Conjugates consisting of or comprising at least one beta-glucan or mannan

Abstract

The present invention relates to the use of beta-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 belonging to the class of C-type lectins (CLECs). Background Art

[0002] Vaccination is considered one of the most effective means of saving lives and reducing the burden of disease. Through active immunization, vaccines are administered, causing the host's immune system to produce a nonspecific innate immune response as well as specific antibodies, B and T memory cells that can act against the immunogen used.

[0003] Polysaccharides are important virulence factors, especially for encapsulated bacteria that present complex carbohydrate structures on their surface. Bacterial, fungal or other polysaccharides are composed of 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, because they do not induce immunological memory, they are not enhanced by subsequent immunization.

[0004] These characteristics are based on the properties of polysaccharides: unlike proteins, polysaccharides constitute T-cell-independent (TI) antigens. Polysaccharide antigens directly activate polysaccharide-specific B cells, which then differentiate into plasma cells to produce antibodies. Memory B cells are not formed. 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 and presented to T lymphocytes together with major histocompatibility complex (MHC) class II molecules. Interaction with T cells induces B cell differentiation into plasma cells and memory B cells. Unlike TI antigens, TD antigens are immunogenic and can be enhanced and promoted by adjuvants (Peltola et al. Pediatrics November 1977, 60(5)730-737, Guttormsen HK et al. INFECTION and IMMUNITY, May 1998, p.2026-2032 and INFECTION and IMMUNITY, Dec. 1999, p.6375-6384, Avci et al. Nature Medicine volume 17, pages 1602-1609 (2011)).

[0005] The limitations of pure polysaccharide vaccines have been overcome by covalently conjugating polysaccharides to carrier proteins as a source of T-cell epitopes. This concept has been successfully applied to several glycoconjugate vaccines currently on the market that were 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 factor.

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

[0007] PRRs are a class of germline-encoded receptors that are essential for initiating innate immunity upon binding / activation, playing a key role in the first line of defense before the development of more specific adaptive immunity. The innate immune response is the first line of defense against infectious diseases and tissue damage. Specialized cells (primarily APCs such as macrophages and dendritic cells) 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. In addition, activation of APCs by innate immune signals is a key prerequisite for the generation of strong adaptive immunity (including antibody responses and memory responses).

[0008] The PRR family that has been identified so far is divided into transmembrane receptors and intracellular receptors. Transmembrane receptors include Toll-like receptors (TLR 1-9) and C-type lectin receptors (CLR), and intracellular receptors include nucleotide-binding oligomerization domain (NOD)-like receptors (NLR), retinoic acid-inducible gene-(RIG-)I-like receptors (RLR), and AIM2-like receptors (ALR).

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

[0010] A prominent example is inulin, a polysaccharide found in the roots of plants in the Asteraceae family. It consists of linear β-D-(2,1) polyfructofuranosyl α-D-glucose with up to 100 fructose moieties attached to a single terminal glucose. Inulin has no immunological activity in its native soluble form, but when crystallized into different stable microcrystalline particles (inulin α-δ), it acquires potent adjuvant activity for use in protein-coupled vaccines. The marketed product for delta inulin particles is Advax TMAdjuvant, which shows consistent spherulite-like disc-shaped particles, 1-2 μm in diameter, composed of a series of lamellar sheets. Delta- and gamma-inulin are believed 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, thereby enhancing T cell activation (Kerekes et al. J Leukoc Biol. 2001 Jan; 69(1): 69-74).

[0011] Another class of polysaccharide-based adjuvant candidates 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 itself is also poorly immunogenic. However, chitosan formulated as dry powder particles or in solution has been widely used as an encapsulant for mucosal and systemic vaccine delivery, and is also used to prepare mucosal DNA vaccines. It is used for mucosal vaccine delivery because it promotes absorption and subsequent phagocytosis, thereby 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 attributed to their cationic properties.

[0012] In vitro experiments showed that bovine serum albumin (BSA) or ovalbumin (OVA) encapsulated in chitosan particles can more effectively stimulate the activation of RAW264.7 macrophages and BMDCs compared with 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 was used with a vaccine against Haemophilus influenzae type b (Act-HIB) as an adjuvant-free vaccine, inducing stronger humoral and cellular responses against these antigens. Serum IgG1 and IgG2a titers were significantly enhanced. The production of Th1, Th2, and Th17 type cytokines was also increased. Unfortunately, Viscogel was shown to be unsuitable for human use (Vaccine. 2014; 32: 5967-5974).

[0014] Both chitin and chitosan particles are readily phagocytosed, supporting recognition via specific receptor-mediated phagocytosis. The receptors on myeloid cells that bind to chitin or chitosan and induce phagocytic responses have not been 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 has also been shown to bind to GlcNac and is therefore a potential receptor for chitosan-based vaccine conjugates. ViscoGel has been reported to trigger an immune response in a similar manner to chitin, as chitin particles have been reported to function as PAMPs and recognize TLR-2 receptors on macrophages to induce an innate immune response.

[0015] Yu et al. (Mol. Pharmaceutics, 2016, DOI: 10.1021 / acs.molpharmaceut.6b00138) also demonstrated that the conjugate of inulin-chitosan and Mycobacterium tuberculosis CFP10-TB10.4 fusion protein (CT) significantly increased the hydrodynamic volume compared with the unadjuvanted protein, and this vaccine stimulated high levels of Th1-type (IFN-γ, TNF-α and IL-2) and Th2-type cytokines (IL-4) and effective CT-specific antibody titers, mainly IgG1 and IgG2b. Pharmacokinetic studies have shown that inulin-chitosan conjugates can prolong the exposure of CT to the immune system in serum (Mol. Pharmaceutics 2016, 13, 11, 3626–3635).

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

[0017] CLRs are divided into transmembrane CLRs (TM-CLRs) and soluble CLRs (Collectins). TM-CLRs are further divided into type I TM-CLRs and type II TM-CLRs. Type I TM-CLRs include mannose receptors (MR) and ENDO180 [mannose receptor type C 2 (MRC 2)] receptors, which bind to mannose, fucose, and N-acetylglucosamine. Type II TM-CLRs include dendritic cell-specific intracellular adhesion molecule-3 grasping non-integrin (DC-SIGN), Langerin, and macrophage galactose-type lectin (MGL) receptors. DC-SIGN is known to bind to N-linked glycans (branched trimannose structures), such as glycans on HIV-I glycoprotein gp120 and other viruses (such as hepatitis C virus, human cytomegalovirus, dengue virus, or Ebola virus). DC-SIGN also recognizes lipoarabinomannan and mannan. Langerin is a Langerhan cell (LC)-associated LR that binds to glycan residues containing mannose and fucose. MGL has binding specificity for terminal N-acetylgalactosamine (GalNAc) residues and has also shown affinity for Campylobacter jejuni and the tumor-associated mucin MUC1, which is involved in controlling adaptive immunity of effector T cells. In addition, macrophage-inducible C-type lectins (Mincle, also known as CLEC4A) and the dectin-1 / dectin-2 receptor family have been reported. Dectin-1 plays an important role in mediating antifungal innate immunity and can bind to β-glucans from fungi (e.g., Saccharomyces cerevisiae β-glucans), lichens (e.g., sphaerocarpin, lichenin), algae (e.g., laminarin), or barley and other cereal species. Dectin-2 contains an EPN (Glu-Pro-Asn) amino acid motif that confers sensitivity to mannose ligands. In addition, dectin-2 interacts with Candida albicans.

