and α1,6-fucose attachment, the fut11 / 12 and FucT6 genes, respectively. Instead, they simultaneously targeted both α1,3- and α1,6-linked fucose by inserting genes for enzymes that consume the intermediate precursor GDP-L-fucose to generate GDP-D-rhamnose. This eliminated any substrate for fucose addition. While this was successful, Mabashi-Asazuma et al. (2014), Glycobiology 24: 325-340, raised questions about the long-term stability of the cell lines.

[0015] The S2 insect cell line was first established by Imogene Schneider in 1971. Since then, approximately 100 Drosophila cell lines have been obtained, 12 of which are easily cultured. However, the primary cell lines used for recombinant protein production are the two original Schneider cell lines, Schneider 2 (S2) and 3 (S3). Unlike BEVS, both S2 and S3 can be genetically modified to express recombinant proteins independent of viral infection. However, only S2 cells have been used to produce vaccine antigens for clinical trials. Stably transfected S2 cells can be grown at high cell densities (up to 50 × 10 6 S2-based production processes are scalable, as S2 cells can be grown in suspension up to 1000 cells / mL. S2 cell recombinant proteins have been well established to have low mannosidic glycans, often containing core α1,6-fucose. Furthermore, we have detected low amounts of high-mannose structures and A1.

[0016] The two most abundant N-linked glycan structures found on proteins secreted from S2 and Sf9 cells are M3 and F(6)M3. (商標) Two additional structures, F(3)M3 and F(3)F(6)M3, were also found in cell lines, which were immunogenic.

[0017] Xylose is not a common component of N-glycans in mammalian or insect cells, but it occurs naturally in plants and helminths. Species-specific glycosylation can pose challenges for the production of therapeutic glycoproteins, but it also opens up the possibility of altering the immune response to vaccines.

[0018] In summary, the various expression systems discussed above are summarized below. [Table 1]

[0019] The most common N-glycosylations in humans, insects, and plants are summarized in Figure 1. The fucose present on the N-glycan can be α1,6- and / or α1,3-linked to the core GlcNAc or (only in plants) α1,4-linked to the GlcNAc preceding the Gal.

[0020] Glycosylation and the immune system In general, protein glycosylation plays an important role in various parts of the vertebrate immune system and is one of the most common post-translational modifications (PTMs).

[0021] Most N-glycans are composed of sugar chains of N-acetylglucosamine (GlcNAc), mannose (Man), galactose (Gal), fucose (Fuc), and sialic acid (Sia), and the complexity varies depending on the organism.

[0022] Antibodies or immunoglobulins (Ig) are glycoproteins produced by the immune system to target foreign invading pathogens. Ig consists of a variable antigen-binding (Fab) fragment and a constant (Fc) fragment.

[0023] The variable Fab region binds to a wide variety of molecular structures in proteins, carbohydrates, and lipids. Antibodies can exist in secreted or membrane-bound forms. Five antibody isotypes exist. IgA is found in mucosal areas such as the intestine, respiratory tract, and genitourinary tract, as well as in saliva, tears, and breast milk. IgD is an antigen receptor on unexposed B cells. IgE acts as a receptor on the surface of mast cells and basophils, triggering their release of histamine upon cross-linking to antigen. While biologically, these actions protect against parasites, this response is also involved in type I allergy. IgG, which consists of four different isotypes, is the primary antibody involved in immunity against invading pathogens. IgM is expressed not only as a monomer on the surface of B cells but also in secreted forms as dimers or pentamers and is involved in the early stages of the B cell-mediated humoral response, eliminating pathogens before IgG levels reach sufficient levels. The core fucose on the glycan structure limits IgG binding to the IgG Fc receptor, resulting in reduced antibody-dependent cell-mediated cytotoxicity.

[0024] Antibodies are produced by the adaptive immune system, more specifically, by B cells. B cells mature in the bone marrow and are released, each expressing a unique antigen-binding receptor on its membrane. When a naive B cell first encounters an antigen that matches its membrane-bound antibody, binding of the antigen to the antibody (a process that normally requires costimulation by T helper lymphocytes, which recognize the processed antigen presented by the B cell on its surface) causes the B cell to rapidly divide into memory B cells and effector B cells. Memory B cells have a longer lifespan than the parent B cells and, like the parent B cells, continue to express membrane-bound antibody. Effector cells produce a secreted form of antibody. Effector cells survive for only a few days, but they secrete large amounts of antibody. Secreted antibodies are the primary effectors of humoral immunity. Some antibodies function simply through binding to target epitopes to block or induce signal transduction. Meanwhile, other antibodies bind to antigens and subsequently recruit circulating lymphoid and myeloid cells to kill invading pathogens through antibody-mediated effector functions (i.e., complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis).

[0025] Dendritic cells (DCs) are the primary link between the innate and adaptive immune systems in mammals. Their primary function is to present digested antigens to T cells. DCs are found in tissues in contact with the environment, such as the skin, nasal lining, lungs, stomach, and intestine. Once activated, DCs migrate to lymph nodes and interact with B cells and T cells. This process shapes the adaptive immune response.

[0026] Immature DCs constantly scan their environment for pathogens via pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), which recognize specific repeating structures found on pathogens. During maturation, immature dendritic cells phagocytose pathogens, degrade them into peptides, and present them on their cell surface. Surface presentation is achieved by major histocompatibility complex (MHC) molecules, which present peptides to T cells. During maturation, DCs upregulate surface receptors, such as CD80, CD86, and CD40, which contribute significantly to T cell activation. Furthermore, activated T cells support the full maturation and antibody production of B cells. DCs possess specific C-type lectin receptors (CLRs) on their surface, which help instruct DCs in inducing immune tolerance rather than immune responses. Examples of these C-type lectins are the mannose receptor (MR, CD206) and dendritic cell-specific intercellular adhesion molecule-3-binding nonintegrin (DC-SIGN, CD209). Blood contains monocytes that can mature into DCs in vitro.

[0027] Innate immune responses are often triggered by macrophage lectins that recognize microbial glycans, resulting in phagocytosis. Circulating lectins, such as serum mannose-binding lectin (MBL) and ficolins, bind to pathogen cell surfaces, thereby activating the complement cascade. When immune cells bind to glycans, they can also activate intracellular signaling pathways that either trigger or suppress cellular responses. For example, binding of trehalose dimycolate, a glycolipid found in the cell wall of Mycobacterium tuberculosis, by the macrophage C-type lectin Mincle induces signaling pathways that cause macrophages to secrete pro-inflammatory cytokines. However, glycans can also have the opposite effect. For example, B lymphocytes possess a lectin called CD22, which, when bound to α2,6-linked sialic acid, triggers signaling that inhibits activation to prevent autoreactivity. Interestingly, α2,6-linked sialic acid is also the gateway through which human influenza viruses enter human cells. Viral lectins, also known as hemagglutinins, facilitate binding to host cell membranes and entry into cells. This interaction is highly specific; human influenza viruses recognize α2,6-linked sialic acid, while avian influenza viruses recognize only α2,3-linked sialic acid.

[0028] The mannose receptor (MR) or cluster of differentiation 206 (CD206) is a C-type lectin found on the surface of macrophages and dendritic cells. MR has eight recognition domains that recognize terminal mannose, GlcNAc, and fucose residues on protein glycans. MR has a higher affinity for branched mannose structures, preferably low-mannose structures, than linear structures. In the adaptive immune system, MR is responsible for antigen uptake and presentation by immature DCs. Upon binding, MR ensures delivery of bound antigen to early endosomes and subsequently to lysosomes, where it is degraded and presented on MHC class II molecules, stimulating and polarizing the adaptive immune response.

[0029] Like MR, dendritic cell-specific intercellular adhesion molecule-3-binding nonintegrin (DC-SIGN) is a C-type lectin found on the surface of macrophages and dendritic cells. DC-SIGN has high specificity for high-mannose structures, preferably M9 structures, over low-mannose structures, but this receptor also recognizes mannose. DC-SIGN has been shown to bind relatively weakly to F(6)M3 and not at all to F(3)M3. Although DC-SIGN contains only one recognition site, it forms tetramers with other DC-SIGN molecules on the DC surface. Once DC-SIGN binds to glycans or microorganisms, it delivers the bound components to late endosomes or lysosomes, where they are destined for degradation. The degraded antigens are presented on MHC class II molecules for T cell presentation. In certain cases, both MR and DC-SIGN appear to deliver antigens to MHC class I molecules.

[0030] Mannose-binding lectin (MBL) is a secreted C-type lectin found in the circulation that recognizes mannose structures. Unlike membrane-bound MR and DC-SIGN, its recognition is not entirely specific; MBL's highly specific binding to high mannose also recognizes fucose and GlcNAc. MBL contains a single receptor and forms trimers as its basic unit. When six trimers aggregate, very strong binding is observed. In contrast to MR and DC-SIGN, MBL can activate the innate immune system. Upon binding to mannose-bearing microorganisms or antigens, MBL activates complement via the lectin pathway, followed by opsonization and phagocytosis.

[0031] Furthermore, a study conducted by Sandig et al. ("Engineering of CHO Cells for the Production of Recombinant Glycoprotein Vaccines with Xylosylated N-glycans," Bioengineering, vol. 4, no. 38, pp. 1-12, 2017, doi: 10.3390 / bioengineering4020038) showed that introducing xylose into N-glycans can positively affect the efficacy of recombinant glycoprotein-based vaccines by increasing immunogenicity. Pathogen recognition is essential for stimulating T cell differentiation. Dendritic cells express various types of pattern recognition receptors (PRRs) that bind to structures present on pathogens, called pathogen-associated molecular patterns (PAMPs). Binding leads to antigen uptake, cytokine secretion, and T cell differentiation. A particular pathogen can have multiple PAMPs that can simultaneously bind to dendritic cells and shape the immune response in a specific manner. The role of non-human N-glycan structures in regulating the immune system has not yet been thoroughly investigated, but much attention has been focused on C-type lectin receptors (CLRs) present on dendritic cells that recognize pathogen-derived sugar structures. For example, Sandig et al. showed that xylosylated F protein of human respiratory syncytial virus produced in glycoengineered CHO cells exhibited a more favorable cytokine profile in human artificial lymph node reactors compared with wild-type, non-xylosylated vaccines, and concluded that the addition of xylose has a clear adjuvant effect.On the other hand, Brzezicka et al. ("Influence of Core β-1,2-Xylosylation on Glycoprotein Recognition by Murine C-type Lectin Receptors and Its Impact on Dendritic Cell Targeting," ACS Chem. Biol., vol. 11, no. 8, pp. 2347-2356, Aug. 2016, doi: 10.1021 / acschembio.6b00265) screened biantennary GlcNAc N-glycans with and without xylose (G0) and found that non-xylosylated glycans led to high uptake by dendritic cells and had high affinity for several CLRs.

[0032] Similar to Sandig et al., Larsen et al. ("Engineering mammalian cells to produce plant-specific N-glycosylation on proteins," Glycobiology, vol. 30, no. 8, pp. 528-538, Aug. 2020, doi: 10.1093 / glycob / cwaa009) showed that expression of a single gene encoding a plant xylosyltransferase from Arabidopsis thaliana or Nicotiana tabacum is sufficient to obtain xylosylated N-glycans in CHO cells, indicating that mammalian cells possess endogenous metabolic pathways for xylose and UDP-xylose synthesis.

[0033] WO 2020 / 144358 provides glycol engineered antigens for use in immunization techniques, including vaccines, with improved antigenicity / immunogenicity compared to their unmodified counterparts.

