Conjugates consisting of or including at least β-glucan or mannan

Abstract

The present invention relates to the use of β-glucan as a C-type lectin (CLEC) polysaccharide adjuvant for B-cell or T-cell epitope polypeptides.

Description

[Technical Field]

[0001] The present invention relates to polysaccharide adjuvants that belong to the class of C-type lectins (CLECs). [Background technology]

[0002] Vaccination is considered one of the most powerful means of saving lives and reducing the burden of disease. Vaccines are administered to induce, through active immunization, the host's immune system to develop nonspecific innate immune responses as well as specific antibodies, B memory cells, and T memory cells that can act against the applied immunogen.

[0003] Polysaccharides constitute important virulence factors, especially for encapsulated bacteria that exhibit complex carbohydrate structures on their surface. Bacterial polysaccharides, as well as fungal and other polysaccharides, are constructed from repeating monosaccharide units linked by glycosidic bonds to form linear or branched polymeric structures. It is well known that antibody responses to various bacterial polysaccharides are weak and do not induce immunological memory, and therefore are not boosted by subsequent immunizations.

[0004] These characteristics are based on the properties of polysaccharides, which, unlike proteins, constitute T-cell-independent (TI) antigens. Polysaccharide antigens directly activate polysaccharide-specific B cells, which then differentiate into plasma cells and produce antibodies. Memory B cells are not formed. Instead, proteins and peptides are T-cell-dependent (TD) antigens; after interaction with antigen-presenting cells (APCs) such as dendritic cells (DCs), macrophages, and B cells, protein antigens are internalized and processed into small peptides, which are then re-exposed to and presented to T lymphocytes in association with major histocompatibility complex (MHC) class II molecules. Interaction with T cells causes B cells to differentiate into plasma cells and memory B cells. Unlike TI antigens, TD antigens are immunogenic and responses can be boosted and enhanced by adjuvants (Peltola et al. Pediatrics November 1977, 60 (5) 730-737; Guttormsen HK et al. INFECTION AND IMMUNITY, May 1998, pp. 2026-2032 and INFECTION AND IMMUNITY, December 1999, pp. 6375-6384; Avci et al. Nature Medicine volume 17, pages 1602-1609 (2011)).

[0005] The limitations of pure polysaccharide vaccines have been overcome by conjugation, which involves the covalent attachment of polysaccharides to carrier proteins as a source of T cell epitopes. This concept has been successfully used in several glycoconjugate vaccines currently on the market, developed against bacterial pathogens such as Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae b, and group B streptococcus. All of these vaccines use pathogen-specific carbohydrates to induce carbohydrate-specific antibodies as the primary protective agent.

[0006] It is well known in the art that carbohydrate-based polysaccharides from plant, bacterial, fungal, and synthetic sources can act as so-called pathogen-associated molecular patterns (PAMPs), which, upon contact with the immune system, are recognized by pattern recognition receptors (PRRs) on specialized immune cells.

[0007] PRRs constitute a class of germline-encoded receptors important for the initiation of innate immunity, which upon binding / activation play a crucial role in the first line of defense until more specific adaptive immunity develops. The innate immune response is the first line of defense against infectious diseases and tissue damage. Specialized cells, primarily APCs such as macrophages and DCs, as well as some non-specialized cells such as epithelial cells, endothelial cells, and fibroblasts, express these PRRs and play a major role in pathogen recognition during the innate immune response. Furthermore, activation of APCs by innate immune signals is a critical prerequisite for the generation of potent adaptive immunity, including antibody and memory responses.

[0008] Currently identified PRR families are divided into transmembrane receptors and receptors located in intracellular compartments. Transmembrane receptors include Toll-like receptors (TLR1-9) and C-type lectin receptors (CLRs). Intracellular receptors include nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene (RIG) I-like receptors (RLRs), and AIM2-like receptors (ALRs).

[0009] The PAMP properties of carbohydrates, particularly polysaccharides, have led to a variety of approaches to develop polysaccharides as successful vaccine adjuvants.

[0010] One prominent example is inulin, a polysaccharide found in the roots of the Asteraceae (Compositae) family. Inulin consists of linear β-D-(2,1) polyfructofuranosyl α-D-glucose chains, with up to 100 fructose moieties linked to a single terminal glucose. Inulin has no immunological activity in its native soluble form, but when crystallized into different stable microcrystalline microparticles (inulin α-δ), it acquires potent adjuvant activity for protein conjugate vaccines. δ-inulin microparticles, commercially available as Advax™ adjuvant, exhibit consistent spherulite-like, discoidal particles 1–2 μm in diameter, composed of a series of lamellar sheets. Delta-inulin and gamma-inulin are thought to act through alternative complement activation, as the adjuvant mechanism of gamma-inulin has been shown to involve increased C3 deposition on the surface of macrophages, which leads to enhanced T-cell activation (Kerekes et al. J Leukoc Biol. 2001 Jan;69(1):69-74).

[0011] Another type of polysaccharide-based adjuvant candidate is chitosan-based adjuvants. Chitosan is a linear β-(1,4)-linked copolymer of D-glucosamine and N-acetyl-D-glucosamine (GlcNAc) prepared by partial alkaline deacetylation of chitin. Soluble chitosan also has low immunogenicity in its native form. However, chitosan formulated as dry powder particles or in solution has been widely used as an encapsulating agent for mucosal and systemic vaccine delivery, as well as in the preparation of mucosal DNA vaccines. Chitosan has been used in mucosal vaccine delivery because it promotes absorption and subsequent phagocytosis, enhancing mucosal immune responses (Dodane et al. International Journal of Pharmaceutics 182 (1999) 21-32, Seferian et al. Vaccine 19 (2001) 661-668). The mucoadhesive properties of chitosan particles are due to their cationic nature.

[0012] In vitro assays have shown that bovine serum albumin (BSA) or ovalbumin (OVA) encapsulated in chitosan particles can stimulate RAW264.7 macrophage activation and BMDC activation more efficiently than soluble antigens (Koppolu B, et al. The effect of antigen encapsulation in chitosan particles on uptake, activation, and presentation by antigen-presenting cells. Biomaterials. 2013; 34:2359-2369.).

[0013] Neimert-Andersson et al. used ViscoGel, a soluble chitosan hydrogel. ViscoGel, along with a vaccine against Haemophilus influenzae type b (Act-HIB), induced stronger humoral and cellular responses to the antigen than the unadjuvanted vaccine. Serum IgG1 and IgG2a titers were significantly enhanced. Production of Th1, Th2, and Th17 cytokines was also increased. Unfortunately, ViscoGel proved unsuitable for human use (Vaccine. 2014; 32:5967-5974).

[0014] Both chitin and chitosan particles are readily phagocytosed, supporting the role of specific receptor-mediated recognition in mediating phagocytosis. The receptors on myeloid cells that bind chitin or chitosan and induce the phagocytic response have not yet been clearly identified. To date, several receptors have been shown to bind to chitin or chitin oligosaccharides, including FIBCD1, a homotetrameric 55 kDa type II transmembrane protein expressed in the gastrointestinal tract; NKR-P1, an activating receptor on rat natural killer cells; RegIIIc, a secreted C-type lectin; and galectin-3, a lectin with affinity for β-galactosides. The mannose receptor also binds to GlcNAc and is therefore a potent receptor for chitosan-based vaccine conjugates. Chitin particles have been reported to function as a potent anti-inflammatory drug (PAMP) and recognize the TLR-2 receptor on macrophages to induce innate immune responses. ViscoGel, like chitin, has also been reported to elicit immune responses.

[0015] Yu et al. (Mol.Pharmaceutics, 2016, DOI:10.1021 / acs.molpharmaceut.6b00138) demonstrated that inulin-chitosan conjugates of Mycobacterium tuberculosis CFP10-TB10.4 fusion protein (CT) had significantly increased hydrodynamic volume compared with the unadjuvanted protein, and that this vaccine induced high levels of Th1 (IFN-γ, TNF-α, and IL-2) and Th2 cytokines (IL-4) as well as efficient CT-specific antibody titers, primarily in the form of IgG1 and IgG2b. Pharmacokinetic studies revealed that inulin-chitosan conjugation could prolong serum exposure of CT to the immune system (Mol.Pharmaceutics 2016, 13, 11, 3626-3635).

[0016] One of the most prominent classes of polysaccharides used as adjuvants is the class of C-type lectins (CLECs), which interact with their receptors, called C-type lectin receptors (CLRs). CLRs are Ca 2+ These receptors are considered to be dependent carbohydrate recognition proteins. These receptors express single or multiple carbohydrate recognition domains (CRDs) on a C-type lectin-like domain (CLECD) that are required for CLEC binding. Members of the CLR family bind different carbohydrates, such as mannose, fucose, glucose, maltose, N-acetyl-D-glucosamine, or other glycans and glucans.

[0017] CLRs are classified as transmembrane CLRs (TM-CLRs) and soluble CLRs (collectins). TM-CLRs are further classified as type I and type II TM-CLRs. Type I TM-CLRs include the mannose receptor (MR) and ENDO180 [mannose receptor C type 2 (MRC2)] receptor, which bind to mannose, fucose, and N-acetylglucosamine. Type II TM-CLRs include the dendritic cell-specific intercellular adhesion molecule-3-binding nonintegrin (DC-SIGN receptor), Langerin receptor, and macrophage galactose-type lectin (MGL) receptor. DC-SIGN is known to bind to N-linked glycans (branched trimannose structures) present in, for example, HIV-1 glycoprotein gp120 and other viruses such as hepatitis C virus, human cytomegalovirus, dengue virus, and Ebola virus. DC-SIGN also recognizes lipoarabinomannan and mannan. Langerin is a Langerhans cell (LC)-associated CLR that binds to mannose- and fucose-containing glycan residues. MGL has binding specificity for terminal N-acetylgalactosamine (GalNAc) residues and has also been shown to have affinity for Campylobacter jejuni (C. jejuni) and tumor-associated mucin MUC1, implicated in the regulation of adaptive immunity by effector T cells. Furthermore, macrophage-inducible C-type lectin (Mincle, also known as CLEC4A) and the Dectin-1 / Dectin-2 receptor family have been described. Dectin-1 plays an important role in mediating innate immunity against fungi and has the ability to bind to β-glucans found in fungi (e.g., Saccharomyces cervisiae β-glucan), lichens (e.g., pustulan, lichenan), algae (e.g., laminarin), and barley and other cereal varieties. Dectin-2 contains an EPN (Glu-Pro-Asn) amino acid motif that confers sensitivity to mannose ligands. Furthermore, dectin-2 interacts with Candida albicans (C. albicans).

[0018] Targeting antigens to endocytic receptors on antigen-presenting cells (APCs) is an attractive approach to enhance vaccine efficacy. In particular, mannose receptor (MR) family members and related C-type lectin receptor (CLR) family members, such as DEC205, DC-SIGN, MGL, Langerin, Dectin-1, and Mincle, have demonstrated excellent carbohydrate antigen capture and processing capabilities.

[0019] Immature dendritic cells (DCs), in particular, express a large panel of CLRs associated with the critical function of sensing and capturing self- and non-self-antigens for processing and presentation on MHC molecules, thereby inducing antigen-specific T cell activation. Thus, these cells link the innate and adaptive immune responses.

[0020] CLECs have been used as nonspecific stimulators of immune responses and as adjuvants for immunization. For example, Vojtek et al. (Food and Agricultural Immunology, 2017, 28:6, 993-1002) were able to demonstrate that orally administered β-(1,3),β-(1,6) glucan in combination with immunization against canine rabies and canine parvovirus-2 resulted in the early development of protective levels of antibodies against both viruses.

[0021] Several studies have demonstrated that the activation state of APCs plays a dominant role in the type of immunity induced: for example, immunization with antigen-antibody conjugates under inflammatory conditions results in a TH1 response, regardless of whether the antigen targets the CLR DEC-205, Langerin, Clec9A, or the Ig superfamily member Treml4.

[0022] In contrast, immunization under non-inflammatory (steady-state) conditions leads to tolerance induction. Langerin+ migratory DCs (but not lymph node-resident DCs) have been identified as the primary Treg inducers, regardless of the source of dendritic cells (skin vs. lung) or target receptor.

[0023] In particular, targeting the mannose receptor (MR) or other mannan-sensing CLRs by using mannans as CLECs has demonstrated effective induction of cellular and humoral immune responses, and as a result, MR-targeted vaccines and other CLR-targeted vaccines are attracting increasing attention for the treatment of cancer, infectious diseases, and specific tolerance induction in autoimmune diseases.

[0024] Mannan, a polysaccharide derived from yeast cell walls, consists of a backbone of primarily β-(1,4)-linked mannose with a few α-(1,6)-linked glucose and galactose side chain residues. Furthermore, approximately 5% protein content has been detected in conventional mannan preparations. As an important component of fungal cell walls, mannan is widely used as a component in carbohydrate-based vaccines for candidiasis (Han and Rhew, Arch Pharm Res 2012, Vol 35, No 11, 2021-2027; Cassone, Nat Rev Microbiol. 2013 Dec;11(12):884-91; Johnson and Bundle, Chem.Soc.Rev., 2013, 42, 4327). Furthermore, various examples of vaccines based on mannan carrier-antigen complexes / conjugates have been developed, including mannan-mucin 1 (MUC1) fusion protein conjugation for tumor therapy, or conjugates of mannan with model allergens such as ovalbumin (OVA), papain, or Betv1.

[0025] Mucins are highly glycosylated proteins expressed on the cell surface. MUC1 is a prototypic mucin that has been found to be overexpressed on a wide range of tumor cells. In line with this, we generated MUC1 fusion proteins containing five tandem repeats of human MUC1 (containing the immunodominant epitope APDTRPAPGSTAPPAHGVTS) and a peptide (Cpl3-32). When conjugated to mannan under oxidizing or reducing conditions, dramatically different immunological responses were observed: oxidized mannan-MUC1 stimulated a Th1-type response mediated by CD8+ T cells, resulting in IFN-γ secretion and a predominantly IgG2a antibody response, whereas reduced mannan-MUC1 stimulated a Th2-type response, resulting in IL-4 production and a high IgG1 antibody response. The fusion proteins used represented a single protein displaying both T and B cell epitopes.

[0026] Recently, protein-carbohydrate / mannan conjugates of papain and OVA have also been prepared to analyze their allergenic potential. It was found that coupling mannan to the protein surface can reduce the binding and cross-linking of IgE antibodies to papain. Interestingly, conjugation of either mannan, dextran, or maltodextrin only reduced the allergenic potential of papain, but not that of OVA in these experiments, demonstrating the importance of carbohydrate selection for vaccine design (Weinberger et al. J. Control. Release 2013; 165:101-109). These experiments also demonstrated that mannan conjugation resulted in the development of elevated IgG titers against OVA after intradermal immunization.

[0027] Similar to the neoglycoconjugate vaccine used for MUC1, Ghochikyan et al. (DNA AND CELL BIOLOGY, Volume 25, Number 10, 2006, pp. 571-580) and Petrushina et al. (Journal of Neuroinflammation, 2008, 5:42) applied amyloid beta (Aβ)28, a 28-aa peptide containing B- and T-cell epitopes of the human Aβ42 peptide linked to mannan, which was able to induce low-level anti-Aβ responses in mice. These responses were also found to attenuate amyloid deposition in the cortical and hippocampal regions of APP transgenic mice after subcutaneous immunization. Immunization also induced increased anti-mannan titers in animals treated with Aβ28-mannan and BSA-mannan. However, the treatment was not further developed due to the occurrence of increased microhemorrhages in the brains of treated animals, likely due to the potential adverse effect of mannan as a trigger of adverse vascular events, highlighting the importance of carbohydrate selection for designing efficient and safe vaccines.

[0028] Conjugates using individual B-cell or T-cell epitope peptides linked to mannan or other related CLECs are not known to date.

[0029] Beta-glucans comprise a group of beta-D-glucose polysaccharides. These polysaccharides are the major structural components of fungal cell walls and are also found in bacteria, yeast, algae, lichens, and plants such as oats and barley. Depending on the source, beta-glucans vary in the type of linkage, degree of branching, molecular weight, and tertiary structure.

[0030] Beta-glucan is a source of soluble, fermentable fiber, also called prebiotic fiber, that provides substrate for the colonic microflora, increases stool bulk, and produces short-chain fatty acids as by-products with a wide range of physiological activities. For example, dietary intake of cereal beta-glucan from oats at a daily dose of at least 3 grams reduces total cholesterol and low-density lipoprotein cholesterol levels by 5-10% in people with normal or elevated blood cholesterol levels.

[0031] β-Glucans typically form a linear backbone with one to three β-glycosidic bonds, but vary in terms of molecular mass, solubility, viscosity, branching structure, and gelling properties. Yeast and fungal β-glucans are typically based on a β-(1,3) backbone and contain β-(1,6) side branches, while cereal β-glucans contain both β-(1,3) and β-(1,4) backbone bonds, with or without side branches.

[0032] β-Glucans are recognized by the innate immune system as pathogen-associated molecular patterns (PAMPs). The PRR dectin-1 has emerged as the primary receptor for these carbohydrates, and binding of β-glucans to dectin-1 induces various cellular responses, including phagocytosis, respiratory burst, and cytokine secretion, via the Syk / CARD9 signaling pathway. Furthermore, complement receptor 3 (CR3, CD11b / CD18) has also been implicated as a receptor for β-glucans. Dectin-1-mediated stimulation has been reported to stimulate Th1, Th17, and cytotoxic T lymphocyte responses.

[0033] Members of the β-glucan family include: Beta-glucan peptide (BGP) is a high-molecular-weight (approximately 100 kDa) branched polysaccharide extracted from the fungus Trametes versicolor. BGP consists of a highly branched glucan moiety containing a beta-(1,4) main chain and beta-(1,3) side chains, with the beta-(1,6) side chains covalently linked to polypeptide moieties rich in aspartic acid, glutamic acid, and other amino acids.

[0034] Curdlan is a high molecular weight linear polymer consisting of β-(1,3) linked glucose residues derived from Agrobacterium species.

[0035] Laminarin, derived from the brown alga Laminaria digitata, is a linear β-(1,3)-glucan with β-(1,6) linkages. It is a low-molecular-weight (5-7 kDa) water-soluble β-glucan that can act as either a dectin-1 antagonist or a dectin-1 agonist. Laminarin can bind to dectin-1 without stimulating downstream signaling and can block dectin-1 binding of particulate β-(1,3)-glucans such as zymosan.

[0036] Pustulan is a medium molecular weight (20 kDa) linear β-(1,6)-linked β-D-glucan from the lichen Lasallia pustulata that also binds to dectin-1 as its primary receptor and can activate signal transduction via dectin-1.

[0037] Lichenan is a high molecular weight (approximately 22-245 kDa) linear β-(1,3)β-(1,4)-β-glucan from Cetraria islandica, which has a structure similar to that of barley and oat β-glucans. Lichenan has a much higher ratio of 1,3-β-D to 1,4-β-D bonds than the other two glucans. The ratio of β-(1,4)-β-D to β-(1,3)-β-D bonds is approximately 2:1.

[0038] β-Glucans from oats and barley are linear β-(1,3)β-(1,4)-β-D glucans that are commercially available in a range of molecular weights (from a medium molecular weight fraction of 35.6 kDa to a high molecular weight fraction of up to 650 kDa).

[0039] Schizophyllan (SPG) is a gel-forming β-glucan derived from the fungus Schizophyllum commune. SPG is a high molecular weight (450 kDa) β-(1,3)-D-glucan with a β-(1,6) monoglucosyl branch at every three β-(1,3)-glucosyl residues on the main chain.

[0040] Scleroglucan is a high-molecular-weight (>1000 kDa) polysaccharide produced by fermentation of the filamentous fungus Sclerotium rolfsii. Scleroglucan consists of a linear β-(1,3)D-glucose backbone with one β-(1,6)D-glucose side chain for every three main residues.

[0041] Whole glucan particles (WGP) are β-glucans known for their ability to modulate immune responses. WGP dispersion (WGP® dispersion, manufactured by Biothera) is a microparticle preparation of budding yeast β-glucan. It consists of hollow yeast cell wall "ghosts" composed primarily of long polymers of β-(1,3) glucose obtained after sequential alkaline and acid extractions from the budding yeast cell wall. In contrast to other dectin-1 ligands, such as zymosan, WGP dispersion lacks TLR-stimulating activity. In contrast, soluble WGP binds to dectin-1 without activating this receptor. This can significantly block the binding of WGP dispersion to macrophages and its immunostimulatory effects.

[0042] Zymosan, an insoluble preparation of yeast cells, activates macrophages via TLR2, which cooperates with TLR6 and CD14 in response to zymosan. Zymosan is also recognized by dectin-1, a phagocytic receptor expressed on macrophages and dendritic cells, which cooperates with TLR2 and TLR6 to enhance the immune response triggered by zymosan recognition by each receptor.

[0043] As major components of fungal cell walls, various β-glucans have been used as antigens to generate anti-glucan antibodies against fungal infections (e.g., Torosantucci et al. J Exp Med. 2005 Sep 5;202(5):597-606; Bromuro et al., Vaccine 28 (2010) 2615-2623; Liao et al., Bioconjug Chem. 2015 Mar 18;26(3):466-76).

[0044] Torosantucci et al. (2005) and Bromuro, et al. (2010) disclosed conjugates of the branched β-glucan laminarin and the linear β-glucan curdlan linked to the diphtheria toxoid CRM197. These conjugate vaccines induced high IgG titers against β-glucan and provided protection against fungal infection in mice. Furthermore, high titers against CRM197 could also be detected using such conjugates (Donadei et al., Mol Pharm. 2015 May 4;12(5):1662-72). The authors also produced β-glucan-CRM197 vaccines using synthetic linear β-(1,3)-oligosaccharides or β-(1,6)-branched β-(1,3)-oligosaccharides formulated with the human-tolerable adjuvant MF59. All conjugates induced high titers of anti-β-(1,3)-glucan IgG and / or anti-β-(1,3)-glucan IgG as well as anti-β-(1,6)-glucan antibodies, demonstrating the immunogenicity of the different glucans in combination with classical carrier proteins. Interestingly, Torosantucci et al. failed to demonstrate superior anti-CRM titers after immunization with CRM-glucan conjugates compared to unconjugated CRM alone.

[0045] Donadei et al. (2015) also analyzed conjugates of the diphtheria toxoid CRM197 linked to linear β-(1,3) glucan curdlan or synthetic β-(1,3) oligosaccharides. The conjugates were immunogenic and induced comparable antibody responses to CRM197. Interestingly, the authors showed that intradermal delivery of the CRM-curdlan conjugate resulted in higher antibody titers compared with intramuscular (i.m.) immunization. However, intradermal administration of CRM-curdlan did not demonstrate differential immunogenicity compared with subcutaneous administration. Furthermore, the in vivo efficacy was comparable between CRM-curdlan and unlinked CRM with Alum adjuvant. Therefore, this system failed to detect any additional benefit of CLEC linkage on the overall immune response.

[0046] Liao et al. (2015) described a series of linear β-(1,3)-β-glucan oligosaccharides (hexa-, octa-, deca-, and dodeca-β-glucans) linked to KLH to generate glycoconjugates. These conjugates were shown to elicit robust T cell responses, were highly immunogenic, and induced high anti-glucan antibody levels. Mice immunized with such vaccines also elicited protective immune responses against the lethal pathogen Candida albicans. Comparison of anti-KLH titers to unconjugated KLH was not performed, and therefore, information regarding the potential benefits of β-glucans is not available in this experimental setting.

[0047] These findings are crucial for the applicability of glucan-based neoglycoconjugates as novel vaccines. Potential anti-glucan antibodies induced during initial glucan conjugate immunization could lead to rapid clearance of the same β-glucan vaccine in subsequent booster immunizations or attenuate immune responses to novel neoglycoconjugate vaccines for other indications, an effect well-known from vector-based vaccines. Thus, as demonstrated above for mannan and β-glucan (Petrushina et al. 2008, Torosantucci et al. 2005, Bromuro et al. 2010, Liao et al. 2015), the presence or even (re)stimulation of high levels of anti-glucan antibodies could reduce or eliminate potential immune responses elicited by conjugate vaccines. Therefore, ensuring that the glucan antibody-inducing potential of the polysaccharides / oligosaccharides used is very low or nonexistent will be crucial for novel and sustainable platforms using CLECs, especially β-glucans, as scaffolds for immunization.

[0048] Glucan particles (GPs) are highly purified 2-4 μm hollow porous cell-wall microparticles composed primarily of β-(1,3)-D-glucan, with minor amounts of β-(1,6)-D-glucan and chitin, typically isolated from Saccharomyces cerevisiae using a series of hot alkaline, acid, and organic extractions. Glucan particles interact with their receptors, Dectin-1 and CR3 (although evidence also implicates interactions with Toll-like receptors and CD5 as additional factors for GP function), upregulating cell surface presentation of MHC molecules, altering the expression of costimulatory molecules, and inducing the production of proinflammatory cytokines. GPs have been investigated for vaccine delivery due to their immunomodulatory properties.

[0049] There are three general approaches to applying GPs in vaccines: (i) as adjuvants co-administered with antigens to enhance T- and B-cell mediated immune responses, (ii) chemically cross-linked to antigens, and most frequently (iii) used as physical delivery vehicles for antigens entrapped within hollow GP cavities to provide targeted antigen delivery to APCs.

[0050] Regarding (i): Antigen-specific adaptive immune responses can be enhanced by coadministering GP with the antigen. This conventional adjuvant strategy activates both innate and adaptive immune responses to exert protective responses against pathogens. For example, Williams et al. (Int J Immunopharmacol. 1989;11(4):403-10) adjuvanted a killed Trypanosoma cruzi vaccine by coadministering GP. The immune response elicited using this formulation resulted in 85% survival of mice challenged with Trypanosoma cruzi. In contrast, controls administered with dextrose, glucan, or vaccine alone experienced 100% mortality.

[0051] Regarding (ii): The carbohydrate surface of GPs can also be covalently modified using NaIO4 oxidation, carbodiimide cross-linking, or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate-mediated conjugation of antigens to the GP shell. Using this approach, the conjugation efficiency is very low (approximately 20%, as described, for example, in Pan et al. Sci Rep 5, 10687 (2015)), significantly limiting the applicability and number of vaccine candidates compared to antigen encapsulation in GPs or the proposed platform technology provided in this application. Such covalently linked antigen-GP conjugates have been used in cancer immunotherapy and infectious disease research. For example, Pan et al. (2015) used OVA cross-linked to periodate-oxidized GPs and subcutaneously immunized mice with this vaccine. When mice were challenged with OVA-expressing E.G7 lymphoma cells, a significant reduction in tumor size was observed. GP-OVA significantly increased the number of DCs (CD11c) in lymph nodes 12 and 36 hours after subcutaneous injection. + MHC-II + Tumor protection was associated with increased total anti-Ova immunoglobulin (Ig)G titers, enhanced expression of MHC-II and costimulatory molecules (CD80, CD86), and elevated cytotoxic lymphocyte responses.

[0052] Regarding (iii): The most effective approach to applying GPs to vaccines is to use them to encapsulate vaccines / antigens into hollow cores. GPs can encapsulate one or more antigens / DNA / RNA / adjuvants / drugs / combinations thereof with high loading efficiency determined by the type of payload and the intended delivery mode.

[0053] Antigens can be encapsulated in the hollow cavities of GPs using polymer nanocomplexation methods, such as payload loading and complexation using bovine serum albumin or mouse serum albumin and yeast RNA / tRNA, or the addition of alginate-calcium or alginate-calcium-chitosan mixtures. Using these strategies, for example, Huang et al. (Clin. Vaccine Immunol. 2013; 20:1585-91) reported that mice vaccinated with GP-OVA exhibited strong CD4+ T cell lymphoproliferation, Th1- and Th17-skewed T cell-mediated immune responses, along with high IgG1- and IgG2c-specific antibody responses to ovalbumin. The noncovalent encapsulation strategy elicited a stronger immune response compared with GPs coadministered with antigens.

[0054] An example of a GP-encapsulated subunit vaccine is a GP encapsulating a soluble alkaline extract of an acapsulated strain of Cryptococcus neoformans (cap59). This GP protected mice (60% survived) from a lethal dose of virulent Cryptococcus neoformans by inducing antigen-specific CD4+ T cell responses (positive for IFN-γ and IL-17A) that reduced fungal colony-forming units (CFU) by more than 100-fold compared to the initial challenge dose (Specht CA et al. Mbio 2015; 6:e01905- e1915. and Specht CA et al., mBio 2017; 8:e01872- e1917.). Furthermore, vaccinating mice with encapsulated GP has been shown to be effective against Histoplasma capsulatum (Deepe GS et al., Vaccine 2018;36:3359-67), F. tularensis (Whelan AO et al., PLOS ONE 2018;13:e0200213), Blastomyces dermatitidis (Wuthrich M et al., Cell Host Microbe 2015;17:452-65), and Coccidioides posadasii (Hurtgen BJ et al., Infect. Immun. 2012;80:3960-74).

[0055] In addition to applications in cancer and infectious diseases, limited studies have also been conducted using GP as an encapsulating agent for vaccine delivery to target autoantigens. In line with this, Rockenstein et al. (J. Neurosci., January 24, 2018: 38(4):1000-1014) described the application of recombinant human a-synuclein protein (containing both B and T cell epitopes suitable for inducing anti-aSyn immune responses) and rapamycin-loaded GP, which is known to induce antigen-specific regulatory T cells (Tregs) in mouse models of synucleinopathy. As expected from previous studies using full-length a-synuclein as an immunogen, application of aSyn-containing GP resulted in the induction of robust anti-a-synuclein antibody titers and attenuated the pathological changes caused by a-synuclein in animals to a similar extent as previously published. The number of such Treg cells significantly increased after rapamycin exposure, and the addition of rapamycin effectively induced the formation of iTreg (CD25+ and FOXP3+) cells. Thus, GP loaded with the antigen α-synuclein and rapamycin induced both neuroprotective humoral and iTreg responses in a mouse model of synucleinopathy, and the combined vaccine (aSyn+rapamycin) was more effective than humoral immunization (GP-aSyn) or cellular immunization (GP-rapamycin) alone. No information has been reported on the equivalence of efficacy to conventional α-synuclein-containing non-GP immunization.

[0056] β-Glucan neoglycoconjugates efficiently target dendritic cells via the C-type lectin receptor Dectin-1 and enhance their immunogenicity. Specifically, certain β-glucans have also been used as potential carriers for vaccination using model antigens such as OVA (Xie et al., Biochemical and Biophysical Research Communications 391 (2010) 958-962; Korotchenko et al., Allergy. 2021; 76:210-222.) or MUC1-based fusion proteins (Wang et al., Chem. Commun., 2019, 55, 253).

[0057] Xie et al. and Korotchenko et al. used the branched β-glucan laminarin as a scaffold for OVA conjugation. These glycoconjugates were then administered to mice via epidermal or subcutaneous routes. Xie et al. showed that laminarin / OVA conjugates, but not the unconjugated mixture of compounds, induced an increase in anti-OVA CD4+ T cell responses compared with ovalbumin alone. Importantly, co-injection of unconjugated laminarin blocked this enhancement, supporting the function of laminarin-mediated APC targeting. As expected, native OVA and a mixture of OVA and laminarin stimulated low levels of anti-OVA antibody production. In contrast, OVA / laminarin conjugates significantly enhanced the antibody response. Similarly, Korotchenko et al. demonstrated that laminarin conjugates of OVA significantly increased BMDC uptake and induced their activation and secretion of pro-inflammatory cytokines. These properties of the LamOVA conjugate also resulted in enhanced stimulation of OVA-specific naive T cells cocultured with BMDCs. In preventive immunization experiments, the authors were able to confirm that immunization with LamOVA reduced its allergenicity and induced approximately threefold higher IgG1 antibody titers compared with OVA after two immunizations. However, while all groups showed similar antibody titers, this effect was lost in all groups treated after the third immunization. Lam / OVA and OVA / Alum conjugates showed comparable therapeutic efficacy in a mouse model of allergic asthma. Therefore, these experiments failed to demonstrate the apparent superiority of glucan-based conjugates compared with conventional vaccines.

[0058] Wang et al. (2019) analyzed the efficacy of a β-glucan-based MUC1 cancer vaccine candidate. The MUC1 tandem repeat sequence GVTSAPDTRPAPGSTPPAH, a well-studied cancer biomarker, was selected as a peptide antigen, providing T cell and B cell epitopes within the repeat sequence. β-glucan and MUC1 peptide were linked to yeast β-(1,3)-β-glucan polysaccharide using an ethylene glycol (i.e., PEG) spacer and 1,1'-carbonyl-diimidazole (CDI)-mediated conditions. The diameter of the β-glucan-MUC1 nanoparticles was in the 150 nm range (actual average 162 nm), while unmodified β-glucan formed particles approximately 540 nm in diameter. The β-glucan-MUC1 conjugates induced significantly higher titers of anti-MUC1 IgG antibodies compared with the control group. Further analysis of the isotype and subtype of the antibodies produced showed that IgG2b was the predominant subtype, indicating activation of a Th1-type response, as the IgG2b / IgG1 ratio was greater than 1. The significant amount of IgM antibodies observed indicates the involvement of the C3 component of the complement system, which often induces cytotoxicity, which can be problematic for the use of such scaffolds for vaccines, e.g., for chronic or degenerative diseases, where the occurrence of cytotoxicity should be avoided.