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

[0019] In particular, immature dendritic cells (DCs) express a large number of CLRs, which are associated with their important function of sensing and capturing self and non-self antigens for processing and presentation on MHC molecules, thereby inducing activation of antigen-specific T cells. Thus, they link innate and adaptive immune responses.

[0020] CLECs have been used as nonspecific stimulators of immune responses and as immune adjuvants. For example, Vojtek et al. (Food and Agricultural Immunology, 2017, 28: 6, 993-1002) were able to demonstrate that oral administration of β-(1,3), β-(1,6) glucans combined with immunization against rabies virus and canine parvovirus 2 in dogs resulted in earlier formation of protective levels of antibodies against both viruses.

[0021] Several studies have shown 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 leads to a TH1 response, regardless of whether the antigen targets CLR DEC-205, langerin, Clec9A, or the Ig superfamily member Treml4.

[0022] In contrast, immunization under non-inflammatory (homeostatic) conditions results in the induction of tolerance. Langerin+ migratory DCs, but not lymph node-resident DCs, have been identified as the major Treg inducers, independent of the source of the DCs (skin or lung) or the receptor targeted.

[0023] In particular, the use of mannans as CLECs to target the mannose receptor (MR) or other mannan-sensitive CLRs has been shown to be effective in inducing cellular- and humoral-immune responses; therefore, MR- and other CLR-targeted vaccines have received increasing attention in the treatment of cancer, infectious diseases, and inducing specific tolerance to autoimmune diseases.

[0024] Mannan is a polysaccharide derived from yeast cell walls, whose backbone is mainly composed of β-(1,4)-linked mannose and a small amount of α-(1,6)-linked glucose and galactose side chain residues. In addition, the protein content detected in conventional mannan preparations is about 5%. As an important component of fungal cell walls, mannan has been widely used as a component of 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). In addition, different examples of vaccines based on mannan carrier-antigen complexes / conjugates have been developed, including mannan-mucin 1 (MUC1) fusion protein conjugates 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 prototypical mucin that has been found to be overexpressed on a wide variety of tumor cells. Along these lines, a MUC1 fusion protein containing five tandem repeats of human MUC1 (containing the immunodominant epitope: APDTRPAPGSTAPPAHGVTS) and a peptide (Cpl3-32) was made and conjugated to mannan under oxidative or reduced conditions, resulting in distinct immune responses: oxidized mannan-MUC1 stimulated a Th1-type response mediated by CD8+ T cells, with IFN-γ secretion and a predominant IgG2a antibody response, whereas reduced mannan-MUC1 stimulated a Th2-type response with IL-4 production and a high level of IgG1 antibody response. The fusion proteins used represent a single protein displaying both T-cell and B-cell epitopes.

[0026] Protein-carbohydrate / mannan complexes of papain and OVA have also recently been generated to analyze their sensitizing potential. It was found that coupling mannan to the protein surface reduced the binding and cross-linking of anti-papain IgE antibodies. Interestingly, in these experiments, coupling of mannan, dextran, or maltodextrin only reduced the sensitizing potential of papain, but not OVA, indicating the importance of carbohydrate choice for vaccine design (Weinberger et al. J. Control. Release 2013;165:101–109). These experiments also showed that mannan coupling resulted in the generation of elevated anti-OVA IgG titers after intradermal immunization.

[0027] Similar to the new sugar conjugate vaccine for MUC1, Ghochikyan et al. (DNA and CELL BIOLOGY, Volume 25, Number 10, 2006, 571-580) and Petrushina et al. (Journal of Neuroinflammation 2008, 5:42) used amyloid beta (Aβ) 28 (a 28 aa residue peptide that carries a combination of B cell epitopes and T cell epitopes of the human Aβ42 peptide) coupled to mannan to induce low-level anti-Aβ responses in mice. These responses were also demonstrated to reduce amyloid deposition in the cortex and hippocampus after subcutaneous immunization of APP transgenic mice. Immunization also resulted in the induction of increased anti-mannan titers in animals treated with Aβ28-mannan and BSA-mannan. However, the treatment failed to progress further, most likely due to increased microbleeding in the brains of treated animals, which was attributed to the potential deleterious effects of mannan as it can induce adverse vascular events, emphasizing the importance of carbohydrate selection for the design of effective and safe vaccines.

[0028] To date, no conjugates of single B-cell epitope peptides or T-cell epitope peptides conjugated to mannan or other related CLECs are known.

[0029] Beta-glucans comprise a group of beta-D-glucose polysaccharides. These polysaccharides are the main structural component 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] β-glucan is a source of soluble, fermentable fiber (also known as prebiotic fiber), which provides substrate for the microflora in the large intestine, increases stool volume, and produces short-chain fatty acids as byproducts with a wide range of physiological activities. For example, people with normal or elevated blood cholesterol levels who consume at least 3 grams of cereal-based β-glucan from oats per day can reduce total cholesterol and low-density lipoprotein cholesterol levels by 5-10%.

[0031] Typically, β-glucans form a linear backbone with 1-3 β-glycosidic bonds, but vary in molecular weight, solubility, viscosity, branching structure, and gel properties. Yeast and fungal β-glucans are usually built on a β-(1,3) backbone with 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 a major receptor for these carbohydrates, and β-glucans bind to dectin-1, inducing multiple cellular responses via the Syk / CARD9 signaling pathway, including phagocytosis, respiratory burst, and cytokine secretion. In addition, complement receptor 3 (CR3, CD11b / CD18) is also considered a receptor for β-glucans. Stimulation via dectin-1 has been reported to elicit responses from Th1, Th17, and cytotoxic T lymphocytes.

[0033] Members of the beta-glucan family include:

[0034] β-Glucan peptide (BGP) is a high molecular weight (~100 kDa) branched polysaccharide extracted from the fungus Trametes versicolor. BGP consists of a highly branched glucan portion containing a β-(1,4) backbone and β-(1,3) side chains, with β-(1,6) side chains covalently linked to a polypeptide portion rich in aspartic acid, glutamic acid, and other amino acids.

[0035] Curdlan is a high molecular weight linear polymer composed of β-(1,3) linked glucose residues from Agrobacterium spp.

[0036] Laminarin is derived from the brown alga Laminaria digitata (Dactyl kelp) and is a linear β-(1,3)-glucan with β-(1,6) linkages. Laminarin is a low molecular weight (5-7 kDa) water-soluble β-glucan that acts as a dectin-1 antagonist or agonist. It binds to dectin-1 without stimulating downstream signaling and can block the binding of dectin-1 to particulate β-(1,3)-glucans such as zymosan.

[0037] Pustulan is a medium molecular weight (20 kDa) linear β-(1,6) linked β-D-glucan from the lichen Lasallia pustulata. It can also bind to dectin-1 as a major receptor and activate signal transduction through dectin-1.

[0038] Lichenan is a high molecular weight (approximately 22-245 kDa) linear β-(1,3)β-(1,4)-β-D glucan from Cetraria islandica (Icelandic lichen), similar in structure to the β-glucans of barley and oats. Lichenan has a much higher ratio of 1,3-phase to 1,4-β-D bonds than the other two glucans. The ratio of β-(1,4)-phase to β-(1,3)-β-D bonds is approximately 2:1.

[0039] β-glucan from oats and barley is a linear β-(1,3)β-(1,4)-β-D glucan, which is available on the market with different molecular weights (medium molecular weight fraction is 35.6 kDa and high molecular weight fraction is up to 650 kDa).