[0034] In conclusion, there is a continuing need to provide recombinant proteins with engineered glycosylation designed for specific purposes, particularly those related to immunotherapy and vaccines. Overcoming the limitations of the native S2 glycosylation machinery is also valuable, as it may lead to new, powerful expression systems for industrial applications that will enable the production of new drugs. Summary of the Invention

[0035] Object of the invention It is an object of embodiments of the present invention to provide glycoengineered antigens for use in immunization techniques, including vaccination, which have improved antigenicity / immunogenicity compared to their unmodified counterparts. It is also an object of other embodiments of the present invention to provide means and methods for producing glycoengineered antigens, as well as methods and uses that utilize glycoengineered antigens.

[0036] Summary of the Invention We have discovered that the Drosophila S2 cell line, stably expressing Arabidopsis thaliana β-1,2-xylosyltransferase, produces xylosylated N-glycans, and that the xylosylated proteins produced by this cell line are potent inducers of antibody responses.

[0037] Thus, in a first aspect, the present invention relates to a non-plant polypeptide or protein comprising an N-linked glycan comprising β-1,2-xylose.

[0038] In a second aspect, the present invention relates to an immunogenic composition comprising a polypeptide of the first aspect of the invention in admixture with at least one immune adjuvant and optionally a pharmaceutically acceptable carrier, and / or diluent, and / or excipient.

[0039] In a third aspect, the present invention relates to a method for inducing or enhancing a specific immune response in an animal, such as a human, which method comprises at least one immunization of the animal with an effective amount of a protein or polypeptide of the first aspect of the invention, or a composition of the second aspect of the invention.

[0040] In a fourth aspect, the present invention relates to a genetically modified non-plant eukaryotic cell, such as a mammalian cell, an insect cell, or a fungal cell such as yeast, that contains at least one heterologous polynucleotide sequence encoding and expressing a β1,2-xylosyltransferase, enabling the cell to produce N-glycosylated proteins having β1,2 xylose, wherein the heterologous polynucleotide sequence preferably corresponds to the XylT gene from Arabidopsis thaliana or a polynucleotide encoding a plant β1,2-xylosyltransferase. This aspect also relates to a cell line, such as a clonal cell line, comprising the cell of the fourth aspect.

[0041] Finally, in a fifth aspect, the present invention relates to a method for producing an N-glycosylated polypeptide or protein having β1,2-xylose, said method comprising culturing a cell according to the fourth aspect, wherein said cell line expresses a polynucleotide encoding the amino acid sequence of the N-glycosylated polypeptide or protein, followed by isolating the N-glycosylated polypeptide or protein from the culture mixture and, optionally, subjecting the N-glycosylated polypeptide or protein to further purification. [Brief explanation of the drawings]

[0042] [Figure 1] This shows the most common N-glycosylation in humans, insects, and plants. The fucose present on the N-glycan can be α1,6- and / or α1,3-linked to the core GlcNAc, or (only in plants) α1,4-linked to the GlcNAc preceding the Gal. [Figure 2]This shows the construction design of a vaccine antigen containing a portion of the spike 1 protein of SARS-CoV-2. [Figure 3] Examples of N-linked glycan structures and nomenclature are shown. Dark squares represent N-acetylglucosamine (GlcNAc), light gray circles represent mannose, triangles represent fucose, light gray circles represent galactose, and diamonds represent sialic acid. [Figure 4] 1 shows the shapes of N-glycans that are abundant in wild-type S2 cells and in S2 cells expressing plant β1,2-xylosyltransferase. [Figure 5a] Figures 5a-5e show that the secretomes of three clones of the S2-xylT cell line (designated S2-Xyl clone 6, S2-Xyl clone 7, and S2-Xyl clone 8) were further analyzed by LS-MS to obtain detailed glycan profiles and compared with the wild-type glycoprofile. Figure 5a shows the annotation of the peaks in Figures 5b, 5c, and 5d. [Figure 5b] Figure 1 shows the glycoprofile of the secretome of S2-WT (LC-MS, 2AB label). [Figure 5c] N-glycoprofile of the secretome of S2-XylT clone 6 (LC-MS, Rapi Fluor label). [Figure 5d] N-glycoprofile of the secretome of S2-XylT-clone 7 (LC-MS, Rapi Fluor label). [Figure 5e] N-glycoprofile of the secretome of S2-XylT-clone 8 (LC-MS, Rapi Fluor label). [Figure 6]Comparison of N-glycosylation at the secretome level between wild-type S2 cells and three S2-Xyl clones is shown, with the relative proportions of free N-glycans analyzed by LC-MS. M3 is low mannose; F(6)M3 is low mannose linked to a core α1,6-fucose; HM is high mannose; A1 is low mannose linked to a single terminal GlcNAc; F(6)A1 is low mannose linked to a single terminal GlcNAc and a core α1,6-fucose; and Xyl-containing N-glycans are primarily M3Xyl and F(6)M3Xyl. [Figure 7A] Figures 7A-D show a comparison of the N-glycosylation of recombinant human erythropoietin expressed in S2-WT cells (A) and S2-Xyl clone 8 (B). [Figure 7B] Same as above. [Figure 7C] N-glycan structures are shown for expression annotation. [Figure 7D] Comparison of the relative proportions of hEPO-derived N-glycans expressed in S2-WT and S2-Xyl clone 8. M3 indicates low mannose, F(6)M3 indicates low mannose linked to a core 1,6-fucose, HM indicates high mannose, A1 indicates one terminal GlcNAc, F(6)A1 indicates one terminal GlcNAc and 1,6-fucose, and Xyl-containing F(6)M3X and F(6)A1X. [Figure 8-1] The relative proportions of N-glycans on the RBD expressed in S2-WT and S2-Xyl clone 8 are shown. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 9A] Figures 9A–C show the RBD of the SARS-CoV-2 spike 1 protein produced in S2-WT and S2-Xyl. The released N-glycans of RBD-WT were analyzed by LC-MS. [Figure 9B] The released N-glycans of S2-Xyl were analyzed by LC-MS. [Figure 9C]The relative proportions of glycans derived from RBD-WT and RBD-Xyl are shown. M3 is low mannose, F(6)M3 is low mannose linked to a core 1,6-fucose, HM is high mannose, A1 is one terminal GlcNAc, F(6)A1 is one terminal GlcNAc and 1,6-fucose, M3Xyl is xylosylated low mannose, and F(6)M3Xyl is xylosylated low mannose linked to a core 1,6-fucose. [Figure 10] The degraded vaccine components are shown on SDS-PAGE. M is the marker; lane 1 is (RBD-WT)-cVLP; lane 2 is (RBD-Xyl)-cVLP. Lanes 1 and 2 contain the 60 kDa antigen-conjugated cVLP (upper band) and the 16.5 kDa uncoupled cVLP (lower band). Lane 3 is uncoupled cVLP (16.5 kDa), lane 4 is RBD-WT (44 kDa), and lane 5 is RBD-Xyl (44 kDa). [Figure 11A] Figures 11A-G show total IgG titers and IgG subclass titers measured against spike 1 protein produced by HEK293. A shows dilution curves of total anti-spike IgG detected in the first bleed of mice administered the four vaccines. [Figure 11B] Dilution curves of total anti-spike IgG from the second bleed of mice administered the four vaccines. [Figure 11C] Total anti-spike IgG shown as AUC for pre-dose, first blood draw, and second blood draw samples. [Figure 11D] AUC of IgG subclasses from the second blood sample. Each point in the AUC represents one individual mouse. Geometric means with geometric SD are shown in each graph. Statistical significance was determined by a two-tailed nonparametric Mann-Whitney test. p>0.05 indicates no significant difference (ns), p≦0.05 indicates *, and p<0.01 indicates **. [Figure 11E]AUC of IgG subclasses from the second blood sample. Each point in the AUC represents one individual mouse. Geometric means with geometric SD are shown in each graph. Statistical significance was determined by a two-tailed nonparametric Mann-Whitney test. p>0.05 indicates no significant difference (ns), p≦0.05 indicates *, and p<0.01 indicates **. [Figure 11F] AUC of IgG subclasses from the second blood sample. Each point in the AUC represents one individual mouse. Geometric means with geometric SD are shown in each graph. Statistical significance was determined by a two-tailed nonparametric Mann-Whitney test. p>0.05 indicates no significant difference (ns), p≦0.05 indicates *, and p<0.01 indicates **. [Figure 11G] AUC of IgG subclasses from the second blood sample. Each point in the AUC represents one individual mouse. Geometric means with geometric SD are shown in each graph. Statistical significance was determined by a two-tailed nonparametric Mann-Whitney test. p>0.05 indicates no significant difference (ns), p≦0.05 indicates *, and p<0.01 indicates **. [Figure 12] Dilution curves of total anti-spike 1 IgG are shown. (A) First blood collection. (B) Second blood collection. [Figure 13] Figure 1 shows the mass spectrum of released N-glycans of secreted proteins from the S2-XylT-clone 8 cell line. "X" indicates an unannotated glycan structure. [Figure 14] Figure 1 shows the mass spectrum of released N-glycans from secreted proteins of the S2 cell line, S2-XylT-UXS. "X" indicates an unannotated glycan structure. Arrows indicate the apparent changes (decreases or increases) in peak area for S2-XylT-UXS relative to the peak area for S2-XylT clone 8 in Figure 13. [Figure 15]Anti-tag Western blots of CMV Gb mutants expressed in WT, S2-XylT-clone 8, and S2-XylT-UXS cell lines are shown. Samples derived from transient transfections are total secreted protein (unpurified protein). [Figure 16] A depiction of the immunization scheme and blood collection schedule is shown. DETAILED DESCRIPTION OF THE INVENTION

[0043] (definition) As used herein, the term "polypeptide" is intended to refer to both short peptides of 2 to 10 amino acid residues, oligopeptides of 11 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. Furthermore, the term is typically intended to include proteins, i.e., functional biomolecules comprising at least one polypeptide, and, when comprising at least two polypeptides, these may be complexed, covalently linked, or non-covalently linked. Polypeptides within a protein may be glycosylated and / or lipidated and / or contain prosthetic groups. In the present application, all polypeptides and proteins are glycosylated.

[0044] By "non-plant" polypeptide or protein is meant an amino acid sequence, protein, that does not naturally occur in members of the plant kingdom. Thus, typically, non-plant proteins are of animal (particularly mammalian), viral (from viruses that do not infect plants), bacterial, or fungal origin. In particular, non-plant polypeptides of interest in the present invention are mammalian polypeptides / proteins that are the expression products of malignant cells, as well as polypeptides / proteins that are the expression products of pathogenic viruses, bacteria, and fungi, particularly human pathogens. Non-plant polypeptides or proteins may also be artificial sequences that do not occur in nature.

[0045] As defined herein, the "origin" of a polypeptide or protein is one or more organisms or viruses in which the polypeptide or protein occurs (if not artificial) as a naturally occurring expression product or fragment thereof.

[0046] The term "subsequence" also refers to any contiguous stretch of at least 3 amino acids or at least 3 nucleotides, respectively, that is derived directly from a naturally occurring amino acid sequence or nucleic acid sequence, as applicable. The term subsequence is used interchangeably with "fragment" in the context of nucleic acids and polypeptides.

[0047] The term "amino acid sequence" refers to the arrangement of amino acid residues joined by peptide bonds and arranged in a chain in peptides and proteins from the free N-terminus to the free C-terminus.

[0048] The term "adjuvant" or "immunoadjuvant" has its conventional meaning in the field of vaccine technology, i.e., a substance or composition of substances that 1) is not capable of initiating a specific immune response to a vaccine immunogen by itself, but 2) can still enhance the immune response to the immunogen. Or, in other words, vaccination with an adjuvant alone does not provide an immune response to the immunogen; vaccination with an antigen may or may not elicit an immune response to the immunogen, but vaccination with an immunogen in combination with an immunoadjuvant will induce a stronger immune response to the immunogen than that induced by the immunogen alone.