[0059] US 2013 / 171187 A1 discloses an immunogenic composition comprising glucan and a pharmaceutically acceptable carrier for eliciting protective anti-glucan antibodies. Metwali et al. (Am. J. Respir. Crit. Care Med. 185 (2012), A4152; poster session C31 Regulation of Lung Inflammation) investigate the immunomodulatory effects of glucan derivatives in pulmonary inflammation. WO 2021 / 236809 A2 discloses a multi-epitope vaccine containing amyloid-beta and tau peptides for treating Alzheimer's disease (AD). US 2017 / 369570 A1 discloses a β-(1,6)-glucan linked to an antibody against cells present in the tumor microenvironment. US 2002 / 077288 A1 discloses synthetic immunogenic but non-amyloidogenic peptides homologous to amyloid beta, alone or conjugated, for treating AD. US 2013 / 171187 A1 discloses anti-glucan antibodies used as protective agents against fungal infections caused by Candida albicans. WO 2004 / 012657 A2 discloses particulate β-glucan as a vaccine adjuvant. CN 113616799 A discloses a vaccine vector composed of oxidized mannan and a cationic polymer. CN 111514286 A discloses a Zika virus E protein conjugate vaccine containing glucan. US 4,590,181 A discloses a viral antigen mixed in solution with pustulan or mycodextran. Lang et al. (Front. Chem. 8 (2020):284) reviewed carbohydrate conjugates in vaccine development. Larsen et al. (Vaccines 8 (2020):226) reported that pustulan activated chicken bone marrow-derived dendritic cells in vitro and ex vivo CD4 agonists against infectious bronchitis virus. +It has been reported that the use of glucans, preferably laminarin and curdlan, as antigens conjugated to adjuvants for immunization against fungi promotes T cell recall responses. US 2010 / 266626 A1 discloses glucans, preferably laminarin and curdlan, as adjuvant-bound antigens for fungal immunization. Mandler et al. (Alzheimer's Disease. 15 (2019), 1133-1148) reported the efficacy of mono- and combination immunotherapy approaches targeting amyloid-beta protein and α-synuclein in a Lewy body dementia-like model. Mandler et al. (Acta Neuropathol. 127 (2014), 861-879) reported a next-generation active immunization approach for synucleinopathies using short immunogenic (B cell response) peptides that are too short to induce T cell responses (autoimmunity), do not contain natural epitopes, but rather have sequences that mimic the original epitopes (e.g., oligomeric α-synuclein), and its impact on Parkinson's disease (PD) clinical trials. Mandler et al. (Mol. Neurodegen. 10 (2015), 10) reported that active immunization against α-synuclein ameliorates degenerative pathology and prevents demyelination in a model of multiple system atrophy (MSA). Jin et al. (Vaccine 36 (2018), 5235-5244) reviewed β-glucans as potential immunoadjuvants, primarily in terms of adjuvanticity, structure-activity relationships, and receptor recognition properties. WO 2022 / 060487 A1 discloses vaccines containing specific α-synuclein peptides for treating neurodegenerative diseases. WO 2022 / 060488 A1 discloses multi-epitope vaccines containing amyloid-beta and α-synuclein peptides for treating AD. US 2009 / 169549 A1 discloses conformers of modified versions of alpha-synuclein made by introducing cysteines into the alpha-synuclein polypeptide and scrambling disulfide bonds to form stable immunogenic isomers. WO 2009 / 103105 A2 discloses vaccines having mimotopes of the alpha-synuclein epitope stretching from amino acid D115 to amino acid N122 in the native alpha-synuclein sequence.

[0060] To date, there have been no published reports demonstrating the construction or use of individual B-cell or T-cell epitope peptides linked to β-glucans, particularly linear β-glucans and / or pustulans, with high binding specificity / capacity for Dectin-1, thereby forming novel gluconeoconjugates as proposed in the present application. Summary of the Invention [Problem to be solved by the invention]

[0061] It is therefore an object of the present invention to provide an improved vaccine in the form of a conjugate vaccine composed of a vaccinating antigen conjugated to a carbohydrate-based CLEC adjuvant, in particular to provide a vaccine that provides an improved immune response in vaccinated individuals compared to current prior art conjugate vaccines, in particular carbohydrate-based CLEC-peptide / protein conjugate vaccines.

[0062] It is a further object of the present invention to provide vaccine compositions that immunize against short, easily exchangeable, highly specific B-cell / T-cell epitopes using CLEC scaffolds with efficacy, specificity, and affinity previously unmet by conventional vaccines.

[0063] A particular object of the present invention is to provide vaccines with improved selectivity and / or specificity of CLEC-based vaccines for the dermal compartment.

[0064] Another object of the present invention is to provide a vaccine that induces, as exclusively as possible, a target-specific immune response, but does not induce a CLEC- or carrier protein-specific antibody response, or only induces a very limited CLEC- or carrier protein-specific antibody response.

[0065] It is a further object of the present invention to provide vaccine compositions that immunize against short, easily exchangeable, highly specific B-cell / T-cell epitopes of alpha-synuclein using a CLEC scaffold with efficacy, specificity, and affinity previously unmet by conventional vaccines to adequately prevent and treat synucleinopathies.

[0066] A particular object of the present invention is to provide an alpha-synuclein vaccine with improved selectivity and / or specificity of CLEC-based vaccines for the dermal compartment.

[0067] Another object of the present invention is to provide a vaccine that induces, as exclusively as possible, an alpha-synuclein-specific immune response, but does not induce a CLEC- or carrier protein-specific antibody response, or induces only a very limited CLEC- or carrier protein-specific antibody response.

[0068] Another object of the present invention is to provide peptide immunogenic constructs of alpha-synuclein protein (aSyn) and formulations thereof for the treatment of synucleinopathies. [Means for solving the problem]

[0069] Accordingly, the present invention provides a β-glucan for use as a C-type lectin (CLEC) polysaccharide adjuvant for a B cell and / or T cell epitope polypeptide, preferably the β-glucan is covalently conjugated to a B cell and / or T cell epitope polypeptide to form a conjugate between the β-glucan and the B cell and / or T cell epitope polypeptide, and the β-glucan is a predominantly linear β-(1,6)-glucan in which the ratio of β-(1,6)-linked monosaccharide moieties to non-β-(1,6)-linked monosaccharide moieties is at least 1:1, preferably at least 2:1, more preferably at least 5:1, and particularly at least 10:1.

[0070] The present invention successfully solves one or more of the above-listed objects, which was unexpected for those skilled in the art, since to date, there have been no reports published in the art demonstrating the construction and applicability or efficacy of compounds similar to the novel, small, modular gluconeoconjugates of the present invention.

[0071] It has now been surprisingly shown that conjugation (i.e., covalent linkage; used synonymously herein) of peptides / proteins to selected CLEC-carriers according to the invention (linkage may be based on prior art chemistry) results in superior pharmaceutical formulations for generating immune responses. A considerable number of different linkage methods are available in the art. In the course of establishing the present invention, linkage via hydrazone formation or heterobifunctional linkers was identified as a particularly preferred method. Generally, activation of the CLEC prior to conjugation (e.g., formation of a reactive aldehyde on the vicinal OH group of the sugar moiety) and the presence of a reactive group on the selected peptide / protein (e.g., an N- or C-terminal hydrazide residue, an SH group (e.g., via an N- or C-terminal cysteine)) are required. The reaction may be a one-step reaction (e.g., mixing an activated CLEC with a hydrazide-peptide, resulting in hydrazone formation) or a multi-step process (e.g., reacting an activated CLEC with a hydrazide from a heterobifunctional linker, followed by linking the peptide / protein via their respective reactive groups).

[0072] Thus, the components of the conjugates according to the invention may be directly linked to each other, for example by linking the B-cell epitope and / or T-cell epitope to the β-glucan and / or carrier protein, or by linking the β-glucan to the carrier protein (in all possible orientations). Reference to a "B-cell epitope polypeptide" or a "T-cell epitope polypeptide" herein by default means the B-cell or T-cell epitope of the "B-cell epitope polypeptide" or "T-cell epitope polypeptide," and does not mean the B-cell or T-cell epitope of the carrier protein (if any), unless the B-cell or T-cell epitope of the carrier protein is explicitly mentioned. According to a preferred embodiment, the B-cell epitope and / or the T-cell epitope is preferably linked via a linker, more preferably a cysteine ​​residue or a linker comprising a cysteine ​​or glycine residue, a linker resulting from hydrazide-mediated linkage, a linker resulting from linkage via a heterobifunctional linker such as N-β-maleimidopropionic acid hydrazide (BMPH), 4-[4-N-maleimidophenyl]butyric acid hydrazide (MPBH), N-[ε-maleimidocaproic acid) hydrazide (EMCH) or N-[κ-maleimidoundecanoic acid] hydrazide (KMUH), The β-glucan or mannan and / or carrier protein is linked to a linker resulting from imidazole-mediated coupling, a linker resulting from reductive amination, a linker resulting from carbodiimide coupling, an -NH-NH linker; a peptide linker such as an NRRA linker, an NRRA-C linker, or an NRRA-NH-NH linker, a dimeric, trimeric, tetrameric (or higher) peptide group such as CG or CG, or a cleavage site such as a cathepsin cleavage site; or a combination thereof, in particular a cysteine ​​linker or an NRRA-NH-NH linker. It is clear that "a linker resulting from (for example) hydrazide-mediated coupling" refers to the resulting chemical structure of the conjugate after coupling, i.e., the one present in the resulting conjugate after coupling.The amino acid linker may be present in conjugate form by a peptide bond (eg, with a glycine-containing linker) or through a functional group of the amino acid (such as a disulfide bond in the case of a cysteine ​​linker).

[0073] The novel class of conjugates according to the invention has been found to confer immunization with short, easily exchangeable, highly specific B / T cell epitopes by using the CLEC scaffold of the invention, which exhibit potency, specificity, and affinity previously unmet by conventional vaccines. Indeed, the conjugates according to the invention represent the first example of the use of short B / T cell epitopes in a CLEC-based vaccine, circumventing the need to present epitopes in the form of fusion proteins involving the formation of tandem repeats of the epitope or the fusion of different tandem repeats to form stable and effective immunogens (as required, for example, for the mannan-based MUC1 approach mentioned above).

[0074] The present invention also avoids the need to use full-length proteins (i.e., payloads in glucan particles (GPs)) for use in CLEC vaccines. Furthermore, when using CLECs, the problem of autoimmune responses induced by (undesirable) T cell epitopes present in immunogens, particularly self-proteins (e.g., T cell epitopes of aSyn, amyloid beta, etc.) or mixed self-epitopes (e.g., MUC1-tandem repeats used as immunogens), can also be avoided.

[0075] According to the present invention, for the first time, short epitopes (B-cell and / or T-cell epitopes, mainly peptides, modified peptides) can be integrated into functional CLEC-based scaffolds using covalent linkages based on well-established chemistries, where possible linkage methods can be adapted to the requirements of a particular epitope based on methods well known in the art.

[0076] The presentation of short peptides according to the invention can be carried out as individually linked moieties in combination with individual foreign T cell epitopes (as short peptides or long proteins) or as complexes / conjugates with larger carrier molecules that provide the T cell epitopes for inducing a sustainable immune response. The design of vaccines according to the invention allows for the preparation of multivalent conjugates as a prerequisite for efficient immune response induction through highly efficient B cell receptor (BCR) cross-linking.

[0077] Furthermore, the present invention can provide CLEC-based vaccines with excellent selectivity / specificity for the dermal compartment. Indeed, the conjugate design according to the present invention is based on CLECs as carriers for target-specific epitopes that exhibit high binding specificity for dermal APCs / DCs, in particular PRRs on Dectin-1 (or MR and DC-SIGN in the case of mannan), to enable skin-selective / skin-specific uptake and receptor-mediated uptake (=targeted vaccine delivery).

[0078] The CLEC polysaccharides used as carriers according to the present invention are preferably used to concentrate carrier-peptide conjugates in dermal / skin DCs and initiate immune responses. This is due, inter alia, to their epidermal or dermal (but not subcutaneous) specificity. The CLEC scaffold and efficient dermal immune response initiation according to the present invention also help to avoid the mandatory use of adjuvants (e.g., the use of alum, MF59, CFA, PolyI:C, or other adjuvants) that are typical of conventional vaccines and are also used in exemplary CLEC-based vaccines. According to preferred embodiments of the present invention, the use of adjuvants can be significantly reduced or omitted, for example, in situations where the addition of an adjuvant is not indicated.

[0079] Although several CLECs have been used in previous applications, none of the proposed conjugate structures were able to confer skin selectivity (i.e., high Dectin-1 binding capacity, highly efficient dermal DC targeting, and superior immunogenicity for dermal administration compared to all other routes (i.e., subcutaneous, intramuscular, and i.p.)).

[0080] The selection of CLECs according to the present invention was undertaken to provide a novel solution for targeting skin-specific DCs and skin-specific immunization with high efficacy. The conjugates according to the present invention also exhibit limited activity in other classical tissues for immunization, such as muscle or subcutaneous tissue, which is in contrast to previous CLEC-based vaccines / vaccine candidates that have been administered im or sc. As a result of experiments conducted during the course of the present invention, vaccines according to the present invention, particularly vaccines using pustulan as a CLEC, were surprisingly identified as selective for skin immunization.

[0081] The present invention relates to any B cell and / or T cell epitope polypeptide and any predominantly linear β-(1,6)-glucan in which the ratio of (1,6)-linked to non-(1,6)-linked monosaccharide moieties is at least 1:1. As also shown in the Examples section below, the present teachings enable and provide support for any B cell and / or T cell epitope polypeptide, and no limitations regarding epitopes were apparent, particularly if such epitopes are already part of prior art and / or established epitopes. While the specific B cell and / or T cell epitope polypeptides shown and referred to herein are preferred epitopes, the present invention is not limited thereto. During the course of this invention, and following the large number of epitopes tested to date (see the Examples section for a highly diverse set of functionally and structurally relevant epitopes (including a significant number of model epitopes) experimentally investigated and validated), no limitations were placed on the nature and structure of the B-cell and / or T-cell epitopes (linear polypeptides, self-peptides, polypeptides with glycostructures or post-translational modifications such as pyroglutamic acid, mimotopes, allergens, structural epitopes, etc.), particularly for pustulan as a β-(1,6)-glucan. In each case, it was experimentally shown that it is the covalent binding to the β-glucan and epitope polypeptides according to the invention that is responsible for the immunological performance, and not the specific structural features of the individual epitopes.

[0082] The term "B cell and / or T cell epitope polypeptide" as used herein is a functional term accepted in the art. That is, T cell epitopes are presented on the surface of antigen-presenting cells, where they are bound to major histocompatibility complex (MHC) molecules. In humans, professional antigen-presenting cells are specialized to present MHC class II peptides, while most nucleated somatic cells present MHC class I peptides. T cell epitopes presented by MHC class I molecules are typically peptides 8-11 amino acids long, while MHC class II molecules present peptides longer than 13-17 amino acids, and non-classical MHC molecules also present non-peptide epitopes, such as glycolipids. A B cell epitope is a portion of an antigen to which an immunoglobulin or antibody binds. B cell epitopes can be, for example, structural or linear.

[0083] According to a preferred embodiment of the present invention, the conjugate comprises a polypeptide comprising at least one B cell epitope and at least one T cell epitope, preferably a B cell epitope+CRM197 conjugate covalently linked to a β-glucan, particularly a peptide+CRM197+linear β-(1,6)-glucan conjugate or a peptide+CRM197+linear pustulan conjugate. The preferred glucan-to-peptide ratio, particularly the pustulan-to-peptide ratio, is 10:1 (w / w) to 0.1:1 (w / w), preferably 8:1 (w / w) to 2:1 (w / w), and particularly 4:1 (w / w). However, when the conjugate comprises a carrier protein, the preferred ratio of β-glucan or mannan to B cell epitope-carrier polypeptide is 50:1 (w / w) to 0.1:1 (w / w), particularly 10:1 to 0.1:1.

[0084] The present invention allows for the focus on inducing target-specific immune responses that do not induce CLEC- or carrier protein-specific antibody responses or induce only very limited CLEC- or carrier protein-specific antibody responses. Thus, the conjugates of the present invention solve the problems posed by classical conjugate vaccines, which must rely on the use of exogenous carrier proteins to induce sustainable immune responses. Current prior art conjugate vaccine development has focused on targeting, for example, Her2-neu positive cancers, α-synuclein for synucleinopathies such as Parkinson's disease, amyloid beta peptide for amyloidosis such as Alzheimer's disease, tau for treating tauopathies including Alzheimer's disease, PCSK9 for hypercholesterolemia, IL23 for psoriasis, TDP43 and FUS for frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), (mutant) huntingtin for Huntington's disease, immunoglobulin G1 (IG1), and IL-16 for psoriasis. These are strongly based on carrier molecules such as KLH, CRM197, tetanus toxoid, or other suitable proteins conjugated to target-specific short antigens that deliver substrates for immune responses against various target diseases such as infectious, degenerative, or neoplastic diseases, including immunoglobulin heavy chain amyloidosis and immunoglobulin heavy chain amyloidosis (AL, AH, AA), islet amyloid polypeptide (IAPP) and amylin for type 2 diabetes, (mutant) transthyretin for ATTR / transthyretin amyloidosis, etc.

[0085] The immunological performance and efficiency of the conjugates according to the invention and vaccines comprising these conjugates are also unexpected and surprising in view of the prior art guidance in which β-glucans, particularly primarily linear β-(1,6)-glucans, have been primarily used as antigens themselves to induce specific immune responses against fungi in which such β-glucans are present (e.g., US 2013 / 171187 A1; Metwali et al., Am. J. Respir. Crit. Care Med. 185 (2012), A4152; poster session C31; US ​​2013 / 171187 A1, US 2010 / 266626 A1, Jin et al. (Vaccine 36 (2018), 5235-5244)). However, the present invention demonstrates that although the conjugates according to the present invention are unable to induce a significant immune response against β-glucan, the structure of the conjugates does shift the immune response to the B cell and / or T cell epitope polypeptides covalently linked to the β-glucan. Conjugating these B cell and / or T cell epitope polypeptides to linear β-glucan appears to mask the ability of the β-glucan to induce an immune response, but exposes the covalently linked B cell and / or T cell epitope polypeptides to the immune system, significantly improving their presentation to the immune system. This teaching was not disclosed in the prior art, nor was it obvious from such prior art.

[0086] US 2017 / 369570 A1 discloses that β-(1,6)-glucan conjugated to antibodies directed against cells present in the tumor microenvironment is based on an entirely different concept and mechanism (tumor therapy).

[0087] On the other hand, glucans have been used as components in vaccines (mainly as "(liposomal) glucan (nano)particles"), but not by covalently linking B cell and / or T cell epitope polypeptides to the glucans (e.g., WO 2004 / 012657 A2, CN 113616799 A, US 4,590,181 A, Lang et al., Front.Chem.8 (2020):284; Larsen et al., Vaccines 8 (2020):226).

[0088] Finally, the improved effect of the predominantly linear β-(1,6)-glucan according to the present invention over constructs and compositions according to WO 2022 / 060487 A1, WO 2022 / 060488 A1, US 2009 / 169549 A1, WO 2009 / 103105, and CN 111514286 A (such as β-(1,2)-glucan or β-(1,3)-glucan) is demonstrated in the Examples section below.

[0089] Considering these advantageous properties of the conjugates of the invention, the conjugates and vaccines according to the invention will be particularly usable for vaccination with active anti-tau proteins, including also truncated, (hyper)phosphorylated, nitrated, glycosylated and / or ubiquitinated variants, for the treatment and prevention of tauopathies, in particular Alzheimer's disease and Down's syndrome, or other tauopathies, including Pick's disease, progressive supranuclear palsy (PSP), corticobasal degeneration, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), and argyrophilic grain dementia. Other emerging entities and pathologies include globuloglial tauopathies, primary age-associated tauopathies (PARTs) (including neurofibrillary tangle dementia), chronic traumatic encephalopathy (CTE), and age-associated tauastrogliopathy. Additionally, other entities such as vacuolar tauopathies, gangliogliomas and gangliocytomas, Ritiko-Bodig disease (Parkinson-dementia complex of Guam), meningioangiomatosis, postencephalitic parkinsonism, and subacute sclerosing panencephalitis (SSPE) are also included.

[0090] Tauopathies often overlap with synucleinopathies, likely due to the interaction between synuclein and tau proteins. Thus, the anti-tau conjugates according to the invention are particularly usable for active anti-tau vaccination against synucleinopathies, particularly Parkinson's disease (PD), dementia with Lewy bodies (DLB), and Parkinson's disease dementia (PDD).

[0091] Anti-tau vaccines can be highly effective when used alone or in combination with existing peptide vaccines against other pathological molecules involved in β-amyloidosis, tauopathy, or synucleinopathy, especially with mixed pathology (i.e., the presence of Aβ pathology with tau pathology and / or aSyn pathology). Thus, a preferred embodiment provides a combination of an anti-tau vaccine with an anti-Aβ vaccine and / or an anti-aSyn peptide vaccine for treating degenerative diseases such as Alzheimer's disease, dementia in Down's syndrome, dementia with Lewy bodies, Parkinson's disease dementia, and Parkinson's disease.

[0092] According to a preferred embodiment, the polypeptide derived from the tau protein is selected from the group consisting of tau2-18, tau176-186, tau181-210, tau200-207, tau201-230, tau210-218, tau213-221, tau225-234, tau235-246, tau251-280, tau256-285, tau259-288, tau275-304, tau260-264, tau267-273, tau294-308, tau295-309, tau296-310, tau297-311, tau298-312, tau299-313, tau299-314, tau299-316, tau299-318, tau299-319, tau299-320, tau299-321, tau299-322, tau299-323, tau299-324, tau299-325, tau299-326, tau299-328, tau299-329, tau299-330, tau299-331, tau299-332, tau299-333, tau299-334, tau299-335, tau299-336, tau299-337, tau299-338, tau299-339, tau299-3 05, native human tau (441 aa isoform; GenBank entry >AAC04279.1; SEQ ID NO: 1), including tau298-304, tau300-317, tau329-335, tau361-367, tau362-366, tau379-408, tau389-408, tau391-408, tau393-402, tau393-406, tau393-408, tau418-426, and tau420-426.

[0093] [ka]

[0094] or a polypeptide comprising or consisting of amino acid residues derived from human tau, including post-translationally modified, phosphorylated, dually phosphorylated, hyperphosphorylated, nitrated, glycosylated, and / or ubiquitinated amino acids.

[0095] According to a preferred embodiment, the tau protein derived polypeptide is selected from the mimetics of the above tau derived polypeptides, including mimotopes and peptides containing amino acid substitutions that mimic phosphorylated amino acids, including substitution of phosphorylated S with D and phosphorylated T with E, including tau176-186, tau200-207, tau210-218, tau213-221, tau225-234, tau379-408, tau389-408, tau391-408, tau393-402, tau393-406, tau418-426, tau420-426, respectively.

[0096] US 2008 / 050383 A1 and Asuni et al. (Journal of Neuroscience 34:9115-9129) disclose antibodies against tau 379-408, which has two phosphorylated aas, pS396 and pS404, suitable for immunotherapy against tau pathology, and Boutajangout et al. (J.Neurosci., December 8, 2010 30(49):16559-16566) disclose the use of the dual-phosphorylated polypeptide tau 379-408, which has the same epitopes, pSp396 and pS404, in combination with AdjuPhos, an effective adjuvant as active immunotherapy to prevent cognitive decline in several studies in the htau / PS1 model, which is associated with a reduction in pathological tau in the brain. Bi et al. (2011, PLoS One 12:e26860) also showed that targeted tau immunization using a 10-mer polypeptide derived from dual-phosphorylated contiguous tau 395-406 (harboring pS396 and pS404) conjugated to KLH and adjuvanted with complete or incomplete Freund's adjuvant prevented the progression of neurofibrillary histopathology in aged P301L tau transgenic mice.

[0097] Boimel M et al. (2010; Exp Neurol 2:472-485) showed that the use of the doubly phosphorylated peptides tau 195-213 [pS202 / pT205], tau 207-220 [pT212 / pS214], and tau 224-238 [pT231] emulsified in complete Freund's adjuvant (CFA), along with pertussis toxin, resulted in the attenuation of tau-associated pathology in animals.

[0098] Troquier et al. (2012 Curr Alzheimer Res 4:397-405) showed that targeting tau by active tau immunotherapy using artificial peptide constructs consisting of an N-terminal YGG linker fused to a CFA-adjuvanted human tau-derived 7-mer (tau 418-426) or 11-mer (tau 417-427) peptide with pS422 linked to KLH in the THY tau22 mouse model may be a suitable therapeutic approach, as a detectable reduction in insoluble tau species (AT100 immunoreactivity and pS422 immunoreactivity) correlated with significant cognitive improvement using the Y-maze.

[0099] US 2015 / 0232524 A1 and Davtyan H et al. (Sci Rep. 2016;6:28912, Vaccine. 2017;35:2015-24 and Alzheimer's Research & Therapy (2019) 11:107) and Joly-Amado et al. (Neurobiol Dis. 2020 February ;134:104636) disclose peptide immunogens and have shown that vaccines AV-1980R and AV-1980D based on the MultiTEP platform, consisting of three repeats of tau 2-18 fused to several promiscuous T cell epitopes as recombinant polypeptide or DNA vaccines, respectively, induce strong immune responses and reduce tau pathology in various tauopathy models.

[0100] EP 3 097 925 B1 discloses a peptide immunogen consisting of a phosphopeptide derived from human tau 441, and Theunis et al. (2013, PLoS ONE 8(8):e72301) based on EP 3 097 925 B1 demonstrates a liposomal vaccine containing the tau peptide tau 393-408 (containing pS396 and pS402) that is able to induce anti-phosphotau antibodies in the brains of tau.P301L mice, which is accompanied by an improvement in the clinical condition and a reduction in indicators of tauopathy.

[0101] Sun et al. (Signal Transduction and Targeted Therapy (2021) 6:61) disclosed various immunogens based on norovirus P particles. The vaccine pTau31 (consisting of particles containing fusion peptides of tau 195-213 with pS202 and pT205, and tau 395-406 with pS396 and pS404) generated robust pTau antibodies, significantly reduced tau pathology, and improved behavioral impairment in a tau Tg animal model.

[0102] EP 2 758 433 B1 discloses peptide-based immunogens for disrupting tau pathology. The present invention discloses their use as peptide conjugate vaccines (e.g., as peptide KLH vaccines). Kontsekova et al. (Alzheimer's Research & Therapy 2014, 6:44) disclose that such a peptide vaccine (i.e., axonal peptide 108 (tau 294-305; KDNIKHVPGGGS); also known as AADvac1) conjugated to KLH and adjuvanted with alum induced a robust protective humoral immune response with antibodies that distinguish between pathological and physiological tau. Active immunotherapy reduced the level of tau oligomers and the extent of neurofibrillary pathology in the brains of transgenic rats.

[0103] Although in principle the present invention can improve all suggested tau vaccination polypeptides, selected epitopes were specifically evaluated for their compatibility with the present platform. For example, tau 294-305, SEQ ID NO: 35+36, was shown to be superior to KLH-based vaccines as suggested in EP2 758 433 B1 and Kontsekova et al.

[0104] Further preferred target sequences include:

[0105] [Table 1]

[0106] [Table 2]

[0107] Considering these advantageous properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention can be particularly used for active immunotherapy of IL12 / IL23-associated diseases and autoimmune inflammatory diseases, which are selected from the group consisting of psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, diabetes, preferably type 1 diabetes, atherosclerosis, inflammatory bowel disease (IBD) / M. Crohn's disease, multiple sclerosis, Behçet's disease, ankylosing spondylitis, Vogt-Koyanagi-Harada disease, chronic granulomatous disease, hidradenitis suppurativa, antineutrophil cytoplasmic antibody (ANCA-) associated vasculitis, neurodegenerative diseases, preferably M. Alzheimer's disease or multiple sclerosis, atopic dermatitis, graft-versus-host disease, cancer, preferably esophageal cancer, colorectal cancer, lung adenocarcinoma, small cell carcinoma, and oral squamous cell carcinoma, in particular psoriasis, neurodegenerative diseases, or IBD. Furthermore, recent data suggest that IL-23-driven inflammation may exacerbate other diseases such as Alzheimer's disease or possibly diabetes, so vaccines against IL-12 / 23 could be used together / in combination with vaccines against other targets.

[0108] According to a preferred embodiment, the IL12 / IL23 protein-derived polypeptide is derived from native human IL12 / IL23 or is a mimetic with one or more aa exchanges forming a mimotope of the respective native sequence.

[0109] According to a preferred embodiment, the IL12 / IL23 protein-derived polypeptide is selected from the heterodimeric protein IL23, a subunit of native human IL-23p19, or a polypeptide comprising or consisting of amino acid residues derived from this subunit or mimotope. In WO 2005 / 108425 A1, peptides derived from IL-23p19, FYEKLLGSDIFTGE, FYEKLLGSDIFTGEPSLLPDSP, VAQLHASLLGLSQLLQP, GEPSLLPDSPVAQLHASLLGLSQLLQP, PEGHHWETQQIPSLSPSQP, PSLLPDSP, LPDSPVAQLHASLLGLSQLLQP, LLPDSP, LLGSDIFTGEPSLLPDSPVAQLHASLLG, FYEKLLGSDIFTGEPSLLPDSPVAQLHASLLG, QPEGHHW, LPDSPVGQLHASLLGLSQLLQ, and QCQQLSQKLCTLAWSAHPLV, were proposed as vaccination peptides against IL-23. WO 03 / 084979 A2 mentioned GHMDLREEGDEETT, LLPDSPVGQLHASLLGLSQ, and LLRFKILRSLQAFVAVAARV derived from IL-23p19 as possible anti-cytokine vaccines. WO 2016 / 193405 A1 mentioned the following amino acid sequences as possible anti-cytokine vaccines: [ka] In particular, we disclose peptide immunogens derived from the IL12 / 23 p19 subunit (Accession Number: Q9NPF7) having aa 136-145, aa 136-143, aa 136-151, aa 137-146, aa 144-154, aa 144-155, etc. thereof, in particular the sequences: QPEGHHWETQQIPSLS, GHHWETQQIPSLSPSQPWQRL, QPEGHHWETQ, TQQIPSLSPSQ, QPEGHHWETQQIPSLSPSQ, QPEGHHWETQQIPSLSPS.

[0110] According to a preferred embodiment, the polypeptide derived from the IL12 / IL23 protein is selected from the heterodimeric protein IL23, a subunit of native human IL12 / 23p40, or a polypeptide comprising or consisting of amino acid residues aa 15-66, aa 38-46, aa 53-71, aa 119-130, aa 160-177, aa 236-253, aa 274-285, aa 315-330 of native human IL12 / 23p40 (Accession Number: P29460.1) having the following amino acid sequence: [ka]

[0111] In WO 03 / 084979 A2, the peptides LLLHKKEDGIWSTDILKDQKEPKNKTFLRCE and KSSRGSSDPQG derived from the IL-12 / 23 p40 subunit were mentioned as possible anti-cytokine vaccines.

[0112] Luo et al. J Mol Biol 2010 Oct 8;402(5):797-812. discloses the structural epitope-aa15-66 of the anti-IL12 / IL23p40-specific antibody ustekinumab, which effectively reduces IL-12 (IL-23)-associated diseases. Guan et al. (Vaccine 27 (2009) 7096-7104) discloses immunogens of murine IL12 / 23 accession numbers: P43432(p40) and Q9EQ14(p19) having the following amino acid sequences recombinantly linked to HBcAg: P43432(p40), aa38-46, aa53-71, aa119-130, aa160-177, aa236-253, aa274-285, aa315-330. [ka]

[0113] Although in principle the present invention can improve all suggested IL12 / IL23-associated disease vaccination polypeptides, selected epitopes were specifically evaluated for their compatibility with the present platform. For example, the WISIT vaccines of SEQ ID NOs: 37 / 38 and 41 / 42 were shown to be superior to KLH-based vaccines. The murine sequences SEQ ID NOs: 39 / 40 showed similar efficacy to KLH-based conjugates in mice and were also active in IL12 / IL23 recognition.

[0114] Given these advantageous properties of the conjugates of the invention, the conjugates and vaccines according to the invention are particularly usable for active anti-EMPD (outer membrane proximal domain as part of membrane IgE-BCR) vaccination to treat and prevent IgE-related diseases. Exclusive targeting and cross-linking of membrane IgE-BCR was achieved by addressing the membrane-anchoring region found only on membrane-IgE and not on soluble serum IgE (the extracellular membrane proximal domain of IgE (EMPD IgE)). IgE-related conditions include seasonal allergies, food allergies, pollen allergies, mold spore allergies, poisonous plant allergies, medication / drug allergies, insect venom allergies, scorpion venom allergies, or spider venom allergies, latex or dust allergies, pet allergies, allergic bronchial asthma, non-allergic asthma, Churg-Strauss syndrome, allergic rhinitis and allergic conjunctivitis, atopic dermatitis, nasal polyps, Kimura's disease, contact with adhesives, antimicrobials, fragrances, hair dyes, metals, rubber components, topical medications, rosin, wax, polish, cement, and leather. These include allergic diseases such as allergic dermatitis, chronic sinusitis, atopic eczema, autoimmune diseases in which IgE plays a role ("autoallergies"), chronic (idiopathic) urticaria and autoimmune urticaria, cholinergic urticaria, mastocytosis, particularly cutaneous mastocytosis, allergic bronchopulmonary aspergillosis, chronic idiopathic angioedema or recurrent idiopathic angioedema, interstitial cystitis, anaphylaxis, particularly idiopathic anaphylaxis and exercise-induced anaphylaxis, immunotherapy, eosinophilic asthma, eosinophilic gastroenteritis, eosinophilic otitis media, and eosinophil-related diseases such as eosinophilic esophagitis (see, e.g., Holgate 2014 World Allergy Organ. J. 7:17., US 8,741,294 B2). Furthermore, the vaccine or conjugate according to the invention is used in the treatment of lymphoma or in the prevention of sensitization side effects of acid-reducing treatment, especially for gastric or duodenal ulcers or reflux. In the present invention, the term "IgE-associated disease" includes or is used synonymously with the terms "IgE-dependent disease" or "IgE-mediated disease".

[0115] According to a preferred embodiment, the EMPD protein-derived polypeptide is derived from the native human IgE-BCR or is a mimetic with one or more aa exchanges forming a mimotope of the respective native sequence.

[0116] The development of dedicated antibodies specifically targeting human or mouse EMPD IgE has enabled clinical and preclinical validation of this targeting strategy in vitro and in vivo (Liour et al., 2016 Pediatr Allergy Immunol Aug;27(5):446-51). The concept of IgE-BCR cross-linking was first demonstrated in vivo by passive administration of anti-EMPD IgE antibodies in wild-type mice (Feichtner et al., 2008 J. Immunol. 180:5499-5505) and in a dedicated mouse model harboring a partially humanized IgE EMPD region (Brightbill et al., 2010 J. Clin. Invest. 120:2218-2229). Chen et al. (2010 Journal of Immunology 184, 1748-1756) demonstrated that mAbs specific for the N-terminal or central segments of CemX can bind to mIgE-expressing B cells and effectively induce their apoptosis and ADCC. CemX refers to the human membrane-bound e chain. This isoform contains an extra 52-aa residue domain located between the CH4 domain and the C-terminal membrane-anchoring peptide, referred to as CemX or M1' peptide. This was specifically demonstrated with antibodies against the CemX N-terminal segment P1 (SVNPGLAGGSAQSQRAPDRVL, where SVNP represents the C-terminal 4 aa residues of the CH4 domain of m) and the central segment P2 (HSGQQQGLPRAAGGSVPHPR), whereas the C-terminal P3 (GAGRADWPGPP) was unsuccessful.

[0117] Furthermore, antibodies generated by active immunization against the human EMPD IgE region were able to mediate apoptosis and ADCC in vitro (Lin et al., Mol. Immunol., 52 (2012), pp. 190-199). Lin et al. disclosed immunogens using CemX or HBcAg containing inserts of its P1, P2, and P1-P2 portions as anti-EMPD vaccines.

[0118] Kirizumab, the first clinical anti-human EMPD IgE monoclonal antibody, demonstrated selective IgE suppression in healthy volunteers combined with clinical benefits in patients with allergic rhinitis and mild asthma in phase I and II trials, respectively (Scheerens et al., 2012 Asthma Therapy: Novel Approaches: p.A6791; Gauvreau et al., 2014 Sci.Transl.Med. 6, 243ra85.), but failed to improve clinical outcomes in patients with severe bronchial asthma (Harris et al., 2016 Respir.Res. 17:29.). The epitope of kiruizumab, which also serves as a potential immunogen, is located within the 11-residue segment SAQSQRAPDRV of CemX.