[0040] Schizophyllan (SPG) is a gel-forming β-glucan from Schizophyllm commune. SPG is a high molecular weight (450 kDa) β-(1,3)-D-glucan with one β-(1,6) monoglucosyl branch for every three β-(1,3)-glucosyl residues on the backbone.

[0041] 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 every three main residues.

[0042] Whole Glucan Particles (WGP) are β-glucans known for their ability to modulate immune responses. Dispersible, Biothera) is a granular preparation of Saccharomyces cerevisiae β-glucan. It consists of hollow yeast cell wall "ghosts" and mainly contains long polymers of β-(1,3) glucose obtained from the cell wall of Saccharomyces cerevisiae after a series of alkali and acid extractions. Compared with other dectin-1 ligands (such as Zymosan), WGP dispersible lacks TLR stimulatory activity. In contrast, soluble WGP binds dectin-1 without activating this receptor. And it can significantly block the binding of WGP dispersible to macrophages and its immunostimulatory effects.

[0043] Zymosan (zymosan) is an insoluble preparation of yeast cells that activates macrophages via TLR2. TLR2 collaborates with TLR6 and CD14 to respond to Zymosan. Zymosan is also recognized by dectin-1, a phagocytic receptor expressed on macrophages and dendritic cells, which collaborates with TLR2 and TLR6 to enhance the immune response triggered by the recognition of Zymosan by each receptor.

[0044] As the main component of fungal cell wall, different β-glucans have been used as antigens to generate anti-glucan antibodies against fungal infection (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).

[0045] Torosantucci et al. (2005) and Bromuro et al. (2010) disclosed conjugates of branched β-glucan laminarin and linear β-glucan caldran gum with diphtheria toxoid CRM197. These conjugate vaccines induced high IgG titers against β-glucan and conferred protection against fungal infection in mice. In addition, high titers against CRM197 were detected using such conjugates (Donadei et al., Mol Pharm. 2015 May 4; 12(5): 1662-72). These authors also generated a β-glucan-CRM197 vaccine with synthetic linear β-(1,3)-oligosaccharides or β-(1,6)-branched β-(1,3)-oligosaccharides, combined with a human-acceptable adjuvant MF59. All conjugates induced high titers of anti-β-(1,3)-glucan IgG and / or also anti-β-(1,6)-glucan antibodies, as well as anti-β-(1,3)-glucan IgG, indicating the immunogenicity of different glucan combinations with traditional carrier proteins. Interestingly, Torosantucci et al. failed to demonstrate higher anti-CRM titers after immunization with CRM-dextran conjugates than with unconjugated CRM alone.

[0046] Donadei et al. (2015) also analyzed conjugates of the diphtheria toxoid CRM197 with either the linear β-(1,3) glucan Curdlan or synthetic β-(1,3) oligosaccharides. These conjugates were immunogenic and produced comparable anti-CRM197 antibody responses. Interestingly, these authors showed that intradermal administration of the CRM Curdlan conjugate produced higher antibody titers than intramuscular (im) immunization. However, intradermal application of CRM-Curdlan did not show different immunogenicity compared to subcutaneous application. Furthermore, the in vivo effects of CRM-Curdlan and non-Curdlan conjugated CRM (with Alum as adjuvant) were comparable. Therefore, no additive benefit of CLEC conjugation on the overall immune response was detected in this system.

[0047] Liao et al. (2015) disclosed a series of linear β-(1,3)-β-glucan oligosaccharides (hexa-, octa-, deca-, and dodeca-β-glucans) that were conjugated to KLH to generate glycoconjugates. These conjugates were shown to elicit strong T cell responses and were highly immunogenic, inducing high levels of anti-glucan antibodies. Mice immunized with this vaccine also elicited a protective immune response against the virulent pathogen Candida albicans. Anti-KLH titers were not compared to unconjugated KLH, so no information is available on the potential benefits of β-glucan in this experimental system.

[0048] These findings are very important for the suitability of glucan-based neoglycoconjugates as novel vaccines: potential anti-glucan antibodies induced by initial glucan conjugate immunization can lead to a rapid disappearance of the same β-glucan vaccine in subsequent booster immunizations or can attenuate the immune response to neoglycoconjugate vaccines for other indications, which is a well-known vector vaccine effect. The presence or even (re)stimulation of high levels of anti-glucan antibodies, as demonstrated above for mannan and β-glucan (Petrushina et al. 2008, Torosantucci et al. 2005, Bromuro et al., 2010, Liao et al., 2015), can therefore reduce or eliminate potential immune responses induced by the conjugate vaccine. Therefore, for new sustainable platforms using CLEC (especially β-glucan) as a backbone for immunization, it is crucial to ensure that the polysaccharides / oligosaccharides used induce very low levels of glucan antibodies or no glucan antibodies at all.

[0049] Glucan particles (GP) are highly purified 2-4 μm hollow porous cell wall microspheres consisting 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. They interact with their receptors dectin-1 and CR3 (there is also evidence implicating toll-like receptors and CD5 as other factors in GP function) and upregulate cell surface presentation of MHC molecules, leading to altered expression of co-stimulatory molecules and induction of proinflammatory cytokine production. Due to their immunomodulatory properties, GP have been developed for vaccine delivery.

[0050] There are three common approaches to using GPs in vaccines: (i) as adjuvants co-administered with antigens to enhance T-cell and B-cell mediated immune responses, (ii) chemically cross-linked with antigens, and most commonly (iii) as physical delivery vehicles for antigens trapped within the hollow GP cavity to provide targeted antigen delivery to APCs.

[0051] Benefit (i): Antigen-specific adaptive immune responses can be enhanced by co-administering GP with antigens. In this traditional adjuvant strategy, both innate and adaptive immune responses are activated to exert a protective response against pathogens. For example, Williams et al. (Int J Immunopharmacol. 1989; 11(4): 403-10) provided an adjuvant for an inactivated Trypanosoma cruzi vaccine by co-administering GP. The immune response elicited using this formulation resulted in 85% survival of mice challenged with Trypanosoma cruzi. In contrast, the mortality rate of the control group that received only glucose, dextran or vaccine was 100%.

[0052] Benefit (ii): The carbohydrate surface of GP can also be covalently modified using NaIO 4 Oxidation, carbodiimide cross-linking or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate-mediated coupling of antigens to the GP shell. Using this method, the coupling efficiency is very low (about 20%, see, for example, Pan et al. Sci Rep 5, 10687 (2015)), which significantly limits the applicability and the number of candidate vaccines, compared with antigen encapsulation in GP or the platform technology proposed in this application. Such covalently linked antigen-GP conjugates have been used in the study of cancer immunotherapy and infectious diseases. For example, Pan et al. (2015) used OVA cross-linked with periodate-oxidized GP to subcutaneously immunize mice. When mice were challenged with E.G7 lymphoma cells expressing OVA, a significant reduction in tumor size was observed. At 12 and 36 hours after subcutaneous injection, DCs (CD11c + MHC-II + GP-OVA was detected in 144 samples of 147 ...

[0053] Benefit (iii): The most effective way to use GPs in 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, depending on the type of payload and the intended mode of delivery.