[0049] In the present specification, "sequence identity" is determined by comparing two optimally aligned sequences of equal length (such as DNA, RNA, or amino acids) according to the following formula: (N ref -N dif )·100 / N ref , where N ref is the number of residues in one of the two sequences, N difrepresents the number of residues that are not identical when the two sequences are aligned in the same direction over their entire length. Therefore, the sequence identity between the two sequences 5'-ATTCGGAAC-3' and 5'-ATACGGGAC-3' is 77.8% (N ref =9 and N dif = 2). It is understood that such a determination of sequence identity requires that the two aligned sequences be aligned such that there are no overhangs between the two sequences; each amino acid in each sequence must match its counterpart in the other sequence.

[0050] An "immunocarrier" is a molecule or moiety that can be attached to an immunogen or hapten to enhance or enable the induction of an immune response to the immunogen / hapten. Classically, immunocarriers are relatively large molecules (e.g., tetanus toxoid, KLH, diphtheria toxoid) that can be fused or conjugated to an immunogen / hapten but are not sufficiently immunogenic by themselves. Typically, immunocarriers can elicit a strong T-helper lymphocyte response against the combined entity formed by the immunogen and immunocarrier, which in turn elicits an enhanced response to the immunogen by B lymphocytes and cytotoxic lymphocytes. More recently, large carrier molecules have been partially replaced by so-called promiscuous T-helper epitopes, i.e., shorter peptides recognized by a large fraction of HLA haplotypes in the population, to elicit a T-helper lymphocyte response.

[0051] A "linker" is an amino acid sequence that is introduced between two different amino acid sequences to spatially separate them. A linker can be "rigid," meaning that it does not substantially allow the two amino acid sequences to be attached in a manner that allows them to move freely relative to each other. Similarly, a "flexible" linker allows the two amino acid sequences to be attached via the linker in a manner that allows them to move substantially freely relative to each other. Both types of linkers are useful in the fusion proteins that are part of the present invention.

[0052] A "T-helper lymphocyte response" is an immune response elicited based on a peptide, which can bind to an MHC class II molecule (e.g., an HLA class II molecule) in an antigen-presenting cell and stimulates T helper lymphocytes in an animal species as a result of T cell receptor recognition of the complex of the peptide and the MHC class II molecule presenting the peptide.

[0053] An "immunogen" is a component of a substance that can induce an adaptive immune response in a host, against which the immune system opposes the immunogen. As such, immunogens are a subset of the larger category of "antigens." An antigen is a substance that can be specifically recognized by the immune system (e.g., when bound by an antibody, or when a fragment of an antigen bound to an MHC molecule is recognized by a T cell receptor), but does not necessarily induce immunity. However, an antigen can always "trigger" immunity; that is, a host that has established immunological memory against the antigen will mount a specific immune response to the antigen.

[0054] A "hapten" is a small molecule that cannot induce or trigger an immune response, but when conjugated to an immunocarrier, antibody, or TCR that recognizes the hapten, the hapten can induce the immune system to fight against the hapten-carrier conjugate.

[0055] An "adaptive immune response" is an immune response that responds to an antigen or immunogen, where the immune response is specific to an antigenic determinant of the antigen / immunogen. Examples of adaptive immune responses are the induction of antigen-specific antibody production or the antigen-specific induction / activation of T helper lymphocytes or cytotoxic lymphocytes.

[0056] A "protective adaptive immune response" is an antigen-specific immune response induced in a subject in response to immunization (artificial or natural) with an antigen, which immune response can protect the subject from subsequent challenge with the antigen or a pathogen containing the antigen. Typically, prophylactic vaccination aims to establish a protective adaptive immune response against one or more pathogens.

[0057] "Immune response stimulation" means that a substance or composition of substances exhibits a general, nonspecific immunostimulatory effect. Many adjuvants and putative adjuvants (such as certain cytokines) share the ability to stimulate the immune system. Use of an immunostimulant results in increased "alertness" of the immune system; that is, simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response compared to use of the antigen alone.

[0058] As used herein, the term "animal" is intended to generally refer to animal species (preferably mammals), such as Homo sapiens, Canis domesticus, etc., and not simply to a single individual animal. However, the term also refers to a population of such animal species, since it is important that individuals immunized by the methods substantially all disclosed herein mount an immune response to the immunogens of the invention.

[0059] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides comprising at least one antibody binding site. An "antibody binding site" is a three-dimensional binding space with an internal surface shape and charge distribution complementary to the nature of the antigen's epitope, allowing the antibody to bind to the antigen. "Antibodies" include, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, human antibodies, altered antibodies, univalent antibodies, Fab proteins, and single-domain antibodies.

[0060] "Specific binding" refers to a bond between two substances that goes beyond the binding of one substance to a randomly selected substance and goes beyond a simple association between substances that tend to aggregate because they share similar overall hydrophobicity or hydrophilicity. As such, specific binding usually involves a combination of electrostatic bonds and other interactions between conformationally complementary regions on the two substances. That is, in a complex mixture, substances can "recognize" each other.

[0061] The term "vector" is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it can be replicated and expressed. This term also refers to certain biological vehicles useful for similar purposes, such as viral vectors and phages, both of which are capable of introducing heterologous nucleic acid sequences into cells.

[0062] An "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a transcribable gene product. If the transcription product is an mRNA molecule, it can then be translated into a protein, polypeptide, or peptide.

[0063] A "glycan" is a sugar or sugar chain attached to a biomolecule (such as a lipid or protein).

[0064] As used herein, "high mannose" refers to a glycosylation pattern of Man5-Man9.

[0065] Glycobiology Glycobiology is described as the biology, biosynthesis, structure, and evolution of sugars widely distributed in nature and the proteins that recognize them. Sugars are also called carbohydrates or glycans. All cells and many macromolecules in nature contain a collection of covalently attached sugars or glycosidic sugar chains, also called "glycans."

[0066] Common monosaccharide units of complex carbohydrates Hundreds of different monosaccharides are found in nature, but the monosaccharide variations in common glycans are limited to the sugars listed in the table below. [Table 2]

[0067] glycosidic bond Monosaccharides are linked to each other via glycosidic bonds. The anomeric carbon of each sugar is a stereogenic center, meaning that each glycosidic bond can be constructed as either an α- or β-linkage. Depending on which carbon atom in the sugar structure forms the bond, for example, if the bond occurs at carbon atom 6 or 3, it can be named either Manα1,6 or Manα1,3, respectively.

[0068] Glycan-processing enzymes Generally, there are two groups of glycan-modifying enzymes: transferases and glycosidases. Glycosyltransferases build branched and linear glycan chains, linking monosaccharide moieties to each other. Glycosidases have the opposite effect; they degrade glycan structures for turnover of used glycans or intermediates to use as substrates in glycan biosynthesis. Glycosyltransferases are generally specific for both donor and acceptor substrates. For example, α2,3-sialyltransferase acts on β-linked galactose, and β1,4-galactosyltransferase acts on β-linked N-acetylglucosamine (GlcNAc).

[0069] Glycosylation types Glycosylation is a broad term that encompasses many different types of oligosaccharides and linkages. Glycosylation is found in all domains of organisms, with significant structural variation across domains. Bacteria possess glycans on their surfaces. The most well-known is lipopolysaccharide (LPS), also known as "endotoxin," found on the outer membrane of Gram-negative bacteria. Gram-positive bacteria possess capsular polysaccharides that are included in other glycans on the cell wall. Archaea also possess glycans on the surface of their cell walls and are capable of N-glycosylation of proteins. Glycosylation in eukaryotic cells has been studied more extensively; the major glycan types in mammalian cells are glycosphingolipids, proteoglycans, N-linked glycans, and O-linked glycans. See Figure 2.

[0070] Glycolipid Glycolipids are lipids with glycans attached by glycosidic bonds. They are commonly found on the extracellular surface of eukaryotic cell membranes, where they extend from the phospholipid bilayer into the extracellular space. Glycolipids maintain membrane stability and aid in cell-cell interactions. Additionally, glycolipids can act as receptors for viruses and other pathogens attempting to enter cells. Glycerolipids and sphingolipids are the two most common types of glycolipids.

[0071] Proteoglycans Proteoglycans are highly glycosylated proteins found on the extracellular side of animal cell membranes. They consist of a core protein and one or more covalently attached linear glycosaminoglycan chains. They fill the intercellular space in multicellular organisms, play important roles in matrix assembly and regulating cell signaling, and serve as reservoirs for small biologically active proteins such as growth factors.

[0072] O-linked glycosylation The broad definition of O-linked glycosylation is the attachment of sugars to the oxygen atoms of amino acid residues in proteins, most commonly serine and threonine. O-linked glycans are constructed by the addition of O-acetylgalactosamine, O-fucose, O-glucose, O-acetylglucosamine, or O-mannose. Hyper-O-glycosylation can result in the formation of mucin-type molecules that coat mucosal surfaces. The initial N-acetylglucosamine (mucin-type O-glycosylation) or mannose (O-mannosylation) is often extended (linear or branched) by 5–10 different monosaccharides, including galactose, N-acetylglucosamine, N-galactosamine, sialic acid, and xylose. Eight distinct core structures of N-acetylglucosamine-based mucin-type O-glycans are currently known.

[0073] N-linked glycosylation The structure, number, and location of N-glycans are known to affect the biological activity, protein stability, clearance rate, and immunogenicity of biopharmaceutical proteins. N-linked glycans are most commonly found on cell surfaces and secreted proteins. Protein N-glycosylation can occur at any amino acid sequence in which an Asn precedes any amino acid other than Pro, followed by a Ser or Thr. The common N-glycan "core" structure shared by all eukaryotic cells is Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr. Different organisms build on this core structure differently, resulting in glycans classified as 1) "oligomannose," in which only mannose residues extend the antennae; 2) "complex," in which the core extends primarily from GlcNAc; and 3) "hybrid," in which Man extends the core Manα1-6 arm and GlcNAc extends the Manα1-3 arm.

[0074] Nomenclature of N-linked glycans in this application The description of N-glycans in this document may cause some confusion. The level of detail and information required may vary depending on the situation. In some cases, it is necessary to know the type of each branch and linkage, while in other cases it is sufficient to simply convey whether the structure has, for example, four or five mannoses. To date, no consensus has been reached, and the authors have devised their own nomenclature or adapted existing nomenclature. To avoid confusion, this application uses the "Oxford notation," which is based on the construction of the N-glycan structure. It can therefore be used to represent even very complex glycans. In summary, the notation is as follows:

[0075] All N-glycans have two core GlcNAc. The initial F in the abbreviation indicates the core fucose. Mx indicates the number of mannose units (x) on the core GlcNAc. Ax indicates the number of antennae (GlcNAc) on the trimannosyl core. "A2" indicates bibranching with both alpha 1-2 linked GlcNAcs. Gx indicates the number of galactose units (x) attached to the antennae. [3]G1 and [6]G1 indicate that the galactose is present on the antennae of either α1-3 mannose or α1-6 mannose. Sx indicates the number of sialic acids (x) attached to the galactose.

[0076] Examples of the most commonly occurring N-linked glycans in this application are provided in FIG.