[0119] WO 2017 / 005851 A1 and Vigl et al. (Journal of Immunological Methods 449 (2017) 28-36) disclose peptides that are located in the membrane proximal domain of EMPD in combination with a suitable protein carrier as active anti-EMPD immunogens. The disclosed sequences include AVSVGLAGGSAQSQRAPDRVLCHSGQQQGLPRAAGGSVP, QQQGLPRAAGG, QQLGLPRAAGG, QQQGLPRAAEG, QQLGLPRAAEG, QQQGLPRAAG, QQLGLPRAAG, QQQGLPRAAE, QQLGLPRAAE, HSGQQQGLPRAAGG, HSGQQLGLPRAAGG, HSGQQQGLPRAAEG, HSGQQLGLPRAAEG, QSQRAPDRVLCHSG, GSAQSQRAPDRVL, and WPGPPELDV.

[0120] Although in principle the present invention can improve all suggested IgE-related disease vaccination polypeptides, selected epitopes were specifically evaluated for their compatibility with the present platform. For example, SEQ ID NO: 43 / 44 (QQQGLPRAAGG) was shown to be superior to KLH-based vaccines.

[0121] Given these advantageous properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention are particularly usable for active anti-human epidermal growth factor receptor 2 (anti-Her2) vaccination to treat and prevent Her2-positive neoplastic diseases. Amplification or overexpression of Her2 occurs in approximately 15-30% of breast cancers and 10-30% of gastric / gastroesophageal cancers and serves as a prognostic and predictive biomarker. Her2 overexpression has also been found in other cancers, such as ovarian cancer, endometrial cancer, and uterine serous endometrial cancer, cervical cancer, bladder cancer, lung cancer, colon cancer, and head and neck cancer.

[0122] According to a preferred embodiment, the Her2 protein-derived polypeptide is derived from native human Her2 or is a mimetic with one or more aa exchanges forming a mimotope of the respective native sequence.

[0123] Dakappagari et al. (JBC (2005) 280, 1, 54-63) described a structural epitope derived from the measles virus fusion protein MVF (amino acids 288-302) that was synthesized colinearly with a promiscuous TH epitope cyclized by a disulfide bridge. The peptide was formulated with the muramyl dipeptide adjuvant nor-MDP (N-acetylglucosamin-3-yl-acetyl-L-alanyl-D-isoglutamine) and emulsified with Montanide ISA 720. The vaccine was immunogenic, and immunization with the vaccine reduced tumor burden in tumor models.

[0124] EP 1 912 680 B1 and Allen et al. (J Immunol 2007; 179:472-482) disclose immunogens using three structural peptide constructs (aa266-296 (LHCPALVTYNTDTFESMPNPEGRYTFGASCV), aa298-333 (ACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEK), and aa315-333 (CPLHNQEVTAEDGTQRCEK)) to mimic the dimerization loop region of the receptor. The vaccine candidate also contained the MVF T cell epitope (aa288-302) KLLSLIKGVIVHRLEGVE and a GPSL linker. All peptides induced high anti-Her2 immune responses, and the construct using peptide aa266-296 was equally effective compared to Herceptin. The aa266-296 peptide of the following Her2 sequence (accession number P04626) as a vaccine statistically reduced tumor incidence in both transplantable tumor models and significantly reduced tumor incidence in two transgenic mouse tumor models. [ka]

[0125] Garret et al. (J Immunol 2007; 178:7120-7131) disclosed that Her2 peptides as immunogens aa563-598, aa585-598, aa597-626, and aa613-626 were colinearly synthesized with a promiscuous Th epitope derived from the measles virus fusion protein (aa288-302) and applied in combination with Montanide ISA 720. The vaccine was immunogenic, and immunization with the vaccine containing the aa597-626 epitope significantly reduced tumor burden in tumor models.

[0126] Jasinska et al. (Int. J. Cancer: 107, 976-983 (2003)) reported that seven peptides derived from the extracellular domain of Her2, namely, P1 aa115-132 AVLDNGDPLNNTTPVTGA, P2 aa149-162 LKGGVLIQRNPQLC, P3 aa274-295 YNTDTFESMPNPEGRYTFGAS, P4 aa378-398 PESFDGDPASNTAPLQPEQLQ, P5 aa489-504 PHQALLHTANRPEDE, P6 aa544-560 CRVLQGLPREYVNARHC, and P7 aa610-623, linked to tetanus toxoid and adjuvanted with Gerbu, induced humoral immune responses with antitumor activity in animal models. (2007 Breast Cancer Res Treat. 2007;106:29-38) disclosed peptide immunogens for immunization studies using single peptides P4 (aa378-394: PESFDGDPASNTAPLQPC), P6 (aa545-560: RVLQGLPREYVNARHC), and P7 (aa610-623: YMPIWKFPDEEGAC) linked to tetanus toxoid and adjuvanted with Gerbu. Vaccination with or without IL-12 resulted in antitumor efficacy in preclinical models. Tobias et al. 2017 (BMC Cancer (2017) 17:118) discloses peptide immunogens for immunization studies, applying single peptides P4 (aa378-394: PESFDGDPASNTAPLQP), P6 (aa545-560: RVLQGLPREYVNARHC), and P7 (aa610-623: YMPIWKFPDEEGAC) combined as hybrid peptides P467 (PESFDGDPASNTAPLQPRVLQGLPREYVNARHSLPYMPIWKFPDEEGAC) and P647 (RVLQGLPREYVNARHSPESFDGDPASNTAPLQPYMPIWKFPDEEGAC).The cysteine ​​(C) of P6 was replaced with "SLP" or "S," respectively. Both constructs were coupled to virosomes or diphtheria toxoid CRM197 (CRM) in combination with either Montanide or aluminum hydroxide (Alum) as adjuvants, and the induced antibodies displayed antitumor properties.

[0127] Riemer et al. (J Immunol 2004; 173:394-401) reported the generation of peptide mimetic of the epitope recognized by trastuzumab on Her-2 / neu using a restricted 10-mer phage display library. The peptide mimotopes were linked to the immunogenic carrier tetanus toxoid (TT) and adjuvanted with aluminum hydroxide. The sequences included C-QMWAPQWGPD-C, C-KLYWADGELT-C, C-VDYHYEGTIT-C, C-QMWAPQWGPD-C, C-KLYWADGELT-C, C-KLYWADGEFT-C, C-VDYHYEGTIT-C, and C-VDYHYEGAIT-C. Similarly, Singer et al. (ONCOIMMUNOLOGY 2016, VOL. 5, NO. 7, e1171446) disclosed mimotopes against trastuzumab epitopes predicted from an AAV-mimotope library platform. The mimotope sequences tested included RLVPVGLERGTVDWV, TRWQKGLALGSGDMA, QVSHWVSGLAEGSFG, LSHTSGRVEGSVSLL, LDSTSLAGGPYEAIE, HVVMNWMREEFVEEF, and SWASGMAVGSVSFEE. QVSHWVSGLAEGSFG and LSHTSGRVEGSVSLL were found to be immunogenic and effective in tumor models.

[0128] Miyako et al. (ANTICANCER RESEARCH 31:3361-3368 (2011)) disclosed peptides derived from the Her-2 / neu extracellular domain (aa 167-175), presented in particular in the form of Her-2 / neu-associated multiple antigenic peptides (MAPs). The Her-2 / neu peptides contained epitopes for CD4+ T cells and CD8+ T cells, which contribute to the suppressive effect on the growth of Her-2 / neu-expressing tumor cells. The disclosed sequences include:

[0129] Peptide sequence (B; t-butoxycarbonyl residue (Boc)). [ka]

[0130] Humoral immune responses were induced, tumor growth was suppressed in immunized mice, and tumor-infiltrating lymphocytes contained more CD8+ T cells and secreted large amounts of interleukin-2 after peptide restimulation.

[0131] Henle et al. (J. Immunol. 2013 January 1; 190(1):479-488) disclose peptide epitopes derived from Her2 that generate cross-reactive T cells. For the HER-2 / neu HLA-A2-binding peptide aa369-377 (KIFGSLAFL), it has been shown that cytotoxic T lymphocytes (CTLs) specific for this epitope can directly kill HER-2 / neu-overexpressing breast cancer cells. Other epitopes disclosed include the HER-2 / neu peptides p368-376, KKIFGSLAF; p372-380, GSLAFLPES; p364-373, FAGCKKIFGS; p373-382, SLAFLPESFD; p364-382, FAGCKKIFGSLAFLPESFD; and p362-384, QEFAGCKKIFGSLAFLPESFDGD. One of these sequences, p373-382 (SLAFLPESFD), binds to HLA-A2 more strongly than p369-377 and was identified as a potential epitope for vaccination.

[0132] Kaumaya et al. (ONCOIMMUNOLOGY 2020, VOL. 9, NO. 1, e1818437) discloses a combination of a Her2-targeting vaccine (aa 266-296 and aa 597-626 combined with the measles virus fusion peptide (MVF) amino acids 288-302 via a four-amino acid residue (GPSL) emulsified with Montanide ISA 720VG) and a novel PD1 immune checkpoint-targeting vaccine (PD-1 B cell peptide epitope (aa 92-110; GAISLAPKAQIKESLRAEL) combined with the viral fusion peptide (MVF) amino acids 288-302 via a four-amino acid residue (GPSL) emulsified with Montanide ISA 720VG) for the combined treatment of Her2-positive diseases. Therefore, providing a combination of an anti-neoplastic disease vaccine, particularly a cancer target-specific vaccine, with an immune checkpoint-targeting vaccine is also a preferred embodiment.

[0133] In principle, the present invention can improve all suggested Her2-associated disease vaccination polypeptides, but selected epitopes were specifically evaluated for their compatibility with the present platform. For example, SEQ ID NO: 47 / 48 (aa610-623: YMPIWKFPDEEGAC) was shown to be superior to CRM-based vaccines.

[0134] Considering these advantageous properties of the conjugates of the invention, the conjugates and vaccines according to the invention are particularly usable for personalized neoantigen-specific therapy, preferably using NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-C1, MAGE-C2, MAGE-C3, survivin, gp100, tyrosinase, CT7, WT1, PSA, PSCA, PSMA, STEAP1, PAP, MUC1, 5 T4, KRAS, or Her2.

[0135] Considering these advantageous properties of the conjugates of the invention, the conjugates and vaccines according to the invention may be particularly useful for active anti-immune checkpoint vaccination for controlling the cancer microenvironment, for the treatment and prevention of neoplastic diseases, and for the treatment and prevention of T cell dysfunction in chronic degenerative diseases, including cancer / neoplastic diseases (e.g., avoiding exhaustion of CD8 T cell infiltrated cancer tissue) and diseases with reduced T cell activity, such as Parkinson's disease.

[0136] It is well accepted in the art that PD patients suffer from various alterations in the T cell compartment compared with healthy controls (e.g., Bas et al., J Neuroimmunol 2001; 113:146-52 or Gruden et al., J Neuroimmunol 2011; 233:221-7). These phenotypic changes in T cells in PD include, for example, a decrease in absolute lymphocyte counts, a decrease in absolute and relative numbers of total T cells, a decrease in absolute and relative numbers of CD4+ lymphocytes and sometimes CD8+ lymphocytes, an increase in the Th1 / Th2 and Th17 / Treg ratios, and an increase in the expression of inflammatory cytokines. However, most of these changes are also seen during healthy aging, making it difficult to discern the impact of diseases such as PD, which exhibit a wide range of onset (approximately 30-90 years) and variable progression rates. Regarding absolute cell counts, there appears to be consensus on a net decrease in CD3+CD4+ T cells in PD. This CD4 decrease is supported by the described changes in the CD4:CD8 ratio.

[0137] Along these lines, for example, Bhatia et al. (J Neuroinflammation (2021) 18:250) demonstrated an overall decline in total CD3+ T cells in PD associated with disease severity (e.g., as measured using H+Y stage). This suggests progressive systemic T cell dysfunction with ongoing disease, likely reflecting the combined effects of ongoing inflammation, medications, and lifestyle changes. Lindestam Arlehamn et al. (2020) also demonstrated that the highest T cell activity is detectable in PD patients in the prodromal or early clinical stage (duration less than 10 years and H+Y stage 0-2).

[0138] It is therefore a preferred embodiment of the present invention to provide a treatment for enhancing or maintaining T cell numbers, particularly T effector cell numbers and T cell function in PD patients, preferably comprising a checkpoint inhibitor or vaccine combination that uses an anti-immune checkpoint inhibitor epitope to induce an anti-immune checkpoint inhibitor immune response in combination with a target-specific vaccine of the present invention to enhance or maintain T cell numbers, particularly T effector cell numbers and T cell function in PD patients.

[0139] Patients who are amenable to treatment are characterized by a global decrease in CD3+ cells at all disease stages, particularly CD3+CD4+ cells, typical of PD patients. The preferred disease stages that define the patient population suitable for this combination are H+Y stages 1-4, preferably H+Y1-3, and most preferably H+Y2-3.

[0140] Examples of such immune checkpoint-targeting vaccines are vaccines that provide epitopes against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4, accession number P16410) and programmed cell death protein 1 (PD-1, accession number Q15116) or its ligands, programmed cell death-ligand 1 (PD-L1 or PD1-L1, accession number Q9NZQ7), CD276 (accession number Q5ZPR3), VTCN1 (accession number Q7Z7D3), LAG3 (accession number P18627), or Tim3 (accession number Q8TDQ0), and have the following amino acid sequences:

[0141] Human CTLA4:>sp|P16410|CTLA4_ Uniprot [ka]

[0142] Human PD1:>sp|Q15116|PDCD1_ Uniprot [ka]

[0143] Human PD1-L1 >sp|Q9NZQ7|PD1L1_ Uniprot [ka]

[0144] Human B7-H3-CD276 >sp|Q5ZPR3|CD276_ Uniprot [ka]

[0145] Human B7-H4-VTCN1 >sp|Q7Z7D3|VTCN1_ Uniprot [ka]

[0146] Human LAG3:>sp|P18627|LAG3_ Uniprot [ka]

[0147] Human Tim3:>sp|Q8TDQ0|HAVR2_ Uniprot [ka]

[0148] Antibodies targeting CTLA-4 inhibit immune responses in several ways, including blocking autoreactive T cell activation at the proximal stage of the immune response, usually in lymph nodes. In contrast, the PD-1 pathway controls T cells at the later stage of the immune response, usually in peripheral tissues. Thus, two major intervention strategies for manipulating immune checkpoints by targeting either CTLA-4 or PD-1 / PD-L1 are currently clinically available. Anti-CTLA-4 is involved in the lymphocyte proliferation process after antigen-specific T cell receptor activation, while anti-PD-1 / PD-L1 acts primarily in peripheral tissues during effector processes. However, CTLA-4 is also expressed on regulatory T lymphocytes and is therefore involved in peripheral inhibition of T cell proliferation.

[0149] Currently, several immune checkpoint blockade antibodies, such as ipilimumab (anti-CTLA-4 antibody), nivolumab and pembrolizumab (both anti-PD-1 antibodies), avelumab (anti-PD-L1 antibody), or atezolizumab and durvalumab (both anti-B7-H1 / PD-L1 antibodies), can induce strong anti-cancer immunity and low side effects.

[0150] According to a preferred embodiment, the CTLA4 protein-derived polypeptide is derived from native human CTLA4 or is a mimetic with one or more aa exchanges that form a mimotope of the respective native sequence.

[0151] According to a preferred embodiment, the PD1 protein-derived polypeptide is derived from native human PD1 or is a mimetic with one or more aa exchanges that form a mimotope of the respective native sequence. The protein sequences correspond to the extracellular domains of mouse PD1 (Q02242; Uniprot) and human PD1 (Q15116; Uniprot).

[0152] According to a preferred embodiment, the PD-L1 protein-derived polypeptide is derived from native human PD-L1 or is a mimetic with one or more aa exchanges that form a mimotope of the respective native sequence.

[0153] Guo et al. (Br J Cancer. 2021;125:152-154) and Kaumaya et al. (Oncoimmunology. 2020;9:1818437) disclosed a PD1-derived peptide (aa92-110:GAISLAPKAQIKESLRAEL) that induced antibodies that reduced tumor growth in a syngeneic BALB / c model bearing CT26 colon cancer cells. Furthermore, the combination of the disclosed PD1 epitope vaccine with a HER-2 peptide vaccine demonstrated enhanced tumor growth inhibition in colon cancer.

[0154] Tobias et al. (Front Immunol. 2020;11:895) disclosed peptide / mimotopes derived from mouse PD-1 and human PD-1 (i.e., the epitopes of the anti-human PD1 mAb nivolumab and the anti-mouse mAb clone 29F.1A12). The peptides, designated JT-N1, JT-N2, and JT-N3, contain the human PD1-derived sequences PGWFLDSPDRPWNPP, FLDSPDRPWNPPTFS, and SPDRPWNPPTFSPA, corresponding to aa 21-35, aa 24-38, and aa 27-41 of human PD1, respectively. Furthermore, a mimotope for mouse PD1 contains ISLHPKAKIEESPGA (JT-mPD1), corresponding to amino acid residues 126-140 of mPD1. The antitumor effect of the mimotope JT-mPD1 was shown to be associated with a significant decrease in tumor growth and an increased apoptosis rate in the Her-2 / neu-expressing syngeneic tumor mouse model used. Furthermore, the antitumor effect of the Her-2 / neu vaccine was shown to be enhanced when combined with JT-mPD1.

[0155] Chen et al. (Cancer 2019, 11, 1909) disclosed a novel PD-L1-targeted vaccine, PDL1-Vax, which is a fusion protein of human PD-L1 (aa 19-220 of human PD-L1) linked to a T-helper epitope sequence and human IgG1 Fc sequence. Jorgensen et al. (Front Immunol. 2020; 11:595035) disclosed a 19-amino acid peptide (FMTYWHLLNAFTVTVPKDL) derived from the signal peptide of human PD-L1 as a novel PD-L1-targeted vaccine. Tian et al. (Cancer Letters 476 (2020) 170-182) disclosed a truncated mouse PDL1 extracellular domain (aa19-239) fused to the NitraTh epitope as a novel PD-L1-targeting vaccine, and hPDL1-NitraTh was also constructed by fusing a truncated human PDL1 extracellular domain (aa19-238) to the NitraTh epitope.

[0156] These anti-immune checkpoint vaccines can be highly effective when used alone or in combination with existing peptide vaccines. Therefore, a preferred embodiment provides a combination of an anti-immune checkpoint vaccine with an existing peptide vaccine for treating neoplastic or degenerative diseases such as Parkinson's disease.

[0157] Although in principle the present invention can improve all suggested PD1- and PD-L1-associated vaccination polypeptides, selected epitopes were specifically evaluated for their compatibility with the present platform, for example, SEQ ID NO: 49 / 50 (GAISLAPKAQIKESLRAEL) were shown to be superior to KLH-based vaccines.

[0158] Considering these advantageous properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention are particularly suitable for active anti-Aβ immunotherapy for use in the prevention, treatment, and diagnosis of diseases associated with β-amyloid formation and / or β-amyloid aggregation. The most prominent form of β-amyloidosis is Alzheimer's disease (AD). Other examples include familial and sporadic AD, familial and sporadic Aβ cerebral amyloid angiopathy, hereditary cerebral hemorrhage with amyloidosis (HCHWA), dementia with Lewy bodies and dementia in Down's syndrome, retinal ganglion cell degeneration in glaucoma, and inclusion body myositis / myopathy.

[0159] Aβ peptides exist in several forms, including full-length Aβ1-42 and Aβ1-40, and various modified forms of Aβ, including truncated, N- or C-terminally truncated, nitrated, acetylated and N-terminally truncated, pyroglutamic Aβ3-40 / 42 (i.e., AβpE3-40 and AβpE3-42), and Aβ4-42, which appear to play a major role in neurodegeneration.

[0160] According to a preferred embodiment, the Aβ peptide derived polypeptide has the following amino acid sequence: [ka] or aa1-6, aa1-7, aa1-8, aa1-9, aa1-10, aa1-11, aa1-12, aa1-13, aa1-14, aa1-15, aa1-21, aa2-7, aa2-8, aa2-9, aa2-10, aa3-8, aa3-9, aa3-10, aa pE3-8, aa pE3-9, aa The polypeptides are selected from polypeptides comprising or consisting of amino acid residues derived from human Aβ1-40 and / or Aβ1-42, including pE3-10, aa11-16, aa11-17, aa11-18, aa11-19, aa12-19, aa13-19, aa14-19, aa14-20, aa14-21, aa14-22, aa14-23, aa30-40, aa31-40, aa32-40, aa33-40, aa34-40, aa30-42, and aa37-42, and containing a pyroglutamic acid residue at aa3 or aa11, truncated, in particular N-terminally truncated, C-terminally truncated, post-translationally modified, nitrated, glycosylated, acetylated, or ubiquitinated peptide amino acids or peptides.

[0161] According to a preferred embodiment, the Aβ1-40 or Aβ1-42 derived polypeptide is selected from the mimetics of Aβ-derived polypeptides described above, including mimotopes and peptides containing amino acid substitutions that mimic the pyroglutamic acid amino acid.

[0162] Schenk et al. (Nature. 1999 Jul 8;400(6740):173-7.) disclosed Aβ1-42 as an immunogen for anti-Aβ immunotherapy, Pride et al. (Neurodegenerative Dis 2008;5:194-196) disclosed peptide epitopes of Aβ1-6 linked to CRM197 adjuvanted with QS21, and Wiesner et al. (J Neurosci. 2011 Jun 22;31(25):9323-31) disclosed Aβ1-6 peptides linked to Qβ virus-like particles as an efficient immunotherapeutic agent.

[0163] Wang et al. (Alzheimer's & Dementia: Translational Research & Clinical Interventions 3 (2017) 262-272) and US 2018 / 0244739 A1 disclose an Aβ1-42 peptide immunogen, specifically UB311, containing two Aβ immunogens, the cationic Aβ1-14-εK-KKK-MvF5 Th[ISITEIKGVIVHRIETILF] peptide and the Aβ1-14-εK-HBsAg3 Th[KKKIITITRIITIITID] peptide, in an equimolar ratio; these were mixed with polyanionic CpG oligodeoxynucleotides (ODN) to form stable immunostimulatory complexes of micron-sized particles, with aluminum inorganic salt (Adju-Phos) added to the final formulation.

[0164] Illouz et al. (Vaccine Volume 39, Issue 34, 9 August 2021, Pages 4817-4829) disclose Aβ1-11 fused to HBsAg as a vaccine in aged mice.

[0165] Davtyan H et al. (J Neurosci. 2013 Mar 13; 33(11):4923-4934) and Petrushina et al. (Molecular Therapy Vol. 25 No. 1 153-164) disclose a vaccine containing two foreign Th cell epitopes, P30 and P2, derived from tetanus toxin, and three copies of a B cell epitope of Aβ1-12 adjuvanted with QuilA. Similarly, Davtyan H et al. (Alzheimer's & Dementia 10 (2014) 271-283) disclose a DNA-based vaccine based on a protein coding region consisting of an immunoglobulin (Ig) k chain signal sequence, three copies of the Aβ1-11 B cell epitope, one synthetic peptide (PADRE), and a series of eight non-self promiscuous Th epitopes from tetanus toxin (TT) (P2, P21, P23, P30, P32), hepatitis B virus (HBsAg, HBVnc), and influenza (MT), or three additional Th epitopes from TT (P7 (NYSLDKIIVDYNLQSKITLP); P17 (LINSTKIYSYFPSVISKVNQ); and P28 (LEYIPEITLPVIAALSIAES)).

[0166] Petrushina et al. (Journal of Neuroinflammation 2008, 5:42) disclose Aβ1-28 with an N-terminal linker (n-CAGA) linked to bromoacetylated Saccharomyces cerevisiae mannan as a potential vaccine, albeit with severe side effects.

[0167] US 2011 / 0002949 A1 discloses a multivalent vaccine construct (Aβ3-10 / Aβ21-28) (MVC) and a monovalent vaccine construct Aβ1-8 (MoVC1-8) that are conjugated to a carrier (KLH) and administered with the saponin-based adjuvant ISCOMATRIX.

[0168] Muhs et al. (Proc Natl Acad Sci US A. 2007 Jun 5;104(23):9810-5), Hickman et al. (J Biol Chem. 2011 Apr 22;286(16):13966-76), and Belichenko et al. (PLoS One. 2016;11(3):e0152471) disclose Aβ1-15 as an array of Aβ1-15 sequences sandwiched between palmitoylated lysines at either end, where the peptides adopt an aggregated β-sheet structure and are anchored to the surface of a liposome to form a structural epitope.

[0169] Ding et al. (Neuroscience Letters, Volume 634, 10 November 2016, Pages 1-6) disclose peptides prepared by linking Aβ3-10 to the immunogenic carrier protein keyhole limpet hemocyanin (KLH) or by linking five Aβ3-10 epitopes in a linear tandem fashion.

[0170] Bakrania et al. (Mol Psychiatry (2021). https: / / doi.org / 10.1038 / s41380-021-01385-7) disclose cyclized Aβ1-14 (thioacetal-bridged Aβ peptide 1-14-KLH conjugate; DAC*FRHDSGYEC*HH[Cys]-amide emulsified in complete Freund's adjuvant (CFA), followed by a booster dose of the protein emulsified in incomplete Freund's adjuvant (IFA)) as a suitable immunogen.

[0171] Lacosta et al. (Alzheimers Res Ther. 2018 Jan 29;10(1):12.) discloses an Aβ peptide immunogen containing multiple repeats of a short C-terminal fragment of Aβ1-40. To generate an immune response, the repeats are conjugated to the keyhole limpet hemocyanin (KHL) carrier protein and formulated with the adjuvant alum hydroxide.

[0172] Axelsen et al. (Vaccine Volume 29, Issue 17, 12 April 2011, Pages 3260-3269) discloses Aβ37-42 linked to keyhole limpet hemocyanin.

[0173] WO 2004 / 062556 A2, WO 2006 / 005707 A2, WO 2009 / 149486 A2, and WO 2009 / 149485 A2 disclose mimotopes of epitopes of Aβ, which have been shown to be capable of inducing the in vivo formation of antibodies against uncleaved Aβ1-40 / 42 and N-terminally truncated AβpE3-40 / 42, Aβ3-40 / 42, Aβ11-40 / 42, AβpE11-40 / 42, and Aβ14-40 / 42, respectively.

[0174] According to a preferred embodiment, the Aβ peptide-derived polypeptide is selected from:

[0175] [Table 3]

[0176] [Table 4]

[0177] These anti-Aβ vaccines are highly effective when used alone or in combination with existing peptide vaccines directed against other pathological molecules involved in β-amyloidosis, tauopathy, or synucleinopathy, especially those with mixed pathology (i.e., the presence of Aβ pathology with tau pathology and / or aSyn pathology). Thus, a preferred embodiment provides a combination of an anti-Aβ vaccine with an anti-tau vaccine and / or an anti-aSyn peptide vaccine for treating degenerative diseases such as Alzheimer's disease, dementia in Down's syndrome, dementia with Lewy bodies, Parkinson's disease dementia, Parkinson's disease, or a tauopathy.

[0178] Although in principle the present invention can improve all suggested Aβ and Aβ-related vaccination polypeptides, selected epitopes were specifically evaluated for their compatibility with the present platform: for example, SEQ ID NO: 32 / 33 (AβpE3-8; pEFRHDS) was shown to be superior to KLH-based vaccines, and SEQ ID NO: 10 (Aβ1-6; DAEFRH) was found to be immunogenic in combination with various CLECs.

[0179] Considering these advantageous properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention can be particularly used for active anti-IL31 vaccination to treat and prevent IL31-associated diseases and autoimmune inflammatory diseases.

[0180] IL31-associated diseases include allergic diseases that cause pruritus, inflammatory diseases that cause pruritus, and autoimmune diseases that cause pruritus in mammals, including humans, dogs, cats, and horses. These diseases include atopic dermatitis, prurigo nodularis, psoriasis, cutaneous T-cell lymphoma (CTCL), and other pruritic disorders such as uremic pruritus, cholestatic pruritus, bullous pemphigoid, and chronic urticaria, allergic contact dermatitis (ACD), dermatomyositis, chronic pruritus of unknown etiology (CPUO), primary localized cutaneous amyloidosis (PLCA), mastocytosis, chronic idiopathic urticaria, bullous pemphigoid, dermatitis herpetiformis, and other dermatological conditions including lichen planus, cutaneous amyloidosis, stasis dermatitis, scleroderma, pruritus associated with wound healing, and non-pruritic diseases such as allergic asthma, allergic rhinitis, inflammatory bowel disease (IBD), osteoporosis, follicular lymphoma, Hodgkin's lymphoma, and chronic myeloid leukemia.

[0181] According to a preferred embodiment, a single IL31 epitope can be used to elicit an immune response against various domains of IL31. In another preferred embodiment, a combination of IL31 epitopes can be used to elicit an immune response against various domains of IL31, particularly involving helix C or helix A, and further involving helix D, thereby preventing IL31 binding to both IL31 receptors, interleukin-31 receptor alpha (IL-31RA) and oncostatin M receptor (OSMR).

[0182] An anti-IL31 vaccine can be highly effective when used alone or in combination with a peptide vaccine directed against other pathological molecules involved in pruritus-causing allergic diseases, pruritus-causing inflammatory diseases, and pruritus-causing autoimmune diseases. Therefore, a preferred embodiment provides a combination of an anti-IL31 vaccine with an anti-IL4 peptide vaccine and / or an anti-IL13 peptide vaccine for treating pruritus-causing allergic diseases, pruritus-causing inflammatory diseases, and pruritus-causing autoimmune diseases.

[0183] According to a preferred embodiment, the IL31 protein-derived polypeptide is a fragment of the IL-31 protein, and / or is preferably a fragment of the native human IL31 (Genbank: AAS86448.1; MASHSGPSTSVLFLFCCLGGWLASHTLPVRLLRPSDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQNYTLPCLSPDAQPPNNIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHECKRFILTISQQFSECMDLALKSLTSGAQQATT); native canine IL31 (Genbank: BAH97742.1; MLSHTGPSRFALFLLCSMETLLSSHMAPTHQLPPSDVRKIILEL QPLSRGLLEDYQKKETGVPESNRTLLLCLTSDSQPPRLNSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVFKSLNSGPQ); natural feline IL31 (UNIPROT: A0A2I2UKP7; MLSHAGPARFALFLLCCMETLLPSHMAPAHRLQPSDVRKIILELRPMSKGLLQDYVSKEIGLPESNHSSLPCLSSDSQLPHINGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKFQREPEAKVSMPADNFERKNFILAVLQQFSACLEHVLQSLNSGPQ); or natural equine IL31 (UNIPROT F7AHG9;MVSHIGSTRFALFLLCCLGTLMFSHTGPIYQLQPKEIQAIIVELQNLSKKLLDDYVSALETSILSCFFKTDLPSCFTSDSQAPGNINSSAILPYFKAISPSLNNDKSLYIIEQLDKLNFQNAPETEVSMPTDNFERKRFILTILRWFSNCLEHRAQ), or any peptide sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity with any of the above, or which differs from the naturally occurring sequence by some point mutations of surface-exposed amino acids (the number of point mutations is 1, 2 or 3).

[0184] According to a preferred embodiment, the IL31 protein-derived polypeptide is selected from the mimetics of the above-mentioned IL31-derived polypeptides, including mimotopes and peptides containing amino acid substitutions.

[0185] Further preferred target sequences include the following (presented as linear or constrained peptides, e.g., cyclized or as peptide bonds with suitable aa linkers, e.g., ggsgg):

[0186] For human IL31: peptides derived from the sequences aa98-145, aa87-150, aa105-113, aa85-115, aa84-114, aa86-117, aa87-116; or fragments thereof, and the peptides SDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQNYTL; DVQKIVEELQSLSKMLLKDV, EELQSLSK, and DVQK, LDNKSVIDEIIEHLDKLIFQDA; and DEIIEH, TDTHECKRFILTISQQFSECMDLALKS, TDTHESKRF, TDTHERKRF HESKRF, HERKRF, HECKRF;SDDVQKIVEELQ, VQKIVEELQSLS, IVEELQSLSKML, ELQSLSKMLLKD, SLSKMLLKDVEE, KMLLKDVEEEKG, LKDVEEEKGVLV, VEEEKGVLVSQN, EKGVLVSQNYTL, LDNKSVIDEIIE, KSVIDEIIEHLD, ID EIIEHLDKLI, IIEHLDKLIFQD, HLDKLIFQDAPE, KLIFQDAPETNI, FQDAPETNISVP, APETNISVPTDT, TNISVPTDTHEC, SVPTDTHESKRF, TDTHECKRFILT, TDTHESKRFILT, TDTHERKRFILT, HECKRFILTISQ, HESKRFILTISQ , HERKRFILTISQ, KRFILTISQQFS, ILTISQQFSECM, ILTISQQFSESM, ILTISQQFSERM, ISQQFSECMDLA, ISQQFSESMDLA, ISQQFSERMDLA, QFSECMDLALKS, QFSESMDLALKS, QFSERMDLALKS, SKMLLKDVEEEKG, EELQSLSK, KGVLVS, SPAIRAYLKTIRQLDNKSVIDEIIEHLDKLI, DEIIEHLDK, SVIDEIIEHLDKLI, SPAIRAYLKTIRQLDNKSVI, TDTHECKRF, HECKRFILT, HERKRFILT, HESKRFILT, SVPTDTHECKRF, SVPTDTHESKRF, and SVPTDTHERKRF.

[0187] For canine IL31: peptides consisting of aa97-144, aa97-133, aa97-122, aa97-114, aa90-110, aa90-144, aa86-144, aa97-149, aa90-149, aa86-149, aa124-135, or fragments thereof, and the peptides: SDVRKIILELQPLSRGLLEDYQKKETGV, DVRKIILELQPLSRGLLEDY ELQPLSR LSDKNIIDKIIEQLDKLKFQHE, LSDKNIIDKIIEQLDKLKFQ, KLKFQHE, LSDKNI, LDKL, LSDKN, ADTFECKSFILTILQQFSACLESVFKS and ADNFERKNF.

[0188] For feline IL31: aa 124-135 of the feline IL-31 sequence and the peptides SDVRKIILELRPMSKGLLQDYVSKEIGL and DVRKIILELRPMSKGLLQDY, LSDKNTIDKIIEQLDKLKFQRE, ADNFERKNFILAVLQQFSACLEHVLQS and ADNFERKNF.

[0189] For equine IL31: aa 118-129 of the equine IL-31 sequence, and peptides: LQPKEIQAIIVELQNLSKKLLDDY, EIQAIIVELQNLSKKLLDDY, SLNNDKSLYIIEQLDKLNFQ and TDNFERKRFILTILRWFSNCLEHRAQ.

[0190] For Mimotope: Canine IL-31 mimotopes comprise the amino acid sequences SVPADTFECKSF, SVPADTFERKSF, NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF, APTHQLPPSDVRKIILELQPLSRG, TGVPES or variants thereof.