[0054] Antigens can be encapsulated in the cavity of GP using polymer nanocomposite approaches, such as using bovine or mouse serum albumin and yeast RNA / tRNA to load and complex the payload, or adding alginate-calcium or alginate-calcium-chitosan mixtures. For example, Huang et al. (Clin. Vaccine Immunol. 2013; 20: 1585–91) reported that using these strategies, mice vaccinated with GP-OVA showed strong CD4+ T cell lymphocyte proliferation against ovalbumin (a Th1- and Th17-biased T cell-mediated immune response) and high levels of IgG1- and IgG2c-specific antibody responses. Non-covalent encapsulation strategies triggered stronger immune responses than GP co-administered with antigens.

[0055] An example of a GP-encapsulated subunit vaccine is GP encapsulating a soluble alkaline extract of the non-capsulated strain of Cryptococcus neoformans (cap59), which provided protection (60% survival) to mice challenged with a lethal dose of highly virulent Cryptococcus neoformans by inducing an antigen-specific CD4+ T cell response (IFN-γ, IL-17A positive) 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.). In addition, vaccination of mice with GP-encapsulated antigens 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 C. posadasii (Hurtgen BJ et al., Infect. Immun. 2012; 80: 3960-74).

[0056] In addition to cancer and infectious diseases, there are still a few studies using autoantigens that use GP as an encapsulation agent for vaccine delivery. Along these lines, Rockenstein et al. (J. Neurosci., 2018-01-24, 38 (4): 1000-1014) describe the application of GP loaded with recombinant human α-synuclein (aSynuclein) (comprising B cell epitopes and T cell epitopes suitable for inducing anti-aSyn immune responses) and rapamycin (known to induce antigen-specific regulatory T cells (Treg) in mouse synuclein disease models). As expected from previous studies using full-length α-synuclein as an immunogen, the use of GP containing aSyn leads to the induction of strong anti-α-synuclein antibody titers and reduces the pathological changes caused by α-synuclein in animals, the extent of which is similar to that previously published. The addition of rapamycin effectively induced the formation of iTregs (CD25 and FOXP3+) cells, because the number of such Treg cells increased significantly after rapamycin exposure. Thus, GP loaded with the antigens α-synuclein and rapamycin triggered neuroprotective humoral and iTreg responses in a mouse model of synucleinopathy, and the combined vaccine (aSyn+rapamycin) was more effective than either humoral immunization (GP aSyn) or cellular immunization (GP rapamycin) alone. Comparative information on the effects of immunization with conventional non-α-synuclein-containing GP has not been reported.

[0057] β-glucan neoglycoconjugates effectively target dendritic cells via the C-type lectin receptor dectin-1, enhancing their immunogenicity. Specifically, certain β-glucans are also 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 fusion proteins based on MUC1 (Wang et al., Chem. Commun., 2019, 55, 253).

[0058] Xie et al. and Korotchenko et al. used the branched β-glucan laminarin as the backbone for OVA conjugation. These new glucose conjugates were then applied to mice via the epidermal or subcutaneous route. Xie et al. showed that laminarin / OVA conjugates, but not unconjugated mixtures of these compounds, induced an anti-OVA CD4+T cell response that was increased compared to ovalbumin alone. Importantly, co-injection of unconjugated laminarin blocked this enhancement, supporting the APC targeting function mediated by laminarin. 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 antibody responses. Similarly, Korotchenko et al. demonstrated that laminarin conjugation to OVA significantly increased the absorption and induced activation of BMDCs, as well as the secretion of proinflammatory cytokines. These properties of LamOVA conjugates also lead to enhanced stimulation of OVA-specific naive T cells co-cultured with BMDCs. In the preventive immunization experiments, the authors could demonstrate that LamOVA immunization had reduced allergenic properties but induced approximately threefold higher IgG1 antibody titers compared to OVA after two immunizations. However, this effect disappeared in all treatment groups after the third immunization, when all groups showed similar antibody titers. Lam / OVA conjugates and OVA / Alum conjugates showed comparable therapeutic effects in the mouse allergic asthma model. Therefore, these experiments failed to provide a clear superiority of the dextran-based conjugates over conventional vaccines.

[0059] Wang et al. (2019) analyzed the efficacy of β-glucan-based MUC1 cancer vaccine candidates. Again, 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. Ethylene glycol (i.e., PEG) spacers were used to link β-glucan and MUC1 peptides to yeast β-(1,3)-β-glucan polysaccharide, applying 1,1'-carbonyl-diimidazole (CDI)-mediated conditions. The size of β-glucan-MUC1 nanoparticles was in the range of 150nm (actual average 162nm), while unmodified β-glucan formed particles of approximately 540nm. The β-glucan-MUC1 conjugate elicited high titers of anti-MUC1 IgG antibodies, which were significantly higher than the control group. Further analysis of the isotype and subtype of the antibodies generated showed that IgG2b was the major subtype, indicating that a Th1-type response was activated, as the IgG2b / IgG1 ratio was > 1. The large amounts of IgM antibodies observed indicated the involvement of the C3 component of the complement system, which typically induces cytotoxic activity and could be problematic for vaccine applications of this type of backbone, as vaccines should avoid cytotoxic activity, for example for chronic or degenerative diseases.

[0060] US2013 / 171187 A1 discloses an immunogenic composition comprising glucan and a pharmaceutically acceptable carrier to elicit protective anti-glucan antibodies. Metwali et al. (Am. J. Respir. Crit. Care Med. 185 (2012), A4152; Poster link C31 Regulation of pulmonary inflammation) studied the immunomodulatory effects of glucan derivatives in pulmonary inflammation. WO 2021 / 236809 A2 discloses a multi-epitope vaccine comprising amyloid-β and tau peptides for the treatment of Alzheimer's disease (AD). US2017 / 369570 A1 discloses β-(1,6)-glucan linked to antibodies against cells present in the tumor microenvironment. US2002 / 077288 A1 discloses synthetic, immunogenic, but non-amyloidogenic peptides homologous to amyloid β, used alone or in combination to treat AD. US2013 / 171187 A1 discloses anti-glucan antibodies as protective agents against Candida albicans fungal infections. WO 2004 / 012657 A2 discloses microparticulate β-glucan as a vaccine adjuvant. CN113616799A discloses a vaccine carrier composed of oxidized mannan and a cationic polymer. CN 111514286A discloses a Zika virus E protein conjugate vaccine with glucan. US 4,590,181 A discloses a solution of viral antigen mixed with mycoglucan or mycoglucan. Lang et al. (Front.Chem.8(2020):284) reviewed carbohydrate conjugates in vaccine development. Larsen et al. (Vaccines 8(2020):226) reported that mycoglucan activated chicken bone marrow-derived dendritic cells in vitro and promoted ex vivo CD4 +T cell memory response. US 2010 / 266626A1 discloses dextran, preferably laminarin and curdlan gum, as antigens coupled to adjuvants for antifungal immunization. Mandler et al. (Alzh.Dement.15(2019), 1133-1148) reported the effects of single and combined immunotherapy targeting amyloid beta protein and alpha synuclein in a Lewy body dementia model. Mandler et al. (Acta Neuropathol.127(2014), 861-879) reported a next-generation active immunization method for synucleinopathy, which uses immunogenic (B cell response) short peptides that are too short to induce T cell responses (autoimmunity) and do not carry natural epitopes, but carry a sequence that mimics the original epitope (e.g., oligomeric alpha synuclein), as well as the application of this method in clinical trials for Parkinson's disease (PD). Mandler et al. (Mol. Neurodegen. 10 (2015), 10) reported that active immunization against alpha-synuclein improved degenerative pathology and prevented demyelination in a multiple system atrophy (MSA) model. Jin et al. (Vaccine 36 (2018), 5235-5244) reviewed β-glucan as a potential immune adjuvant, mainly on adjuvant properties, structure-activity relationship and receptor recognition properties. WO 2022 / 060487 A1 discloses a vaccine containing a specific α-synuclein peptide for the treatment of neurodegenerative diseases. WO 2022 / 060488 A1 discloses a multi-epitope vaccine containing amyloid β protein and α-synuclein peptide for the treatment of AD. US2009 / 169549 A1 discloses a modified conformational isomer of α-synuclein, which is produced by introducing cysteine ​​into α-synuclein polypeptide and disrupting disulfide bonds to form a stable and immunogenic isomer. WO 2009 / 103105 A2 discloses a vaccine having a mimetic epitope of an α-synuclein epitope (extending from amino acid D115 to amino acid N122 of the natural α-synuclein sequence).