[0077] The Oxford notation is relatively intuitive. The "core" consists of two GlcNAc residues and three mannose residues. The first GlcNAc is linked to an Asn amino acid via a β-linkage. The next GlcNAc is linked to a mannose via a β1,4-linkage, followed by a β1,4-linked mannose. From here, the glycan structure branches, with the remaining two mannoses being linked via either α1,3- or α1,6-linkages. This core is widespread in N-glycans and is designated "M3." If a core fucose is present, it is called "FM3." When the position is known, it is indicated in parentheses. For example, if the fucose is α1,6-linked, it is designated "F(6)M3," if the core fucose is α1,3-linked, it is designated "F(3)M3," or if the core contains both α1,3- and α1,6-linked fucose, it is designated "F(3)F(6)M3." The name depends on the sugars added to this core. A core with one GlcNAc attached is called A1. If the position is known, square brackets are added. For example, if the GlcNAc is on an α1,3-linked mannose branch, it is called "A1[3]." If a glycan falls into the "high mannose" category, some structures are "fixed" both structurally and notationally, such as "M5," while others, such as "M6," allow for the position of the attached mannose residue to vary. Names also exist for complex triantennary and tetraantennary structures, where all linkages and positions are defined. An example of a more complex structure is "A2G(4)2S(3)1," where "A2" describes two β1,2-linked GlcNAcs, "G(4)2" describes two galactoses, both in a β1,4-linkage (not α1,3-linkage, etc.), and "S(3)1" describes one sialic acid linked by an α2,3-linkage. When the location is known, it is indicated by adding "[3]" or "[6]" to indicate α1,3-linked or α1,6-linked mannose branches.

[0078] N-glycan biosynthesis The category of N-glycans found on proteins depends on the organism from which they originate. Whether in yeast, insect cells, or mammalian cells, all N-glycans begin with the same structure in the lumen of the endoplasmic reticulum. N-glycan biosynthesis occurs in two steps: 1) synthesis and transfer of a dolichol-linked precursor, and 2) processing of the Glc3Man9GlcNAc2Asn glycan.

[0079] Synthesis and transfer of dolichol-linked precursors The first part of protein N-glycosylation is the construction of a dolichol precursor and its attachment to an asparagine residue in the protein, as described in more detail below.

[0080] Dolichol phosphate is located on the cytoplasmic side of the endoplasmic reticulum (ER) membrane. It accepts GlcNAc-1-P from UDP-GlcNAc to generate Dol-PP-GlcNAc, which is then elongated to Dol-PP-M5. At this point, an enzyme called a "flippase" flips the structure toward the inside of the ER lumen, adding four Man residues from Dol-P-Man and three Glc residues from Dol-P-Glc. These oligosaccharides are then transferred to Asn residues of proteins in an NXS / T sequence pattern by oligosaccharide transferases, which covalently attach glycans to proteins. See Figure 5.

[0081] Processing steps of M9Glc3 glycans Processing begins when a protein receives an M9Glc3 glycan at the N-glycan site. Protein folding and transport to the Golgi apparatus for further N-linked glycan processing is common in most eukaryotes. Different pathways and enzymes determine the final N-linked glycan structure, depending on the organism. Initial deglycosylation is performed by α-glucosidase I, which removes the first α1,2-linked glucose residue. The next glucose, an α1,3-linked glucose, is removed by α-glucosidase II. After these two glucose residues are removed, the N-linked glycan processing pathway intersects with the protein quality control pathway, which ensures proper folding of newly synthesized proteins bearing M9Glc1. The quality control pathway is primarily mediated by the ER chaperones, carnesin and calreticulin. These two chaperones require the presence of an α1,3-linked glucose residue attached to the protein. As soon as the last glucose residue is removed, the chaperones terminate the folding process. This process leaves either a properly folded protein with M9 attached or an improperly folded protein. The improperly folded protein is deglycosylated by glycosyltransferases, resulting in a monoglycosylated form that can re-associate with chaperones. If the protein ultimately fails to fold properly, it is degraded in a separate ER compartment via the ER-associated degradation pathway (ERAD). Properly folded glycoproteins are finally processed by class I α-mannosidases, which remove the α1,2-mannose residues on the B branch. Here, the glycoprotein bearing M8 is transported to the Golgi apparatus, where the remaining glycosylation takes place. The protein is transported to the cis side of the Golgi apparatus and modified for transit inside the trans-Golgi cisternae. From here, the pathway follows a pathway that depends on whether N-linked glycosylation occurs in yeast, plants, insects, or mammals.

[0082] Here, N-linked glycans are classified as "oligomannose," "complex," or "hybrid" as shown in Figure 3. The biosynthesis of complex and hybrid N-linked glycans begins with the mid-Golgi N-acetylglucosaminyltransferase I (GlcNAcT I), which adds GlcNAc to the second carbon atom of α1,3-Man in the core or M5. Next, two mannoses on the 6-branch are cleaved by α-mannosidase II to produce A1. Because α-mannosidase II has substrate specificity for A1M5, it can only function after the action of GlcNAcT I. The resulting A1 is the point at which invertebrates and plants begin to diverge from mammals. The genomes of plants and invertebrates, including insects, encode a hexoaminidase via the fused lobes gene (fdl), which removes the terminal GlcNAc residue to form M3. Mammalian cells, on the other hand, encode N-acetylglucosaminyltransferase II (GlcNAcT II), which adds GlcNAc onto the 6-branch to form A2. This structure is further extended to include galactose and sialic acid (see Figure 6). Triantennary or tetraantennary structures are also found in some mammalian glycoproteins. The primary core modification in all mammalian, invertebrate, and plant glycans is the attachment of a core fucose. In plants and some insect cells, the core fucose is often attached via an α1,3-linkage, while in other insect and mammalian cells, it is attached via an α1,6-linkage. Like α-mannosidase II, α1,6-fucosyltransferase also requires the prior action of GlcNAcT I to function. The addition of β-1,2 xylose to core β-Man is also common in plants.

[0083] Plant glycosylation is reviewed by Strasser R. 2016 (Glycobiology 26, 926-939). Like mammalian glycosylation, the pathway proceeds to an intermediate Golgi cistern, yielding A1M5. Here, as in most other eukaryotes, the A1M5 precursor is trimmed to A1. In plants, processing can continue to xylT, which uses either A1M5 or A1 as substrates, followed by trimming of the mannose, yielding A1, which xylosylate the core β-Man. As in mammals, GlcNAcTII then adds β1,2-GlcNAc to the 6-branch, yielding A2, which has a xylose attached to the β-Man via a β1,2 linkage (Kajiura et al. 2012, J. Biosci. Bioeng. 113, 48-54). However, unlike mammals and insects, plant glycans do not elongate or branch beyond a biantennary structure. Furthermore, glycans can be α1,3-fucosylated by fut11 or fut12 in the trans-Golgi cisternae. Plant glycosylation also involves several unknown components. For example, post-Golgi sugar modifications in the vacuole have been proposed, and the function of glycosylation in plants grown under normal conditions is unclear. Glycosyltransferases are conserved across organisms, from mosses to higher plants, suggesting that evolutionary constraints have prevented their removal. However, deletion studies of glycosyltransferases, including α1,3-fucosyltransferase and β1,2-xylosyltransferase, have shown no phenotype changes or only detectable under stress conditions (Koiwa et al. 2003, Plant Cell 15, 2273-2284, Strasser R2016).

[0084] The following abbreviations are used throughout this application: α-gal: galactose-α1,3-galactose BEVS: Baculovirus Expression Vector System BHK21: baby hamster kidney cells Cas9: CRISPR-associated protein 9 CE: Capillary electrophoresis CHO: Chinese hamster ovary cells CLR: C-type lectin receptor ConA: Concanavalin A CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats DC: dendritic cell DC-SIGN: dendritic cell-specific intercellular adhesion molecule-3-binding nonintegrin Ebola GP1: Ebola glycoprotein 1 ESI: electrospray ionization FAB: Fast atom bombardment Fab: antigen-binding fragment (of an antibody) Fc: constant fragment (of an antibody) fdl: fused lobes gene FucT6: α1,6-fucosyltransferase gene (α1,6-fucosylatransferase) fut11: α1,3-fucosyltransferase gene (α1,3-fucosylatransferase) GalNAc: N-acetylgalactosamine GlcNAc: N-acetylglucosamine GlcNAcT I:N-acetylglucosaminyltransferase I gene GlcNAcT II:N-acetylglucosaminyltransferase II gene HA: hemagglutinin hEPO: human erythropoietin HER2: Human epidermal growth factor receptor 2 HILIC: Hydrophilic Interaction Chromatography HM: high mannose ID1-ID2a: Inter-domain 1-Inter-domain 2a (VAR2CSA) Indel: insertion / deletion LC-MS: Liquid chromatography mass spectrometry LCA: lentil agglutinin LPS: lipopolysaccharide M3 (or "Man3"): refers to the "core" structure of the glycan mAb: monoclonal antibody MALDI-TOF: matrix-assisted laser desorption ionization time of flight MGAT4: N-acetylglucosaminyltransferase IV gene MGAT5: N-acetylglucosaminyltransferase V gene MHC: major histocompatibility complex mo-DC: monocyte-derived dendritic cells MPLA: Monophosphoryl lipid A MR: mannose receptor MS: Mass spectrometry Neu5Gc: N-glycolylneuraminic acid NHEJ: Non-homologous end joining PAM: protospacer adjacent motif PM: Placental malaria PRR: Pattern recognition receptor PTM: Post-translational modification QIT: Quadrupole ion trap S2: Drosophila melanogaster Schneider 2 cells S3: Drosophila melanogaster Schneider 3 cells SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis SPR: surface plasmon resonance TLR: Toll-like receptor VAR2CSA: a placental cell-associated erythrocyte-transmitted malaria receptor VLP: virus-like particle WT: wild type

[0085] First aspect of the present invention and its embodiments A first aspect of the present invention relates to a non-plant polypeptide or protein comprising xylosylated N-linked glycans comprising β1,2-xylose, as set forth above, wherein at least 25% of individual protein species comprise xylosylated N-glycans.

[0086] In some embodiments, the non-plant polypeptide or protein is selected from the group consisting of VAR2CA, HER2, and hEPO, viral proteins or polypeptides from HIV, Ebola virus, Zika virus, Chikungunya virus, Dengue virus, Hepatitis A virus, Influenza virus, Poliovirus, Rabies virus, Measles virus, Mumps virus, Rubella virus, Rotavirus, Smallpox virus, Chickenpox virus, Hepatitis B virus, Human papillomavirus, Varicella-zoster virus, Yellow fever virus, SARS-CoV-1, Cytomegalovirus (CMV), and SARS-CoV-2, and viral proteins or polypeptides from Clostridium tetanii, Corynebacterium diphtheria, Haemophilus influenzae, Bordetella pertussis, Streptococcus pneumoniae, and the like. The proteins and polypeptides of the present invention are selected from the group consisting of bacterial proteins or polypeptides from Bacillus subtilis, Bacillus pneumoniae, and Neisseria meningitidis. As described herein, all of these proteins and polypeptides are associated with disease, and therefore the proteins and polypeptides of the present invention also offer the potential to treat or reduce the risk of developing disease associated with the wild-type forms of the proteins and polypeptides.

[0087] In some embodiments, the non-plant polypeptide or protein comprises F(6)M3, F(6)A1, F(6)A2, and / or F(3)F(6)M3 glycan structures and / or increased Man-5-Man9 structures compared to its non-plant wild-type protein counterpart (if such wild-type protein exists).

[0088] As indicated above, the non-plant polypeptides or proteins of the first aspect and embodiment discussed above are of mammalian, viral, fungal or bacterial origin, but may also be of crustacean, insect, arachnid, helminth or protozoan origin.

[0089] The glycosylation obtained according to the present invention is such that at least 25% of the individual protein / peptide species in the non-plant polypeptide or protein of the first aspect contain xylosylated N-glycans, such as at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, 75%, and at least 90%. This means that while glycosylation patterns naturally vary among molecules with the same amino acid sequence, preferred polypeptides or proteins of the invention have a high proportion of β1,2-xylose (and preferably high mannose glycans and / or fucosylated glycans, see below) among molecules with the same amino acid sequence.