[0191] The feline IL-31 mimotope comprises the amino acid sequence SMPADNFERKNF, NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF, APAHRLQPSDIRKIILELRPMSKG, IGLPES or a variant thereof.

[0192] The equine IL-31 mimotope comprises the amino acid sequence SMPTDNFERKRF, NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF, GPIYQLQPKEIQAIIVELQNLSKK, KGVQKF or a variant thereof.

[0193] Human IL-31 mimotopes include the amino acid sequences SVPTDTHECKRF, SVPTDTHERKRF, HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF, LPVRLLRPSDDVQKIVEELQSLSKM, KGVLVS or variants thereof that retain anti-IL-31 binding.

[0194] According to a preferred embodiment, the IL31 epitope may be a structural epitope comprising at least two amino acids or amino acid sequences that are spatially distinct from each other but in close proximity to form a respective paratope, which is typically bound by an anti-IL31 antibody, e.g., a polyclonal anti-IL31 antibody obtained by vaccinating a mammal with a vaccine that specifically recognizes naturally occurring IL31.

[0195] IL-31 is a protein with a four-helix bundle structure found in the gp30 / IL-6 cytokine family. The receptor for IL-31 is a heterodimer of interleukin-31 receptor alpha (IL-31 RA, also known as GPL or gp130-like receptor) and oncostatin M receptor (OSMR). Both heterodimeric structures are called IL-31 receptors or IL-31 coreceptors. The putative interaction site between human IL-31 and its receptor has been described by Saux et al. (J Biol Chem 2010, 285, 3470-34). Targeting of IL-31 can be achieved by antibodies targeting IL-31 and / or its receptor. The development of dedicated monoclonal antibodies that specifically target IL31 has enabled clinical and preclinical validation of this targeting strategy in vitro and in vivo (Front Med (Lausanne. 2021 Feb 12;8:638325)).

[0196] BMS-981164 is an anti-IL-31 monoclonal antibody targeting circulating IL-31 being developed by Bristol-Myers Squibb. A two-part, single-dose ascending study was conducted between 2012 and 2015 to investigate the safety and pharmacokinetic profile of BMS-981164 (NCT01614756). The study design was randomized, double-blind, and placebo-controlled. The drug was administered as both SC and IV formulations (0.01-3 mg / kg) to healthy volunteers (Part 1) and adults with atopic dermatitis (Part 2). Adult subjects in Part 2 were required to have at least moderate atopic dermatitis (Physician's Global Assessment rating of 3 on a scale of 0-5) and a visual analogue scale (visual analogue scale) of at least 7 out of 10. To date, the results of this study have not been published. As of 2016, BMS-981164 is no longer listed in Bristol-Myers Squibb's development pipeline, and no new trials have been announced.

[0197] US 8,790,651 B2 describes a monoclonal antibody that binds to IL-31 for treating immunological disorders such as atopic dermatitis. A monoclonal antibody against canine IL-31 (lokivetmab, Zoetis) is commercially available for the treatment of canine atopic dermatitis. Lokivetmab presumably blocks the binding of IL-31 to the coreceptor GPL.

[0198] EP 4 019 546 A1 discloses monospecific and multispecific antibodies in which the antibody variable domains block the binding of IL-31 to the interleukin-31 receptor alpha (IL-31RA) / oncostatin M receptor (OSMR) complex (IL-31RA / OS-MR complex).

[0199] Bachmann et al. disclose a vaccine utilizing whole canine IL-31 linked to virus-like particles for immunization of dogs to treat atopic dermatitis (Bachmann, M.F.; Zeltins, A.; Kalnins, G.; Balke, I.; Fischer, N.; Rostaher, A.; Tars, K.; Favrot, C. Vaccination against IL-31 for the Treatment of Atopic Dermatitis in Dogs. J. Allergy Clin. Immunol. 2018, 142, 279-281 .e1). Similarly, US11,324,836 B2, US11,207,390 B2, and US10,556,003, and Fettelschloss et al (doi:10.1111 / eve.13408) disclose VLP-based immunogens for targeting IL31 from various species, including human, canine, equine, or porcine IL31, and IL31-associated diseases. These VLP-based immunogens feature anti-IL31 immunogens with full-length IL31-derived sequences, native IL31-derived sequences, and full-length modified IL31-derived sequences, respectively.

[0200] US2021 / 0079054A1 discloses peptide-based immunogens based on UbiTh platform technology targeting IL31 for treating and / or preventing pruritic conditions or allergic conditions such as atopic dermatitis. Along these lines, for canine IL31: peptides consisting of aa97-144, aa97-133, aa97-122, aa97-114, aa90-110, aa90-144, aa86-144, aa97-149, aa90-149, aa86-149; for human IL31: peptides derived from the sequences aa98-145, aa87-150, aa105-113, aa85-115, aa84-114, aa86-117, aa87-116, where appropriate, with modifications, e.g., substitutions of serine with cysteine, canine IL31 (Genbank: BAH97742.1; MLSHTGPSRFALFLLCSMETLLSSHMAPTHQLPPSDVRKIILELQPLSRGLLEDYQ B cell epitope-based immunogens derived from KKETGVPESNRTLLLCLTSDSQPPRLNSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVFKSLNSGPQ) and human IL31 (Genbank: AAS86448.1; MASHSGPSTSVLFLFCCLGGWLASHTLPVRLLRPSDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQNYTLPCLSPDAQPPNNIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHECKRFILTISQQFSECMDLALKSLTSGAQQATT) are presented. The B cell epitope may be linear or restricted and fused to a promiscuous T helper epitope and formulated in the presence of an adjuvant (e.g., different CpG molecules, Alhydrogel, AdjuPhos, Montanide such as ISA50V2, ISA51, ISA720).

[0201] US2019 / 0282704 A1 discloses a vaccine composition for immunizing and / or protecting a mammal against IL-31-mediated disorders, comprising a carrier polypeptide (e.g., CRM197) in combination with at least one mimotope against an IL31-derived epitope selected from a feline IL-31 mimotope, a canine IL-31 mimotope, an equine IL-31 mimotope, or a human IL-31 mimotope; and an adjuvant. The mimotope can be linear or constrained (e.g., cyclized).

[0202] The canine IL-31 mimotope comprises the amino acid sequence SVPADTFECKSF, SVPADTFERKSF, NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF, APTHQLPPSDVRKIILELQPLSRG, TGVPES, or a variant thereof.

[0203] The feline IL-31 mimotope comprises the amino acid sequence SMPADNFERKNF, NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF, APAHRLQPSDIRKIILELRPMSKG, IGLPES, or a variant thereof.

[0204] The equine IL-31 mimotope comprises the amino acid sequence SMPTDNFERKRF, NS SAILPYFKAISPSLNNDKSLYIIEQLDKLNF, GPIYQLQPKEIQAIIVELQNLS KK, KGVQKF, or a variant thereof.

[0205] Human IL-31 mimotopes include the amino acid sequences SVPTDTHECKRF, SVPTDTHERKRF, HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF, LPVRLLRPSDDVQKIVEELQSLSKM, KGVLVS, or variants thereof that retain anti-IL-31 binding.

[0206] Furthermore, the region of approximately amino acid residues 124 to 135 of the feline IL-31 sequence represented by (UNIPROT: A0A2I2UKP7); the region of approximately amino acid residues 124 to 135 of the canine IL-31 sequence represented by (Genbank: BAH97742.1); and the region of approximately amino acid residues 118 to 129 of the equine IL-31 sequence represented by (UNIPROT F7AHG9) have been disclosed as suitable epitopes.

[0207] WO 2019 / 086694 A1 discloses peptide-based immunogens targeting IL31, achieved by an IL31 antigen comprising an unpacked IL31 helix peptide derived from canine, human, feline, equine, porcine, bovine, or camel IL31, or an epitope contained therein. The antigen can be linked to a conventional carrier molecule (e.g., KLH), adjuvanted with Imject alum, or linked to an anti-CD32 scFv construct potentially containing the TLR9 agonist CpG or the TLR7 / 8 agonist imidazoquinoline. Specifically, the IL31 peptide comprises or consists of any one of the following amino acid sequences, alone or in combination, fused using the disclosed linker sequences:

[0208] Helix A: Human: SDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQNYTL; and DVQKIVEELQSLSKMLLKDV, EELQSLSK, and DVQK Dog: SDVRKIILELQPLSRGLLEDYQKKETGV, and DVRKIILELQPLSRGLLEDY, and ELQPLSR Cat:SDVRKIILELRPMSKGLLQDYVSKEIGL and DVRKIILELRPMSKGLLQDY Horse: LQPKEIQAIIVELQNLSKKLLDDY and EIQAIIVELQNLSKKLLDDY

[0209] Helix C Human: LDNKSVIDEIIEHLDKLIFQDA; and DEIIEH Dog: LSDKNIIDKIIEQLDKLKFQHE, LSDKNIIDKIIEQLDKLKFQ, KLKFQHE, LSDKNI, LDKL, LSDKN, Cat:LSDKNTIDKIIEQLDKLKFQRE Horse:SLNNDKSLYIIEQLDKLNFQ

[0210] and / or Helix D: Human: TDTHECKRFILTISQQFSECMDLALKS, TDTHESKRF, and HESKRF Dog: ADTFECKSFILTILQQFSACLESVFKS and ADNFERKNF Cat: ADNFERKNFILAVLQQFSACLEHVLQS and ADNFERKNF Horse:TDNFERKRFILTILRWFSNCLEHRAQ.

[0211] WO 2022 / 131820 A1 discloses immunomodulatory or anti-inflammatory IL31-derived peptides as active ingredients for preventing or treating atopic dermatitis as pharmaceuticals or cosmetics. This document also describes that, in order to increase the stability and skin permeability of the peptide, the IL31 peptide or a fragment thereof is mixed with a biocompatible polymer, such as pullulan, chondroitin sulfate, hyaluronic acid (HA), glycol chitosan, starch, chitosan, dextran, pectin, curdlan, poly-L-lysine, polyaspartic acid (PAA), polylactic acid (PLA), polyglycol Ride (polyglycolide, PGA), polycaprolactone (poly(ε-caprolactone), PCL), poly(caprolactone-lactide) random copolymer (PCLA), poly(caprolactone-glycolide) random copolymer (PCGA), poly(lactide-co-glycolide) random copolymer (PLGA), polyethylene glycol (PEG), pluronic F-68, and pluronic F-127 (pluronic F-127), or fatty acids such as hexanoic acid (hexanoic acid), caprylic acid (caprylic acid, C8), capric acid (capric acid, C10), lauric acid (lauric acid, C12), myristic acid (myristic acid, C14), palmitic acid (C16), stearic acid (C18), and cholesterol (cholesterol). Neither the peptide nor the conjugate is suggested as an immunogen in this disclosure.

[0212] Although in principle the present invention can improve all suggested IL31-associated disease vaccination polypeptides, selected epitopes (see SEQ ID NOs) were specifically evaluated for their compatibility with the present platform in comparison to CRM197-based vaccines.

[0213] Selected Array [ka]

[0214] Considering these advantageous properties of the conjugates of the invention, the conjugates and vaccines according to the invention are particularly usable for the active immunotherapy of calcitonin gene-related peptide (CGRP)-related diseases.

[0215] The CGRP-related disorder is selected from the group consisting of episodic migraine and chronic migraine, and cluster headache, hyperalgesia, e.g., hyperalgesia in dysfunctional pain states such as rheumatoid arthritis, osteoarthritis, visceral hyperalgesia syndrome, fibromyalgia, inflammatory bowel syndrome, neuropathic pain, chronic inflammatory pain, and headache.

[0216] According to a preferred embodiment, the CGRP-derived polypeptide is derived from native human CGRP alpha (ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF; a 37-aa peptide fragment covering aa 83-119 of the calcitonin isoform α-CGRP preproprotein, accession number NP_001365879.1) or native human CGRP beta (ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF; a 37-aa peptide fragment covering aa 82-118 of the calcitonin gene-related peptide 2 precursor, accession number NP_000719.1), or their precursor molecules (NP_001365879.1 and NP_000719.1). The CGRP-derived polypeptide may also be a mimetic with one or more aa exchanges forming a mimotope of the respective native sequence.

[0217] According to a preferred embodiment, the CGRP-derived polypeptide is selected from a functional portion of native human CGRP, including the central region of CGRP (e.g., aa 8-35) or a fragment thereof, the C-terminal CGRP receptor binding region (e.g., aa 11-37) or a fragment thereof, or the N-terminal region (e.g., aa 1-20) or a fragment thereof, which potentially also contains the cyclic C2-C7 loop within CGRP, consisting of amino acid residues or mimotopes derived from the functional portion of native human CGRP.

[0218] Further preferred target sequences include ACDTATCVTH;ACDTATCVTHRLAGL;ACDTATCVTHRLAGLLSR;ACDTATCVTHRLAGLLSRSG;ACDTATCVTHRLAGLLSRSGGVVKN;TATCCVTHRLAGLL;ATCVTHRLAGLLSR;RLAGLLSR;RLAGLLSRSGGVVKN;RSGGVVKN;RLAGLLSRSGGVVKNNFVPT;RLAGLLSRSGGVVKNNFVPTNVG;RLAGLLSRSGGVVKNNFVPTNVGSKAF;RLAGLLSRSGGVVKNNFVPTNVGSKAF;LLSRSGGVVKNNFVPTNVGSKAF;RSGGVVKNNFVPTNVGSKAF;GGVVKNNFVPTNVGSKAF;VVKNNFVPTNVGSKAF;NNFVPTNVGSKAF;VPTNVGSKAF;NVGSKAF;GSKAF.

[0219] In US 2022 / 0073582 A1, native human CGRP (accession number: NP_001365879.1) has the following amino acid sequence: MGFQKFSPFLALSILVLLQAGSLHAAPFRSALESSPADPATLSEDEARLLLAALVQDYVQMKASELEQEQEREGSRIIAQKRACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAFGRRRRDLQA, and the CGRP-derived peptides are: aa1-10 ACDTATCVTH; aa1-15 ACDTATCVTHRLAGL; aa1-18 ACDTATCVTHRLAGLLSR; aa1-20 ACDTATCVTHRLAGLLSRSG; aa1-25 ACDTATCVTHRLAGLLSRSGGVVKN; aa4-16 TATCVTHRLAGLL; aa5-18 ATCVTHRLAGLLSR; aa11-18 Polypeptide constructs containing RLAGLLSR; aa11-25 RLAGLLSRSGGVVKN; aa11-30 RLAGLLSRSGGVVKNNFVPT; aa11-33 RLAGLLSRSGGVVKNNFVPTNVG; aa11-35 RLAGLLSRSGGVVKNNFVPTNVGSK; aa11-37 RLAGLLSRSGGVVKNNFVPTNVGSKAF; aa15-37 LLSRSGGVVKNNFVPTNVGSKAF; aa18-37 RSGGVVKNNFVPTNVGSKAF; aa20-37 GGVVKNNFVPTNVGSKAF; aa22-37 VVKNNFVPTNVGSKAF; aa25-37 NNFVPTNVGSKAF; aa28-37 VPTNVGSKAF; aa31-37 NVGSKAF have been disclosed. The peptide immunogen constructs disclosed in US 2022 / 0073582 A1 require a CGRP-derived B cell epitope linked to one or more promiscuous T cell epitopes in order to be functional as peptide immunogen constructs for targeting GCRP.

[0220] In addition to active immunotherapy, humanized anti-calcitonin gene-related peptide (CGRP) monoclonal antibodies have been suggested as a paradigm for targeting CGRP. Antibodies have been found to be effective in reducing the frequency of chronic migraines (Dodick DW et al. (2014) Lancet Neurol. 13:1100-1107; Dodick DW et al. (2014) Lancet Neurol. 13:885-892; Bigal ME et al. (2015) Lancet Neurol. 14:1081-1090; Bigal ME et al. (2015) Lancet Neurol. 14:1091-1100; and Sun H et al. (2016) Lancet Neurol. 15:382-390).

[0221] Along these lines, US 8,597,649 B2, EP 1957106 B1, and US 9,266,951 B2 disclose clinically used monoclonal antibodies targeting aa 25-37 and / or aa 33-37 in human CGRP for treating migraine, cluster headache, and tension headache. US 20120294797 A1 discloses a clinically used CGRP-targeting monoclonal antibody that is also specific for the C-terminal epitope aa 26-37, following co-crystallization results showing that this epitope is suitable for immunotherapy (https: / / doi.org / 10.1080 / 21655979.2021.2006977). US 9,505,838 B2 also discloses clinically used monoclonal antibodies against CGRP that bind to a C-terminal fragment having amino acids 25-37 of CGRP or a C-terminal epitope within amino acids 25-37 of CGRP.

[0222] Although in principle the present invention can improve all suggested CGRP-related disease vaccination polypeptides, selected epitopes (see SEQ ID NOs: 152 to 162) were specifically evaluated for their compatibility with the present platform in comparison to CRM197-based vaccines.

[0223] Sequences selected for the experiment: [ka]

[0224] Considering these advantageous properties of the conjugates of the present invention, the CLEC-based conjugates and CLEC-based vaccines according to the present invention can be particularly used for specific allergen immunotherapy (AIT) for treating IgE-mediated allergic diseases type I. Allergic diseases typically refer to a number of conditions caused by hypersensitivity of the immune system to normally harmless substances in the environment. These diseases include, but are not limited to, hay fever, seasonal allergies, food allergies, pollen allergies, mold spore allergies, poisonous plant allergies, medication / drug allergies, insect, scorpion, or spider venom allergies, latex or dust allergies, pet allergies, allergic bronchial asthma, allergic rhinitis and allergic conjunctivitis, atopic dermatitis, contact dermatitis to adhesives, antimicrobial agents, fragrances, hair dyes, metals, rubber components, topical medications, rosin, wax, polish, cement, and leather, chronic sinusitis, atopic eczema, autoimmune diseases in which IgE plays a role ("autoallergies"), chronic (idiopathic) urticaria and autoimmune urticaria, anaphylaxis, particularly idiopathic anaphylaxis and exercise-induced anaphylaxis.

[0225] To date, specific AIT is the only curative approach to allergies and is mediated by repeated injections of allergens containing extracts from various sources, such as food, pollen, animal dander, dust mites, or insect venom. However, specific AIT paradigms currently used in clinical practice are characterized by very long treatment periods, the need for frequent injections, and limited efficacy, all of which result in low patient compliance (Musa et al. Hum Vaccin Immunother. 2017 Mar; 13(3):514-517. doi:10.1080 / 21645515.2016.1243632).

[0226] The main mechanism of AIT is the induction of so-called blocking antibodies, preferably of the IgG4 isotype, but also of other isotypes (e.g., IgG1 or IgA). Naturally occurring IgA and IgG have been shown to target epitopes on the surface of allergens that are different from the epitopes specifically recognized by IgE (so-called IgE epitopes) (Shamji, Valenta et al. 2021; Allergy 76(12):3627-3641). However, the latter epitopes crosslink IgE bound to mast cells via the high-affinity FcεRI receptor, thus inducing an immediate allergic immune response.

[0227] In contrast, AIT-inducing blocking antibodies (mainly IgG and IgA types) are directed against the IgE epitopes. Their binding to allergens prevents the cross-linking of cell-bound IgE, thereby inhibiting the initiation of allergic reactions. IgG4 exhibits favorable characteristics as a blocking antibody because it cannot cross-link allergens, and it has low affinity for activating IgG Fc receptors (FcγR) while retaining high affinity for FcγRIIb. These characteristics allow IgG4 to be an efficient inhibitor of IgE-dependent reactions without the untoward inflammation associated with IgG immune complex formation and complement activation (Shamji, Valenta et al. 2021). However, the blocking capacity of IgG4 is not necessarily superior to other IgG subclasses {Ejrnaes et al. 2004; Molecular Immunology Vol. 41, Issue 5, .2004, P. 471-478}, and blocking activity is also conferred by other IgG types, especially IgG1, especially in early AIT (Strobl, Demir et al. 2023, Journal of Allergy and Clinical Immunology doi:10.1016 / j.jaci.2023.01.005).

[0228] According to a preferred embodiment, a single allergen epitope can be used to elicit an immune response against the respective allergen (e.g., an IgE epitope as set out in Tables A and B). In another preferred embodiment, a combination of epitopes from one allergen can be used to elicit an immune response against different domains of the allergen.

[0229] These anti-monoallergen vaccines are highly effective when used alone or in combination with peptide vaccines directed against other allergen molecules involved in allergic diseases. Therefore, providing a combination of epitopes of different allergens to elicit immune responses against different allergens is a preferred embodiment.

[0230] According to a preferred embodiment, the allergen-derived polypeptide is a fragment of a particular allergen protein, in particular one of those listed in Tables A and B, and / or is preferably selected from naturally occurring proteins, in particular those listed in Tables A and B.

[0231] According to a preferred embodiment, the allergen-derived polypeptide is a linear fragment of a particular allergen protein, including those set out in Tables A and B.

[0232] According to a preferred embodiment, the allergen-derived polypeptide is selected from mimetics of the allergen-derived polypeptides mentioned above, including mimotopes and peptides containing amino acid substitutions.

[0233] According to a preferred embodiment, the allergen-derived polypeptide is derived from a natural allergen or is a mimetic with one or more aa exchanges forming a mimotope of the respective natural sequence.

[0234] According to a preferred embodiment, the allergen epitopes may be structural epitopes comprising at least two amino acids or stretches of amino acids that are spatially distinct from each other but in close proximity to form the respective paratopes, which are usually bound by anti-allergen antibodies, such as polyclonal anti-allergen antibodies obtained after vaccinating a mammal with a vaccine that specifically recognizes naturally occurring allergens.

[0235] According to preferred embodiments, the respective structural epitopes or mimotopes can be obtained from the literature or identified using predictive algorithms (as disclosed in Dall'Antonia and Keller 2019, Nucleic Acids Research 47(W1):W496-W501) or publicly available databases (e.g.: https: / / www.iedb.org / ). Selected examples of potential target antigens and their respective epitopes / mimotopes for use in the present invention are summarized in Tables A and B.

[0236] According to a preferred embodiment, further preferred target sequences comprise a constrained peptide, such as a cyclized peptide or a peptide bond with a suitable aa linker known to those skilled in the art, such as a (G)n linker, a (K)n linker, GGSGG, etc.

[0237] Table A: Preferred allergens for use in CLEC-based vaccines [Table 5]

[0238] [Table 6]

[0239] [Table 7]

[0240] [Table 8]

[0241] Table B: Preferred allergen epitopes for use in CLEC-based vaccines [Table 9]

[0242] [Table 10]

[0243] [Table 11]

[0244] [Table 12]

[0245] Positive outcomes of AIT are associated with the induction of high-affinity IgG antibodies capable of neutralizing allergens (Svenson, Jacobi et al. 2003, Molecular Immunology 39(10):603-612; Zha, Leoratti et al. 2018, Journal of Allergy and Clinical Immunology 142(5):1529-1536.e1526.). However, during classical AIT, the initial avidity of the induced blocking IgG did not further increase over time (Strobl et al. 2023; Jakobsen CG et al., 2005, Clinical & Experimental Allergy, 35:193-198. doi:10.1111 / j.1365-2222.2005.02160.x), supporting the idea that AIT-induced inhibition of allergen binding to IgE can be primarily or solely explained by the induction of increased amounts of specific IgG (Svenson et al., 2003, Molecular Immunology 39(10):603-612; Jakobsen et al., 2005). Therefore, the rather limited success of classical AIT may be primarily due to the low immunogenicity of existing AIT compounds and the lack of further avidity maturation during long-term AIT application.

[0246] In contrast, vaccines or conjugates according to the present invention induce IgE epitope-specific immune responses with high antibody levels (compared to conventional vaccines) that exhibit long-term affinity maturation after repeated immunizations, making them particularly suitable for the necessary induction of AIT and high avidity IgG (see, e.g., Figures 13 and 21), which results in higher avidity immune sera compared to classical vaccines, including alum-adjuvanted vaccines and conjugate vaccines (with and without adjuvant).

[0247] Currently, AIT exclusively uses allergen extracts from natural sources, which represent complex heterogeneous mixtures of allergenic and non-allergenic proteins, glycoproteins, and polysaccharides (Cox et al. 2005, Expert Review of Clinical Immunology 1(4):579-588.). The resulting products are difficult to standardize and can induce undesirable side effects, including anaphylaxis and delayed T-cell-based reactions (Mellerup, Hahn et al. 2000, Experimental Allergy 30(10):1423-1429).

[0248] Therefore, novel vaccine concepts in clinical development use platforms that provide universal T cell help (virus-like particles {Shamji, 2022 #14} or carrier proteins such as KLH or hepatitis preS fusion proteins (Marth et al. 2013, The Journal of Immunology 190(7):3068-3078)) and recombinant allergenic proteins or peptides (containing allergenic epitopes or mimotopes thereof) to increase immunogenicity and affinity maturation (Bachmann et al. 2020, Trends in Molecular Medicine 26(4):357-368).

[0249] The latter approach, applying peptide-carrier conjugates containing allergenic epitopes or mimotopes thereof, would be particularly preferable for the novel AIT paradigm in patients, as it would focus the immune response on the desired target epitopes (i.e., IgE epitopes) and completely avoid immediate side effects (i.e., cross-linking of cell-bound IgE by the vaccine) and delayed side effects (i.e., activation of allergen-specific T cell responses).

[0250] Marth et al. (2013) disclosed AIT compounds based on fusion proteins of two nonallergenic peptides, PA and PB, derived from the IgE-reactive region of the major birch pollen allergen Bet v 1, fused to the hepatitis B surface protein PreS in four recombinant fusion proteins containing different numbers of peptides and peptide combinations. Similarly, the clinically tested AIT vaccine, BM32, used four fusion proteins consisting of peptides derived from four major timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Phl p 6) fused to the hepatitis B-derived PreS carrier protein. Weber et al. (2017; doi:10.1016 / j.jaci.2017.03.048) were able to demonstrate similar immunogenicity of alum-adjuvanted BM32-mediated AIT and conventional extract-mediated AIT in rabbits. However, despite initially promising clinical results (Eckl-Dorna, 2019 EBioMedicine. 2019 Dec;50:421-432. doi:10.1016 / j.ebiom.2019.11.006.), further development of the BM32 approach was discontinued after a Phase IIb trial. To date, no peptide-carrier conjugate or fusion protein AIT approaches have been licensed, nor have any other novel recombinant vaccines for AIT been licensed (Pavon-Romero, 2022, Cells. 2022 Jan 8;11(2):212. doi:10.3390 / cells11020212).

[0251] Along these lines, a single injection of two monoclonal antibodies against two epitopes within the major cat allergen Fel d 1 in allergic patients has been shown to be equally effective compared to long-standing conventional AIT (Orengo, Radin et al. 2018, Nature Communications 9(1):1421), demonstrating that a small number of target epitopes within a given allergen may be sufficient to provide complete protection from an allergic immune response. Vaccines or conjugates according to the invention are particularly suitable for combining universal T cell epitopes with such IgE epitopes or mimotopes on a CLEC scaffold to treat allergies.

[0252] In principle, the present invention can improve all suggested allergic disease vaccination polypeptides, but selected epitopes (see Tables A and B and SEQ ID NOs: 45 / 46) are particularly preferred. For example, SEQ ID NOs: 45 / 46 have been shown to be superior to KLH-based vaccines.

[0253] Given these advantageous properties of the conjugates of the invention, the CLEC-based conjugates and CLEC-based vaccines according to the invention will be particularly useful for enhancing the immunogenicity of commercially available peptide / glycoconjugate vaccines, in particular glycoconjugate vaccines used to prevent infectious diseases such as microbial or viral infections caused by Haemophilus influenzae type b (Hib), Streptococcus pneumoniae, Neisseria meningitidis, and Salmonella Typhi, or other infectious agents including those that cause hepatitis A or B, human papillomavirus infection, influenza, typhoid fever, measles, mumps, and rubella. Additionally, there are infections caused by Neisseria meningitidis group B bacteria, cytomegalovirus (CMV), respiratory syncytial virus (RSV), Clostridioides difficile, extraintestinal pathogenic Escherichia coli (Expec), Klebsiella pneumoniae, Shigella, Staphylococcus aureus, Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2), Ebola virus, Borrelia burgdorferi, and HIV.

[0254] To date, several carrier proteins, namely, recombinant cross-reacting material of diphtheria toxin (CRM197), tetanus toxoid (TT), meningococcal outer membrane protein complex (OMPC), diphtheria toxoid (DT), Haemophilus influenzae protein D (HiD), and recombinant Pseudomonas aeruginosa exotoxin A (rEPA), have been used in licensed conjugate vaccines. Clinical trials have demonstrated the efficacy of these conjugate vaccines in preventing infectious diseases and altering the spread of Haemophilus influenzae type b, Streptococcus pneumoniae, Neisseria meningitidis, and typhoid fever. All carrier proteins have been effective in enhancing vaccine immunogenicity, but they differ in the amount and avidity of antibodies they elicit, their ability to contain multiple polysaccharides in the same product, and their ability to be administered simultaneously with other vaccines.

[0255] According to preferred embodiments, conjugate vaccines amenable to CLEC modification and immunogenicity enhancement include, but are not limited to, Haemophilus influenzae b conjugate vaccines (e.g., PedvaxHIB®, ActHIB®, Hiberix®), recombinant hepatitis B vaccines (e.g., Recombivax HB®, PREHEVBRIO®, Engerix-B, HEPLISAV-B®), human papillomavirus vaccines (e.g., Gardasil®, Gardasil®), and recombinant human papillomavirus vaccines (e.g., Gardasil®). 9®, Cervarix®), Meningococcal (groups A, C, Y, and W-135) oligosaccharide diphtheria CRM197 conjugate vaccines (e.g., Menveo®), Meningococcal (groups A, C, Y, and W-135) polysaccharide diphtheria toxoid conjugate vaccines (e.g., Menactra®), Meningococcal (groups A, C, Y, and W) TT-conjugate vaccines (e.g., MenQuadfi®), multivalent pneumococcal conjugate vaccines (e.g., Prevnar-13®, Prevnar 20®, Pneumovax-23®, Vaxneuvance®), anti-typhoid vaccines (e.g., Typhim V®, Typhim Currently available vaccines include VI®, Typherix®, Vi polysaccharide bound to non-toxic recombinant Pseudomonas aeruginosa exotoxin A, or Vi-rEPA, or polysaccharide tetanus toxoid conjugate vaccine Typbar-TCV®), varicella-zoster virus vaccines (e.g., Shingrix®), and other anti-infective conjugate vaccines with a genetically engineered cross-reacting part of diphtheria toxin (CRM197), or tetanus toxin (TT), or meningococcal outer membrane protein complex (OMPC), or diphtheria toxoid (DT), or Haemophilus influenzae protein D (HiD), or recombinant Pseudomonas aeruginosa exotoxin A (rEPA) as the carrier molecule.

[0256] According to a further aspect, the novel conjugates of the present invention can be used in the prevention of infectious diseases, such as microbial or viral infections caused by Haemophilus influenzae type b (Hib), Streptococcus pneumoniae, Neisseria meningitidis, and Salmonella typhi, or other infectious agents, including those that cause hepatitis A or B, human papillomavirus infection, influenza, typhoid fever, measles, mumps, and rubella. These include infections caused by Neisseria meningitidis group B bacteria, cytomegalovirus (CMV), respiratory syncytial virus (RSV), Clostridioides difficile, extraintestinal pathogenic Escherichia coli (Expec), Klebsiella pneumoniae, Shigella, Staphylococcus aureus, Plasmodium species, coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2), Ebola virus, Borrelia burgdorferi, HIV, and the like.

[0257] While in principle the present invention can improve all proposed anti-infective conjugate vaccines, selected vaccines were specifically analyzed. For example, the CLEC-modified Meningococcal (groups A, C, Y, and W-135) oligosaccharide diphtheria CRM197 conjugate vaccine (i.e., Menveo®) and the Haemophilus influenzae b conjugate vaccine ActHIB® were shown to be superior to the commercially available Menveo® and ActHIB® vaccines.

[0258] Considering these advantageous properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention may be particularly useful for the active immunotherapy of proprotein convertase subtilisin / kexin type 9 (PCSK9)-associated diseases, including but not limited to hyperlipidemia, hypercholesterolemia, atherosclerosis, elevated serum levels of low-density lipoprotein cholesterol (LDL-C), and cardiovascular events, stroke, or various forms of cancer.

[0259] According to a preferred embodiment, the PCSK9 protein-derived polypeptide has the following amino acid sequence: [ka] or a fragment thereof, or a mimetic with one or more aa exchanges that form a mimotope of the respective native sequence. Further preferred target sequences include linear or constrained peptides (e.g., cyclized) or peptide bonds with a suitable aa linker (e.g., ggsgg, etc.).

[0260] According to a preferred embodiment, the polypeptide derived from the PCSK9 protein is selected from polypeptides comprising or consisting of amino acid residues from the regions: aa150-170, aa153-162, aa205-225, aa211-223, aa368-382, or subunits thereof, or polypeptides comprising or consisting of mimotopes.

[0261] According to a preferred embodiment, the PCSK9 protein-derived polypeptide is a PCSK9-derived sequence: NVPEEDGTRFHRQASK, NVPEEDGTRFHRQASKC, PEEDGTRFHRQASK, CPEEDGTRFHRQASK, PEEDGTRFHRQASKC, AEEDGTRFHRQASK, TEEDGTRFHRQASK, PQEDGTRFHRQASK, PEEDGTRFHRRASK, PEEDGTRFHRKASK, PEEDGTRFHRQASR, PEEDGTRFHRTASK, SIPWNLERITPPR, PEEDGTRFHRQASK, PEEDGTRFHRQA, EEDGTRFHRQASK, EEDGTRFHRQAS, SIPWNLERITP, SIPWNLERITPC, SIPWNLERIT, SIPWNLERITC, LRPRGQPNQC, SRHLAQASQ, SRHLAQASQC, SRSGKRRGER, SRSGKRRGE RC, IIGASSDCSTCFVSQ, IIGASSDSSTSFVSQ, IIGASSDSSTSFVSQC, CIGASSDSSTSFVSC, IGASSDSSTSFVSC, CDGTRFHRQASKC, DGTRFHRQASKC, CDGTRFHRQASK, AGRDAGVAKGAC, RDAGVAKC, RDAGVAK, SRHLAQASQLEQC;SRHLAQASQLEQ, GDYEELVLALRC;GDYEELVLALR, LVLALRSEEDC;LVLALRSEED, AKDPWRLPC;AKDPWRLP, AARRGYLTKC, AARRGYLTK, FLVKMSGDLLELALKLPC;FLVKMSGDLLELALKLP, EEDSSVFAQC, EEDSSVFAQ, NVPEEDGTRFHRQASKC, NVPEEDGTRFHRQASK, CKSAQRHFRTGDEEPVN, KSAQRHFRTGDEEPVN.