[0061] So far, no one has reported the construction or use of coupling a single B cell or T cell epitope peptide to β-glucan, especially linear β-glucan and / or pyruvic acid with high binding specificity / capacity to dectin-1 to form the novel glucose conjugate of the present application. Summary of the invention

[0062] It is therefore an object of the present invention to provide improved vaccines, being conjugate vaccines of a vaccination antigen and a carbohydrate-based CLEC adjuvant, particularly said vaccines which provide an improved immune response in vaccinated individuals over existing conjugate vaccines, particularly carbohydrate-based CLEC-peptide / protein conjugate vaccines.

[0063] Another object of the present invention is to provide vaccine compositions that utilize the CLEC backbone to confer immunity to short, readily interchangeable, highly specific B / T cell epitopes with potency, specificity, and affinity not previously achieved by traditional vaccines.

[0064] It is a particular object of the present invention to provide CLEC-based vaccines with improved selectivity and / or specificity for the dermal compartment.

[0065] Another object of the present invention is to provide a vaccine which induces target-specific immune responses as exclusively as possible, while inducing no or only very limited CLEC- or carrier protein-specific antibody responses.

[0066] Another object of the present invention is to provide vaccine compositions that utilize the CLEC backbone to confer immunity to short, readily interchangeable, highly specific B / T cell epitopes of alpha synuclein with potency, specificity, and affinity previously unmet by traditional vaccines for appropriate prevention and treatment of synucleinopathies.

[0067] A specific object of the present invention is to provide an alpha synuclein vaccine for use in the dermis which has improved selectivity and / or specificity over CLEC-based vaccines.

[0068] Another object of the present invention is to provide a vaccine which induces as exclusively as possible an alpha-synuclein-specific immune response while inducing no or only very limited CLEC- or carrier protein-specific antibody responses.

[0069] Another object of the present invention is to provide peptide immunogen constructs of alpha synuclein (aSyn) and their preparations for use in the treatment of synucleinopathies.

[0070] Therefore, the present invention provides a β-glucan for use as a C-type lectin (CLEC) polysaccharide adjuvant for B cell and / or T cell epitope polypeptides, preferably, wherein the β-glucan is covalently coupled to the B cell and / or T cell epitope polypeptide to form a conjugate of β-glucan and the B cell and / or T cell epitope polypeptide, wherein the β-glucan is mainly linear β-(1,6)-glucan, and the ratio of β-(1,6)-coupled monosaccharide part to non-β-(1,6)-coupled monosaccharide part is at least 1:1, preferably at least 2:1, more preferably at least 5:1, and especially at least 10:1.

[0071] The present invention successfully solves one or more of the above-mentioned goals. This is surprising to those skilled in the art, because no one in the art has reported the construction and use or efficacy of compounds similar to the novel, small, modular glucose conjugates of the present invention.

[0072] Surprisingly, the present invention shows that by coupling (i.e. covalently binding; synonymous herein) a peptide / protein to a selected CLEC-carrier of the present invention, wherein the coupling can be based on current chemistry, a superior pharmaceutical preparation is obtained that achieves an immune response. In the prior art, a large number of different coupling methods are available. In the process of establishing the present invention, hydrazone formation or coupling via a heterobifunctional linker is a particularly preferred method. Generally, activation of the CLEC prior to coupling (e.g. formation of a reactive aldehyde on the ortho-OH group of the sugar moiety) and the presence of a reactive group (e.g. N- or C-terminal hydrazide residue, SH group (e.g. via N- or C-terminal cysteine)) on the selected peptide / protein are necessary. The reaction can be a single step reaction (e.g. mixing the activated CLEC with the hydrazide peptide leads to hydrazone formation), or a multi-step process (e.g. reaction of the activated CLEC with the hydrazide from the heterobifunctional linker followed by coupling of the peptide / protein via the respective reactive groups).

[0073] Thus, the individual components of the conjugate of the invention can be directly coupled to each other, for example by coupling the B cell epitope and / or T cell epitope to β-glucan and / or the carrier protein, or by coupling β-glucan to the carrier protein (in all possible orientations). "B cell epitope polypeptide" or "T cell epitope polypeptide" as used herein refers by default to the B cell or T cell epitope of the "B cell epitope polypeptide" or "T cell epitope polypeptide", rather than the B cell or T cell epitope of the carrier protein (if present), unless the B cell or T cell epitope of the carrier protein is explicitly mentioned. According to a preferred embodiment, the attachment of the B-cell epitope and / or T-cell epitope to the β-glucan or mannan and / or the carrier protein is preferably achieved via a linker, more preferably a linker comprising a cysteine ​​residue or a cysteine ​​or glycine residue, a linker resulting from a hydrazide-mediated coupling, a linker resulting from a coupling 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), by imidazole-mediated coupling, by reductive amination, by carbodiimide coupling of -NH-NH 2 Connector; NRRA, NRRA-C or NRRA-NH-NH 2 A linker, a peptide linker, such as a bi-, tri-, tetra- (or more)-mer peptide group, such as CG or CG, or a cleavage site, such as a cathepsin cleavage site; or a combination thereof, in particular cysteine ​​or NRRA-NH-NH 2Linkers. Obviously, "linkers resulting from, for example, hydrazide-mediated coupling" refers to the chemical structure in the resulting conjugate after coupling, i.e., the chemical structure that appears in the resulting conjugate after coupling. Amino acid linkers can have peptide bonds (e.g., glycine-containing linkers) or via functional groups of amino acids (e.g., disulfide bonds of cysteine ​​linkers) in the coupled form.

[0074] By using the CLEC backbone of the invention, the novel conjugates of the invention have been shown to confer immunity to short, easily interchangeable, highly specific B / T cell epitopes, exhibiting potency, specificity and affinity previously unmet by conventional vaccines: in fact, the conjugates of the invention are the first to utilize short B cell / T cell epitopes in a CLEC-based vaccine, obviating the need to present these epitopes in the form of fusion proteins, including the formation of tandem repeats of these epitopes or fusions of different tandem repeats to form a stable and effective immunogen (e.g., as required for the above-mentioned MUC1 approach with mannan).

[0075] By means of the present invention, the necessity of using full-length proteins (i.e. payloads in glucan particles (GP)) in CLEC vaccines can also be avoided. Furthermore, autoimmune reactions caused by (unwanted) T-cell epitopes present in the immunogen, like self-proteins (e.g. T-cell epitopes in aSyn, amyloid β, etc.) or mixed self-epitopes (e.g. tandem repeats of MUC1 used as immunogen)) can also be avoided when using CLEC.