[0090] As detailed in the Examples, non-plant polypeptides or proteins of the first aspect of the present invention have been produced in S2 cells. Thus, in one preferred embodiment, the non-plant polypeptide or protein can be obtained or has been obtained by a method comprising expressing a polynucleotide encoding the polypeptide or protein in S2 cells transformed to produce active β1,2-xylosyltransferase. Such S2 cells can include the S2-Xyl·S2 cell line, M3Xyl·S2 cell line, or F(6)M3Xyl·S2 cell line disclosed herein. Furthermore, as outlined in Example 2, the cell line can be further genetically modified to produce active UDP-xylose synthase or to exhibit elevated expression of the UDP-xylose synthase gene. The latter may be obtained by introducing an active (optionally heterologous) gene encoding UDP-xylose synthase or by modifying the expression control of a UDP-xylose synthase gene already present in the cell line's genome. For example, the expression control region of an existing UDP-xylose synthase gene can be genetically modified by introducing a strong promoter / enhancer region. Alternatively, the transcript can be made more stable so that it has a longer half-life in vivo, thereby increasing the amount of translation product. Site-specific recombinant cell modification methods, such as CRISPR-Cas9 technology, are well known to those skilled in the art.

[0091] In addition to containing xylosylated N-linked glycans containing β1,2-xylose, the non-plant polypeptide or protein of the first embodiment may have additional glycosylation modifications. For example, the non-plant polypeptide or protein may also contain α-1,3-linked fucose. In this case, the protein or polypeptide may contain an F(3)M3 glycan structure.

[0092] Details regarding the fact that non-plant polypeptides or proteins may also contain α1,3-linked fucose as well as increased Man-5-Man9 structures are disclosed in WO 2020 / 144358, the disclosure of which is incorporated herein by reference.

[0093] It has been found that the non-plant polypeptides or proteins of the first aspect of the invention can be used, for example, as immunization antigens in mammals (see Examples). Thus, in some embodiments, the non-plant polypeptides or proteins of the first aspect of the invention further comprise or are linked via a non-peptide bond to a heterologous moiety, such as a purification tag, an immunogenic carrier molecule, or a T-helper lymphocyte epitope, a solubility modifying group, an elongating group, a targeting moiety, a virus-like particle, and an immunomodulatory moiety, optionally fused to the polypeptide or protein via a peptide linker. Such heterologous moieties confer additional functionality to the polypeptide or protein, such as increased immunogenicity, ease of purification, or increased biological half-life. In some cases, it is practical to use a (rigid or flexible, as appropriate) "linker," i.e., a relatively short stretch of amino acids, instead of directly linking the moiety to the protein / polypeptide. This may be, for example, to avoid steric interactions between the moiety and the protein / polypeptide.

[0094] Expression system Eukaryotic-based systems can be employed to produce N-glycosylated polypeptides, proteins, and peptides for use according to the present invention, and many such systems are widely available commercially.

[0095] As described, for example, in U.S. Pat. Nos. 5,871,986 and 4,879,236, which are incorporated herein by reference, insect cell / baculovirus expression systems are capable of high levels of protein expression of heterologous nucleic acid segments, as described, for example, in INVITROGEN (登録商標) By MAXBAC (登録商標) Under the name CLONTECH (登録商標) By BACPACK (商標) It is available commercially under the name baculovirus expression system.

[0096] In addition to the expression systems disclosed herein, other exemplary expression systems include STRATAGENE(登録商標) COMPLETE CONTROL (商標) The Inducible Mammalian Expression System includes the Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor. Another example of an inducible expression system is the T-REX inducible mammalian expression system, which uses the full-length CMV promoter. (商標) INVITROGEN with (tetracycline-regulated expression) system (登録商標) Available from INVITROGEN (登録商標) also provides a yeast expression system called the Pichia methanolica Expression System, which is designed to produce recombinant proteins at high levels in the methylotrophic yeast Pichia methanolica. Those of skill in the art will know how to express vectors, such as expression constructs, to produce nucleic acid sequences or their cognate polypeptides, proteins, or peptides.

[0097] Second aspect of the invention and its embodiments According to the present invention, immunogenic compositions, particularly vaccines, may be prophylactic (i.e., suitable to prevent infection) or therapeutic (i.e., to treat disease after infection), or may be useful for inducing antibody production in animals used for that purpose.

[0098] Immunogenic compositions of the invention comprise a non-plant polypeptide or protein of the first aspect of the invention (or a polypeptide or protein produced by the method of the fifth aspect of the invention) in admixture with at least one immune adjuvant and, optionally, a pharmaceutically acceptable carrier and / or diluent and / or excipient. See below for a further discussion of immune adjuvants and other materials in compositions.

[0099] Typically, the immunogenic compositions of the invention are in liquid form (suitable for injection or oral ingestion), such as a solution, suspension, emulsion, or suspoemulsion, or in solid or semi-solid form, such as a powder, tablet, suppository, pill, gel, cream, or ointment. For convenience, the immunogenic compositions of the invention are included in unit dosage form, such as lyophilized form.

[0100] The vaccines disclosed herein typically include an immunizing N-glycosylated polypeptide, protein, or peptide in combination with a "pharmaceutically acceptable carrier." A "pharmaceutically acceptable carrier" also includes any carrier that does not itself induce the production of antibodies harmful to the individual to whom the composition is administered or to which the protein / pathogen is targeted. Suitable carriers are typically large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.

[0101] Such carriers are well known to those skilled in the art. Additionally, these carriers may function as immunostimulants ("adjuvants"). Additionally, antigens or immunogens may be conjugated to bacterial toxoids, such as toxoids derived from pathogens such as diphtheria, tetanus, cholera, Helicobacter pylori, etc. See the discussion of immunogenic carriers above.

[0102] The pharmaceutical compositions disclosed herein therefore typically include an immunological adjuvant, which is generally an aluminum-based adjuvant (including aluminum salts), an oil-in-water emulsion, a saponin, complete or incomplete Freund's adjuvant, and a cytokine, or one of the other adjuvants described below.

[0103] Preferred adjuvants that enhance the effectiveness of the composition include, but are not limited to: (1) Aluminum salts (alum), aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc. (2) Oil-in-water emulsion formulations (with or without muramyl peptides (see below) or other specific immunostimulants such as bacterial cell wall components). For example, (a) MF59 (WO 90 / 14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995) containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally, but not necessarily, containing various amounts of MTP-PE (see below)) formulated into submicron particles using a microfluidizer such as a Model 110Y microfluidizer (Microfluidics, Newton, MA), and (b) 10% squalane, 0.4% Tween 80, 5% pluronic-blocked polymer that is microfluidized into a submicron emulsion or vortexed to form a large particle emulsion. polymer) L121, and thr-MDP (see below) containing SAF, (c) AddaVax (商標) (a squalene-based oil-in-water nanoemulsion similar to MF59), (d) Ribi Adjuvant System (RAS) (Ribi Immunochem, Hamilton, MT) containing 2% squalene, 0.2% Tween 80, and one or more cell wall components selected from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox™). (3) Stimulant (商標) Saponin adjuvants such as (Cambridge Bioscience, Worcester, Mass.) or particles made therefrom such as ISCOMs (immunostimulating complexes) may also be used. (4) Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) (5) cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon), macrophage colony-stimulating factor (M-SCF), and tumor necrosis factor (TNF); and (6) Other substances that act as immunostimulants and enhance the effects of the composition, such as alum and MF59. (商標) and Addavax (商標) Adjuvants are preferred, but MPLS / LPS, TLR4 agonists are other examples.

[0104] Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2"-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE).

[0105] Immunogenic compositions (e.g., immunizing antigen or immunogen or polypeptide or protein or nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically include a diluent, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

[0106] Typically, immunogenic compositions are prepared as injectable solutions or suspensions. Solid forms suitable for dissolution or suspension in liquid vehicles prior to injection may also be prepared. As discussed above in relation to pharmaceutically acceptable carriers, they may also be emulsified or encapsulated in liposomes for enhanced adjuvant effect.

[0107] Immunogenic compositions used as vaccines contain an immunologically effective amount of an antigenic or immunogenic polypeptide and, optionally, any other components described above. By "immunologically effective amount" is meant an amount administered to an individual, either as a single dose or as part of a series, that is effective for treatment or prophylaxis. This amount will vary depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated (e.g., non-human primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies or generally mount an immune response, the degree of protection desired, the composition of the vaccine, the treating physician's evaluation in the clinical setting, and other relevant factors. It is expected that the amount will fall within a relatively broad range that can be determined by routine testing. However, for purposes of protein vaccination, the dosage per vaccination typically ranges from 0.5 μg to 500 mg (but often not more than 5000 μg), and most often ranges from 10 to 200 μg.

[0108] Immunogenic compositions have traditionally been administered parenterally, for example, by subcutaneous, intramuscular, or transdermal / transdermal injection (e.g., as in WO 98 / 20734). Additional formulations suitable for other modes of administration include oral, pulmonary, and nasal formulations, suppositories, and transdermal treatments.

[0109] Dosage treatment may be administered as a single dose schedule or a multiple dose schedule. The vaccine may be administered in combination with other immunomodulatory agents.

[0110] The third aspect of the present invention and its embodiments As noted above, the proteins and polypeptides of the first aspect of the invention enable the method of the third aspect for inducing / enhancing a specific immune response in an animal, such as a human, which method comprises at least one immunization of the animal with an effective amount of the protein or polypeptide of the first aspect or the composition of the second aspect.

[0111] Regardless of the precise protein or polypeptide, it is preferred that the active ingredient be administered in both a priming immunization followed by at least one booster immunization. Alternatively, a somewhat more recent approach utilizes cluster immunization (i.e., an immunization scheme in which repeated administration of an immunogen occurs at short intervals early in the immunization regimen, before a memory immune response is established. This is followed by a delayed immunization similar to a traditional booster immunization using a prime-boost immunization regimen).

[0112] The disease targeted by immunization naturally depends on the origin of the immunogen. For example, in some embodiments of the present invention, at least one immunization reduces the risk of developing a disease caused by an infectious agent in the vaccinated animal, or the immunization modulates the existing immune response to a protein or polypeptide. The latter is relevant, for example, in the treatment of allergies by certain immunotherapy. In this case, the unwanted Th2-dependent IgE immune response is modulated to a harmless Th1-dependent IgG immune response.

[0113] In other embodiments of the third aspect, at least one immunization can treat, ameliorate, or reduce the risk of disease caused by self-protein or the cell that produces this self-protein.This is relevant for example to cancer immunotherapy.In cancer immunotherapy, not only can target cancer-related or cancer-specific antigens, but also actively fight against the self-protein that contributes to the progression of the disease.

[0114] The route of immunization is typically selected from parenteral routes such as subcutaneous, intradermal, subdermal, intraperitoneal, intrathecal, and intramuscular routes, or oral or buccal routes, see below for details.

[0115] In conclusion, the method of the third aspect typically involves: a) methods for the prevention of diseases; b) methods of treating or ameliorating disease, and c) Methods for antibody production is selected from.

[0116] The third aspect also relates to a protein or polypeptide of the first aspect of the invention or a composition of the second aspect for use in the prophylactic or therapeutic method of the third aspect. Similarly, a related aspect is the use of a protein or polypeptide of the first aspect for the preparation of a pharmaceutical composition (of the second aspect) for use in the therapeutic or prophylactic method of the third aspect.

[0117] The third aspect of the invention relates generally to the induction of immunity and therefore also involves methods related to the treatment, prevention, and amelioration of disease as well as methods directed to the production of antibodies in an animal host.