[0262] According to a preferred embodiment, a single PCSK9-derived epitope can be used to elicit an immune response against different regions within three distinct domains of PCSK9 (i.e., the inhibitory pro-domain (aa 1-152), the catalytic domain (aa 153-448), and the C-terminal domain (449-692)). In another preferred embodiment, a combination of PCSK9-derived epitopes can be used to elicit an immune response against different epitopes within domains of PCSK9, particularly involving the catalytic domain (aa 153-449), and further involving the inhibitory pro-domain (aa 1-152) and / or the C-terminal domain (449-692).

[0263] Hyperlipidemia, hypercholesterolemia, atherosclerosis, coronary heart disease, and vascular disorders such as stroke are among the leading causes of death worldwide, and elevated levels of LDL-C play a key role in their pathogenesis. Therefore, LDL-C control is a crucial factor for the successful treatment of hyperlipidemia, hypercholesterolemia, and atherosclerosis. Therefore, PCSK9 plays an important role in LDL catabolism through its direct action on LDLR. Inhibition of PCSK9 has been found to be beneficial to LDL-C levels. Therefore, anti-PCSK9 therapy is a promising approach for beneficially regulating LDL-C levels and treating PCSK9-related diseases.

[0264] WO2015128287A1 and EP2570135A1 disclose PCSK9 mimotope-carrier conjugate vaccines (e.g., KLH or CRM197 as carrier) and disclose the sequences PEEDGTRFHRQASK, AEEDGTRFHRQASK, TEEDGTRFHRQASK, PQEDGTRFHRQASK, PEEDGTRFHRRASK, PEEDGTRFHRKASK, PEEDGTRFHRQASR, PEEDGTRFHRTASK and aa150-170 and / or aa205-225 of PCSK9, in particular SIPWNLERITPPR, PEEDGTRFHRQASK, PEEDGTRFHRQA, EEDGTRFHRQASK, EEDGTRFHRQAS, SIPWNLERITP, and SIPWNLERIT.

[0265] Patent document CN105085684A discloses a recombinant vaccine containing a PCSK9 epitope and diphtheria toxin DTT. The epitope peptide is linked to the C-terminus of the transmembrane domain DTT of the carrier protein diphtheria toxin. Patent document CN106822881A discloses a protein vaccine featuring a recombinant PCSK9 protein fragment polypeptide (catalytic domain and C-terminal domain).

[0266] WO2022150661A2 discloses viruses (including bacteriophage viruses or plant viruses) or virus-like particles for PCSK9 immunotherapy, which in particular contain the PCSK9-derived sequence NVPEEDGTRFHRQASKC.

[0267] EP3434279A1 discloses an OSK-1-PCSK9 conjugate vaccine using the PCSK9-derived sequences LRPRGQPNQC, SRHLAQASQ, and SRSGKRRGER. WO2021 / 154947 A1 discloses an anti-PCSK9 immunogen based on Ubith technology, i.e., a conjugate vaccine comprising a PCSK9 epitope fused to a promiscuous T cell epitope. The disclosed sequences include aa153-162, aa368-382, aa211-223, and SIPWNLERIT, CIGASSDSSTSFVSC, and CDGTRFHRQASKC.

[0268] WO2011 / 027257 A2 and WO 2012 / 131504 A1 disclose PCSK9-derived peptide-VLP and PCSK9-derived peptide-carrier vaccines targeting PCSK9, comprising the sequences SIPWNLERITPC, SIPWNLERITC, SIPWNLERITP, AGRDAGVAKGA, RDAGVAK;SRHLAQASQLEQ;GDYEELVLALR;LVLALRSEED;AKDPWRLP-;AARRGYLTK;FLVKMSGDLLELALKLP;EEDSSVFAQ. WO2015 / 123291 A1 discloses a peptide-VLP(Qb)-targeted PCSK9 vaccine comprising the sequences NVPEEDGTRFHRQASKC and CKSAQRHFRTGDEEPVN, and WO2018 / 189705 discloses peptide-carrier conjugates targeting PCSK9 based on the sequence SIPWNLERITPC and modified derivatives thereof.

[0269] A preferred polypeptide immunogen construct according to the invention contains a B-cell epitope derived from alpha-synuclein and a heterologous T-helper cell (Th) epitope linked to a CLEC. The present invention provides novel conjugates that are surprisingly superior to conventional vaccines in immunogenicity, cross-reactivity to alpha-synuclein, selectivity for alpha-synuclein species / aggregates, affinity, affinity maturation, and inhibitory potency.

[0270] Covalent conjugation of alpha-synuclein polypeptides to beta-glucan or mannan in accordance with the present invention allows for a surprising and unexpected enhancement of the immune response to such polypeptides, which is particularly impressive in direct comparison with traditional vaccine formulations such as those described by Rockenstein et al. (J. Neurosci., January 24, 2018; 38(4):1000-1014), as also demonstrated in the Examples section below.

[0271] Rockenstein et al. (2018) disclosed the application of yeast whole glucan particles (GPs) noncovalently complexed with aSyn and rapamycin as immunotherapeutic agents for Parkinson's disease. These GPs were produced from Saccharomyces cerevisiae following a series of hot alkaline, organic, and aqueous extraction steps, resulting in a final product consisting of a highly purified yeast cell wall preparation, 3–4 μm in diameter, free of cytoplasmic contents and bound by a porous, insoluble shell of β-glucan (primarily β1-3 β-glucan).

[0272] Importantly, the vaccine compositions disclosed by Rockenstein et al. (2018) consisted of GP non-covalently complexed with either ovalbumin and mouse serum albumin (MSA), human aSyn and MSA, or human aSyn, MSA, and rapamycin. This complexation method relied on co-incubation of different payloads with GP and subsequent diffusion into the hollow GP cavity without covalent conjugation, and is therefore similar to the set of vaccines disclosed in Example 28 provided within this application, which formulated vaccines using only mixing of components and no covalent conjugation, and was found to be inefficient and inadequate compared to the vaccines of the present invention.

[0273] 1) Rockenstein et al. showed that non-covalent mixing of aSyn with GP resulted in a detectable immune response to aSyn, thus indicating that GP can act as an adjuvant. However, Rockenstein et al. also showed that non-covalent addition / co-complexation of rapamycin was necessary to induce significantly enhanced functionality of such vaccines compared to controls. In this regard, a mixture of various adjuvants (GP and the mTOR inhibitor rapamycin) is necessary to provide a fully functional vaccine, such as the vaccine disclosed by the present invention.

[0274] 2) The vaccine disclosed by Rockenstein et al. is active in this aSyn-overexpression model because it provides aSyn-specific T cell epitopes (among other T cell epitopes, such as MSA-derived epitopes) to exert its full functionality, i.e., induction of neuroprotective anti-aSyn-directed cellular (i.e., T cell-mediated) and humoral (i.e., antibody / B cell-based) immune responses. This is in direct contrast to the teachings of the present invention, where it is already sufficient for only aSyn-specific B cell responses to be elicited by the selected vaccine.

[0275] 3) The use of full-length aSyn also poses the risk of inducing / enhancing autoreactive aSyn-specific T cells, which may exacerbate the neuropathology underlying PD and other synucleinopathies. Therefore, the GP-aSyn-rapamycin vaccine proposed by Rockenstein et al. is also unsuitable for human use due to this issue.

[0276] 4) As shown in Example 5, non-covalent mixing of aSyn-derived peptides (e.g., SEQ ID NO: 2, i.e., a B cell epitope) and promiscuous T cell epitopes (e.g., SEQ ID NO: 7) with β-glucan particles (e.g., unoxidized pustulan) can also induce low-level antibody responses to aSyn, similar to Rockenstein et al. However, vaccines according to the present invention based on the covalent attachment of such peptides to the appropriate glucan exert significantly different and superior immune responses (see also Figure 5).

[0277] Furthermore, as also disclosed in Example 6 and Figure 7, such covalent vaccines also show a significant beneficial lack of anti-glucan antibody responses compared to non-covalent combination vaccines based on glucan particles and peptides disclosed by the present invention.

[0278] Thus, the prior art disclosure by Rockenstein et al. does not suggest the claimed subject matter disclosed by the present invention.

[0279] Particularly preferred aSyn polypeptides to be conjugated in the present invention are native alpha-synuclein or the following amino acid sequence of native human alpha-synuclein: [ka] and a polypeptide comprising or consisting of amino acid residues 1-5, 1-8, 1-10, 60-100, 70-140, 85-99, 91-100, 100-108, 102-108, 102-109, 103-129, 103-135, 107-130, 109-126, 110-130, 111-121, 111-135, 115-121, 115-122, 115-123, 115-124, 115-125, 115-126, 118-126, 121-127, 121-140, or 126-135 of a polypeptide comprising or consisting of: 3-135, 107-130, 110-130, 115-121, 115-122, 115-123, 115-124, 115-125, 115-126, 118-126, 121-127, or 121-140; or a mimotope selected from the group consisting of DQPVLPD, DQPVLPDN, DQPVLPDNE, DQPVLPDNEA, DQPVLPDNEAY, DQPVLPDNEAYE, DSPVLPDG, DHPVHPDS, DTPVLPDS, DAPVTPDT, DAPVRPDS, and YDRPVQPDR.

[0280] All current prior art CLEC vaccines induce high titers against the carrier proteins used (e.g., CRM197 or OVA). However, this high immunogenicity and the structural complexity and heterogeneity of the carrier protein components can result in the induction of high levels of carrier / protein-specific antibodies at the expense of target-specific responses, which may be under-represented compared to the induced carrier responses.

[0281] Affinity maturation of target-specific responses induced upon repeated immunization with carrier conjugates is also impaired due to over-representation of carrier-specific epitopes in the conjugates. Affinity maturation in immunology, as used and understood herein, refers to the development of T FHCell activation is the process by which B cells produce antibodies with increasing affinity to antigens during the course of an immune response. Repeated exposure to the same antigen causes the host to produce antibodies with successively higher affinities. Secondary responses can elicit antibodies with several-fold higher affinities than the primary response. Affinity maturation occurs primarily in the surface immunoglobulins of germinal center B cells, through somatic hypermutation (SHM) and T cell differentiation. FH It occurs as a direct result of selection by the cell (see also https: / / en.wikipedia.org / wiki / Affinity_maturation). Affinity maturation according to Segen's Medical Dictionary (https: / / medical-dictionary.thefreedictionary.com / affinity+maturation”>affinity maturation) is the increase in the average affinity of antibodies for an antigen following immunization. Affinity maturation results in the increase of specific, more homogeneous IgG antibodies, following the less specific, more heterogeneous initial response by IgM molecules.

[0282] Furthermore, a high anti-carrier response also poses the risk of immunological rejection and related safety issues.

[0283] Therefore, the identification of effective constructs with low or non-existent carrier reactivity (i.e., against protein carriers), high immunogenicity, high target specificity, and high tolerability / safety according to the present invention successfully addresses this challenge with an innovative solution. Furthermore, it is important that the novel vaccines according to the present invention provide immunotherapeutic agents that induce no / very weak immune responses against the sugar backbone. This is particularly important because high anti-CLEC antibody levels induced upon immunization may inhibit or reduce the efficacy of repeated immunizations using the same CLEC-based vaccine due to vaccine neutralization or may adversely affect the use of this type of vaccine for sequential immunizations against various different targets.

[0284] The vaccine platform according to the present invention also meets the need to combine different epitopes against one or several targets in one formulation without the risk of reducing efficacy due to unintended epitope spreading, as reported for classical vaccines. The modular design of the platform according to the present invention allows for easy exchange of B- and T-cell epitopes without the adverse effects of carrier-induced responses.

[0285] The present invention is based on CLECs that exhibit highly specific binding to their cognate receptors. This binding is important as only strong binders are efficient as vaccine carriers / scaffolds.

[0286] According to the present invention, CLEC conjugate formation enables an efficient immune response with novel characteristics. The conjugate formation according to the present invention prevents the formation of anti-CLEC antibodies, especially against pustulan, and such prevention can be impressively demonstrated in the course of the present invention. This lack of induction of anti-CLEC antibodies is crucial for the reusability and re-booster stability of individual vaccines designed on the platform according to the present invention, whether with the same or different antigens.

[0287] In contrast to the conjugate embodiments of the present invention, simply mixing a CLEC polysaccharide adjuvant with a B-cell epitope peptide or a T-cell epitope peptide does not produce the same effect in vivo. However, when conjugated, the orientation of the peptide does not significantly affect the performance of the compounds according to the present invention; therefore, CLEC conjugation is substantially independent of the peptide orientation in the construct. In the course of the present invention, CLEC conjugation, particularly to pustulan, can be shown to result in novel peptide immunogens / antigens and improvements of existing peptide immunogens / antigens: this improvement is accompanied by a higher, more target-specific, and more affine antibody response (as can be demonstrated by antibody selectivity and functionality). This effect was most pronounced with pustulan or similar β-glucans, which are primarily linear β-(1,6)-glucans with a ratio of β-(1,6)-linked to non-β-(1,6)-linked monosaccharide moieties of at least 1:1, preferably at least 2:1, more preferably at least 5:1, and especially at least 10:1, which surprisingly performed significantly better than KLH or CRM in direct comparisons, and even better than conjugates of mannan or lichenan, or conjugates containing barley β-glucan.

[0288] As used herein, the term "predominantly linear" β-(1,6)-glucan refers to a β-(1,6)-D-glucan that is free or largely free of cross-linked sugar monomer entities, i.e., where less than 1%, preferably less than 0.1%, and in particular less than 0.01% of the monosaccharide moieties have three or more covalently linked monosaccharide moieties.

[0289] As already mentioned above, pustulan is the most preferred CLEC according to the present invention. Because pustulan typically does not contain cross-linked sugar moieties and is predominantly β-(1,6) linked, typical pustulan preparations used to prepare conjugates according to the present invention contain less than 1%, preferably less than 0.1%, and in particular less than 0.01% of monosaccharide moieties having three or more covalently linked monosaccharide moieties, and contain no more than 10% impurities including β-(1,3)-linked or β-(1,4)-linked monosaccharides.

[0290] Various references have shown that pustulan is not very effective at binding dectin-1 (e.g., Adams et al., J Pharmacol Exp Ther. 2008 Apr;325(1):115-23), and the literature has reported that linear 1,3 and branched (1,3 backbone and 1,6 branches) dectin-1 binders are the most effective. Therefore, the fact that pustulan was found to be the most effective CLEC during the course of this invention was unexpected. For example, Adams et al., 2008, reported that mouse recombinant dectin-1 recognizes and interacts only with polymers containing a β-(1,3)-linked glucose backbone. Dectin-1 did not interact with glucans composed exclusively of a β-(1,6)-glucose backbone (pustulan), nor did it interact with non-glucan carbohydrate polymers such as mannan.

[0291] Thus, according to a preferred embodiment of the present invention, the β-glucan of the conjugate is a dectin-1-binding β-glucan. The ability of any compound, particularly a glucan, to bind to dectin-1 can be readily determined by the methods disclosed herein, particularly in the Examples section. In case of doubt, a "dectin-1-binding β-glucan" is a β-glucan that binds to the soluble mouse Fc-Dectin-1a receptor with an IC50 value of less than 10 mg / ml, as determined by competitive ELISA, e.g., as disclosed in the Examples.

[0292] Dectin-1-binding β-glucans (such as linear β-(1,6)-glucans) according to the present invention are advantageous compared to other glucans, such as DC-SIGN β-glucans (such as β-(1,2)-glucans), because such dectin-1-binding glucans can address a wider range of DCs (immature, mature, myeloid, and plasmacytoid; also: APCs), significantly increasing the likelihood of inducing an effective immune response in vivo compared to non-dectin-1-binding glucans (immature DCs, myeloid DCs), which have limited applicability.

[0293] WO 2022 / 060487 A1 and WO 2022 / 060488 A1 disclose conjugates linking peptide immunogens to immunostimulatory polymer molecules (e.g., β-(1,2) glucans). β-(1,2) glucans, including cyclic variants, have previously been suggested as potential adjuvants (Martirosyan A et al., doi:10.1371 / journal.ppat.1002983). They are a class of glucans that primarily bind to a specific PRR, DC-SIGN (Zhang H et al., doi:10.1093 / glycob / cww041), and specifically bind to N-linked high-mannose oligosaccharides and branched fucosylated structures. Importantly, β-1,2-glucans are unable to bind to Dectin-1 (Zhang H et al., doi:10.1093 / glycob / cww041), thereby restricting their activity to DC-SIGN-positive cells.

[0294] DC-SIGN (CD209) was the first SIGN molecule identified and found to be highly expressed only on a restricted subset of DCs, including immature (CD83-negative) DCs, as well as specialized macrophages in the placenta and lung (Soilleux EJ et al., doi:10.1189 / jlb.71.3.445). In the periphery, e.g., skin or mucosal sites, expression, and therefore the potential biological activity of the receptor according to the present invention, is detectable only in a subset of immature DCs. Mature plasmacytoid DCs and other APCs, such as epithelial DC-like Langerhans cells, do not express DC-SIGN (Engering A, et al., doi:10.4049 / jimmunol.168.5.2118).

[0295] In contrast, the target receptor for the β-glucan-based immunogens provided herein is Dectin-1, which is expressed by a variety of different DC types, including immature DCs, myeloid DCs, as well as plasmacytoid DCs, which express Dectin-1 at both the mRNA and protein levels, and DC-like Langerhans cells in the skin (Patente et al., doi:10.3389 / fimmu.2018.03176; Joo et al. doi:10.4049 / jimmunol.1402276).

[0296] Therefore, while the biological activity of DC-SIGN-targeting polymers such as β-(1,2) glucan is limited to a specific DC target cell population, the Dectin-1-targeting polymers applied in the present invention can exert their function on a variety of additional DC types. Therefore, these novel conjugates can exert significantly different and superior immune responses compared to other conjugates. Therefore, the prior art disclosures do not suggest the claimed subject matter disclosed by the present invention.

[0297] According to a particularly preferred embodiment, the conjugate of the present invention comprises a potent Dectin-1-binding β-glucan, preferably a β-glucan that binds to soluble mouse Fc-Dectin-1a receptor with an IC50 value of less than 10 mg / ml, more preferably less than 1 mg / ml, even more preferably less than 500 μg / ml, and in particular less than 200 μg / ml, when determined by competitive ELISA, e.g., as disclosed in the Examples. Particularly preferred are conjugates that bind to soluble mouse Fc-Dectin-1a receptor with an IC50 value of less than 1 mg / ml, more preferably less than 500 μg / ml, even more preferably less than 200 μg / ml, and in particular less than 100 μg / ml, when determined by competitive ELISA; and / or

[0298] a β-glucan that binds to the soluble human Fc-Dectin-1a receptor with an IC50 value of less than 10 mg / ml, more preferably less than 1 mg / ml, even more preferably less than 500 μg / ml, and especially less than 200 μg / ml, as determined by competitive ELISA; and / or

[0299] The conjugate binds to the soluble human Fc-Dectin-1a receptor with an IC50 value of less than 1 mg / ml, more preferably less than 500 μg / ml, even more preferably less than 200 μg / ml, and especially less than 100 μg / ml, as determined by competitive ELISA, e.g., as disclosed in the Examples.

[0300] Furthermore, conjugates according to the invention also showed a proportionally higher increase in the ratio of antibodies reacting with the target polypeptide rather than the carrier molecule, as was the case with CLEC-free vaccines, particularly pustulan-free vaccines, which significantly increased the specific focusing of the antibody immune response against the target rather than the carrier, thereby increasing the efficacy and specificity of the response.

[0301] CLEC conjugation according to the present invention, particularly to pustulan, also results in increased affinity maturation (AM) towards the target protein (largely increased AM, whereas KLH / CRM conjugates show only limited AM upon repeated immunization).

[0302] In the field of vaccines, suitable vaccines have been disclosed that contain only B cell epitopes or only T cell epitopes. There are certain situations in which it is appropriate and preferable to have a vaccine that contains exclusively T cell epitopes or exclusively B cell epitopes. However, most commercially available vaccines contain both types of epitopes, i.e., T cell epitopes and B cell epitopes.

[0303] For example, vaccines containing only B cell epitopes are in most cases less effective, despite producing a detectable antibody immune response. However, in most cases, this immune response is usually much less effective than vaccines containing B cell and T cell epitopes. This is also consistent with the examples given in the Examples section of the present invention, where lower levels of responses were detectable.

[0304] On the other hand, vaccines containing only T cell epitopes (e.g., in vaccines where a specific T cell response is the active component of the response) are of particular interest for certain applications, particularly cancer, where only cancer-specific cytotoxic T lymphocyte and T helper cell epitopes or CTL epitopes are combined with the vaccine platform according to the invention. In this case, only T cell epitopes are provided with the CLEC polysaccharide adjuvant according to the invention. This is particularly preferred, for example, when somatic mutations in cancer affect protein-coding genes that can generate potential therapeutic neoepitopes. These neoepitopes can guide adoptive cell therapy and peptide (and RNA-based) neoepitope vaccines to selectively target tumor cells using the patient's autologous cytotoxic T cells. This can be used in accordance with the present invention for general antigen as well as personalized neoantigen-specific therapy (e.g., with NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-C1, MAGE-C2, MAGE-C3, survivin, gp100, tyrosinase, CT7, WT1, PSA, PSCA, PSMA, STEAP1, PAP, MUC1, 5 T4, KRAS, Her2, etc.). The use of vaccines bearing only T cell epitopes may also be preferred for specific autoimmune diseases. The therapeutic effect of conjugates of the respective T cell epitopes alone is the reduction of effector T cells and regulatory T cells (T cells), which leads to the suppression of the respective autoimmune disease (e.g., multiple sclerosis or similar diseases). reg Related to the development of cell populations.

[0305] Since most conventional vaccine settings contain both B and T cell epitopes, the CLEC conjugates according to the invention also preferably contain both individual B and T cell epitopes (at a minimum: at least one B cell epitope and at least one T cell epitope) for a sustained B cell immune response. However, if desired, a weaker effect may be demonstrated to demonstrate T cell-independent immunity.

[0306] Thus, the conjugates according to the present invention are not limited with respect to possible vaccine antigens, however, it is preferred that the vaccine antigens (i.e., B cell and / or T cell epitope polypeptides) have a length of 6-50 amino acid residues, preferably 7-40 amino acid residues, and especially 8-30 amino acid residues.

[0307] B cell receptor crosslinking is also possible using vaccines according to the present invention. In specific embodiments, conjugates according to the present invention are used for T cell-independent immunization. T cell-independent responses are well known for polysaccharide vaccines. These vaccines / polysaccharides generate immune responses through direct stimulation of B cells without T cell assistance. T cell-independent antibody responses are short-lived. Antibody concentrations to pneumococcal capsular polysaccharides typically decline to baseline within 3 to 8 years, depending on the serotype. Polysaccharide vaccines typically do not confer immunological memory, so booster doses cannot be used to boost vaccine responses. Polysaccharide vaccines have low immunogenicity in children under 2 years of age. Here, direct stimulation may be due to B cells expressing a molecule called CR3 (complement receptor type 3). Macrophage-1 antigen, or CR3, is a human cell surface receptor found on B and T lymphocytes, polymorphonuclear leukocytes (mainly neutrophils), NK cells, and mononuclear phagocytes such as macrophages. CR3 also recognizes iC3b when bound to the surface of foreign cells and β-glucan, meaning that direct uptake of vaccines by B cells via Pus-CR3 interactions can lead to cellular stimulation and the generation of low-level TI immune responses.

[0308] The adjuvants, conjugates, and vaccines of the present invention are capable of fixing complement and can be opsonized. Opsonized conjugates of the present invention can have increased B cell activation capacity, which can lead to higher antibody titers and affinity. This effect is known for C3d conjugates (Green et al., J. Virol. 77 (2003), 2046-2055) and can unexpectedly be used in the process of the present invention.

[0309] Another unexpected advantage of the present invention is that the CLEC structure of the present invention allows for modular vaccine design. For example, epitopes can be freely combined, and the platform is independent of conventional carrier molecules. While the main emphasis of the present invention is on peptide-only vaccines, the present invention also works with the independent attachment of proteins and peptides, as well as the attachment of peptide-protein conjugates to the CLEC scaffolds of the present invention, particularly pustulan. As shown in the Examples section using pustulan, significantly superior immune responses can be obtained according to the present invention compared to classical vaccines.

[0310] As already outlined above, the conjugates according to the invention, when provided in a pharmaceutical preparation (e.g. as a vaccine intended to be administered to a (human) subject in order to induce an immune response against the specific polypeptide epitope attached to the CLEC scaffold against which an immune response should be elicited), can be administered without the need to use (by co-administration) a (further) adjuvant in this preparation. According to a preferred embodiment, the pharmaceutical formulation comprising the conjugates according to the invention does not comprise an adjuvant.

[0311] A particularly preferred class of CLEC polysaccharide adjuvants according to the present invention are β-glucans, particularly pustulan. Another preferred CLEC polysaccharide adjuvant is mannan. In contrast to the present invention, pustulan has only been used in antifungal vaccines in the prior art (pustulan was used as an antigen, not as a carrier as in the present invention). Pustulan also presents a different backbone, as it is composed exclusively of β-(1,6)-linked sugar moieties.

[0312] Pustulan is a medium-sized linear β-(1,6) glucan. β-glucans are typically composed of branched glucan chains (preferably a β-(1,3) backbone with β-(1,6) side chains, such as those found in yeast extract, GP, laminarin, schizophyllan, and scleroglucan), or linear glucans that rely solely on β-(1,3) glucans, such as synthetic β-glucans, curdlan, and Saccharomyces cerevisiae β-glucan (150 kDa), or linear β-(1,3:1,4) glucans, such as barley β-glucan, oat β-glucan, and lichenan. Pustulan and synthetic forms of linear β-(1,6) glucans differ from all other glucans used in the art, as they typically consist of linear glucans that rely solely on branched glucan chains (preferably a β-(1,3) backbone with β-(1,6) side chains, such as those found in yeast extract, GP, laminarin, schizophyllan, and scleroglucan), or on synthetic β-glucans, such as curdlan and Saccharomyces cerevisiae β-glucan (150 kDa), or on linear β-(1,3:1,4) glucans, such as barley β-glucan and oat β-glucan, and lichenan.

[0313] As shown for the first time in this study, in vitro binding of glucan conjugates to the Dectin-1 receptor is a proxy for subsequent in vivo efficacy: low-binding molecules can only elicit a low immune response, moderate binders are better, and highly efficient binders induce a highly effective response (oat / barley BG < licanan < pustulan).

[0314] According to the present invention, CLECs are linked (e.g., by standard techniques) to individual polypeptides to create small nanoparticles with low polydispersity (hydrodynamic radius (HDR) range: 5-15 nm) that are not crosslinked and aggregate to form larger particles similar to conventional CLEC vaccines, such as glucan particles (2-4 μm) or β-glucan particles disclosed in the literature, characterized by a size range typically exceeding 100 nm (typical range (diameter; 150-500 nm; e.g., Wang et al. (2019) provided particles with a diameter of 160 nm (assessed by DLS) and a size of approximately 150 nm as assessed by TEM; Jin et al. (Acta Biomater. 2018 Sep 15;78:211-223) provided β-glucan particles (aminated β-glucan-ovalbumin nanoparticles) with a size of 180-215 nm (assessed by DLS and SEM, respectively)).

[0315] By definition, the DLS-measured hydrodynamic radius is the radius of a hypothetical hard sphere diffusing at the same rate as the particle under test. The radius is calculated from the diffusion coefficient, assuming a spherical shape for the molecule / particle and a given viscosity of the buffer. The HDR is also called the Stokes radius and is calculated from the diffusion coefficient using the Stokes-Einstein equation (see https: / / en.wikipedia.org / wiki / Stokes_radius).

[0316] The preferred diameter range of the nanoparticles according to the present invention may be that typically provided in the prior art, i.e., diameters of 1 to 5000 nm, preferably 1 to 200 nm, and particularly 2 to 160 nm, as determined by dynamic light scattering (DLS) as the hydrodynamic radius (HDR). According to preferred embodiments of the present invention, the particle size is smaller, e.g., 1 to 50 nm, preferably 1 to 25 nm, and particularly 2 to 15 nm, as determined by DLS as the HDR. Therefore, these preferred particles, including the peptide-only conjugate (average HDR of about 5 nm) and the CRM-pustulan conjugate (average HDR of about 10 to 15 nm), are smaller. Therefore, preferred particles according to the present invention are less than 100 nm, which distinguishes us from Wang et al.

[0317] Thus, the present invention also relates to a vaccine product designed to vaccinate an individual against a specific antigen, comprising a compound comprising β-glucan or mannan as a C-type lectin (CLEC) polysaccharide adjuvant covalently linked to the specific antigen.

[0318] Preferably, a vaccine product according to the invention comprises a conjugate disclosed herein or obtainable or obtained by a method according to the invention.

[0319] According to a preferred embodiment, the vaccine product according to the invention comprises an antigen comprising at least one B cell epitope and at least one T cell epitope, preferably the antigen is a polypeptide comprising one or more B cell epitopes and T cell epitopes.

[0320] According to a preferred embodiment, the covalently linked antigen and CLEC polysaccharide adjuvant in the vaccine product according to the invention are present as particles having a diameter of 1 to 5000 nm, preferably 1 to 200 nm, especially 2 to 160 nm, as determined as the hydrodynamic radius (HDR) by dynamic light scattering (DLS). As used herein, all particle sizes are median particle sizes, the median being the value that separates the half of particles having a larger diameter from the half having a smaller diameter. The median particle size is the determined particle size at which half of the particles are smaller and half are larger.

[0321] According to a preferred embodiment, the covalently linked antigen and CLEC polysaccharide adjuvant in the vaccine product according to the invention are present as particles with a diameter of 1 to 50 nm, preferably 1 to 25 nm, in particular 2 to 15 nm, determined as HDR by DLS.

[0322] Preferably, the covalently linked antigen and CLEC polysaccharide adjuvant in the vaccine product according to the invention are present as particles with a diameter, determined as HDR by DLS, of less than 100 nm, less than 50 nm, preferably less than 70 nm, especially less than 50 nm.

[0323] The vaccine product according to the invention exhibits high storage stability: it can be determined that the particle size does not increase significantly (i.e., by more than 10%) upon storage, so that virtually no aggregation occurs when stored as liquid or frozen material (storage temperatures: -80°C, -20°C, 2-8°C, or at room temperature for extended periods of at least 3 months).

[0324] The extremely high efficacy of such small particles produced by using the medium molecular weight component pustulan according to the present invention is surprising: for example, according to Adams et al. (J Pharmacol Exp Ther. 2008 Apr;325(1):115-23), the best Dectin-1 substrates are linear β(1,3) glucan phosphate (approximately 150 kDa) and branched glucans (containing a β(1,3) main chain and β(1,6) side chains) such as scleroglucan, or a glucan derived from Candida albicans, or laminarin. Furthermore, data from Adams et al., Palma et al. (J Biol Chem. 281(9) (2006) 5771-5779), and Willment et al. (J Biol Chem. 276(47) (2001), 43818-23) indicate that dectin-1 does not interact, or only weakly interacts, with pustulan, and that dectin-1 also does not interact with non-glucan carbohydrate polymers such as mannan. Indeed, various references have reported pustulan as being less effective at binding dectin-1. However, in general, linear 1,3 and branched (1,3 backbone and 1,6 side branches) dectin-1 are the most effective dectin-1 binders; Adams et al. (2008) showed that only mouse recombinant dectin-1 recognized and interacted with polymers containing a β(1,3)-linked glucose backbone. Dectin-1 did not interact with glucans composed exclusively of a β(1,6)-glucose backbone (such as pustulan), nor did it interact with non-glucan carbohydrate polymers such as mannan.

[0325] In contrast to these findings, it was shown in the course of this invention that pustulan-based conjugates can bind strongly to dectin-1 and induce cellular responses in vitro.

[0326] According to a preferred embodiment of the present invention, β-(1,6)-glucan is used. Because the prior art has generally reported that larger particles are more effective at activating PRRs than smaller ("soluble") monomeric formulations, particles containing larger glucans are superior (and therefore preferred), and small, soluble glucans can be used to block DC activation, thereby preventing the intended effect. It is well accepted that particulate β-glucans, such as the widely used yeast cell wall fraction zymosan, bind to and activate Dectin-1, thereby inducing cellular responses. In contrast, the interaction of soluble β-glucans with Dectin-1 has been the subject of debate. However, the general consensus is that soluble β-glucans, such as the small branched glucan laminarin (β-(1,3) and β-(1,6) side chains), bind to dectin-1 but are unable to initiate signaling and induce cellular responses in DCs (Willment et al., J Biol Chem. 276(47) (2001), 43818-23; Goodridge et al. Nature. 2011, 472(7344):471-475).

[0327] According to the present invention, conjugates using high-molecular-weight glucans (10 times the size of pustulan; e.g., oat / barley 229 kDa / lichenan 245 kDa) have been shown to be less effective than pustulan particles (20 kDa). Korotchenko et al. demonstrated that OVA / Lam conjugates with a diameter of approximately 10 nm bind to dectin-1 and induce DC activation in vitro, but are branched glucans that are not skin-specific and are not superior in in vivo efficacy to cutaneously administered OVA or subcutaneously administered OVA / Alum. Wang et al. reported β-glucan particles with a diameter of over 100 nm (mean diameter: 160 nm). Jin et al. (2018) demonstrated aminated β-glucan-ovalbumin nanoparticles with diameters of 180-215 nm.

[0328] According to the present invention, pustulan-based particles have been shown to be potent Dectin-1 binders, activate DCs in vitro (changes in surface marker expression), and induce very strong immune responses a) superior to other routes, b) comparable to KLH / CRM conjugate vaccines (usually much larger particles) and c) larger glucans and mannans. This is true for Pep+Padre+pustulan (5 nm diameter) and Pep+CRM+pustulan (11 nm diameter).

[0329] For optimal immune responses, the degree of activation of CLECs, especially pustulan, and the resulting peptide / sugar ratio are crucial. Activation of each CLEC is achieved by mild periodate oxidation. Therefore, the degree of oxidation is determined based on adding periodate solution at a defined molar ratio, i.e., periodate:sugar subunit; 100% = 1 mol of periodate per mol of sugar monomer.

[0330] According to a preferred embodiment, the conjugate according to the invention comprises CLECs activated in a ratio of periodate to β-glucan or mannan (monomer) moieties of 1 / 5 (i.e. 20% activation) to 2.6 / 1 (i.e. 260% activation), preferably 60% to 140%, in particular 70% to 100%.

[0331] The optimal range of oxidation (directly proportional to the number of epitope polypeptides in the final conjugate), from low / medium to high oxidation, can be defined as the reactivity with Schiff's Fuscin reagent that is similar to the reactivity of an equivalent amount of a given carbohydrate (e.g., pustulan) oxidized with periodate at molar ratios (sugar monomer:periodate) of 0.2-0.6 (low / medium), 0.6-1.4 (optimal range), and 1.4-2.6 (high), respectively.

[0332] Preferred glucan to peptide ratios are in the range of 10:1 (w / w) to 1:1 (w / w), preferably 8:1 (w / w) to 2:1 (w / w), in particular 4:1 (w / w), i.e., a sugar monomer to peptide molar ratio of 24:1, except that when the conjugate includes a carrier protein, preferred ratios of β-glucan or mannan to B cell-epitope-carrier polypeptide are 50:1 (w / w) to 0.1:1 (w / w), in particular 10:1 to 0.1:1, which are lower than the effective vaccines reported elsewhere (e.g., Liang et al., Bromuro et al.).