[0076] According to the present invention, for the first time short epitopes (B- and / or T-cell epitopes, mainly peptides, modified peptides) can be linked to functional CLEC-based scaffolds using covalent coupling techniques based on well-established chemistry, wherein the possible coupling methods can be adapted to the requirements of the specific epitope based on methods well known in the art.

[0077] The presentation of the short peptides of the present invention can be made as individually coupled moieties, in combination with a single foreign T cell epitope (as a short peptide or long protein), or as a complex / conjugate with a larger carrier molecule, providing the T cell epitope to induce a sustainable immune response. The vaccine design of the present invention allows the preparation of multivalent conjugates, which is a prerequisite for inducing an effective immune response by efficient B cell receptor (BCR) cross-linking.

[0078] Furthermore, by means of the present invention, a CLEC-based vaccine with excellent dermal selectivity / specificity can be provided. In fact, the conjugate design of the present invention is built on CLEC, with CLEC as a carrier of target-specific epitopes, which exhibit high binding specificity for PRRs on dermal APC / DC, especially for dectin-1 (or MR and DC-SIGN in the case of mannan), to allow skin-selective / specific and receptor-mediated uptake (= targeted vaccine delivery).

[0079] The CLEC polysaccharide used as a carrier according to the present invention is used to concentrate the carrier-peptide conjugate into the preferred dermal / skin DC and elicit an immune response. This is mainly due to epidermal or dermal (rather than subcutaneous) specificity. The CLEC backbone of the present invention and the effective dermal immune response elicitation also help to avoid the mandatory use of adjuvants, which are commonly used in conventional vaccines and are also used in exemplary CLEC-based vaccines (e.g., using Alum, MF59, CFA, PolyI:C or other adjuvants). According to a preferred embodiment of the present invention, the use of adjuvants can be significantly reduced or omitted, for example, when the addition of adjuvants is not specified.

[0080] Several CLECs have been used in previous applications, but none of the proposed conjugated structures could confer skin selectivity (i.e., high dectin-1 binding capacity for dermal application compared to all other routes (i.e., subcutaneous, intramuscular, and ip (intraperitoneal) injection), efficient dermal DC targeting, and excellent immunogenicity).

[0081] The present invention selects CLEC in order to provide a novel solution to target skin-specific DC and skin-specific immunity in an efficient manner. The conjugates of the present invention also exert limited activity in other classical tissues (such as muscle or subcutaneous tissue) immunity, which is contrary to the previously described CLEC-based vaccines / candidates applied im (muscle) or sc (subcutaneous). As a result of the experiments conducted during the present invention, the vaccines of the present invention, especially the vaccines with pyruvic polysaccharides as CLEC, have been shown to be surprisingly selective for skin immunity.

[0082] The present invention relates to any B cell and / or T cell epitope polypeptide and any predominantly linear β-(1,6)-glucan having a ratio of (1,6)-conjugated monosaccharide moieties to non-(1,6)-conjugated monosaccharide moieties of at least 1:1. As shown in the Examples section, the teachings herein support any B cell and / or T cell epitope polypeptide, and such epitopes do not find any limitation, especially when these epitopes are already part of a previous and / or existing epitope. The specific B cell and / or T cell epitope polypeptides shown and mentioned herein are preferred epitopes, but the present invention is not limited thereto. In the course of the present invention and after testing numerous epitopes to date (see the very diverse functional and structural epitopes investigated and experimentally confirmed in the Examples section (including a large number of model epitopes)), no restrictions were found on the nature and structure of the B-cell and / or T-cell epitopes (linear polypeptides, self-peptides, polypeptides with post-translational modifications, such as sugar structures or pyroglutamate, mimotopes, allergens, structural epitopes, conformational epitopes, etc.), especially for pyruvic sphaeroides as β-(1,6)-glucans. In one case, the experiments showed that it was the β-glucan of the present invention and the covalent binding to the epitope polypeptide that determined the immunological manifestation, rather than the specific structural features of the individual epitopes.

[0083] The term "B cell and / or T cell epitope polypeptide" herein is a functional term recognized in the art: 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 exclusively 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 of 8 to 11 amino acids in length, while MHC class II molecules present longer peptides (13 to 17 amino acids), and non-classical MHC molecules also present non-peptide epitopes such as glycolipids. B cell epitopes are portions of an antigen that are bound by an immunoglobulin or antibody. B cell epitopes can be, for example, conformational or linear.

[0084] According to a preferred embodiment of the present invention, the conjugate of the present invention comprises a polypeptide having at least one B cell epitope and at least one T cell epitope, preferably a B cell epitope + CRM197 conjugate covalently linked to β-glucan, especially a peptide + CRM197 + linear β-(1,6)-glucan or a peptide + CRM197 + linear pyralidin conjugate. The preferred ratio of glucan to peptide, especially the ratio of pyralidin to peptide, is 10 to 1 (w / w) to 0.1 to 1 (w / w), preferably 8 to 1 (w / w) to 2 to 1 (w / w), especially 4 to 1 (w / w). When the conjugate contains a carrier protein, the preferred ratio of β-glucan or mannan to B cell epitope-carrier polypeptide is 50 to 1 (w / w) to 0.1 to 1 (w / w), especially 10 to 1 to 0.1 to 1.

[0085] With the present invention, it is possible to focus on inducing a target-specific immune response without inducing or inducing only a very limited CLEC- or carrier-protein-specific antibody response. The conjugate of the present invention thus solves the problem of traditional conjugate vaccines, which must rely on the use of foreign carrier proteins to induce a sustainable immune response. Existing conjugate vaccine development is mainly based on carrier molecules, such as KLH, CRM197, tetanus toxoid or other suitable proteins, which form complexes with target-specific short antigens and provide them to immune responses to resist different target diseases like infectious diseases, degenerative or neoplastic diseases, including, for example, Her2-neu positive cancers, alpha synuclein for synuclein diseases such as Parkinson's disease, amyloid beta peptide for amyloidosis such as Alzheimer's disease, Tau for the treatment of tau diseases (tau diseases) including Alzheimer's disease. auopathies), 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 light and heavy chain amyloidosis (AL, AH, AA), islet amyloid polypeptide (IAPP) and amylin for type 2 diabetes, (mutant) transthyretin for ATTR / transthyretin amyloidosis, and many more.

[0086] The immunological performance and efficacy of the conjugates of the present invention and vaccines containing these conjugates are also unexpected and surprising in view of the guidance of the prior art, in which β-glucans, especially mainly linear β-(1,6)-glucans, are mainly used as antigens themselves to induce specific immune responses against fungi in which such β-glucans are present (see, for example, US2013 / 171187 A1; Metwali et al., Am. J. Respir. Crit. Care Med. 185 (2012), A4152; poster session C31; US2013 / 171187 A1, US2010 / 266626A1, Jin et al. (Vaccine 36 (2018), 5235-5244)). However, the present invention demonstrates that the conjugates of the present invention are unable to elicit a significant immune response to β-glucan, but - due to the construction of the present conjugates - the immune response is shifted to the B cell and / or T cell epitope polypeptides covalently coupled to the β-glucan. Coupling these B cell and / or T cell epitope polypeptides to linear β-glucan appears to hide the immune response eliciting ability of the β-glucan, but exposes and significantly improves the presentation of the covalently bound B cell and / or T cell epitope polypeptides to the immune system. This teaching is neither disclosed in the prior art nor obvious from the prior art:

[0087] US2017 / 369570 A1 discloses that β-(1,6)-glucan is linked to antibodies against cells in the tumor microenvironment, based on a completely different concept and mechanism (tumor therapy).