[0118] When the immunization regimen includes a polypeptide disclosed herein or a composition comprising such a polypeptide is administered, the animal (e.g., a human) typically receives between 0.5 and 5000 μg of a polypeptide disclosed herein per administration. See supra.

[0119] In a preferred embodiment of this aspect, the immunization scheme involves the animal (eg, a human) receiving a priming dose and one or more booster doses.

[0120] Preferred embodiments of this aspect disclosed herein include those in which administration is for the purpose of inducing therapeutic immune protection against infectious agents. Alternatively, administration is for the purpose of preventing or treating diseases caused by self-proteins or cells. Such diseases include (malignant) neoplastic diseases, as well as diseases in which self-proteins induce undesirable side effects.

[0121] The compositions disclosed herein are capable of inducing humoral immunity, and therefore, in some embodiments, administration is for the purpose of inducing antibodies specific to the antigen, cell, or organism from which the glycosylated polypeptide or protein is derived, and the antibodies or the B lymphocytes producing the antibodies are then recovered from the animal for use as pharmaceuticals, diagnostics, or research reagents themselves.

[0122] As mentioned above, pharmaceutical compositions may comprise the polypeptides / proteins disclosed herein, and the pharmaceutical compositions comprise a therapeutically effective amount thereof.

[0123] As used herein, the terms "therapeutically effective amount" or "prophylactically effective amount" refer to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or prophylactic effect. The effect may be detectable, for example, by chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as a decrease in body temperature. The precise effective amount for a subject will depend on the subject's size and health, the nature and extent of the condition, and the therapeutic agent or combination selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, ranges for the administration of immunologically effective amounts of polypeptides have been mentioned (see above).

[0124] However, the effective amount in a given situation can be determined by routine experimentation and is within the judgment of the clinician.

[0125] Pharmaceutically acceptable salts can be used herein, for example, inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and the like, and organic acid salts such as acetate, propionate, malonate, benzoate, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0126] Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol, and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like may also be present in such vehicles. Typically, therapeutic compositions are prepared as injectable solutions or suspensions; solid forms suitable for dissolving or suspending in liquid vehicles prior to injection may also be prepared. Liposomes are included in the definition of pharmaceutically acceptable carriers.

[0127] Targeted diseases or infections are, for example, SARS-CoV-2 infection (i.e., COVID-19), influenza, and CMV infection. Generally, each of the microorganisms described as proteins of the first aspect of the invention is a target for immunization with the respective protein and composition of the first aspect of the invention.

[0128] Fourth aspect of the present invention and its embodiments The genetically modified cells disclosed herein are useful as organisms for producing proteins and polypeptides, typically proteins and polypeptides of the first aspect of the invention.

[0129] The genetically modified non-plant eukaryotic cell of the fourth aspect, such as an insect cell or a mammalian cell, or a fungal cell such as a yeast cell, comprises at least one heterologous polynucleotide sequence encoding and expressing a β1,2-xylosyltransferase, such that the cell is capable of producing N-glycosylated proteins having β1,2 xylose groups, the heterologous polynucleotide sequence preferably corresponding to the XylT gene from Arabidopsis thaliana or a polynucleotide encoding a plant β1,2-xylosyltransferase.

[0130] As shown in Example 2, embodiments of the fourth aspect involve genetically modified non-plant eukaryotic cells that contain at least one (optionally heterologous) polynucleotide sequence that encodes and expresses a UDP-xylose synthase, or that contain at least one additional (optionally heterologous) UDP-xylose synthase coding sequence such that the cells exhibit increased UDP-xylose synthase activity. For example, the encoded UDP-xylose synthase can have SEQ ID NO:6, and one example of a coding sequence is SEQ ID NO:5.

[0131] In some embodiments, the genetically modified non-plant eukaryotic cell of the fourth aspect further comprises at least one heterologous polynucleotide sequence encoding and expressing a heterologous α1,3-fucosyltransferase, optionally with reduced or eliminated expression of an α-Man-Ia gene or optionally with increased expression of a gene encoding an enzyme that elongates glycans longer than Man3, such as longer than Man5, such that the cell is capable of producing N-glycosylated proteins bearing α1,3-fucosyl groups, and the cell preferably exhibits reduced or eliminated function of at least one gene encoding an α-1,6 fucosyltransferase, such that the heterologous polynucleotide sequence preferably corresponds to the fuc11 gene from Arabidopsis thaliana or a polynucleotide encoding a plant α1,3-fucosyltransferase. For details regarding these modifications, see WO 2020 / 144358.

[0132] The most preferred non-plant eukaryotic cells of the fourth aspect are insect cells (such as Drosophila S cells), although the specific modifications introduced are also relevant to a range of other types of non-plant cells, including mammalian cells and fungal cells, such as filamentous fungal cells, such as yeast.

[0133] The production of proteins and polypeptides requires that the genetically modified cells further express a (heterologous) gene encoding a polypeptide or protein, preferably one of the non-plant polypeptides or proteins of the first aspect of the invention.

[0134] As noted above, the genetically modified cells of the preferred fourth aspect are insect cells, preferably Drosophila melanogaster cells such as S2 or S3, or insect cells such as Sf9, SF21, High5, and C6-36, although if the cells are mammalian they may be CHO or HEK cells.

[0135] Generally useful cells are discussed below.

[0136] Eukaryotic cells can take the form of yeast (such as Saccharomyces cerevisiae) and protists. Alternatively, transformed eukaryotic cells can be derived from multicellular organisms such as fungi, insect cells, or mammalian cells.

[0137] For production purposes, the genetically modified cells disclosed herein are advantageously stably transformed by stably integrating the above-described nucleic acids into their genome, and in certain embodiments, the genetically modified cells preferably secrete or have on their surface the glycosylated polypeptides disclosed herein, as this facilitates recovery of the produced polypeptides.

[0138] As mentioned above, stably genetically modified cells are preferred. In particular, these cells allow the construction of cell lines comprising genetically modified cells as defined herein. Such cell lines are a particularly preferred embodiment of the present invention.

[0139] It is noted that the genetically modified cells of the fourth aspect may be established as a cell line comprising the genetically modified cells. One example of such a cell line is a clonal cell line.

[0140] A more detailed description of cells and cell lines is provided below. Techniques for recombinant gene production, cell transformation, and recombinant gene expression are well known in the art. Examples of such techniques are provided in references such as Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-2002, and Greene and Sambrook, "Molecular Cloning: A Laboratory Manual (Fourth Edition)," Cold Spring Harbor Laboratory Press (ISBN-10: 9781936113422).

[0141] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms include their progeny in any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutation. In the context of heterologous nucleic acid sequence expression, a "host cell" refers to a eukaryotic cell and includes any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by the vector. A host cell can be, or has been, used as a recipient for a vector or virus. A host cell may be "transfected" or "transformed," which refers to a process by which exogenous nucleic acid, such as a recombinant protein-coding sequence, is delivered or introduced into the host cell. Genetically modified cells include the primary subject cell and its progeny.

[0142] As used herein, host cells are derived from non-plant eukaryotic organisms, including yeast cells, insect cells, and mammalian cells, for replicating vectors or expressing part or all of the nucleic acid sequences. Numerous cell lines and cultures are available as host cells and are available through the American Type Culture Collection (ATCC), an organization that provides archives of living cultures and genetic resources or archives derived from other depository organizations, such as the Deutsche Sammlung vor Microorganismen und Zellkulturen (DSM). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired results. For example, plasmids or cosmids can be introduced into prokaryotic host cells for replicating many vectors or expressing their encoded proteins.

[0143] Examples of eukaryotic host cells for vector replication and / or expression include HeLa, NIH3T3, Jurkat, HEK293, Cos, CHO, Saos, and PC12. Numerous host cells derived from a variety of cell types and organisms are available and known to those skilled in the art. Similarly, viral vectors may be used in combination with host cells, particularly those that are permissive for vector replication or expression.

[0144] Some vectors may employ regulatory sequences that allow replication and / or expression in both prokaryotic and eukaryotic cells. Culture conditions for all of the above-mentioned host cells that maintain the host cells and allow the vectors to replicate are further understood by those of skill in the art. Techniques and conditions that allow for large-scale production of vectors, as well as production of the nucleic acids encoded by the vectors and their cognate polypeptides, proteins, or peptides, are also well known.

[0145] Fifth aspect of the present invention and its embodiments In the method for producing an N-glycosylated polypeptide or protein having β-1,2-xylose described in the Summary above, any of the proteins disclosed in the first aspect may be prepared, and any of the cell lines of the fourth aspect may function as host cells. Generally, the method of the fifth aspect relies on methods commonly known in the art of cell culture, recombinant expression, and protein purification. [Example]

[0146] Establishment of an S2 cell line producing proteins with xylosylated N-glycans and immunization of mice with the recombinant proteins produced by the cell line

[0147] Plasmid construction The β1,2-xylosyltransferase gene from Arabidopsis thaliana (accession number NP_568825, SEQ ID NO: 1) was cloned into pExpreS carrying the G418 resistance gene. 2-2 plasmid (ExpreS2ion Biotechnologies, Denmark).

[0148] The construct was designated as "XylT, pExpreS 2 The human erythropoietin gene (accession number AGW15567) carrying an N-terminal BiP secretion signal (MKLCILLAVVAFVGLSLG, SEQ ID NO: 1), a double Strep-tag (WSHPQFEKGGGSGGGSGGSSAWSHPQFEK, SEQ ID NO: 2) at the C-terminus, and flanked by NotI and EcoRI sites was codon-optimized for Drosophila and ordered from GeneArt. 2 -1 vector (ExpreS 2 The construct was cloned into pExpreS (Plant Biotechnologies, Denmark). 2 It was named "-1".

[0149] We engineered the receptor-binding domain, comprising aa 319–591 of SARS-CoV-2 spike 1 (SEQ ID: QIA20044.1), with a Bip secretion signal and Catcher at the N-terminus and a C-tag (EPEA) at the C-terminus, as described by C. Fougeroux et al. (“Capsid-like particles decorated with the SARS-CoV-2 receptor-binding domain elicit strong virus neutralization activity,” Nat. Commun., vol. 12, no. 1, pp. 1–11, 2021, doi: 10.1038 / s41467-020-20251-8). This construct was codon-optimized for expression in Drosophila and flanked by EcoRI and NotI restriction sites for cloning into the pExpreS2-2 vector with the geneticin selectable marker.

[0150] S2 cell handling, transfection, and cloning Wild-type Drosophila S2 cells (ExpreS 2 ion Biotechnologies, Denmark) was used as the initial cell line, and we first 2 The clones were stably transfected with "hEPO, pExpreS-2" and cloned, and the selected clones were labeled "hEPO, pExpreS-2". 2 Stable gene transfer was again performed using "-1".

[0151] Stable transfection was performed in a 25 mL tissue culture flask with 2 x 10 6 The incubation was carried out in EX-CELL 420 serum-free medium (Sigma) with 50 μL of Pen / Strep (Sigma-Aldrich cat. no. P4333-100M) at a concentration of 100 cells / ml. 2 Insect TRx5 transfection reagent was added, the culture medium was swirled, and 12.5 μg of purified DNA was added and swirled. Stable cell lines were obtained after 4 weeks of selection with 4000 μg / ml Geneticin (G418, InvivoGen) or 1500 μg / ml Zeocin (ThermoFisher). Cell cloning was performed by limiting dilution in 96-well plates with feeder cells, 10% serum (fetal bovine serum, Cat. no. 10100-147, ThermoFisher), and G418 selection agent.