[0333] The degree of oxidation and the amount of reactive aldehydes available for sugar conjugation can be determined using prior art methods, such as: 1) gravimetric determination, which allows for the determination of the total mass of the sample; 2) the anthrone method (Laurentin et al. 2003), which determines the concentration of intact, non-oxidized sugars in a sample; in this case, glucan is dehydrated with concentrated H2SO4 to form furfural, which is then condensed with anthrone (0.2% in H2SO4) to form a green complex that can be measured colorimetrically at 620 nm; or 3) the Schiff assay, which assesses the oxidation state of the carbohydrate used in conjugation using Schiff's fuchsin-bisulfite reagent. Briefly, the fuchsin dye is decolorized with sulfur dioxide. Reaction with aliphatic aldehydes (on the glucan) restores the purple color of fuchsin, which can then be measured at 570-600 nm. The resulting color reaction is proportional to the degree of carbohydrate oxidation (the amount of aldehyde groups). Other suitable analytical methods are also possible. Peptide ratios can be assessed using appropriate methods, including UV analysis (205 nm / 280 nm) and amino acid analysis (aa hydrolysis, derivatization, and RP-HPLC analysis).

[0334] The conjugates according to the invention can further be used to induce a target-specific immune response but not a CLEC- or carrier protein-specific antibody response, or to induce only a very limited CLEC- or carrier protein-specific antibody response. As also shown in the Examples section below, the present invention also allows for an improved and focused target-specific immune response, as it induces an immune response away from a response to the carrier protein or CLEC (e.g., in conventional peptide-carrier conjugates or in a non-conjugate comparative setting, especially with the application of non-oxidized CLECs such as pustulan).

[0335] Unless otherwise indicated, as used herein, "peptide" refers to a shorter polypeptide chain (2-50 amino acid residues long), and "protein" refers to a longer polypeptide chain (more than 50 amino acid residues long). Both are referred to as "polypeptides." The B cell and / or T cell epitope polypeptides bound to the CLECs of the present invention include not only polypeptides having amino acid residues naturally used in normal gene expression and protein translation, but also all other forms of such polypeptide-based B cell and / or T cell epitopes, particularly their natural or artificially modified forms such as glycopeptides, and all other post-translationally modified forms thereof (e.g., the pyro-Glu form of Aβ disclosed in the Examples). Furthermore, the CLECs of the present invention are particularly suited to presenting structural epitopes, e.g., structural epitopes that are part of larger naturally occurring polypeptides, mimotopes, cyclic polypeptides, or surface-bound constructs.

[0336] According to a preferred embodiment, the conjugate according to the present invention comprises a CLEC polysaccharide backbone and a B-cell epitope. A "B-cell epitope" is a portion of an antigen to which an immunoglobulin or antibody binds. B-cell epitopes can be divided into two groups: structural and linear. There are two main approaches to epitope mapping: structural and functional studies. Structurally mapping methods for epitopes include X-ray crystallography, nuclear magnetic resonance, and electron microscopy. Functionally mapping methods for epitopes often use binding assays such as Western blots, dot blots, and / or ELISAs to determine antibody binding. Competitive methods determine whether two monoclonal antibodies (mAbs) can simultaneously bind to an antigen or compete with each other for binding at the same site. Another technique involves high-throughput mutagenesis, an epitope mapping strategy developed to improve the rapid mapping of structural epitopes on structurally complex proteins. Mutagenesis uses random or site-directed mutations at individual residues to map epitopes. B cell epitope mapping can be used to develop antibody therapeutics, peptide-based vaccines, and immunodiagnostic tools (Sanchez-Trincado et al., J. Immunol. Res. 2017-2680160). For many antigens, B cell epitopes are known and can be used in this CLEC platform.

[0337] According to a particularly preferred embodiment, the conjugates according to the invention comprise a CLEC polysaccharide backbone and one or more T cell epitopes, preferably promiscuous T cell epitopes and / or MHC II epitopes known to act on several MHC alleles of a given species and other species.

[0338] According to another aspect, the present invention also relates to the use of the present CLEC technology to improve known T cell epitopes. Thus, the present invention also encompasses β-glucan or mannan for use as a C-type lectin (CLEC) polysaccharide adjuvant for T cell epitope polypeptides, covalently linked to the T cell epitope polypeptide to form a conjugate of β-glucan or mannan and the T cell epitope polypeptide.

[0339] A single T cell epitope that binds to two or more HLA alleles is called a "promiscuous T cell epitope." Preferred promiscuous T cell epitopes bind to five or more, preferably ten or more, and particularly fifteen or more HLA alleles. Promiscuous T cell epitopes are suitable for various species, and most importantly, for several MHC / HLA haplotypes of a given species (referring to both MHC I and MHC II epitopes, which are known to function at some / all MHC alleles) and other species. For example, as mentioned in the Examples section, the MHC II epitope PADRE (= non-natural pan-DR epitope (PADRE)) functions at several human MHC alleles and in mice (C57 / B16, although less effective in Balb / c). For example, as mentioned in the Examples section, the MHCII epitope PADRE (=non-natural pan-DR epitope (PADRE)) is functional in some human MHC alleles and in mice (C57 / B16, although less effective in Balb / c). According to a preferred embodiment, the conjugates of the invention comprise a T-cell epitope, preferably a T-cell epitope comprising the amino acid sequence AKFVAAWTLKAAA ("PADRE(polypeptide)") or a PADRE(polypeptide) variant.

[0340] Preferred PADRE polypeptides or PADRE polypeptide variants include linkers such as a cysteine ​​residue or a linker containing a cysteine ​​residue ("-C" or "C-"; particularly for maleimide linkages), NRRA, NRRA-C, or NRRA-NH-NH2 linker (also preferred for other polypeptide epitopes used herein).Preferred PADRE polypeptide variants include those disclosed in the prior art (e.g., Alexander et al., Immunity 1 (1994), 751-761; US ​​9,249,187 B2), preferably truncated variants without a C-terminal A residue (AKFVAAWTLKAA), variants in which the first residue alanine is replaced with an aliphatic amino acid residue (e.g., glycine, valine, isoleucine, and leucine), variants in which the third residue phenylalanine is replaced with L-cyclohexylalanine, variants in which the thirteenth (last) amino acid residue alanine is replaced with an aliphatic amino acid residue (e.g., glycine, valine, isoleucine, and leucine), and variants in which the thirteenth (last) amino acid residue alanine is replaced with an aliphatic amino acid residue (e.g., glycine, valine, isoleucine, and leucine). a variant comprising aminocaproic acid, preferably linked to the C-terminus of a PADRE variant, or a variant having the amino acid sequence AX1FVAAX2TLX3AX4A (X1 is selected from the group consisting of W, F, Y, H, D, E, N, Q, I, and K; X2 is selected from the group consisting of F, N, Y, and W; X3 is selected from the group consisting of H and K; and X4 is selected from the group consisting of A, D, and E) (provided that the oligopeptide sequence is not AKFVAAWTLKAAA; US 9,249,187 B2); in particular, T cell epitopes include AKFVAAWTLKAAANRRA-(NH-NH), AKFVAAWTLKAAAN-C, AKFVAAWTLKAAA-C, AKFVAAWTLKAAANRRA-C, aKXVAAWTLKAAaZC, aKXVAAWTLKAAaZCNRRA (SEQ ID NOs: 7, 8, 87, 88, 89, 90, 91, 92), aKXVAAWTLKAAa, aKXVAAWTLKAAaNRRA, aA(X)AAAKTAAAAa, aA(X)AAATLKAAa, aA(X)VAAATLKAAa, aA(X)IAAATLKAAa, aK(X)VAAWTLKAAa, and aKFVAAWTLKAAa (Alexander et al., 1994 sequences 760.5, 760.57, 906.09, 906.11, 965.10, 1024.03) (wherein X is L-cyclohexylalanine, Z is aminocaproic acid, and a is an aliphatic amino acid residue selected from alanine, glycine, valine, isoleucine, and leucine).

[0341] T cell epitopes are presented on the surface of antigen-presenting cells, where they are bound to major histocompatibility complex (MHC) molecules. In humans, professional antigen-presenting cells are specialized to present MHC class II peptides, while most nucleated somatic cells present MHC class I peptides. T cell epitopes presented by MHC class I molecules are typically peptides 8–11 amino acids long, while MHC class II molecules present longer peptides 13–17 amino acids long. Non-classical MHC molecules also present non-peptide epitopes such as glycolipids. MHC class I and MHC class II epitopes can be reliably predicted by computational means alone, but not all in silico T cell epitope prediction algorithms are equivalent in their accuracy. There are two main methods for predicting peptide-MHC binding: data-driven and structure-based. Structure-based methods model peptide-MHC structures and require significant computational power. Data-driven methods have higher predictive performance than structure-based methods. Data-driven methods predict peptide-MHC binding based on the peptide sequence that binds to the MHC molecule (Sanchez-Trincado et al., 2017). Identifying T cell epitopes allows scientists to track, phenotype, and stimulate T cells. For many antigens, T cell epitopes are known and can be used in this CLEC platform.

[0342] Interestingly, recent breakthrough studies have demonstrated an increase in α-synuclein-specific T cells in PD patients, possibly associated with HLA risk haplotypes, suggesting the involvement of T cells in autoimmunity in PD [Sulzer et al., Nature 2017;546:656-661 and Lindestamn Arlehamn et al., Nat Commun. 1875;2020:11]. The causal role of α-synuclein-reactive T cells has also been recently strengthened by animal model studies [Williams et al., Brain. 2021;144:2047-2059]. The occurrence of α-synuclein-reactive T cells increased years before motor onset in case studies, and their frequency was highest near and immediately after motor onset in a larger cross-sectional cohort of PD patients [Lindestam Arlehamn et al.]. After motor onset, T cell responses to α-synuclein decreased with increasing disease duration. Thus, anti-aSyn T cell responses are highest before or shortly after diagnosis of motor PD and decline thereafter (i.e., maximal detectable activity <10 years after diagnosis; and favor Horne-Yahne (H+Y) stages 1 and 2) (Lindestamn Arlehamn et al. 2020).

[0343] Thus, there are commonly known T cell epitopes contained within the sequence of human alpha-synuclein. Examples are provided in Benner et al. (PLoS ONE 3(1):e1376.60), Sulzer et al., (2017) and Lindestam Arlehamn et al. (2020).

[0344] Benner et al. (Benner et al., (2008) PLoS ONE 3(1):e1376.) used a 60-aa-long nitrated (at Y residues) polypeptide containing the C-terminal portion of α-synuclein T cell epitope aa71-86 (VTGVTAVAQKTVEGAGNIAAATGFVK) emulsified with an equal volume of CFA containing 1 mg / ml of Mycobacterium tuberculosis as an immunogen in a PD model.

[0345] Sulzer et al. (Nature 2017;546:656-661) identified two T cell antigenic regions in the N- and C-terminal regions of α-synuclein from human PD patients. The first region is located near the N-terminus and consists of MHC II epitopes aa 31-45 (GKTKEGVLYVGSKTK) and aa 32-46 (KTKEGVLYVGSKTKE), and also contains a 9-mer polypeptide aa 37-45 (VLYVGSKTK) as a potential MHC I class epitope. The second antigenic region disclosed by Sulzer et al. is located near the C-terminus (aa 116-140) and requires phosphorylation of amino acid residue S129. Three phosphorylated aaS129 epitopes, aa116-130 (MPVDPDNEAYEMPSE), aa121-135 (DNEAYEMPSEEGYQD), and aa126-140 (EMPSEEGYQDYEPEA), induced significantly higher responses in PD patients than in healthy controls. The authors also demonstrated that the naturally occurring immune response to PD-associated α-synuclein has both MHC class I and MHC class II restriction components.

[0346] Furthermore, Lindestam Arlehamn et al. (Nat Commun. 1875; 2020: 11) also disclosed α-synuclein peptide aa61-75 (EQVTNVGGAVVTGVT) as a T cell epitope (MHCII) in PD patients.

[0347] Thus, preferred T cell epitopes according to the invention include the alpha-synuclein polypeptides GKTKEGVLYVGSKTK (aa 31-45), KTKEGVLYVGSKTKE (aa 32-46), EQVTNVGGAVVTGVT (aa 61-75), VTGVTAVAQKTVEGAGNIAAATGFVK (aa 71-86), DPDNEAYEMPSE (aa 116-130), DNEAYEMPSEEGYQD (aa 121-135), and EMPSEEGYQDYEPEA (aa 126-140).

[0348] Regulatory T cells ("Treg cells" or "Tregs") are a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Treg cells are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs produced by the normal thymus are called "natural." Natural Tregs are selected by radiation-resistant hematopoietic-derived MHC class II-expressing cells in the medulla oblongata or Hassall's corpuscles in the thymus. The Treg selection process is determined by the affinity of their interaction with self-peptide-MHC complexes. Selection to become Tregs is a "Goldilocks" process—i.e., T cells that receive a very strong signal undergo apoptotic death, while cells that receive a weak signal survive and are selected to become effector cells. T cells become regulatory cells when they receive an intermediate signal. Due to the stochastic nature of the T cell activation process, all T cell populations bearing a given TCR end up as a mixture of Teff and Treg (the relative proportions are determined by the affinity of the T cell for self-peptide MHC). Tregs formed outside the thymus, i.e., by differentiation of peripheral naive T cells, or in cell culture, are called "adaptive" or "inducible" (i.e., iTregs).

[0349] Natural Tregs are characterized by the expression of both the CD4 T cell coreceptor and CD25, a component of the IL-2 receptor. Thus, Tregs are CD4+CD25+. Expression of the nuclear transcription factor forkhead box P3 (FoxP3) is a defining characteristic that determines the development and function of natural Tregs. Tregs are thought to suppress the activation, proliferation, and cytokine production of CD4+ and CD8+ T cells and to suppress B cells and dendritic cells, thereby dampening autoimmune responses.

[0350] Along these lines, several studies have shown that the number and function of Tregs are reduced in PD patients. For example, Hutter Saunders et al. (J Neuroimmune Pharmacol (2012) 7:927-938) and Chen et al. (MOLECULAR MEDICINE REPORTS 12:6105-6111, 2015) showed that regulatory T cells (Tregs) from PD patients exhibited impaired ability to suppress effector T cell function, demonstrating increased proportions of Th1 and Th17 cells, while decreased proportions of Th2 and Treg cells. Thome et al. (npj Parkinson's Disease (2021) 7:41) demonstrated that reduced PD Treg function correlated with increased pro-inflammatory T cell activation, which may directly result in subsequent increased pro-inflammatory signaling by other immune cell populations. Treg suppression of T cell proliferation was significantly correlated with a peripheral pro-inflammatory immune cell phenotype. The suppressive capacity of PD Tregs against T effector cell (e.g., CD4+) proliferation decreased with increasing PD disease burden using the H&Y disease scale, with highest activity in stages H+Y 1 and 2. Importantly, Lindestam Arlehamn et al. (2020) showed that anti-aSyn T cell responses were highest before or shortly after the diagnosis of motor PD and subsequently decreased (i.e., maximal detectable activity less than 10 years after diagnosis; and favored Hohn-Yarr (H+Y) stages 1 and 2) (Lindestamn Arlehamn et al. 2020).

[0351] Therefore, the vaccine of the present invention and 1) vaccines containing alpha-synuclein-specific Treg epitopes (e.g., CD4 epitopes such as those disclosed by Brenner et al., Sulzer et al., and Lindestam Arlehamn et al. (aa31-45 (GKTKEGVLYVGSKTK), aa32-46 (KTKEGVLYVGSKTKE), aa61-75 (EQVTNVGGAVVTGVT), aa71-86 (VTGVTAVAQKTVEGAGNIAAATGFVK), aa116-130 (MPVDPDNEAYEMPSE), aa121-135 (DNEAYEMPSEEGYQD), and aa126-140 (EMPSEEGYQDYEPEA)); and / or 2) Treg inducers such as rapamycin, low-dose IL-2, TNF receptor 2 (TNFR2) agonists, anti-CD20 antibodies (e.g., rituximab), prednisolone, inosine pranobex, glatiramer acetate, and sodium butyrate Combination with aSyn is preferred in the early stages of disease (i.e., less than 10 years after diagnosis; and Hoehn-Yahr stages 1 and 2 are preferred) to enhance the waning / decline in Treg numbers and activity, thereby reducing the autoimmune reactivity of aSyn-specific T effector cells and attenuating the autoimmune response in PD patients.

[0352] In addition, Tregs have been found to be reduced and / or dysfunctional in several chronic degenerative or autoimmune diseases, particularly in (active) systemic lupus erythematosus (SLE, aSLE), type 1 diabetes (T1D), autoimmune diabetes (AID), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and Alzheimer's disease (AD), among other degenerative diseases (ALS: Beers et al., JCI Insight 2, e89530 (2017); AD: Faridar et al., Brain Commun. 2, fcaa112 (2020); ALS: Beers et al., JAMA Neurol. 75, 656-658 (2018); MS: Haas et al., Eur. J. Immunol. 35, 3343-3352 (2005); T1D: Lindley et al., Diabetes 54, 92-99 (2005):AID:Putnamet al., J.Autoimmun.24, 55-62 (2005);Autoimmune diseases: Ryba-Stanislawowska et al., Expert Rev.Clin.Immunol.15, 777-789 (2019);aSLE:Valencia et al., J.Immunol.178, 2579-2588 (2007);MS:Vigliettaet al., J.Exp.Med.199, 971-979 (2004);sLE:Zhang et al., Clin.Exp.Immunol.153, 182-187 (2008);AD+MS:Ciccocioppo et al., Sci.Rep.9, 8788 (2019)).

[0353] Therefore, it is also preferred to provide suitable T cell epitopes as Treg epitopes or Treg inducers in diseases with reduced or dysfunctional Treg populations in combination with the vaccine according to the present invention in order to enhance the number and activity of depleted / reduced Tregs, thereby reducing the autoimmune reactivity of disease-specific T effector cells and attenuating the autoimmune response in patients. Suitable Treg epitopes are defined as self-MHC epitopes (MHC type II) characterized by their ability to induce intermediate signals during the T cell selection process.

[0354] According to a preferred embodiment, the conjugate according to the invention comprises a polypeptide comprising or consisting of the amino acid sequences SEQ ID NOs: 7, 8, 22-29, 87-131, GKTKEGVLYVGSKTK, KTKEGVLYVGSKTKE, ​​EQVTNVGGAVVTGVT, VTGVTAVAQKTVEGAGNIAAATGFVK, MPVDPDNEAYEMPSE), DNEAYEMPSEEGYQD, EMPSEEGYQDYEPEA, or combinations thereof.

[0355] Thus, preferred T cell epitopes are: [Table 13]

[0356] [Table 14]

[0357] According to another preferred embodiment, the conjugate according to the invention comprises a B-cell epitope and a T-cell epitope, preferably a pan-specific / promiscuous T-cell epitope, independently linked to a CLEC polysaccharide scaffold according to the invention, in particular pustulan.

[0358] According to another preferred embodiment, the conjugate according to the invention comprises a B-cell epitope linked to a "classical" carrier protein such as CRM197, this construct being further linked to a CLEC carrier according to the invention, in particular pustulan.

[0359] For example, in the first step, CRM conjugate formation can be achieved by activation of CRM via GMBS or sulfo-GMBS, etc.; then, the maleimide group of the activated CRM is reacted with the SH group of the peptide (cysteine). The CRM conjugate is then treated with DTT to reduce the disulfide bond and generate an SH group on the cysteine. A one-pot reaction can then be performed to prepare a CLEC-based vaccine by mixing the reduced CRM conjugate with BMPH (N-β-maleimido-propionic acid hydrazide) and activated pustulan (oxidized). The mechanism in the one-pot reaction can be to react oxidized pustulan (with respect to pustulan) with BMPH (containing a hydrazide residue) to form a BMPH-hydrazone. The reduced CRM conjugate then reacts with the maleimide of the BMPH-activated pustulan via the SH group on the CRM conjugate.

[0360] According to another preferred embodiment, the conjugate according to the invention comprises a "classical" carrier protein, such as CRM197, containing multiple T cell epitopes. The conjugate according to the invention also comprises a B cell epitope covalently linked to the polysaccharide moiety. In this embodiment, both polypeptides (B cell epitope and carrier molecule) are independently linked to a CLEC carrier according to the invention, in particular pustulan.

[0361] According to another preferred embodiment, the conjugates of the present invention also comprise a "classical" carrier protein, such as CRM197, containing multiple T cell epitopes. The conjugates of the present invention also comprise a B cell epitope covalently linked to the "classical" carrier protein. The peptide-carrier conjugates of the present invention are also covalently linked to a polysaccharide moiety. In this embodiment, both polypeptides (B cell epitope and carrier molecule) are linked to a CLEC carrier of the present invention, in particular pustulan, as a single conjugate. The carrier protein thus represents the link between the β-glucan or mannan and the B cell and / or T cell epitope polypeptide in the conjugates of the present invention. Therefore, the covalent conjugation between the β-glucan or mannan and the B cell and / or T cell epitope polypeptide is achieved by the carrier protein (as a functional linking moiety).

[0362] A preferred conjugate according to the invention may comprise a B-cell epitope linked to CRM197, this construct being further linked to a CLEC polymer according to the invention, in particular a β-glucan, wherein the β-glucan is pustulan, lichenan, laminarin, curdlan, β-glucan peptide (BGP), schizophyllan, scleroglucan, whole glucan particle (WGP), zymosan or lentinan, preferably pustulan, laminarin, lichenan, lentinan, schizophyllan or scleroglucan, in particular pustulan.

[0363] According to the present invention, a novel B cell epitope-CRM197 conjugate linked to pustulan was shown to be a potent Dectin-1 binder and induces a very strong immune response, superior to conventional CRM conjugate vaccines.

[0364] According to the present invention, conjugation of CLECs to novel B cell epitope-CRM197 conjugates, in particular the creation of B cell epitope-CRM197-glucan, more preferably B cell epitope-CRM197-linear β-(1,6)-glucan or B cell epitope-CRM197-pustulan conjugates, has been shown to be essential for the induction of superior immunogenicity described for various peptide-CRM197-CLECs, in particular peptide-CRM197-β-glucan, more preferably peptide-CRM197-linear β-(1,6)-glucan or peptide-CRM197-linear pustulan conjugates, compared to conventional CRM conjugate vaccines, adjuvanted or unadjuvanted, when mixed with β-glucan / pustulan.

[0365] According to a preferred embodiment of the present invention, the CLEC conjugates according to the present invention comprise a CLEC conjugate comprising a CLEC-specific nucleotide sequence as a source of T cell epitopes (e.g., CRM197, KLH, diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae protein D (HipD), and outer membrane protein complex (OMPC) of serogroup B Neisseria meningitidis, a recombinant non-toxic form of Pseudomonas aeruginosa exotoxin A (rEPA), flagellin, Escherichia coli heat-labile enterotoxin (LT), cholera toxin (CT), mutant toxins (e.g., LTK63 and LTR72)). The conjugate comprises an oligosaccharide / polysaccharide as a B cell epitope linked to a carrier protein, and the construct is further linked to a CLEC polymer according to the present invention, in particular a β-glucan, where the β-glucan is pustulan, lichenan, laminarin, curdlan, β-glucan peptide (BGP), schizophyllan, scleroglucan, whole glucan particle (WGP), zymosan, or lentinan, preferably pustulan, laminarin, lichenan, lentinan, schizophyllan, or scleroglucan, in particular pustulan. When the conjugate comprises a carrier protein, it is a preferred embodiment of the present invention that the conjugate according to the present invention comprises at least an additional independently linked T cell epitope or B cell epitope. This preferred embodiment further clarifies that the present invention is not directed to eliciting specific antibodies against predominantly linear β-(1,6)-glucans, such as pustulan, in which the ratio of (1,6)-linked to non-β-(1,6)-linked monosaccharide moieties is at least 1:1. Therefore, since conjugates according to the present invention significantly reduce or eliminate the induction of a strong de novo immune response against the glucan backbone in vivo when the conjugate contains an additional T cell epitope or B cell epitope (see, for example, Example 7 and Figure 7 below), conjugates containing primarily linear β-(1,6)-glucan with a ratio of (1,6)-linked to non-β-(1,6)-linked monosaccharide moieties of at least 1:1, which contain only a sugar as an antigen and a carrier protein, are excluded from the present invention. In contrast, repeated application of unconjugated glucan (or glucan bound only to a carrier protein) leads to the induction of a strong anti-glucan immune response by enhancing antibody levels against the glucan polysaccharide.This indicates that the conjugates of the present invention require an additional T cell or B cell epitope polypeptide covalently bound to the conjugate of a predominantly linear β-(1,6)-glucan and a carrier protein. This also explains why the conjugates of the present invention do not encompass the prevention or treatment of diseases caused directly or indirectly by fungi, particularly Candida albicans, by providing a predominantly linear β-(1,6)-glucan as an antigen (finally linked to a carrier protein) having a ratio of (1,6)-linked to non-β-(1,6)-linked monosaccharide moieties of at least 1:1.

[0366] According to the present invention, it has been shown that such oligosaccharide / polysaccharide conjugate vaccines linked to pustulan are potent Dectin-1 binders and induce beneficial / efficient immune responses when applied in vivo.

[0367] The present invention therefore also relates to the improvement and / or optimization of a carrier protein (which already contains one or more T cell antigens, optionally in a post-translationally modified form, as part of its polypeptide sequence) by covalently linking the carrier protein to a CLEC polysaccharide adjuvant according to the present invention, i.e., a β-glucan or mannan, preferably pustulan, lichenan, laminarin, curdlan, β-glucan peptide (BGP), schizophyllan, scleroglucan, whole glucan particle (WGP), zymosan, or lentinan. The present invention therefore relates to a β-glucan or mannan for use as a C-type lectin (CLEC) polysaccharide adjuvant for B-cell and / or T-cell epitope polypeptides, wherein the β-glucan or mannan is covalently linked to the B-cell and / or T-cell epitope polypeptide to form a conjugate of the β-glucan or mannan and the B-cell and / or T-cell epitope polypeptide, and the carrier protein is covalently linked to the β-glucan or mannan.

[0368] This improvement / optimization results in a significant reduction or elimination of B cell responses against the CLEC and / or carrier protein and / or an enhancement (or at least maintenance) of T cell responses against T cell epitopes of the carrier protein, which allows for a reduction or elimination of antibody responses against the CLEC and / or carrier (which then results in only a T cell response) and a specific enhancement of antibody responses against the actual target polypeptide conjugated to the carrier and / or CLEC.

[0369] Therefore, a particularly preferred embodiment of the present invention is (a) β-glucan (b) at least a B cell epitope polypeptide or a T cell epitope polypeptide, and (c) Carrier protein consisting of or comprising: The three components (a), (b), and (c) are covalently bonded to one another in a conjugate.

[0370] This combination of these three components can be provided in any orientation or order, i.e., (a)-(b)-(c), (a)-(c)-(b), or (b)-(a)-(c), with (b) and / or (c) being covalently conjugated N- to C- or C- to N-terminus, or via a functional group within the polypeptide (e.g., via a functional group in a lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, tyrosine, tryptophan, or histidine residue, particularly via the ε-ammonium group of a lysine residue). Of course, β-glucan can be linked to one or more of each of components (b) and (c), preferably by the methods disclosed herein. Preferably, these components are linked by a linker, particularly by a linker between at least all three components. Preferred linkers are disclosed herein, such as a cysteine ​​residue, or a linker containing a cysteine ​​or glycine residue; a linker obtained by hydrazide-mediated ligation; a linker obtained by ligation via a heterobifunctional linker such as BMPH, MPBH, EMCH, or KMUH; a linker obtained by imidazole-mediated ligation; a linker obtained by reductive amination; a linker obtained by carbodiimide ligation; an -NH-NH linker; a peptide linker such as an NRRA linker, an NRRA-C linker, or an NRRA-NH-NH linker; a dimeric, trimeric, or tetrameric (or longer) peptide group such as CG or CG. For established carrier proteins, particularly CRM, CRM197, and KLH, the preferred order of the at least three components is (a)-(c)-(b), i.e., β-glucan and at least one B cell epitope polypeptide or one T cell epitope polypeptide are linked to the carrier protein.

[0371] According to another preferred embodiment, the conjugate according to the invention comprises a T cell epitope and no B cell epitope, preferably the conjugate comprises two or more T cell epitopes, in particular two, three, four or five T cell epitopes. This construct is particularly suitable for cancer vaccines. This construct is also particularly suitable for autoantigens, in particular autoantigens associated with autoimmune diseases. The therapeutic effect of the respective conjugate is achieved by the reduction of effector T cells, as shown for example for multiple sclerosis, and the increase of regulatory T cells (T) leading to the suppression of the respective disease, e.g. autoimmune disease or allergic disorder. reg Remarkably, these Treg cells exert potent bystander immunosuppression, thus ameliorating disease induced by cognate and non-cognate self-antigens.

[0372] Preferred CLECs for use as polysaccharide backbones according to the present invention are pustulan or other β-(1,6) glucans (including synthetic forms of such glucans); others that may be used are mannans, β-glucan family members, in particular linear β-(1,3) (e.g., Saccharomyces cerevisiae β-glucan (150 kDa), curdlan) or glucans containing branched β-(1,3) and branched β-(1,6) such as: laminarin (4.5-7 kDa), scleroglucan, schizophyllan, more preferably linear glucans (e.g., β(1,3): Saccharomyces cerevisiae β-glucan (150 kDa), curdlan (75-80 kDa or more), β-(1,3) + β-(1,4) lichenan (22-250 kDa), β-(1,6) pustulan (20 kDa). Thus, preferred CLECs according to the present invention are mannans and β-glucans, including linear and branched β-glucans characterized by the presence of a β-(1,3)-backbone, a β-(1,3)+β-(1,4)-backbone, and a β(-1,6) backbone, with attached side chains having β-(1,6) residues, more preferably linear β-glucans containing β-(1,3) chains, β-(1,3)+β-(1,4) chains, and β-(1,6) chains, more preferably linear β-(1,6) β-glucans, in particular pustulan, fragments thereof, or synthetic variants thereof consisting of polymeric β-(1,6)-glucan sugars (e.g., 4-mer, 5-mer, 6-mer, 8-mer, 10-mer, 12-mer, 15-mer, 17-mer, or 25-mer).

[0373] Preferably, the minimum length of a CLEC according to the present invention is a 6-mer, since smaller polysaccharides present problems with the oxidation reaction performed in the present invention (ultimately, other linking mechanisms may be used for end-linking by adding such smaller and / or reactive forms). CLECs with six or more monomer units (i.e., 6-mers and longer) exhibit good dectin binding. Typically, the longer the CLEC, the better the dectin binding. A degree of polymerization (i.e., the amount of a single glucose molecule within one glucan entity, DP) of 20-25 (i.e., DP 20-25) clearly confirms good binding and in vivo efficacy (e.g., laminarin is a typical example of a DP 20-30).

[0374] While the molecular weight of synthetic CLECs may be smaller, e.g., as low as 1-2 kDa, the preferred molecular weight range for glucans and their fragments is 1-250 kDa (e.g., laminarin, lichenan, Saccharomyces cerevisiae β-glucan, pustulan, curdlan, and barley glucan), preferably 4.5-80 kDa (e.g., laminarin, pustulan, curdlan, and low-molecular-weight lichenan), and particularly 4.5-30 kDa (e.g., laminarin, pustulan, and low-molecular-weight lichenan). Mannans are polysaccharides that are linear polymers of the sugar mannose. Plant mannans have β-(1,4) linkages. These are a form of storage polysaccharide. Mannan cell wall polysaccharides found in yeast have an α-(1,6)-linked backbone and α-(1,2)- and α-(1,3)-linked branches. This is serologically similar to the structure found in mammalian glycoproteins.

[0375] To prepare the conjugates of the present invention, CLECs, particularly pustulan, must be activated (e.g., by using mild periodate-mediated oxidation), and the degree of oxidation is important for the immune response. As already disclosed above, a practical oxidation range, particularly for pustulan, is approximately 20-260% oxidation. In many cases, the optimal oxidation range is between low / moderate oxidation (i.e., 20-60% oxidation) and high oxidation (i.e., 140-260% oxidation), i.e., 60-140% oxidation. Optimization of other CLECs can be easily adapted by those skilled in the art; for example, for lichenan, more than 200% oxidation is required to obtain a similar amount of aldehyde groups.

[0376] Therefore, these ranges may alternatively be defined as reactivity with Schiff's fuchsin reagent, which for example for pustulan can be defined as low / medium oxidation levels of 0.2-0.6 molar ratio (sugar monomer:periodic acid), an optimum range of 0.6-1.4, and a high oxidation level of 1.4-2.6, respectively.

[0377] In any case, the degree of oxidation should be defined to meet the optimal range for each specific CLEC. Preferably, linear β-glucan, more preferably β-(1,6) β-glucan, particularly pustulan, pustulan fragments, or synthetic variants thereof consisting of polymeric β(1,6)-glucan sugars (e.g., 4-mer, 5-mer, 6-mer, 8-mer, 10-mer, 12-mer, 15-mer, 17-mer, or 25-mer), is activated by mild periodate oxidation, which results in cleavage of vicinal OH groups and thus generates reactive aldehydes. Mild periodate oxidation refers to the use of sodium periodate (NaIO), a well-known mild agent for effectively oxidizing vicinal diols in carbohydrate sugars to generate reactive aldehyde groups. Carbon-carbon bonds are cleaved between adjacent hydroxyl groups. By varying the amount of periodate used, aldehydes can be stoichiometrically introduced into a few or many sugar moieties of a given polysaccharide.

[0378] Other exemplary methods for activating carbohydrates are well known in the art and include cyanylation of hydroxyls (e.g., by use of organic cyanylating reagents such as 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP) or N-cyanotriethylammonium tetrafluoroborate (CTEA)), reductive amination of carbohydrates, or activation and conjugation using carboxylic acid reactive chemical groups such as carbodiimides.

[0379] The activated carbohydrate is then reacted with the polypeptide to link it to the activated CLEC, forming a conjugate between the CLEC and the B cell epitope polypeptide or the T cell epitope polypeptide.

[0380] Therefore, the present invention also relates to a method for producing a conjugate according to the invention, which comprises activating a β-glucan or mannan by oxidation and contacting the activated β-glucan or mannan with a B-cell and / or T-cell epitope polypeptide, thereby obtaining a conjugate of the β-glucan or mannan with a B-cell and / or T-cell epitope polypeptide.

[0381] Preferably, the β-glucans or mannans are obtained by periodate oxidation at the vicinal hydroxyl groups, by reductive amination, or by cyanylation of the hydroxyl groups.

[0382] According to a preferred embodiment, the β-glucan or mannan is oxidized to a degree of oxidation, defined as the reactivity with Schiff's fuchsin reagent, that corresponds to the degree of oxidation of an equivalent amount of pustulan oxidized with periodate in a molar ratio of 0.2 to 2.6, preferably 0.6 to 1.4, in particular 0.7 to 1.