[0088] On the other hand, dextran is used as a component of a vaccine (most commonly as “(liposome) dextran (nano)particles”), but B cell and / or T cell epitope polypeptides are not covalently coupled to the dextran (e.g. WO 2004 / 012657A2, CN113616799 A, US 4,590,181 A, Lang et al., Front. Chem. 8(2020):284; Larsen et al., Vaccines 8(2020):226).

[0089] Finally, the improved effects of the predominantly linear β-(1,6)-glucans of the present invention relative to the constructs and compositions described in WO 2022 / 060487 A1, WO 2022 / 060488 A1, US 2009 / 169549 A1, WO 2009 / 103105 and CN 111514286 A (e.g., β-(1,2)-glucans or β-(1,3)-glucans) have been demonstrated in the Examples section below.

[0090] In view of these favorable properties of the conjugates of the present invention, the conjugates and vaccines of the present invention are particularly suitable for active anti-Tau vaccination, including truncated, (hyper)phosphorylated, nitrated, glycosylated and / or ubiquitinated variants, for the treatment and prevention of Tau diseases, in particular Alzheimer's disease and Down syndrome or other Tau diseases, including Pick's disease, progressive supranuclear palsy (PSP), cortical basal degeneration, frontotemporal dementia and Parkinson's disease associated with chromosome 17 (FTDP-17) and argyrophilic grain disease. Other emerging diseases and conditions include globular cell tauopathy, primary senile tauopathy (PART), which includes neurofibrillary tangles, chronic traumatic encephalopathy (CTE), and senile tau astrocytopathy. In addition, other diseases are included, such as vacuolar tauopathies, gangliogliomas and gangliocytomas, lytico-bodig disease (Guam Parkinsonism and dementia syndrome), meningioangiomatosis, postencephalitic Parkinsonism, and subacute sclerosing panencephalitis (SSPE).

[0091] Tauopathies often overlap with synucleinopathy, probably due to the interaction between synuclein and tau protein. Therefore, the anti-Tau conjugates of the present invention are particularly suitable for active anti-Tau vaccination against synucleinopathy, especially Parkinson's disease (PD), Lewy body dementia (DLB) and Parkinson's disease dementia (PDD).

[0092] Anti-Tau vaccines may be very effective when used alone or in combination with existing peptide vaccines against other pathological molecules involved in β-amyloidosis, tauopathy or synucleinopathy, especially mixed pathologies (i.e., the presence of Aβ pathology and Tau pathology and / or aSyn pathology). Therefore, a preferred embodiment is to provide a combination of anti-Tau vaccines with anti-Aβ and / or anti-aSyn peptide vaccines to treat degenerative diseases such as Alzheimer's disease, Down syndrome dementia, Lewy body dementia, Parkinson's disease dementia, Parkinson's disease.

[0093] According to a preferred embodiment, the polypeptide derived from Tau protein is selected from natural human Tau (441aa isoform; GenBank accession number> AAC04279.1; Seq ID No

[0094]

[0095] or a polypeptide comprising or consisting of amino acid residues derived from human Tau, including post-translationally modified, phosphorylated, di-phosphorylated, hyper-phosphorylated, nitrated, glycosylated and / or ubiquitinated amino acids, including Tau2-18, Tau 176-186, Tau 181-210, Tau 200-207, Tau 201-230, Tau 210-218, Tau 213-221, Tau 225-234, Tau 235-246, Tau 251-280, Tau 256-285, Tau 259-288, Tau 275-304, Tau 260-264, Tau 267-273, Tau 294-305, Tau 298-304, Tau 300-317, Tau 329-335,Tau 361-367,Tau 362-366,Tau379–408,Tau 389-408,Tau 391-408,Tau 393-402,Tau 393-406,Tau393-408,Tau418-426,Tau 420-426.

[0096] According to a preferred embodiment, the Tau protein-derived polypeptide is selected from the mimetics of the above-mentioned Tau-derived polypeptide, including mimetic epitopes and peptides containing amino acid substitutions (to simulate phosphorylated amino acids, including phosphorylated S replaced by D and phosphorylated T replaced by 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.

[0097] US2008 / 050383 A1 and Asuni et al. (Journal of Neuroscience 34:9115–9129) disclosed antibodies against Tau379–408 with two phosphorylated aas: pS396 and pS404, which are suitable for immunotherapy against Tau pathology, while Boutajangout et al. (J. Neurosci., December 8, 2010, 30(49):16559–16566) disclosed the use of the same epitope: a dual phosphorylated peptide Tau379–408 with pSp396 and pS404 combined with the adjuvant AdjuPhos, which was as effective as an active immunotherapy agent and prevented cognitive decline in several tests of the htau / PS1 model, which was associated with a reduction in pathological tau in the brain. Bi et al. (2011, PLoS One 12:e26860.) also showed that Tau-targeted immunization using a 10-mer peptide from the dual phosphorylated sequence Tau395-406 (carrying pS396 and pS404) coupled to KLH and adjuvanted with complete or incomplete Freund's adjuvant hindered the progression of neurofibrillary histopathology in aged P301L Tau transgenic mice.

[0098] Boimel M et al. (2010; Exp Neurol 2:472–485) demonstrated that the use of dual phosphorylated peptides Tau195-213 [pS202 / pT205], Tau207-220 [pT212 / pS214], and Tau224-238 [pT231] and pertussis toxin emulsified in complete Freund's adjuvant (CFA) resulted in reduced Tau-related pathology in animals.

[0099] Troquier et al. (2012 Curr Alzheimer Res 4:397–405) demonstrated that targeting Tau by active Tau immunotherapy with artificial peptide constructs consisting of an N-terminal YGG linker fused to a 7-peptide (Tau418-426) or 11-peptide (Tau417-427) derived from human Tau carrying pS422, coupled to KLH and adjuvanted with CFA, could be a suitable therapy in the THYTau22 mouse model, as a decrease in insoluble Tau species (AT100- and pS422-immunoreactive species) was detected that was associated with significant cognitive improvements using a Y-maze.

[0100] US2015 / 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) disclosed peptide immunogens and showed that vaccines AV-1980R and AV-1980D are both based on the MultiTEP platform, consisting of 3 repeats of Tau2-18 fused to several promiscuous T cell epitopes as recombinant polypeptides or as DNA vaccines, which induce strong immune responses and reduce tau pathology in different tau disease models.

[0101] EP 3 097 925 B1 discloses a peptide immunogen composed of a phospho-peptide derived from human Tau441. Theunis et al. (2013, PLoS ONE 8(8):e72301) demonstrated a liposome vaccine based on EP 3 097 925 B1 carrying the Tau peptide Tau 393-408 (carrying pS396 and pS402), which can induce anti-phosphorylated Tau antibodies in the brain of Tau.P301L mice, accompanied by improvement in clinical condition and reduction in the index of tauopathy.

[0102] Sun et al. (Signal Transduction and Targeted Therapy (2021) 6:61) disclosed various immunogens based on norovirus P particles. The vaccine pTau31 (composed of particles containing fusion peptides of Tau195-213 (carrying pS202 and pT205) and Tau395-406 (carrying pS396 and pS404)) produced a powerful pTau antibody that could significantly reduce tau pathology and improve behavioral deficits in Tau Tg animal models.