[0152] Production and purification of hEPO and SARS-CoV-2 RBD The strep-tagged hEPO, pExpreS, was identified as having the highest percentage of xylosylated glycans. 2The transfected clones at 1 were expanded to 0.5 L. The supernatant was collected and passed through a 0.22 μm cutoff filter. The supernatant was concentrated 4-fold using tangential flow filtration (TFF), buffer exchanged into 1 L of buffer W (100 mM Tris / HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0), and eluted with buffer BXT (buffer W containing 50 mM biotin) according to the manufacturer's instructions (Iba). For RBD antigen production, the selected clones with the highest xylosylation levels, S2-Xyl clone 8 and S2-WT, were transiently transfected with a plasmid encoding the RBD. Three days after transfection, cells were harvested and washed with wash buffer A (25 mM Tris, 100 mM NaCl, pH 7.5) using CaptureSelect™. (商標) C-tagged proteins were purified using a C-tagXL Pre-packed Column (Thermo Fisher, 494307201) and eluted using a stepwise elution method with Buffer B (Buffer A containing 2 M MgCl). The supernatant was concentrated, buffer exchanged into 10x Buffer A, and purified using TFF. The captured RBD was concentrated, and monomeric RBD was separated into 1x PBS using a Superdex-200pg 26 / 600 (Cytvia) SEC column.

[0153] Anti-xylose Western blot The xylose signal of samples from the supernatant was analyzed by Western blot. Briefly, samples were reduced, run on a 4-12% SDS-PAGE gel, and transferred to a nitrocellulose membrane (Invitrogen (商標)The membrane was blocked with casein blocking buffer (Sigma), incubated with a primary rabbit polyclonal anti-β1,2-xylose antibody (Agrisera, AS07267, 1:5000), and detected with a secondary HRP-conjugated polyclonal goat anti-rabbit antibody (Dako, Denmark, P0448). The signal was detected using an ECL Chemiluminescent Substrate Reagent Kit (Novex). (商標) Detection was performed using a Thermo Fisher WP20005.

[0154] Glycoprofiling N-glycan analysis was performed as described in Link A. et al., J. Immunol., vol. 188, no. 8, pp. 3724–3733, 2012, DOI: 10.4049 / jimmunol.1103312. N-glycans were released from purified proteins and fluorescently labeled using the GlycoPrep Rapid N-glycan Kit (ProZyme Inc.) or the GlycoWorks RapiFluor-MS N-glycan Kit (Waters). Labeled glycans were analyzed by liquid chromatography-mass spectrometry (LC-MS) on a Thermo Ultimate 3000 HPLC (fluorescence detector coupled to a Thermo Velos Pro Ion Trap MS).

[0155] cVLP design, expression and purification CLP was engineered and purified as described in C. Fougeroux et al., Nat. Commun., vol. 12, no. 1, pp. 1–11, 2021, doi: 10.1038 / s41467-020-20251-8. Briefly, Acinetobacter phage AP205 coat protein (GeneID: 956335) with a proprietary peptide-binding tag and a GSGTAGGSGS (SEQ ID NO: 2) linker attached to its N-terminus was inserted into the pET28a(+) vector (Novagen) and expressed in BL21 (DE3) E. coli cells (New England Biolabs).

[0156] Coupling and isolation of RBD-cVLP vaccines The RBD glycomutants were mixed with cVLPs at a 1:1 molar ratio in coupling buffer (1.2 mM KH2HPO4, 8.1 mM Na2HPO4*H2O, 136.9 mM NaCl, 2.7 mM KCl, 10 mM Tris, 200 mM sucrose, pH 8.5) and incubated overnight at room temperature. The coupled sample was concentrated using a 15 mL Amicon concentrator with a 30 kDa MWCO, loaded onto a Superdex-200 pg 26 / 600 (Cytvia) SEC column, and eluted with 1x PBS (Gibco) to separate the coupled cVLPs from unbound proteins. The coupled RBD-cVLP-containing fractions were concentrated again, and the concentration of RBD-CLP was measured by Bradford reaction. Endotoxin levels were measured using the Endosafe-PTS Portable System equipped with an LAL cartridge (Charles River).

[0157] Immunization of mice Mouse studies were conducted in accordance with the guidelines of the Danish National Animal Experiments Inspectorate (Dyreforsoegstilsynet, license number 2018-15-0201-01541). 14- to 16-week-old BALB / c mice (Janvier, Denmark) were injected with Addavax. (商標) Mice were immunized intramuscularly (in the thigh) with 15 μg of soluble antigen glycovariants (RBD-WT or RBD-Xyl) formulated with adjuvant (vac-adx-10, InvivoGen), 1 μg of either cVLP-displayed glycovariant (RBD-WT)-cVLP or (RBD-Xyl)-cVLP. A total of four groups, five mice per group, were immunized. Immunization consisted of a prime immunization followed by a booster 3 weeks later. Serum samples were collected 1 week before the prime immunization (pre-dose), and the first and second bleeds were collected 2 weeks after each immunization.

[0158] Measuring antibody responses by ELISA Antibody responses from immunized mice were measured by ELISA. A 96-well Nunc Maxisorp plate (Invitrogen, 44-2404-21) was coated with 50 μl of 2 μg / ml SARS-CoV-2 spike protein S1 (aa14-683, His-Avi tagged recombinant protein, Sigma RP-87681) produced in HEK293 cells and incubated overnight at 4°C. The next day, 200 μl of casein blocking solution (Merck, B6429-500ML) was added to the plate and incubated for 2 hours at room temperature. Serum was diluted 50-fold (for total IgG measurement) or 100-fold (for IgG subclass measurement) and further diluted 3-fold with 1x PBS. 50 μl of the diluted solution was added to each well and incubated for 1.5 hours at room temperature. For total IgG antibody measurements, plates were incubated with HRP-conjugated polyclonal goat anti-mouse antibody (Dako, P0447) in casein blocking solution. Antibodies used for IgG class measurements were goat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (Sigma A10551, M32207, M32407, and M32707, respectively). Between each step, plates were washed three times with 1x PBS containing 0.05% Tween 20. Plates were developed with 100 μl of TMB Xnd substrate (Kem-En-Tec Nordic cat. no. 5280A) and stopped after 10 minutes by adding 100 μl of 0.35 M sulfuric acid. OD was measured using a Biotek ELx808 plate reader. 450 was measured.

[0159] result

[0160] Establishment, cloning, and glycoprofiling of the S2-XylT cell line A polyclonal cell line, designated S2-Xyl, expressing β1,2-xylosyltransferase from A. thaliana was established.

[0161] The cell lines were cloned by serial dilution, and the entire secretomes of 10 clones were analyzed for the presence of xylose by Western blot using an anti-β1,2-xylose antibody. All clones showed a signal, but the wild-type strain did not.

[0162] The secretomes of three clones of the S2-xylT cell line (designated S2-Xyl clone 6, S2-Xyl clone 7, and S2-Xyl clone 8) were further analyzed by LC-MS to obtain detailed glycan profiles and compared with the wild-type glycoprofile (see Figures 5a–5e).

[0163] Figure 6 shows the relative proportions of free N-glycans from the total secreted protein. S2-Xyl clone 6 showed approximately 56% xylosylated N-glycans, S2-Xyl clone 7 showed 44%, and S2-Xyl clone 8 showed 58%.

[0164] Glycosylation profile of purified hEPO To confirm the establishment of a cell line stably expressing Strep-tagged hEPO, we compared the xylosylation levels of purified proteins in wild-type S2 cells (S2-WT) and S2-Xyl clone 8, an S2-Xyl clone with a high relative xylose content. Purified S2-WT hEPO showed a major single peak of F(6)M3, accounting for approximately 92% of the total area. In hEPO derived from S2-Xyl clone 8, the xylose content shifted to 42% of the F(6)M3Xyl, and F(6)M3 decreased to 49% (Figure 7A-C).

[0165] A comparison of the relative abundance of hEPO-WT and hEPO-Xyl-derived N-glycans is shown in Figure 7D.

[0166] Analysis of xylosylated N-glycans on SARS-CoV-2 RBD antibodies After constructing cell lines with significant xylosylation levels, the ability of xylosylated glycans to enhance immunogenicity in vaccine formulations was examined using the RBD of the SARS-CoV-2 spike 1 protein transiently expressed in S2-WT and S2-Xyl clone 8 cell lines, which contain two glycosylation sites in the RBD portion and two glycosylation sites in the Catcher portion.

[0167] The glycosylation of RBD-WT was less uniform than that of hEPO-WT, with approximately 14% less low-mannosidic glycans. hEPO-WT contained only fucosylated low-mannose N-glycans, whereas RBD-WT contained approximately 19% nonfucosylated glycans and 60% fucosylated low-mannose glycans. All relative percentages are shown in Figure 8.

[0168] Interestingly, RBD-WT contained 9% M5 glycans, which was negligible in hEPO-WT (see Figure 9A). The free glycans of RBD-Xyl were approximately 31% xylosylated low-mannose glycans, approximately 38% nonfucosylated low-mannose glycans, and approximately 21% M5 glycans (see Figure 9B). The other xylosylated glycans in RBD-Xyl accounted for approximately 36% of the total xylosylated glycans. A comparison of the relative abundance of N-glycans derived from RBD-WT and RBD-Xyl is shown in Figure 9C.

[0169] Coupling of SARS-CoV-2 RBD antigen to cVLPs The cVLP-displayed vaccine formulation was made by mixing a catcher-containing antigen with a tag-containing cVLP in coupling buffer. Excess antigen was separated from the coupled cVLP by size exclusion chromatography. Coupling of the RBD antigen to the cVLP was confirmed by the size shift of the 16.5 kDa cVLP-Tag monomer on SDS-PAGE gels. The soluble antigen vaccines were designated RBD-WT and RBD-Xyl, and the cVLP vaccines were designated (RBD-WT)-cVLP and (RBD-Xyl)-cVLP. The reduced vaccine components were visualized by SDS-PAGE as shown in Figure 10.

[0170] Antibody responses of mice to immunization with RBD glycomutations Mice (n=5 per group) were treated with Addavax in a prime-boost regimen at 3-week intervals. (商標) Immunizations were performed with 15 μg of adjuvanted soluble RBD glycovariants (RBD-WT and RBD-Xyl) and 1 μg of unadjuvanted cVLP-displayed forms ((RBD-WT)-cVLP and (RBD-Xyl)-cVLP). Vaccine immunogenicity was tested by measuring total IgG and IgG subclass responses by ELISA against spike 1 protein produced in human HEK293 cells. The HEK293-produced protein was selected based on the assumption that human cell line-produced spike 1 most closely resembles the native spike 1 protein. Figures 12A and 12B show dilution curves of total anti-spike 1 IgG titers from the four vaccinated groups measured after the first and second vaccinations. Figure 12C presents these dilution curves as area under the curve (AUC). After the first vaccination, RBD-WT elicited very low levels of IgG, while the remaining three vaccines elicited higher levels. After the second vaccination, the total IgG levels induced by RBD-WT increased to the levels observed after the first vaccination of the other vaccines. RBD-Xyl induced the highest levels of IgG, significantly higher than (RBD-WT)-cVLP (p = 0.0159) and (RBD-Xyl)-cVLP (p = 0.0079), with no significant difference from the cVLP-displayed glycovariant (p = 0.0952). The IgG subclass profiles were examined in serum for the four vaccine groups after the second vaccination and can be seen in Figures 12D-12G. RBD-WT induced only the IgG1 subclass, while the IgG subclasses varied between IgG2a and IgG2b in the other three vaccines, with no significant differences between them. The IgG1 subclass patterns for the four vaccines appeared similar to those measured for total IgG (see Figures 12C and 12D). All dilution curves (not geometric means) of total IgG antibodies from the first and second blood draws are shown in Figure 12.