[0383] Preferably, the conjugates are made by hydrazone-based linkages to attach hydrazides to carbonyls (aldehydes), or by using heterobifunctional maleimide- and -hydrazide linkers (e.g., BMPH (N-β-maleimidopropionic acid hydrazide, MPBH (4-[4-N-maleimidophenyl]butyric acid hydrazide), EMCH (N-[ε-maleimidocaproic acid] hydrazide)) or KMUH (N-[κ-maleimidoundecanoic acid] hydrazide) to attach sulfhydryls (e.g., cysteine) to carbonyls (aldehydes).

[0384] The polypeptides linked to CLECs according to the present invention are or contain at least one B cell epitope or at least one T cell epitope. Preferably, the polypeptides linked to CLECs contain a single B cell or T cell epitope (even in embodiments in which two or more polypeptides are linked to the CLEC polysaccharide backbone). As also shown in the Examples section, the preferred length of the polypeptides is 5 to 29 amino acid residues, preferably 5 to 25 amino acid residues, more preferably 7 to 20 amino acid residues, even more preferably 7 to 15 amino acid residues, and especially 7 to 13 amino acid residues. In this regard, it is important to note that these length ranges focus only on the epitope sequence and do not include linkers containing peptide linkers such as cysteine ​​or glycine, or dimeric, trimeric, or tetrameric (or longer) peptide groups such as CG or CG, or cleavage sites such as cathepsin cleavage sites, or combinations thereof (e.g., NRRAC). Illustrative examples of epitopes are examined in the Examples section. These results indicate that the platform according to the present invention is not limited to any particular polypeptide, and therefore virtually all possible epitopes are eligible for the present invention, including epitopes already known in the art, particularly epitopes already described as being capable of being incorporated into presentation platforms (e.g., together with "classical" carrier molecules or adjuvants).

[0385] It is particularly preferred if the epitope can be linked to the activated β-glucan based on prior art linking methods, including hydrazide-mediated linkage, linkage via a heterobifunctional linker (e.g., BMPH, MPBH, EMCH, KMUH, etc.), imidazole-mediated linkage, reductive amination, carbodiimide linkage, etc. (further additions may be made). The epitopes used may include individual peptides, be contained within a peptide or protein, or be provided as a peptide-protein conjugate prior to linkage to the CLEC.

[0386] Thus, preferred linking methods used to provide conjugates according to the invention are hydrazide linkages or linkages using thioester formation (e.g., maleimide linkages using BMPH (N-β-maleimidopropionic acid hydrazide), MPBH, EMCH, KMUH, particularly where pustulan is linked to BMPH via hydrazone formation and the polypeptide is linked via a thioester).

[0387] In this embodiment, a polypeptide having two preferred linkers, such as hydrazide polypeptides / epitopes for hydrazone linkage, i.e. N-terminal linkage of peptides: H2N-NH-CO-CH2-CH2-CO-polypeptide-COOH, preferably in combination with succinic acid or an alternative suitable linker, such as other suitable dicarboxylic acids, especially glutaric acid, used as spacer / linker; C-terminal linkage (which is the preferred linkage orientation according to the present invention): NH2-polypeptide-NH-NH2 It is preferred to provide:

[0388] Alternatively, unmodified polypeptides / epitopes may be applied in the present invention, e.g., polypeptides containing an (extra) cysteine ​​residue or alternative source of SH group at either the C- or N-terminus for heterobifunctional linker-mediated linkage (especially BMPH, MPBH, EMCH, KMUH): NH2-Cys-Pep-COOH or NH2-Pep-Cys-COOH.

[0389] Preferred B cell polypeptides for use in accordance with the present invention are polypeptides having a length of 5 to 19 amino acid residues, preferably 6 to 18 amino acid residues, and particularly 7 to 15 amino acid residues. B cell epitopes are preferably short linear polypeptides, glycopolypeptides, lipopolypeptides, other post-translationally modified polypeptides (e.g., phosphorylated, acetylated, nitrated, containing pyroglutamic acid residues, glycosylated, etc.), cyclic polypeptides, etc.

[0390] Preferred B cell epitopes include B cell epitopes representing autoantigens, B cell epitopes representing antigens present in neoplastic diseases, B cell epitopes representing antigens present in allergic IgE-mediated diseases, B cell epitopes representing antigens present in autoimmune diseases, B cell epitopes representing antigens present in infectious diseases, B cell epitopes representing structural epitopes, B cell epitopes representing carbohydrate epitopes, CRM197, KLH, tetanus toxoid, or other commercially available carrier proteins or carriers known to those skilled in the art, preferably CRM197 and KLH, most preferably CRM197. B cell epitopes anchored / linked to polypeptides or proteins forming multivalent B cell epitope-protein / polypeptide conjugates suitable for CLEC ligation, including carrier molecules such as; non-peptidic antigens amenable to ligation to reactive aldehydes present on pustulan / CLEC (including linear polypeptides, polypeptides representing structural epitopes, mimotopes, or polypeptide variants from native epitopes / sequences, glycopolypeptides, lipopolypeptides, other post-translationally modified peptides (e.g., phosphorylated, acetylated, containing pyroglutamic acid residues, etc.), cyclic polypeptides, etc.).

[0391] Preferred T cell polypeptides for use in accordance with the invention have a length of 8 to 30 amino acid residues, preferably 13 to 29 amino acid residues, more preferably 13 to 28 amino acid residues.

[0392] Preferred specificities of T cell epitopes for use in the present invention are short linear peptides suitable or known to be suitable for presentation via MHC1 and MHCII (known to those skilled in the art), in particular MHCII epitopes for CD4 effector T cells and CD4 Treg cells, cytotoxic T cells (CD8+) and CD8 T cells, useful for cancer, autoimmune diseases, or infectious diseases, with known efficacy in humans or animals. an MHC1 epitope for Treg cells; a short linear peptide suitable for presentation via MHC1 and MHCII, appended at its N- or C-terminus (as known to those skilled in the art) with a lysosomal protease cleavage site, particularly a cathepsin protease family member-specific site, with known efficacy in humans or animals, more particularly a site for a cysteine ​​cathepsin such as cathepsin B, C, F, H, K, L, O, S, V, X, and W, particularly cathepsin S- or L-, most preferably cathepsin L, which promotes efficient endosomal / lysosomal release of the peptide for MHC presentation, particularly MHCII presentation. Cathepsin cleavage sites in various proteins have been identified and are well known in the art. This includes disclosures of sequences or methods for identifying such sequences, for example: Biniossek et al., J. Proteome Res. 2011, 10, 12, 5363-5373; Adams-Cioaba et al., Nature Comm. 2011, 2:197; Ferrall-Fairbanks PROTEIN SCIENCE 2018 VOL 27:714-724; Kleine-Weber et al., Scientific Reports (2018) 8:1659, https: / / en.wikipedia.org / wiki / Cathepsin_S. Specifically, the adaptation of peptide sequences using artificial protease cleavage sites as described in the present invention is based on the surprising effect of these sequence extensions in eliciting a more efficient immune response after dermal administration of a CLEC vaccine according to the present invention when the antigen is linked to a CLEC. The vaccine according to the present invention is taken up by DCs, and the peptide antigen is then processed in lysosomes and presented on MHC.

[0393] Lysosomes are intracellular membrane-bound organelles characterized by an acidic interior and contain a variety of hydrolytic enzymes, including lipases, proteases, and glycosidases, involved in cellular catabolism. Among the various enzymes contained in lysosomes, cathepsins are a family of lysosomal proteases with a wide range of functions. All cathepsins are classified into three distinct protease families: serine proteases (cathepsin A and cathepsin G), aspartic proteases (cathepsin D and cathepsin E), and 11 cysteine ​​cathepsins. In humans, 11 cysteine ​​cathepsins with structures similar to those of papain are known: B, C (J, dipeptidyl peptidase I, or DPPI), F, H, K (O2), L, O, S, V (L2), X (P, Y, Z), and W (lymphopain).

[0394] Cathepsins show similarities in their cellular localization and biosynthesis, but some differences in their expression patterns. Among all lysosomal proteases, cathepsins L, B, and D are the most abundant, with their lysosomal concentrations equal to 1 mM. Cathepsins B, H, L, C, X, V, and O are ubiquitously expressed, while cathepsins K, S, E, and W show cell- or tissue-specific expression. Cathepsin K is expressed in osteoclasts and epithelial cells. Cathepsins S, E, and W are primarily expressed in immune cells.

[0395] In addition to their primary function in lysosomal protein recycling, cathepsins play important roles in various physiological processes. Cathepsin S is the primary protease involved in the processing and presentation of MHC II antigens. Cathepsin S-deficient mice show marked alterations in the generation and presentation of MHC II-associated antibody fragments, due to a substantial reduction in antibody degradation in professional APCs, where cathepsin S is abundantly expressed. Furthermore, endocytosis selectively targets exogenous materials to cathepsin S in human DCs. Enrichment of MHC II molecules within late endocytic structures has also been consistently observed in splenic DCs from cathepsin S-deficient mice. Recent studies have suggested that both cathepsin B and cathepsin D are involved, but not essential, in MHC II-mediated antibody presentation. Cathepsin L also plays a role in a wide variety of cellular processes, including antigen processing, tumor invasion and metastasis, bone resorption, and the turnover of intracellular and secreted proteins involved in growth regulation. Although generally recognized as a lysosomal protease, cathepsin L is also secreted. This broad-spectrum protease is potent in degrading several extracellular proteins (laminin, fibronectin, collagen I and collagen IV, elastin, and other structural proteins of basement membranes), as well as serum proteins, cytoplasmic proteins, and nuclear proteins.

[0396] As a novel means to enhance the efficacy of T cell epitopes in vaccines, particularly CLEC-based vaccines, N- or C-terminal addition of lysosomal protease cleavage sites is provided as a preferred embodiment of the present invention.

[0397] Such cleavage sites according to the present invention can be characterized as follows.

[0398] Cathepsin L-like cleavage site: The intended cathepsin L-like cleavage site is defined based on protease cleavage site sequences known by those skilled in the art, specifically those disclosed in Biniossek et al. (J. Proteome Res. 2011, 10, 5363-5373) and Adams-Cioaba et al. (Nature Comm. 2011, 2:197). The orientation of the site can be N-terminal or C-terminal, preferably C-terminal. A preferred consensus sequence for a C-terminal cathepsin L site is of the following formula:

[0399] X n -X1-X2-X3-X4-X5-X6-X7-X8 X n : 3 to 27 amino acids derived from immunogenic peptides X1: Any amino acid X2: Any amino acid X3: Any amino acid X4: N / D / A / Q / S / R / G / L; preferably N / D, more preferably N X5: F / R / A / K / T / S / E; preferably F or R, more preferably R X6: F / R / A / K / V / S / Y; preferably F or R, more preferably R X7: any amino acid, preferably A / G / P / F, more preferably A X8: cysteine ​​or a linker such as NHNH2 Most preferred sequence: X n -X1X2X3NRRA-linker

[0400] Cathepsin S-like cleavage site: The contemplated cathepsin S cleavage sites are based on protease cleavage site sequences known by those skilled in the art, in particular those disclosed in Biniossek et al. (J. Proteome Res. 2011, 10, 5363-5373) and also at https: / / en.wikipedia.org / wiki / Cathepsin_S, and are characterized by the following consensus sequence:

[0401] Xn -X1-X2-X3-X4-X5-X6-X7-X8 X is characterized by X n : 3 to 27 amino acids derived from immunogenic peptides X1: Any amino acid X2: Any amino acid X3: any amino acid, preferably V, L, I, F, W, Y, H, more preferably V X4: any amino acid, preferably V, L, I, F, W, Y, H, more preferably V X5: K, R, E, D, Q, N, preferably K, R, more preferably R X6: Any amino acid X7: any amino acid, preferably A X8: Preferably A X8: cysteine ​​or a linker such as NHNH2 Most preferred sequence: X n -X1X2VVRAA-Linker

[0402] The protein is a carrier protein, particularly a non-toxic cross-reacting material (CRM) of diphtheria toxin, 197 , KLH, diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae protein D (HipD), and outer membrane protein complex (OMPC) of serogroup B Neisseria meningitidis, recombinant non-toxic form of Pseudomonas aeruginosa exotoxin A (rEPA), flagellin, Escherichia coli heat-labile enterotoxin (LT), cholera toxin (CT), mutant toxins (e.g., LTK63 and LTR72), virus-like particles, albumin-binding proteins, bovine serum albumin, ovalbumin, synthetic peptides, T cell epitopes contained within proteins suitable for conjugation to CLECs including tidodendrimers, such as multiple antigenic peptides (MAPs), or other commercially available carrier proteins, preferably CRM197 and KLH, most preferably CRM197, wherein the ratio of carrier protein to β-glucan in the conjugate is 1 / 0.1 to 1 / 50, preferably 1 / 0.1 to 1 / 40, more preferably 1 / 0.1 to 1 / 20, especially 1 / 0.1 to 1 / 10.

[0403] According to a preferred embodiment of the present invention, the CLEC conjugates according to the present invention comprise: (a) CLECs conjugated to individual B-cell and / or T-cell epitopes, including mixtures of B-cell or T-cell epitopes, in particular these epitopes linked to pustulan; (b) CLECs conjugated to polypeptide-carrier protein conjugates linked to pustulan, preferably polypeptide-KLH conjugates or polypeptide-CRM197 conjugates, most preferably polypeptide-CRM197 conjugates linked to pustulan; (c) CLECs, most preferably CLECs conjugated to individual B-cell and T-cell epitopes derived from self-proteins (cancer) or pathogens (infectious diseases), which are not promiscuous MHC / HLA-specific but known disease-specific T-cell epitopes, linked to pustulan; (d) polypeptides or to B-cell epitopes and T-cell epitopes contained within a protein, such as a carrier protein, a self-protein, a foreign protein from a pathogen, an allergen, etc. (where "individually" means that the polypeptide chains are present as independent entities, not as a fusion protein, tandem repeat polypeptide, or peptide-protein conjugate; i.e., independent B-cell epitope-containing polypeptides and independent T-cell epitope-containing polypeptides); (e) to T-cell epitopes contained within a protein, such as a carrier protein or target protein, representing a linear MHC1 epitope and a linear MHCII epitope, for treating, for example, a neoplastic disease, or an autoimmune disease ("individually" has the same meaning as (d)).

[0404] In view of these advantageous properties of the conjugates of the invention, the conjugates and vaccines according to the invention can be particularly used for active anti-Aβ, active anti-tau, and / or active anti-α-synuclein vaccines for the treatment and prevention of β-amyloidoses, tauopathies, or synucleinopathies, preferably Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), Parkinson's disease dementia (PDD), neuroaxonal dystrophy, Alzheimer's disease (AD), amygdala-restricted AD with Lewy bodies (AD / ALB), dementia in Down's syndrome, Pick's disease, progressive supranuclear palsy (PSP), corticobasal degeneration, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), and argyrophilic grain dementia.

[0405] Thus, the conjugates according to the present invention are particularly useful for the prevention or treatment of diseases, for example in humans, mammals, or birds, in particular for the treatment and prevention of human diseases. Accordingly, one aspect of the present invention is the use of the conjugates in the medical field as a medical application. The present invention therefore relates to the conjugates according to the present invention for use in the treatment or prevention of diseases. Accordingly, the present invention also relates to the use of the conjugates according to the present invention for the manufacture of a medicament for the prevention or treatment of diseases, preferably for the prevention or treatment of infectious diseases, chronic diseases, allergies, or autoimmune diseases. Accordingly, the present invention also relates to a method for the prevention or treatment of diseases, preferably for the prevention or treatment of infectious diseases, chronic diseases, allergies, or autoimmune diseases, comprising administering an effective amount of the conjugates according to the present invention to a patient in need thereof.

[0406] According to a further aspect, the novel glycoconjugates according to the invention can be used in the prevention of infectious diseases, preferably by providing a predominantly linear β-(1,6)-glucan as an antigen (ultimately linked to a carrier protein) with a ratio of (1,6)-linked to non-β-(1,6)-linked monosaccharide moieties of at least 1:1, but excluding their use in the prevention or treatment of diseases caused directly or indirectly by fungi, particularly Candida albicans, such as microbial infections caused by Haemophilus influenzae type b (Hib), Streptococcus pneumoniae, Neisseria meningitidis, and Salmonella typhi, or other infectious agents.

[0407] According to a further aspect, the present invention also relates to a pharmaceutical composition comprising a conjugate or a vaccine as defined above and a pharmaceutically acceptable carrier.

[0408] Preferably, the pharmaceutically acceptable carrier is a buffer, preferably a phosphate or TRIS-based buffer.

[0409] According to a preferred embodiment of the present invention, the pharmaceutical composition is contained in a needle-based delivery system, preferably a syringe, a mini-needle system, a hollow needle system, a solid microneedle system, or a system including a needle adapter; an ampoule, a needle-free injection system, preferably a jet injector; a patch, a transdermal patch, a microstructured transdermal system, a microneedle array patch (MAP), preferably a solid MAP (S-MAP), a coated MAP (C-MAP), or a dissolved MAP (D-MAP); an electrophoresis system, an iontophoresis system, a laser-based system, particularly an Erbium YAG laser system; or a gene gun system. The conjugate according to the present invention is not limited to any form in which it is prepared, stored, or delivered. Therefore, all traditional and typical forms are compatible with the present invention. Preferably, the composition according to the present invention may contain the conjugate or vaccine contained as a solution or suspension, a deep-frozen solution or suspension, a lyophilized product, a powder, or granules.

[0410] The present invention is further illustrated by the following examples and figures, but is not limited thereto. [Brief explanation of the drawings]

[0411] [Figure 1A] Figures 1A-1F show the in vitro binding activity of CLEC conjugates to ConA and DC receptors (i.e., dectin-1). Compared with lichenan (Lich), pustulan (Pus) demonstrated higher binding efficacy to dectin-1. [Figure 1B] β-glucans derived from oats (oat_BG265, oat_BG391) and barley (barley_BG229) showed limited binding effectiveness compared to pustulan. [Figure 1C] Different glucan types (i.e., pustulan, mannan, and barley glucan (229 kd)) retain high or intermediate receptor-binding activity after glucan oxidation, as assessed by competitive binding assays. "20% oxidized and 40% oxidized" indicate the oxidation state of the glucan moiety used in conjugate formation. Inhibition (%) indicates the inhibition of binding of soluble Dectin-1 receptor (pustulan and barley_BG229) or ConA (mannan) to plate-bound β-glucan or mannan in the presence of the indicated concentrations of test CLEC. [Figure 1D] Pustulan-conjugates retain approximately 50% of the Dectin-1 binding capacity compared to unconjugated β-glucan as assessed by competitive binding assays. [Figure 1E] The lichenan-conjugates retain approximately 50% of the Dectin-1 binding capacity compared to unconjugated β-glucan, as assessed by competitive binding assays. [Figure 1F]Pustulan-conjugates prepared via heterobifunctional linkers maintain high Dectin-1 binding efficacy. Data are shown as relative light units (RLU) from luminescence ELISA. Pus70 conjugates 1-3 refer to three different CLEC peptide conjugates (SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 16), respectively. Pus 70% and Lich 200% refer to pustulan and lichenan with their respective oxidation states. BMPH Pus refers to activated pustulan. BMPH conjugate 2 refers to the CLEC-SEQ ID NO: 10 conjugate.

[0412] [Figure 2A] Figures 2A-2D show flow cytometry analysis of dendritic cell activation by lipopolysaccharide (LPS) and different pustulan preparations. Immature bone marrow-derived murine dendritic cells (BMDCs) were generated in vitro using granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF-BMDCs were stimulated for 24 hours with LPS (equal doses were included in the oxidized pustulan and pustulan conjugate preparations), SEQ ID NO:2 + SEQ ID NO:7 + pustulan conjugate, or oxidized pustulan alone. Pustulan conjugates and pustulan alone were used at increasing doses of each sugar (up to 500 μg / mL), starting at 62.5 μg / mL. DCs were identified based on CD11c / CD11b expression, and surface expression of CD80 by SEQ ID NO:2 + SEQ ID NO:7 + pustulan conjugates was measured by flow cytometry. DCs treated with pustulan preparations (=measured) and DCs treated with an equivalent amount of LPS (=predicted) were analyzed for the expression of activation markers by CytExpert software. [Figure 2B]Immature bone marrow-derived murine dendritic cells (BMDCs) were generated in vitro using granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF-BMDCs were stimulated for 24 hours with LPS (equal doses contained in the oxidized pustulan and pustulan conjugate preparations), SEQ ID NO:2 + SEQ ID NO:7 + pustulan conjugate, or oxidized pustulan alone. Pustulan conjugates and pustulan alone were used at increasing doses of each sugar (up to 500 μg / mL), starting at 62.5 μg / mL. DCs were identified based on CD11c / CD11b expression, and surface expression of CD80 with oxidized pustulan alone was measured by flow cytometry. Expression of activation markers was analyzed using CytExpert software for DCs treated with the pustulan preparations (measured) and DCs treated with an equivalent amount of LPS (predicted). [Figure 2C] Immature bone marrow-derived murine dendritic cells (BMDCs) were generated in vitro using granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF-BMDCs were stimulated for 24 hours with LPS (equal doses contained in the oxidized pustulan and pustulan conjugate preparations), SEQ ID NO:2 + SEQ ID NO:7 + pustulan conjugate, or oxidized pustulan alone. Pustulan conjugates and pustulan alone were used at increasing doses of each sugar (up to 500 μg / mL), starting at 62.5 μg / mL. DCs were identified based on CD11c / CD11b expression, and major histocompatibility complex (MHC) class II surface expression by the SEQ ID NO:2 + SEQ ID NO:7 + pustulan conjugate was measured by flow cytometry. DCs treated with the pustulan preparation (measured) and DCs treated with an equivalent amount of LPS (predicted) were analyzed for activation marker expression using CytExpert software. [Figure 2D]Immature bone marrow-derived murine dendritic cells (BMDCs) were generated in vitro using granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF-BMDCs were stimulated for 24 hours with LPS (equal doses contained in the oxidized pustulan and pustulan conjugate preparations), SEQ ID NO:2 + SEQ ID NO:7 + pustulan conjugate, or oxidized pustulan alone. Pustulan conjugates and pustulan alone were used at increasing doses of each sugar (up to 500 μg / mL), starting at 62.5 μg / mL. DCs were identified based on CD11c / CD11b expression, and major histocompatibility complex (MHC) class II surface expression with oxidized pustulan alone was measured by flow cytometry. DCs treated with the pustulan preparation (measured) and DCs treated with an equivalent amount of LPS (predicted) were analyzed for activation marker expression using CytExpert software.

[0413] [Figure 3A] Figures 3A-3C show particle size determination of CLEC conjugates by dynamic light scattering (DLS). Particle size was determined by DLS by measuring random changes in the intensity of light scattered from a suspension or solution. Regularization analysis and corresponding cumulant radius analysis over 24 hours are shown for the SEQ ID NO:5 + SEQ ID NO:7 + pustulan (80% oxidation state) conjugate. [Figure 3B] Particle size was determined by DLS by measuring random changes in the intensity of light scattered from a suspension or solution. A regularization analysis and corresponding cumulant radius analysis over a 24-hour period for the SEQ ID NO:6 + CRM + pustulan conjugate is shown. [Figure 3C] Particle size was determined by DLS by measuring random changes in the intensity of light scattered from a suspension or solution. A regularization analysis and corresponding cumulant radius analysis over a 24-hour period for unmodified pustulan are shown.

[0414] [Figure 4A]Figures 4A and 4B show a comparison of the immunogenicity of different CLEC-based vaccines. Female BALB / c mice aged 8 to 12 weeks received a total of three intradermal vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected two weeks after the third vaccination and analyzed for anti-peptide responses to the mannan-based vaccine, barley-based vaccine, and pustulan-based vaccine (SEQ ID NO:2 + SEQ ID NO:7 + CLEC). [Figure 4B] Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected two weeks after the third vaccination and analyzed for anti-peptide responses (SEQ ID NO:3 and SEQ ID NO:11) to the pustulan-based vaccine and the lichenan-based vaccine (SEQ ID NO:2 + SEQ ID NO:7 + CLEC and SEQ ID NO:10 + SEQ ID NO:7 + CLEC).

[0415] [Figure 5] Figure 5 shows a comparative analysis of the immunogenicity of a peptide-pustulan conjugate and a vaccine consisting of unconjugated peptide and CLEC. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two weeks after the third vaccination, samples were collected and analyzed for anti-peptide responses (SEQ ID NO: 3). Vaccines used were: SEQ ID NO: 2 + SEQ ID NO: 7 + CLEC, or a mixture of unconjugated SEQ ID NO: 2, SEQ ID NO: 7, and CLEC.

[0416] [Figure 6]Figure 6 shows a comparative analysis of the immunogenicity of pustulan conjugates containing B and T cell epitopes compared with conjugates containing only B or T cell epitopes. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Vaccines used were: SEQ ID NO:5 + SEQ ID NO:7 + CLEC, or SEQ ID NO:5 + CLEC, and SEQ ID NO:7 + CLEC. Samples were collected two weeks after the third vaccination and analyzed for anti-peptide responses (SEQ ID NO:6).

[0417] [Figure 7A] Figures 7A-7C show comparative analysis of anti-pustulan antibody responses in mice after repeated immunizations using vaccines containing peptide-pustulan conjugates or the individual unconjugated components. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Pre-plasma and t1-t3 represent detectable immune responses before immunization (pre-plasma) or after the first (t1), second (t2), or third (t3) immunization. Samples were collected two weeks after the third administration and analyzed for anti-pustulan responses. Analysis of anti-pustulan responses induced by various vaccines. Vaccines used: SEQ ID NO:2 + SEQ ID NO:7 + CLEC, or a mixture of unconjugated SEQ ID NO:2, SEQ ID NO:7, and CLEC. [Figure 7B] Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Pre-plasma and t1-t3 represent detectable immune responses before immunization (pre-plasma) or after the first (t1), second (t2), or third (t3) immunization. Kinetics of immune response. Vaccines used: SEQ ID NO:2 + SEQ ID NO:7 + CLEC, or a mixture of unconjugated SEQ ID NO:2, SEQ ID NO:7, and CLEC. [Figure 7C] Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Pre-plasma and t1-t3 represent detectable immune responses before immunization (pre-plasma) or after the first (t1), second (t2), or third (t3) immunization. Inhibition ELISA demonstrated the specificity of the ELISA system. Vaccines used were SEQ ID NO:2 + SEQ ID NO:7 + CLEC, or a mixture of unconjugated SEQ ID NO:2, SEQ ID NO:7, and CLEC.

[0418] [Figure 8A] Figures 8A and 8B show a comparative analysis of the immune responses elicited by CLEC-based vaccines using different peptide linkage orientations. Female BALB / c mice aged 8 to 12 weeks received a total of three intradermal vaccinations, administered at two-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Four different CLEC-based prototype vaccine candidates (two different peptides linked to pustulan via the C-terminus or N-terminus) were tested. Samples were collected two weeks after the third administration and analyzed for anti-peptide responses. Vaccines used: SEQ ID NO: 1 / 2 / 4 / 5 + SEQ ID NO: 7 + CLEC. [Figure 8B] Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Four different CLEC-based prototype vaccine candidates (two different peptides linked to pustulan via the C-terminus or N-terminus) were tested. Samples were collected two weeks after the third vaccination and analyzed for anti-aSyn protein responses. Vaccines used: SEQ ID NO:1 / 2 / 4 / 5 + SEQ ID NO:7 + CLEC.

[0419] [Figure 9A]Figures 9A and 9B show comparative immunogenicity analyses of CLEC-based vaccines using different promiscuous T helper cell epitopes. Female BALB / c mice aged 8 to 12 weeks received a total of three intradermal vaccinations, administered at two-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The immune responses elicited by nine different CLEC-based vaccines (vaccines 1-9) containing the same B cell epitope and different T helper epitopes (i.e., SEQ ID NO: 7, SEQ ID NOs: 22-29) were evaluated against the respective peptide-KLH conjugate (vaccine 10). Samples were collected two weeks after the third administration and analyzed for anti-peptide responses. [Figure 9B] Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Immune responses elicited by nine different CLEC-based vaccines (vaccines 1-9) containing the same B cell epitope and different T helper epitopes (i.e., SEQ ID NO:7, SEQ ID NOs:22-29) were evaluated against the respective peptide-KLH conjugate (vaccine 10). Samples were collected two weeks after the third vaccination and analyzed for anti-aSyn protein responses.

[0420] [Figure 10A]Figures 10A and 10B show comparative analyses of target-specific and carrier protein-specific immunogenicity induced by CLEC-based vaccines and conventional peptide-protein conjugate vaccines using the carrier protein KLH as a source of T helper cell epitopes. Female BALB / c mice aged 8 to 12 weeks received a total of three intradermal or subcutaneous (sc) vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of subsequent immune responses. The immune responses elicited by two peptide-protein conjugate vaccines using KLH as a source of T helper epitopes in combination with CLEC modifications (SEQ ID NO:3 + KLH + pustulan and SEQ ID NO:6 + KLH + pustulan, respectively) were evaluated against responses induced by conventional peptide-KLH conjugates (i.e., SEQ ID NO:3 + KLH and SEQ ID NO:6 + KLH) administered sc with alum / Alhydrogel or i.d. without additional adjuvant. Samples were taken two weeks after the third dose and analyzed for anti-peptide and anti-aSyn protein responses by ELISA. [Figure 10B] Female BALB / c mice aged 8–12 weeks received a total of three intradermal or subcutaneous (sc) vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The immune responses elicited by two peptide-protein conjugate vaccines (SEQ ID NO:3 + KLH + pustulan and SEQ ID NO:6 + KLH + pustulan, respectively) using KLH as a source of T-helper epitopes in combination with CLEC modifications were evaluated against responses induced by conventional peptide-KLH conjugates (i.e., SEQ ID NO:3 + KLH and SEQ ID NO:6 + KLH) administered sc with alum / Alhydrogel or i.d. without additional adjuvant. Samples were collected 2 weeks after the third vaccination and analyzed for anti-KLH responses by ELISA.

[0421] [Figure 11A]Figures 11A and 11B show comparative analyses of target-specific and carrier protein-specific immunogenicity induced by a CLEC-based vaccine and a conventional peptide-protein conjugate vaccine using the carrier protein CRM197 as a source of T helper cell epitopes. Female BALB / c mice aged 8 to 12 weeks received a total of three intradermal or sc vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two different CRM-based vaccine types were used in this study: SEQ ID NO:6 + CRM + Pus represents a peptide-CRM conjugate subsequently linked to pustulan, while SEQ ID NO:5 + CRM + Pus represents a conjugate in which the peptide component and carrier molecule were separately linked to CLEC. The immune responses induced by both types were evaluated against the respective conventional peptide-CRM conjugates (i.e., SEQ ID NO:6 + CRM administered sc with alum / Alhydrogel adjuvant). Samples were taken two weeks after the third dose and analyzed for anti-peptide and anti-aSyn protein responses by ELISA. [Figure 11B] Female BALB / c mice aged 8–12 weeks received three intradermal or sc vaccinations, administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two different CRM-based vaccine types were used in this study: SEQ ID NO:6 + CRM + Pus represents a peptide-CRM conjugate subsequently linked to pustulan, while SEQ ID NO:5 + CRM + Pus represents a conjugate in which the peptide component and carrier molecule were individually linked to CLEC. The immune responses induced by both types were evaluated against the respective conventional peptide-CRM conjugates (i.e., SEQ ID NO:6 + CRM administered sc in alum / Alhydrogel adjuvant). Samples were collected two weeks after the third vaccination and analyzed for anti-CRM responses by ELISA.

[0422] [Figure 12]Figure 12 shows a comparative analysis of the selectivity of immune responses elicited by CLEC-based vaccines in vivo against two different aSyn forms. Female BALB / c mice aged 8 to 12 weeks received three doses of intradermal or sc vaccination, administered at two-week intervals. CLEC-based vaccines (SEQ ID NO:2 + SEQ ID NO:7 + Pus and SEQ ID NO:5 + SEQ ID NO:7 + Pus; id administration) and alternative CLEC-based vaccines (SEQ ID NO:3 + KLH + Pus and SEQ ID NO:6 + CRM + Pus; id administration) were evaluated against the conventional peptide-component vaccine (SEQ ID NO:3 + KLH + Alum and SEQ ID NO:6 + CRM + Alum; s.c. administration). Two weeks after the third dose, samples were collected and subjected to an aSyn selectivity assay (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition.

[0423] [Figure 13] Figure 13 shows a comparative avidity analysis of the immune responses elicited by CLEC-based vaccines. Eight- to 12-week-old female BALB / c mice received a total of three intradermal or sc vaccinations, administered at two-week intervals. CLEC-based vaccines (SEQ ID NO:2 + SEQ ID NO:7 + Pus and SEQ ID NO:5 + SEQ ID NO:7 + Pus, administered i.d.) and alternative CLEC-based vaccines (SEQ ID NO:3 + KLH + Pus and SEQ ID NO:6 + CRM + Pus, administered i.d.) were evaluated against conventional peptide-component vaccines (SEQ ID NO:3 + KLH + Alum and SEQ ID NO:6 + CRM + Alum, administered sc.). Samples were collected two weeks after the second immunization (T2) or two weeks after the third immunization (T3), and antibody avidity to aSyn was assessed using an ELISA-based avidity assay.

[0424] [Figure 14]Figure 14 shows a comparative analysis of the affinity of the immune response elicited by CLEC-based vaccines. Eight to 12-week-old female BALB / c mice received three doses of intradermal or sc vaccination, administered two weeks apart. CLEC-based vaccines (SEQ ID NO:2 + SEQ ID NO:7 + Pus and SEQ ID NO:5 + SEQ ID NO:7 + Pus, administered i.d.) and alternative CLEC-based vaccines (SEQ ID NO:3 + KLH + Pus and SEQ ID NO:6 + CRM + Pus, administered i.d.) were evaluated against the conventional peptide-component vaccine (SEQ ID NO:3 + KLH + Alum and SEQ ID NO:6 + CRM + Alum, administered sc.). Samples were collected two weeks after the third dose, and the antibody equilibrium dissociation constant (Kd) for aSyn was assessed by aSyn displacement ELISA assay.

[0425] [Figure 15A] Figures 15A and 15B show comparative analyses of the in vitro functionality of immune responses elicited by CLEC-based vaccines. Female BALB / c mice aged 8 to 12 weeks received three doses of intradermal and sc vaccinations, administered at 2-week intervals. Samples were collected two weeks after the third vaccination, and modulation of aSyn aggregation in the presence of aSyn-specific Abs was assessed by ThT fluorescence assay. aSyn was aggregated for 0 to 72 hours in the presence of CLEC vaccine-induced Abs (SEQ ID NO:2 + SEQ ID NO:7 + Pus; administered intravenously), conventional peptide component-induced Abs (SEQ ID NO:3 + KLH + Alum; administered sc), or mouse plasma. Kinetic curves were calculated by normalizing ThT fluorescence at t0, and the slope value extracted from linear regression analysis during the exponential growth phase of ThT kinetics was used to calculate the percent inhibition of aSyn aggregation. [Figure 15B]Female BALB / c mice aged 8–12 weeks received three doses of intradermal and sc vaccinations, administered at 2-week intervals. Samples were collected two weeks after the third vaccination, and modulation of aSyn aggregation in the presence of aSyn-specific Abs was assessed by ThT fluorescence assay. aSyn or aSyn bearing preformed fibrils was allowed to aggregate for 0–92 h in the presence of CLEC vaccine-induced Abs (SEQ ID NO:5 + SEQ ID NO:7 + Pus and SEQ ID NO:6 + CRM + Pus, both administered i.d.), conventional peptide component-induced Abs (SEQ ID NO:6 + CRM + Alum, administered sc), or mouse plasma. Kinetic curves were calculated by normalizing ThT fluorescence at t0, and the slope value extracted from linear regression analysis during the exponential growth phase of ThT kinetics was used to calculate the percent inhibition of aSyn aggregation.

[0426] [Figure 16A] Figures 16A and 16B show a comparative analysis of the effect of immunization route on the immune response elicited by the CLEC-based vaccine. Female BALB / c mice aged 8 to 12 weeks received a total of three vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two alternative routes, including subcutaneous (sc) and intramuscular (im), were compared with intradermal (id) administration for the CLEC-based vaccine. Three doses of the CLEC-based vaccine (SEQ ID NO:2 + SEQ ID NO:7 + Pus) were administered per route. Samples were collected two weeks after the third vaccination and analyzed for anti-peptide responses. [Figure 16B] Female BALB / c mice aged 8-12 weeks received a total of three vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two alternative routes, including subcutaneous (sc) and intramuscular (im), were compared with intradermal (id) administration of the CLEC-based vaccine. Three doses of the CLEC-based vaccine (SEQ ID NO:2 + SEQ ID NO:7 + Pus) were administered per route. Samples were collected two weeks after the third vaccination and analyzed for anti-aSyn protein responses.

[0427] [Figure 17A] Figures 17A and 17B show a comparative analysis of the immune responses elicited by CLEC vaccines containing B cell epitopes characterized by post-translationally modified peptides: Aβ. Female BALB / c mice aged 8 to 12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO: 33 + SEQ ID NO: 7 + Pus, id) was evaluated against a conventional peptide conjugate-based vaccine (SEQ ID NO: 32 + KLH + Alum, sc). Samples were collected two weeks after the third vaccination and analyzed for anti-peptide, anti-AβpE3-40, anti-AβpE3-42, and anti-Aβ1-42 responses. [Figure 17B] Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:33 + SEQ ID NO:7 + Pus, id) was evaluated against a conventional peptide conjugate-based vaccine (SEQ ID NO:32 + KLH + Alum, sc). Samples were collected two weeks after the third vaccination, and antibody binding activity to AβpE3-42 was assessed by an ELISA-based avidity assay.

[0428] [Figure 18A]Figures 18A and 18B show a comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes derived from intracellular proteins and autoantigens: tau. Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc), administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:36 + SEQ ID NO:7 + Pus, id) was evaluated against a conventional peptide component-based vaccine (SEQ ID NO:35 + KLH + Alum, sc). Samples were collected two weeks after the third vaccination and analyzed for anti-peptide and anti-recombinant tau441 protein responses. [Figure 18B] Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:36 + SEQ ID NO:7 + Pus, id) was evaluated against a conventional peptide component-based vaccine (SEQ ID NO:35 + KLH + Alum, sc). Samples were collected two weeks after the third vaccination, and antibody binding activity to SEQ ID NO:35 was assessed by an ELISA-based avidity assay.

[0429] [Figure 19]Figure 19 shows a comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes derived from secreted proteins, self-antigens, and structural epitopes: IL23. Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Three CLEC-based vaccines (all SEQ ID NO:7 and SEQ ID NO:38 / SEQ ID NO:40 / SEQ ID NO:42 conjugated to pustulan, id) were evaluated against a conventional peptide conjugate-based vaccine (SEQ ID NO:37 / SEQ ID NO:39 / SEQ ID NO:41 conjugated to KLH and Alhydrogel (Alum), sc). Samples were collected two weeks after the third vaccination and analyzed for anti-peptide and anti-IL23 protein responses.

[0430] [Figure 20A] Figures 20A and 20B show a comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes derived from self-epitopes present in transmembrane proteins: the extracellular membrane-proximal domain of membrane-bound IgE (EMPD). Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO: 44 + SEQ ID NO: 7 + Pus, id) was evaluated against a conventional peptide component-based vaccine (SEQ ID NO: 43 + KLH + Alum, sc). Samples were collected two weeks after the third vaccination and analyzed for anti-injection peptide and anti-EMPD peptide responses. [Figure 20B]Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:44 + SEQ ID NO:7 + Pus, id) was evaluated against a conventional peptide component-based vaccine (SEQ ID NO:43 + KLH + Alum, sc). Samples were collected two weeks after the third vaccination, and antibody binding activity to the EMPD peptide was assessed by an ELISA-based avidity assay.

[0431] [Figure 21A] Figures 21A and 21B show a comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes derived from allergens, mimotopes, and structural epitopes: Bet v 1. Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO: 46 + SEQ ID NO: 7 + Pus, id) was evaluated against a conventional peptide component-based vaccine (SEQ ID NO: 45 + KLH + Alum, sc). Samples were collected two weeks after the third vaccination and analyzed for anti-peptide and anti-Bet v 1 protein responses. [Figure 21B] Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:46 + SEQ ID NO:7 + Pus, id) was evaluated against a conventional peptide component-based vaccine (SEQ ID NO:45 + KLH + Alum, sc). Samples were collected two weeks after the third vaccination, and antibody binding activity to Bet v 1 was assessed by an ELISA-based avidity assay.

[0432] [Figure 22]Figure 22 shows a comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes present in various forms of cancer / neoplastic disease (i.e., oncogenes): Her2. Female BALB / c mice, 8-12 weeks old, received a total of three vaccinations (id and sc), administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:48 + SEQ ID NO:7 + Pus, id) was evaluated against a conventional peptide component-based vaccine (SEQ ID NO:47 + KLH + Alum, sc). Samples were collected two weeks after the third vaccination and analyzed for anti-peptide and anti-Her2 protein responses.

[0433] [Figure 23A] Figures 23A and 23B show a comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes present in various forms of neoplastic disease / cancer (i.e., oncogenes): PD1. Female BALB / c mice, 8-12 weeks old, received a total of three vaccinations (id and sc), administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:50 + SEQ ID NO:7 + Pus, id) was evaluated against a conventional peptide component-based vaccine (SEQ ID NO:49 + KLH + Alum, sc). Samples were collected two weeks after the third vaccination and analyzed for A) anti-peptide responses and anti-PD1 protein responses. [Figure 23B]Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:50 + SEQ ID NO:7 + Pus, id) was evaluated against a conventional peptide component-based vaccine (SEQ ID NO:49 + KLH + Alum, sc). Samples were collected two weeks after the third vaccination, and antibody binding activity to SEQ ID NO:49 was assessed by an ELISA-based avidity assay.

[0434] [Figure 24] Figure 24 shows a comparative analysis of target protein-specific immunogenicity induced by CLEC-based peptide-CRM197 conjugate vaccines using different peptide-CRM197 / CLEC ratios. Female BALB / c mice aged 8 to 12 weeks received a total of three intradermal vaccinations, administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Five different peptide-CRM-based vaccines were used in this study, applying different peptide-CRM / pustulan ratios (w / w). All five groups were immunized using the SEQ ID NO:6 + CRM + Pus conjugate. 1:1, 1:2.5, 1:5, 1:10, and 1:20 represent conjugates with w / w peptide-CRM conjugate / CLEC ratios of 1 / 1, 1 / 2.5, 1 / 5, 1 / 10, and 1 / 20. The induced immune response was assessed using samples collected 2 weeks after the third dose and analyzed for anti-aSyn protein responses by ELISA. Titer determination was based on ODmax / 2 calculation.

[0435] [Figure 25A]Figures 25A and 25B show a comparative analysis of immune responses elicited by CLEC vaccines containing aSyn-derived B cell epitopes (aa1-8). Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO: 12 + SEQ ID NO: 7 + pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO: 13 conjugated to KLH and Alhydrogel, sc). Samples were collected two weeks after the third vaccination and analyzed for A) anti-peptide and anti-aSyn protein responses. Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition. [Figure 25B] Female BALB / c mice aged 8–12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:12 + SEQ ID NO:7 + pustulan, id) was evaluated against a conventional peptide-component conjugate-based vaccine (SEQ ID NO:13 conjugated to KLH and Alhydrogel (Alum), sc). Samples were collected two weeks after the third vaccination and analyzed for A) anti-peptide and anti-aSyn protein responses. Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition.

[0436] [Figure 26A]Figures 26A and 26B show a comparative analysis of the immune response elicited by a CLEC vaccine containing the aSyn-derived B cell epitope (aa100-108). Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO: 16 + SEQ ID NO: 7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO: 17 conjugated to KLH and Alhydrogel, sc). Samples were collected two weeks after the third vaccination and analyzed for A) anti-peptide and anti-aSyn protein responses. Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition. [Figure 26B] Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:16 + SEQ ID NO:7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO:17 conjugated to KLH and Alhydrogel (Alum), sc). Samples were collected two weeks after the third vaccination and analyzed for aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition.

[0437] [Figure 27]Figure 27 shows a comparative analysis of immune responses elicited by CLEC vaccines containing aSyn-derived B cell epitope (aa91-97). Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO: 14 + SEQ ID NO: 7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO: 15 conjugated to KLH and Alhydrogel (Alum), sc). Samples were collected two weeks after the third vaccination and analyzed for anti-peptide and anti-aSyn protein responses.

[0438] [Figure 28A] Figures 28A and 28B show a comparative analysis of immune responses elicited by CLEC vaccines containing a B cell epitope (aa130-140) derived from aSyn. Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:20 + SEQ ID NO:7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO:21 conjugated to KLH and Alhydrogel, sc). Samples were collected two weeks after the third dose and analyzed for anti-peptide and anti-aSyn protein responses. Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition. [Figure 28B]Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:20 + SEQ ID NO:7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO:21 conjugated to KLH and Alhydrogel (Alum), sc). Samples were collected two weeks after the third vaccination and analyzed for aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition.

[0439] [Figure 29A] Figures 29A and 29B show a comparative analysis of immune responses elicited by CLEC vaccines containing the aSyn-derived B cell epitope (aa115-122). Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO: 51 + SEQ ID NO: 7 and pustulan, id) was evaluated against a conventional peptide-component conjugate-based vaccine (SEQ ID NO: 52 conjugated to CRM and Alhydrogel, sc). Samples were collected two weeks after the third vaccination and analyzed for anti-peptide and anti-aSyn fibril responses. Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition. [Figure 29B]Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:51 + SEQ ID NO:7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO:52 conjugated to CRM and Alhydrogel, sc). Samples were collected two weeks after the third vaccination and analyzed for aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition.

[0440] [Figure 30A] Figures 30A and 30B show a comparative analysis of immune responses elicited by CLEC vaccines containing a B cell epitope (aa115-124) derived from aSyn. Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO: 67 + SEQ ID NO: 7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO: 68 conjugated to CRM and Alhydrogel, sc). Samples were collected two weeks after the third vaccination and analyzed for anti-peptide and anti-aSyn fibril responses. Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition. [Figure 30B]Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:67 + SEQ ID NO:7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO:68 conjugated to CRM and Alhydrogel, sc). Samples were collected two weeks after the third vaccination and analyzed for aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition.

[0441] [Figure 31A] Figures 31A and 31B show a comparative analysis of immune responses elicited by CLEC vaccines containing the aSyn-derived B cell epitope (aa107-113). Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO: 73 + SEQ ID NO: 7 and pustulan, id) was evaluated against a conventional peptide-component conjugate-based vaccine (SEQ ID NO: 74 conjugated to CRM and Alhydrogel, sc). Samples were collected two weeks after the third dose and analyzed for anti-peptide and anti-aSyn fibril responses. Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition. [Figure 31B]Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:73 + SEQ ID NO:7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO:74 conjugated to CRM and Alhydrogel, sc). Samples were collected two weeks after the third vaccination and analyzed for aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition.

[0442] [Figure 32A] Figures 32A-32D show comparative in vitro functional analyses of immune responses elicited by CLEC-based vaccines. Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected two weeks after the third vaccination, and ThT kinetic measurements (i.e., aSyn fibrillar fraction) were assessed in the presence of CLEC vaccine-induced Abs (SEQ ID NO: 67 + SEQ ID NO: 7 and pustulan, id) or conventional peptide component-induced Abs (SEQ ID NO: 68 bound to CRM and Alhydrogel, sc). [Figure 32B] Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected 2 weeks after the third vaccination, and ThT kinetics (i.e., aSyn fibrillar fraction) was assessed in the presence of (A-C) CLEC vaccine-induced Abs (SEQ ID NO: 71 + SEQ ID NO: 7 and pustulan, id) or conventional peptide component-induced Abs (SEQ ID NO: 72 bound to CRM and Alhydrogel, sc). [Figure 32C]Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected 2 weeks after the third vaccination, and ThT kinetics (i.e., aSyn fibrillar fraction) was assessed in the presence of (A-C) CLEC vaccine-induced Abs (SEQ ID NO: 73 + SEQ ID NO: 7 and pustulan, id) or conventional peptide component-induced Abs (SEQ ID NO: 74 bound to CRM and Alhydrogel, sc). [Figure 32D] Female BALB / c mice aged 8–12 weeks received a total of three vaccinations (id and sc) administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected 2 weeks after the third vaccination, and ThT kinetics (i.e., the fibrillar fraction of aSyn) was assessed in the presence of the aSyn-specific monoclonal Ab LB09 or naive mouse plasma.

[0443] [Figure 33A] Figures 33A-33D show the in vitro binding activity of CRM197-CLEC conjugates to mouse DC receptors (i.e., Dectin-1). Comparative analysis of Dectin-1 binding ability as determined by ELISA is shown. Pus refers to unmodified pustulan, and pus oxi refers to activated pustulan. CRM-pus conjugate 1 refers to the SEQ ID NO:6 + CRM197 + pustulan conjugate, and CRM conjugate 1 refers to the CRM197 + SEQ ID NO:6 conjugate without β-glucan modification. Negative control refers to a sample without inhibitor. [Figure 33B] Figure 1 shows a comparative analysis of Dectin-1 binding capacity as determined by ELISA. SEQ ID NOs: 52 / 66 / 68 / 70 / 72 refer to CRM197-pustulan conjugates with the indicated B-cell epitopes. [Figure 33C]Figure 1 shows a comparative analysis of Dectin-1 binding ability as determined by ELISA. Lich oxi refers to activated lichenan, and CRM-Lich conjugate 1 refers to SEQ ID NO: 6 + CRM197 + lichenan conjugate. [Figure 33D] Figure 1 shows a comparative analysis of Dectin-1 binding capacity as determined by ELISA. Lam oxi refers to activated laminarin, CRM-Lam conjugate 1 refers to SEQ ID NO: 6 + CRM197 + laminarin conjugate.

[0444] [Figure 34] Figure 34 shows the in vitro binding activity of CRM197-CLEC conjugates to human DC receptors (i.e., Dectin-1). A comparative analysis of Dectin-1 binding ability as determined by ELISA is shown. Lich conjugate refers to the SEQ ID NO: 6 + CRM197 + lichenan conjugate, Pus conjugate refers to the SEQ ID NO: 6 + CRM197 + pustulan conjugate, and Lam conjugate refers to the SEQ ID NO: 6 + CRM197 + laminarin conjugate. The negative control refers to a sample without inhibitor.

[0445] [Figure 35A] Figures 35A and 35B show a comparison of the immunogenicity of different CRM-pustulan-based vaccines. Female BALB / c mice aged 8 to 12 weeks received a total of three intradermal vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected two weeks after the third vaccination and analyzed for anti-peptide responses. [Figure 35B] Female BALB / c mice aged 8–12 weeks received three intradermal vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two weeks after the third vaccination, samples were collected and analyzed for antiaggregatory aSyn fiber responses.

[0446] [Figure 36]Figure 36 shows a comparative analysis of the selectivity of the immune response elicited by the peptide + CRM + pustulan-based vaccine in vivo against aSyn fiber. Female BALB / c mice aged 8 to 12 weeks received a total of three intradermal or sc vaccinations administered at 2-week intervals. The CRM-pustulan-based vaccine was evaluated against the conventional CRM vaccine. Samples were collected two weeks after the third dose and subjected to an aSyn selectivity assay (inhibition ELISA). IC50 values ​​for antibodies inhibited by increasing doses of aSyn fiber are shown.

[0447] [Figure 37] Figure 37 shows the avidity of antibodies induced by peptide + CRM197 + pustulan vaccine. The stability of aSyn-antibody complexes induced by peptide + CRM197 + pustulan vaccine or peptide + CRM197 vaccine after challenge with different concentrations of the chaotropic agent sodium thiocyanate (NaSCN) and the determined avidity index are shown.

[0448] [Figure 38A] Figures 38A and 38B show a comparison of the immunogenicity of different CLEC-based vaccines. Female BALB / c mice aged 8 to 12 weeks received three intradermal vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two weeks after the third vaccination, samples were collected and analyzed for anti-SEQ ID NO:6 peptide responses induced by the peptide + carrier + glucan-based vaccine or the CLEC-unmodified vaccine adjuvanted with alum; dose: 20 μg peptide equivalents / injection; pustulan indicates SEQ ID NO:6 + CRM + pustulan; lichenan indicates SEQ ID NO:6 + CRM + lichenan; laminarin indicates SEQ ID NO:6 + CRM + laminarin; and sc + alum indicates the CLEC-unmodified vaccine adjuvanted with alum, SEQ ID NO:6 + CRM. [Figure 38B]Female BALB / c mice aged 8–12 weeks received three intradermal vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two weeks after the third vaccination, samples were collected and analyzed for anti-aSyn fiber responses induced by the peptide, carrier, and glucan-based vaccine or the CLEC-unmodified vaccine adjuvanted with alum; dose: 20 μg peptide equivalents per injection; pustulan indicates SEQ ID NO:6 + CRM + pustulan; lichenan indicates SEQ ID NO:6 + CRM + lichenan; laminarin indicates SEQ ID NO:6 + CRM + laminarin; and sc + alum indicates the CLEC-unmodified vaccine adjuvanted with alum, SEQ ID NO:6 + CRM.

[0449] [Figure 39A] Figure 39A shows the in vitro binding activity of peptide-CLEC conjugates to mouse DC receptors (i.e., Dectin-1). A comparative analysis of Dectin-1 binding ability as determined by ELISA is shown. Lich conjugate refers to SEQ ID NO:5 + SEQ ID NO:7 + lichenan conjugate, Pus conjugate refers to SEQ ID NO:5 + SEQ ID NO:7 + pustulan conjugate, and Lam conjugate refers to SEQ ID NO:5 + SEQ ID NO:7 + laminarin conjugate. Negative control refers to a sample without inhibitor. [Figure 39B] Figure 39B shows the in vitro binding activity of peptide-CLEC conjugates to human DC receptors (i.e., Dectin-1). A comparative analysis of Dectin-1 binding ability as determined by ELISA is shown. Lich conjugate refers to SEQ ID NO:5 + SEQ ID NO:7 + lichenan conjugate, Pus conjugate refers to SEQ ID NO:5 + SEQ ID NO:7 + pustulan conjugate, and Lam conjugate refers to SEQ ID NO:5 + SEQ ID NO:7 + laminarin conjugate. Negative control refers to a sample without inhibitor.

[0450] [Figure 40A]Figures 40A and 40B show a comparison of the immunogenicity of different CLEC-based vaccines. Female BALB / c mice aged 8 to 12 weeks received a total of three intradermal vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two weeks after the third vaccination, samples were collected and analyzed for anti-peptide responses (SEQ ID NO: 6, shown as peptide) and anti-aSyn responses (shown as protein) induced by the peptide-glucan-based vaccines (i.e., SEQ ID NO: 5 + SEQ ID NO: 7 + CLEC, dose: 5 μg / injection; lichenan indicates SEQ ID NO: 5 + SEQ ID NO: 7 + lichenan; laminarin indicates SEQ ID NO: 5 + SEQ ID NO: 7 + laminarin; and pustulan indicates SEQ ID NO: 5 + SEQ ID NO: 7 + pustulan). [Figure 40B] Female BALB / c mice aged 8–12 weeks received three intradermal vaccinations, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two weeks after the third vaccination, samples were collected and analyzed for anti-peptide (SEQ ID NO: 6, shown as peptide) and anti-aSyn (shown as protein) responses induced by the peptide-glucan-based vaccine (i.e., SEQ ID NO: 5 + SEQ ID NO: 7 + CLEC, dose: 20 μg / injection; lichenan (SEQ ID NO: 5 + SEQ ID NO: 7 + lichenan); laminarin (SEQ ID NO: 5 + SEQ ID NO: 7 + laminarin); and pustulan (SEQ ID NO: 5 + SEQ ID NO: 7 + pustulan).

[0451] [Figure 41]Figure 41 shows the in vitro DC receptor (i.e., Dectin-1) binding activity of glycoconjugate-pustulan conjugates. Both the CLEC-modified vaccines, oligosaccharide + CRM197 + pustulan conjugate and polysaccharide + TT + pustulan conjugate, maintain high Dectin-1 binding efficacy. Comparative analysis of Dectin-1 binding ability as determined by ELISA is shown. Act-Pus refers to the pustulan-modified Haemophilus influenzae type b capsular polysaccharide (polyribosylribitol phosphate, PRP) tetanus toxoid (TT) conjugate ActHIB®; Act refers to the ActHIB® conjugate vaccine without β-glucan modification; Men refers to the CRM197 conjugate vaccine Menveo® containing Neisseria meningitidis oligosaccharides (A, C, W135, and Y) without β-glucan modification; Men-Pus refers to the pustulan-modified Menveo® vaccine; and pus oxi refers to the activated pustulan used for modification.

[0452] [Figure 42A] Figures 42A and 42B show a comparison of the immunogenicity of different CLEC-based glycoconjugate vaccines. Female BALB / c mice aged 8 to 12 weeks received a total of three id / im vaccinations, administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected two weeks after the third vaccination and analyzed for anti-vaccine responses induced by oligosaccharide / polysaccharide-carrier-glucan-based vaccines or non-glucan-modified conjugate vaccines. Responses induced by Menveo® conjugated to pustulan (Menveo® + pustulan): N. meningitidis (A, C, W135, Y) + CRM197 + pustulan (80%) or unmodified Menveo®: N. meningitidis (A, C, W135, Y) + CRM197 (dose: 5 μg) are shown. [Figure 42B]Female BALB / c mice aged 8–12 weeks received three doses of i.d. / i.m. vaccination, administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected two weeks after the third dose and analyzed for anti-vaccine responses induced by oligosaccharide / polysaccharide-carrier-glucan-based vaccines or non-glucan-modified conjugate vaccines. Responses induced by ActHIB® conjugated to pustulan (ActHIB® + pustulan): H. influenzae (b) PRP + TT + pustulan (80%), or unmodified ActHIB® H. influenzae (b) PRP + TT (dose: 2 μg) are shown.

[0453] [Figure 43A] Figures 43A-C show a comparative analysis of the immunogenicity of CLEC-based vaccines using different IL31 peptide epitopes. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The immune responses elicited by eight different CLEC-based vaccines (SEQ ID NO:132 + SEQ ID NO:7 + pustulan; SEQ ID NO:134 + SEQ ID NO:7 + pustulan; SEQ ID NO:136 + SEQ ID NO:7 + pustulan; SEQ ID NO:138 + SEQ ID NO:7 + pustulan; SEQ ID NO:140 + SEQ ID NO:7 + pustulan; SEQ ID NO:142 + SEQ ID NO:7 + pustulan; SEQ ID NO:144 + SEQ ID NO:7 + pustulan; and SEQ ID NO:146 + SEQ ID NO:7 + pustulan) were evaluated against the respective peptide-CRM197 conjugates (i.e., SEQ ID NO:133 + CRM197; SEQ ID NO:135 + CRM197; SEQ ID NO:137 + CRM197; SEQ ID NO:139 + CRM197; SEQ ID NO:141 + CRM197; SEQ ID NO:143 + CRM197; SEQ ID NO:145 + CRM197; and SEQ ID NO:147 + CRM197) adjuvanted with alum. Samples were taken two weeks after the third dose and analyzed for anti-peptide responses. [Figure 43B]Female BALB / c mice aged 8–12 weeks received a total of three intradermal vaccinations administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The immune responses elicited by eight different CLEC-based vaccines (SEQ ID NO:132 + SEQ ID NO:7 + pustulan; SEQ ID NO:134 + SEQ ID NO:7 + pustulan; SEQ ID NO:136 + SEQ ID NO:7 + pustulan; SEQ ID NO:138 + SEQ ID NO:7 + pustulan; SEQ ID NO:140 + SEQ ID NO:7 + pustulan; SEQ ID NO:142 + SEQ ID NO:7 + pustulan; SEQ ID NO:144 + SEQ ID NO:7 + pustulan; and SEQ ID NO:146 + SEQ ID NO:7 + pustulan) were evaluated against the respective peptide-CRM197 conjugates (i.e., SEQ ID NO:133 + CRM197; SEQ ID NO:135 + CRM197; SEQ ID NO:137 + CRM197; SEQ ID NO:139 + CRM197; SEQ ID NO:141 + CRM197; SEQ ID NO:143 + CRM197; SEQ ID NO:145 + CRM197; and SEQ ID NO:147 + CRM197) adjuvanted with alum. Samples were taken two weeks after the third dose and analyzed for anti-IL31 protein responses [Figure 43C]Female BALB / c mice aged 8–12 weeks received a total of three intradermal vaccinations administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The immune responses elicited by eight different CLEC-based vaccines (SEQ ID NO:132 + SEQ ID NO:7 + pustulan; SEQ ID NO:134 + SEQ ID NO:7 + pustulan; SEQ ID NO:136 + SEQ ID NO:7 + pustulan; SEQ ID NO:138 + SEQ ID NO:7 + pustulan; SEQ ID NO:140 + SEQ ID NO:7 + pustulan; SEQ ID NO:142 + SEQ ID NO:7 + pustulan; SEQ ID NO:144 + SEQ ID NO:7 + pustulan; and SEQ ID NO:146 + SEQ ID NO:7 + pustulan) were evaluated against the respective peptide-CRM197 conjugates (i.e., SEQ ID NO:133 + CRM197; SEQ ID NO:135 + CRM197; SEQ ID NO:137 + CRM197; SEQ ID NO:139 + CRM197; SEQ ID NO:141 + CRM197; SEQ ID NO:143 + CRM197; SEQ ID NO:145 + CRM197; and SEQ ID NO:147 + CRM197) adjuvanted with alum. Figure 1 shows the binding activity of antibodies induced by SEQ ID NO: 132 + SEQ ID NO: 7 + Pustulan vaccine or SEQ ID NO: 133 + CRM vaccine as determined by challenging with three different concentrations of the chaotropic agent sodium thiocyanate (NaSCN).

[0454] [Figure 44A]Figures 44A-44C show a comparative analysis of the immunogenicity of CLEC-based vaccines using different IL31 peptide epitopes. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Ten different CLEC-based vaccines (SEQ ID NO: 133 + CRM197 + pustulan; SEQ ID NO: 135 + CRM197 + pustulan; SEQ ID NO: 137 + CRM197 + pustulan; SEQ ID NO: 139 + CRM197 + pustulan; SEQ ID NO: 141 + CRM197 + pustulan; SEQ ID NO: 143 + CRM197 + pustulan; SEQ ID NO: 145 + CRM197 + pustulan; SEQ ID NO: 147 + CRM197 + pustulan; SEQ ID NO: 149 + CRM197 + pustulan; and SEQ ID NO: 151 + CRM197 + pustulan) were administered. The immune response elicited by the suturanium (S. suturanium) was evaluated against each of the unmodified peptide-CRM197 conjugates (i.e., SEQ ID NO: 133 + CRM197; SEQ ID NO: 135 + CRM197; SEQ ID NO: 137 + CRM197; SEQ ID NO: 139 + CRM197; SEQ ID NO: 141 + CRM197; SEQ ID NO: 143 + CRM197; SEQ ID NO: 145 + CRM197; SEQ ID NO: 147 + CRM197; SEQ ID NO: 149 + CRM197; and SEQ ID NO: 151 + CRM197) adjuvanted with alum. Samples were collected two weeks after the third administration and analyzed for anti-peptide responses. [Figure 44B]Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Ten different CLEC-based vaccines (SEQ ID NO: 133 + CRM197 + pustulan; SEQ ID NO: 135 + CRM197 + pustulan; SEQ ID NO: 137 + CRM197 + pustulan; SEQ ID NO: 139 + CRM197 + pustulan; SEQ ID NO: 141 + CRM197 + pustulan; SEQ ID NO: 143 + CRM197 + pustulan; SEQ ID NO: 145 + CRM197 + pustulan; SEQ ID NO: 147 + CRM197 + pustulan; SEQ ID NO: 149 + CRM197 + pustulan; and SEQ ID NO: 151 + CRM197 + pustulan) were administered. The immune response elicited by the IL-31 peptide (S. suturanium) was assessed against each of the unmodified peptide-CRM197 conjugates (i.e., SEQ ID NO: 133 + CRM197; SEQ ID NO: 135 + CRM197; SEQ ID NO: 137 + CRM197; SEQ ID NO: 139 + CRM197; SEQ ID NO: 141 + CRM197; SEQ ID NO: 143 + CRM197; SEQ ID NO: 145 + CRM197; SEQ ID NO: 147 + CRM197; SEQ ID NO: 149 + CRM197; and SEQ ID NO: 151 + CRM197) adjuvanted with alum. Samples were taken two weeks after the third dose and analyzed for anti-IL31 protein responses. [Figure 44C]Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Ten different CLEC-based vaccines (SEQ ID NO: 133 + CRM197 + pustulan; SEQ ID NO: 135 + CRM197 + pustulan; SEQ ID NO: 137 + CRM197 + pustulan; SEQ ID NO: 139 + CRM197 + pustulan; SEQ ID NO: 141 + CRM197 + pustulan; SEQ ID NO: 143 + CRM197 + pustulan; SEQ ID NO: 145 + CRM197 + pustulan; SEQ ...

Claims

1. β-glucan and a B cell and / or a T cell epitope polypeptide, the β-glucan is covalently conjugated to the B cell and / or T cell epitope polypeptide to form a conjugate between the β-glucan and the B cell and / or T cell epitope polypeptide; The β-glucan is a predominantly linear β-(1,6)-glucan having a ratio of (1,6)-linked monosaccharide moieties to non-β-(1,6)-linked monosaccharide moieties of at least 1:1, preferably at least 2:1, more preferably at least 5:1, especially at least 10:

1.

2. The conjugate of claim 1, wherein the β-glucan is pustulan.

3. The conjugate of claim 1 or claim 2, wherein the polypeptide comprises at least one B cell epitope and at least one T cell epitope.

4. The conjugate according to any one of claims 1 to 3, wherein the ratio of β-glucan to B cell and / or T cell epitope polypeptide in the conjugate is from 10:1 (w / w) to 1:1 (w / w), preferably from 8:1 (w / w) to 2:1 (w / w), in particular 4:1 (w / w).

5. The conjugate according to any one of claims 1 to 4, wherein a B cell epitope and a pan-specific / promiscuous T cell epitope are independently coupled to the β-glucan.

6. The conjugate according to any one of claims 1 to 5, wherein the B-cell epitope polypeptide has a length of 5 to 20 amino acid residues, preferably 6 to 19 amino acid residues, in particular 7 to 15 amino acid residues.

7. The conjugate according to any one of claims 1 to 6, wherein the T cell epitope polypeptide has a length of 8 to 30 amino acid residues, preferably 13 to 29 amino acid residues, in particular 13 to 28 amino acid residues.

8. The conjugate is preferably a carrier protein, preferably a non-toxic cross-reacting substance (CRM) of diphtheria toxin (especially CRM １９７ ), KLH, diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae protein D (HipD), and serogroup B meningococcal outer membrane protein complex (OMPC), recombinant non-toxic form of Pseudomonas aeruginosa exotoxin A (rEPA), flagellin, Escherichia coli heat labile enterotoxin (LT), cholera toxin (CT), mutant toxins (e.g., LTK63 and LTR72), virus-like particles, albumin binding proteins, bovine serum albumin, ovalbumin, synthetic peptide dendrimers, e.g., multiple antigenic peptides (MAPs); with the proviso that if the conjugate comprises a carrier protein, the conjugate comprises at least a further independently conjugated T cell or B cell epitope polypeptide, in particular the ratio of carrier protein to β-glucan in the conjugate is from 1 / 0.1 to 1 / 50, preferably from 1 / 0.1 to 1 / 40, more preferably from 1 / 0.1 to 1 / 20, in particular from 1 / 0.1 to 1 / 10; The conjugate according to any one of claims 1 to 7.

9. The conjugate according to any one of claims 1 to 8, wherein the polypeptide is or comprises a B cell or T cell epitope polypeptide, preferably wherein the polypeptide is or comprises a B cell epitope and a T cell epitope.

10. The conjugate according to any one of claims 1 to 8, wherein the conjugate comprises a T cell epitope and no B cell epitope, preferably the conjugate comprises two or more T cell epitopes, in particular two, three, four or five T cell epitopes.

11. A conjugate according to any one of claims 1 to 10 for use in the prevention or treatment of a disease, preferably for use in the prevention or treatment of an infectious disease, a chronic disease, an allergy or an autoimmune disease.

12. 12. The conjugate of any one of claims 1 to 11 for use as an active anti-Aβ, active anti-tau and / or active anti-α-synuclein vaccine for the treatment and prevention of β-amyloidoses, tauopathies or synucleinopathies, preferably Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), Parkinson's disease dementia (PDD), neuroaxonal dystrophy, Alzheimer's disease (AD), amygdala-restricted AD with Lewy bodies (AD / ALB), dementia in Down's syndrome, Pick's disease, progressive supranuclear palsy (PSP), corticobasal degeneration, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and argyrophilic grain dementia.

13. A method for producing a conjugate according to any one of claims 1 to 12, comprising activating the β-glucan by oxidation and contacting the activated β-glucan with the B cell and / or T cell epitope polypeptide, thereby obtaining a conjugate of the β-glucan and the B cell and / or T cell epitope polypeptide.

14. The method of claim 13, wherein the β-glucan is obtained by periodate oxidation at vicinal hydroxyl groups, by reductive amination or by cyanylation of hydroxyl groups.

15. 15. The method according to claim 13 or claim 14, wherein the β-glucan is oxidized to a degree of oxidation, defined as the reactivity with Schiff's fuchsin reagent, which corresponds to the degree of oxidation of an equivalent amount of pustulan oxidized with periodic acid in a molar ratio of 0.2 to 2.6, preferably 0.6 to 1.4, in particular 0.7 to 1.

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