[0171] conclusion RBD-Xyl was more immunogenic than RBD-WT in both formulations (free soluble antigen and VLP-displayed formulations), and a 15 μg dose of soluble RBD-Xyl elicited greater amounts of anti-Spike 1 antibodies than its VLP counterpart (Xyl-RBD)-VLP. [Example]

[0172] Establishment of a further cell line (S2-XylT-UXS) capable of producing proteins with xylosylated N-glycans

[0173] The "S2-XylT-UXS" cell line was established by transfecting S2-XylT clone 8 from Example 1 with a plasmid containing the Drosophila melanogaster UDP-xylose synthase gene (UXS, whose genomic sequence is provided by Flybase ID: FBgn0035848, SEQ ID NO: 4, and whose mRNA is provided by SEQ ID NO: 5) and selecting for stable polyclonal cells. UDP-xylose synthase (SEQ ID NO: 6) is an enzyme that converts UDP-glucuronic acid to UDP-D-xylose. The rationale for expressing this gene is to potentially increase the cellular pool of UDP-D-xylose available for incorporation into N-glycans, ultimately increasing the level of N-glycan xylosylation in glycoengineered S2 cells.

[0174] LS-MS analysis of released and labeled N-glycans was performed for comparison of S2-XylT clone 8 (also referred to herein as "Xylose1") and S2-XylT-UXS cell lines and can be seen in Figures 13 and 14.

[0175] Comparison of the N-glycosylation profiles of secreted proteins shown in Figures 13 and 14 shows that the S2-XylT-UXS S2 cell line exhibits increased xylosylation compared to the S2 cell line from Example 1.

[0176] Furthermore, all three novel vaccine target cytomegalovirus (CMV) glycoprotein B (Gb) mutants expressed in S2-XylT clone 8 and S2-XylT-UXS cell lines exhibited slightly reduced electrophoretic mobility compared with that expressed in wild-type S2 cells and other identical proteins. Mutants 2 and 3 also appeared to have a larger apparent mass when produced in S2-XylT-UXS than in S2-XylT clone 8, suggesting a higher xylose content (see Figure 15). [Example]

[0177] Further testing of expression products from genetically modified cell lines

[0178] Vaccine protein candidate Several proteins will be compared for immunogenicity in the S2 cell line, a high-mannose cell line (the S2 cell line that produced high-mannose proteins as disclosed in WO 2020 / 144358), the S2-XylT clone 8 strain, and the S2-XylT-UXS cell line. Results are expected to confirm increased immunogenicity for full-length, trimer-forming hemagglutinin influenza A virus (HA) proteins from H1N1, H5N1, H7N9, and the H5N1 HA stem alone. Further proteins tested will include influenza NP protein and M2 protein (strain: Brisbane, PR_8). Additionally, Nipah virus G protein will be tested for increased immunogenicity when produced in the glycomodified cell lines compared to wild-type S2.

[0179] In vivo glycosylated protein testing The glycosylated proteins were purified by C-tag affinity chromatography and then purified by gel filtration chromatography. The concentration was measured at OD 280 Endotoxin levels are measured and, if necessary, endotoxin is removed.

[0180] In vivo studies are performed in rodents (mice, CD1) according to the schedule shown in Figure 16 and the table below for each of the three glycosylated cell lines and the wild-type S2 cell line. A total of 140 (4 x 35) mice are therefore immunized. Administration is intramuscular at 100 μl per immunization (2 x 50 μl administrations in the thigh). Blood is collected before immunization and as indicated in Figure 16i. [Table 3]

[0181] All proteins were prepared in 1x PBS using AddaVax (商標) It is prepared in a mixture with an adjuvant.

[0182] End points: body weight (BW) change from the first immunization and antigen-specific antibody titers by ELISA.

[0183] Expected Results The purified new glycoproteins will be used to immunize mice to determine the impact of xylosylation on vaccine efficacy across multiple targets, and to examine the potential advantages of vaccine candidates produced in the S2-XylT-UXS cell line compared to the S2-XylT clone 8 cell line, due to the increased immunogenicity caused by the higher xylosylation. Immunogenicity will be determined by analyzing mouse blood and quantified for polyclonal responses by ELISA or AlphaLisa.

Claims

1. Non-plant polypeptides or proteins comprising xylosylated N-linked glycans comprising β1,2-xylose, wherein at least 25% of individual protein species comprise xylosylated N-glycans, the polypeptides or proteins preferably comprising VAR2CA, HER2, and hEPO; viral proteins or polypeptides from HIV, Ebola virus, Zika virus, Chikungunya virus, Dengue virus, Hepatitis A virus, Influenza virus, Poliovirus, Rabies virus, Measles virus, Mumps virus, Rubella virus, Rotavirus, Smallpox virus, Chickenpox virus, Hepatitis B virus, Human papillomavirus, Varicella-zoster virus, Yellow fever virus, SARS-CoV-1, Cytomegalovirus (CMV), and SARS-CoV-2; and Clostridium tetani (Clostridium tetanii), Corynebacterium diphtheria, Haemophilus influenzae, Bordetella pertussis, Streptococcus pneumoniae, and Neisseria meningitidis.

2. 2. The non-plant polypeptide or protein of claim 1, wherein the protein comprises F(6)M3, F(6)A1, F(6)A2, and / or F(3)F(6)M3 glycan structures.

3. 3. The non-plant polypeptide or protein of claims 1-2, further comprising increased Man-5-Man9 structures compared to the wild-type protein.

4. 10. A non-plant polypeptide or protein according to any one of the preceding claims, derived from a mammal, crustacean, insect, arachnid, virus, bacterium, fungus, helminth or protozoan.

5. 10. The non-plant polypeptide or protein of any one of the preceding claims, wherein at least 25% of individual protein / peptide species comprise xylosylated N-glycans, such as at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, 75%, and at least 90%.

6. 10. The non-plant polypeptide or protein of any one of the preceding claims, obtainable by a method comprising expressing a polynucleotide encoding the polypeptide or protein in an S2 cell that has been genetically modified to produce an active β1,2-xylosyltransferase and optionally an active UDP-xylose synthase or an increased amount of active UDP-xylose synthase.

7. 10. The non-plant polypeptide or protein of any one of the preceding claims, further comprising α1,3-linked fucose.

8. 10. The non-plant polypeptide or protein of any one of the preceding claims, further comprising or linked via a non-peptide bond to a heterologous moiety such as a purification tag, an immunogenic carrier molecule, or a T-helper lymphocyte epitope, a solubility modifying group, an elongating group, a targeting moiety, a virus-like particle, and an immunomodulatory moiety, optionally fused to the polypeptide or protein via a peptide linker.

9. An immunogenic composition comprising a non-plant polypeptide or protein according to any one of the preceding claims in admixture with at least one immune adjuvant and optionally a pharmaceutically acceptable carrier, and / or diluent, and / or excipient.

10. 10. The immunogenic composition of claim 9, preferably in liquid form such as a solution, suspension, emulsion, or suspoemulsion, or in solid or semi-solid form such as a powder, tablet, suppository, pill, gel, cream, or ointment.

11. The immunogenic composition of claim 9 or 10, wherein the immunoadjuvant is selected from the group consisting of aluminum salts, oil-in-water emulsions, saponins, complete and incomplete Freund's adjuvants, and cytokines.

12. 12. The immunogenic composition of claim 11, wherein the emulsion is a squalene-based oil-in-water emulsion.

13. The immunogenic composition of any one of claims 9 to 12, which is contained in a unit dosage form, such as a lyophilized form.

14. A non-plant polypeptide or protein according to any one of claims 1-8, a composition according to any one of claims 9-13, or a non-plant polypeptide or protein comprising xylosylated N-linked glycans comprising β1,2-xylose, 14. A method for inducing or enhancing a specific immune response in an animal, such as a human, wherein at least 25% of individual protein species comprise xylosylated N-glycans, the method comprising at least one immunization of an animal with an effective amount of N-linked glycans comprising β1,2-xylose, the polypeptide or protein of any one of claims 1-8, or the composition of any one of claims 9-13, the method being for the prevention of, or the treatment or amelioration of, a disease such as SARS-CoV-2, influenza, or pr CMV infection, wherein a priming immunization and at least one booster immunization are preferably administered.

15. 15. The non-plant polypeptide, protein or composition for use of claim 14, further comprising N-linked glycans comprising α1,3-linked fucose, wherein at least 25% of the individual protein / peptide species comprise α1,3-linked fucose.

16. 16. The non-plant protein, polypeptide, or composition for use according to claim 14 or 15, wherein at least one immunization reduces the risk, or immunization modulates an existing immune response to the protein or polypeptide, or at least one immunization treats, ameliorate, or reduce the risk of a disease caused by or associated with a self-protein, or a disease caused by cells that produce the self-protein, in an animal suffering from a disease caused by an infectious organism.

17. 1. A genetically modified non-plant eukaryotic cell, such as a mammalian cell, an insect cell, or a fungal cell such as yeast, comprising at least one heterologous polynucleotide sequence that encodes and expresses a β1,2-xylosyltransferase, wherein the cell is capable of producing N-glycosylated proteins having β1,2 xylose groups, and wherein the heterologous polynucleotide sequence preferably corresponds to the XylT gene from Arabidopsis thaliana or a polynucleotide encoding a plant β1,2-xylosyltransferase.

18. 18. The genetically modified non-plant eukaryotic cell of claim 17, further comprising at least one polynucleotide sequence that encodes and expresses a UDP-xylose synthase, or comprising at least one additional UDP-xylose synthase coding sequence such that the cell exhibits increased UDP-xylose synthase activity.

19. 19. The genetically modified non-plant eukaryotic cell of claim 18, wherein the UDP-xylose synthase has the amino acid sequence of SEQ ID NO:6, and is optionally encoded by SEQ ID NO:

5.

20. 20. The genetically modified non-plant eukaryotic cell of any one of claims 17-19, further comprising at least one heterologous polynucleotide sequence encoding and expressing a heterologous α1,3-fucosyltransferase, optionally with reduced or eliminated expression of an α-Man-Ia gene or optionally with increased expression of a gene encoding an enzyme that elongates glycans greater than Man3, such as greater than Man5, wherein the cell is capable of producing N-glycosylated proteins bearing α1,3-fucosyl groups, and wherein the cell preferably exhibits reduced or eliminated function of at least one gene encoding an α-1,6 fucosyltransferase, and wherein the heterologous polynucleotide sequence preferably corresponds to the fuc11 gene from Arabidopsis thaliana or a polynucleotide encoding a plant α1,3-fucosyltransferase.

21. 21. The genetically modified cell of any one of claims 17-20, further expressing a heterologous gene encoding a polypeptide or protein of any one of claims 1-8, wherein preferably more than 25% of the polypeptides or proteins exhibit β1,2-xylose glycans, such as more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, and more than 85% of the polypeptides or proteins.

22. 22. The genetically modified cell of any one of claims 17-21, which is an insect cell, preferably a Drosophila melanogaster cell such as S2 or S3, or other insect cell such as Sf9, SF21, High5, and C6-36, or a mammalian cell such as CHO or HEK.

23. A cell line, such as a clonal cell line, comprising a genetically modified cell according to any one of claims 17-22.

24. 22. A method for producing an N-glycosylated polypeptide or protein having β1,2-xylose, comprising culturing a cell according to any one of claims 17 to 22 or a cell line according to claim 23, wherein the cell line expresses a polynucleotide encoding the amino acid sequence of the N-glycosylated polypeptide or protein, followed by isolating the N-glycosylated polypeptide or protein from the culture mixture and, optionally, subjecting the N-glycosylated polypeptide or protein to further purification.

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