[0103] EP 2 758 433 B1 discloses peptide-based immunogens for interfering with Tau pathology. The present invention discloses peptide conjugate vaccines (e.g., peptide KLH vaccines). Kontsekova et al. (Alzheimer's Research & Therapy 2014, 6:44) disclose that such peptide vaccines (i.e., Axon peptide 108 (Tau294-305; KDNIKHVPGGGS) coupled to KLH and adjuvanted with Alum; also known as AADvac1) induce a strong protective humoral immune response, in which antibodies can distinguish between pathological tau and physiological tau. Active immunotherapy reduces the level of tau oligomers and the extent of neurofibrillary pathology in the brain of transgenic rats.

[0104] Although in principle the present invention can improve all proposed Tau vaccination peptides, selected epitopes were specifically evaluated for their suitability with the present platform. For example, Tau294-305, SeqID35+36 was shown to be superior to KLH-based vaccines, as proposed by EP2 758 433B1 and Kontsekova et al.

[0105] Further preferred target sequences include:

[0106]

[0107]

[0108] In view of these favorable properties of the conjugate of the present invention, the conjugate and vaccine of the present invention are particularly suitable for active immunotherapy of IL12 / IL23 related diseases and autoimmune inflammatory diseases. IL-23 related diseases are selected from psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, diabetes (preferably type 1 diabetes), atherosclerosis, inflammatory bowel disease (IBD) / M.Crohn's disease, multiple sclerosis, Behcet's disease, ankylosing spondylitis, Vogt-Koyanagi-harada disease, chronic granulomatous disease, hidradenitis suppurativa (hidratenitis suppurtiva), anti-neutrophil cytoplasmic antibody (ANCA-) associated vasculitis, neurodegenerative diseases (preferably 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), especially psoriasis, neurodegenerative diseases or IBD. In addition, IL-12 / 23 directed vaccines can be used together / in combination with vaccines against other targets, as recent data suggest that IL-23 driven inflammation can exacerbate other diseases, such as Alzheimer's disease or possibly diabetes.

[0109] According to a preferred embodiment, the IL12 / IL23 protein-derived polypeptide is derived from natural human IL12 / IL23 or a mimetic having one or more aa (amino acid) exchanges to form a mimotope of the corresponding natural sequence.

[0110] According to a preferred embodiment, the polypeptide derived from the IL12 / IL23 protein is selected from a subunit of the heterodimeric protein IL23, native human IL-23p19 or a polypeptide comprising or consisting of amino acid residues derived from the subunit or a mimotope. In WO 2005 / 108425 A1, the peptides FYEKLLGSDIFTGE, FYEKLLGSDIFTGEPSLLPDSP, VAQLHASLLGLSQLLQP, GEPSLLPDSPVAQLHASLLGLSQLLQP, PEGHHWETQQIPSLSPSQP, PSLLPDSP, LPDSPVA, FYEKLLGSDIFTGEPSLLPDSPVAQLHASLLGLSQLLQP, LLPDSP, LLGSDIFTGEPSLLPDSPVAQLHASLLG, FYEKLLGSDIFTGEPSLLPDSPVAQLHASLLG, QPEGHHW, LPDSPVGQLHASLLGLSQLLQ and QCQQLSQKLCTLAWSAHPLV derived from IL-23 are proposed as vaccination peptides for IL-23. In WO 03 / 084979A2, GHMDLREEGDEETT, LLPDSPVGQLHASLLGLSQ and LLRFKILRSLQAFVAVAARV from IL-23p19 are mentioned as possible anti-cytokine vaccines. WO 2016 / 193405 A1 discloses a peptide immunogen derived from the IL12 / 23p19 subunit (accession number: Q9NPF7) having the following amino acid sequence

[0111]

[0112] As possible anti-cytokine vaccines, in particular aa136-145, aa136-143, aa 136-151, aa137-146, aa144-154, aa144-155 and others, in particular the sequences: QPEGHHWETQQIPSLS, GHHWETQQIPSLSPSQPWQRL, QPEGHHWETQ, TQQIPSLSPSQ, QPEGHHWETQQIPSLSPSQ, QPEGHHWETQQIPSLSPS.

[0113] According to a preferred embodiment, the polypeptide derived from the IL12 / IL23 protein is selected from a subunit of the heterodimeric protein IL23, native human IL12 / 23p40, or a polypeptide comprising or consisting of amino acid residues aa15-66, aa38-46, aa53-71, aa119-130, aa160-177, aa236-253, aa274-285, aa315-330 of native human IL12 / 23p40 (Accession No.: P29460.1), and has the following amino acid sequence:

[0114]

[0115] In WO 03 / 084979A2, peptides LLLHKKEDGIWSTDILKDQKEPKNKTFLRCE and KSSRGSSDPQG from the IL-12 / 23p40 subunit are mentioned as possible anti-cytokine vaccines.

[0116] Luo et al., J Mol Biol 2010-10-8; 402(5): 797-812 disclosed the conformational epitope of anti-IL12 / IL23p40 specific antibody Ustekinumab - aa15-66, which effectively reduces IL12 (IL23) related diseases. Guan et al. (Vaccine 27 (2009) 7096-7104) disclosed the immunogens aa38-46, aa53-71, aa119-130, aa160-177, aa236-253, aa274-285, aa315-330 of mouse IL12 / 23, accession number: P43432 (p40) and Q9EQ14 (p19)) having the following amino acid sequence: P43432 (p40):

[0117]

[0118] ; Recombinant HBcAg was added.

[0119] Although in principle, the present invention can improve all proposed IL12 / IL23-related disease vaccine peptides, selected epitopes were specifically evaluated for their suitability with this platform. For example, SeqID37 / 38 and SeqID41 / 42 WISIT vaccines were shown to be superior to KLH-based vaccines. Murine sequences SeqID39 / 40 showed similar potency to KLH-based conjugates in mice and were also active in IL12 / 23 recognition.

[0120] In view of these favorable properties of the conjugate of the present invention, the conjugate and vaccine of the present invention are particularly suitable for active anti-EMPD (extracellular proximal domain, as part of membrane IgE-BC) vaccination for the treatment and prevention of IgE-related diseases. Exclusive targeting and cross-linking of membrane IgE-BCR are achieved by touching the membrane anchoring region (i.e., extracellular membrane proximal domain IgE (EMPD IgE)) found only on membrane IgE but not on soluble serum IgE. IgE-related diseases include allergic diseases such as seasonal, food, pollen, mold spores, poisonous plants, medical / drug, insect-, scorpion- or spider-venom, latex or dust allergies, pet allergies, allergic bronchial asthma, non-allergic asthma, Churg-Strauss syndrome, allergic rhinitis and-conjunctivitis, atopic dermatitis, nasal polyps, Kimura's disease, contact dermatitis to adhesives, antimicrobials, fragrances, hair dyes, metals, rubber components, topical medications, rosin, wax, polishes, cement and leather, chronic sinusitis, Atopic eczema, autoimmune diseases in which IgE plays a role ("autoallergy"), chronic (idiopathic) and autoimmune urticaria, cholinergic urticaria, mastocytosis, especially cutaneous mastocytosis, allergic bronchopulmonary aspergillosis, chronic or recurrent idiopathic angioedema, interstitial cystitis, allergic reactions, especially idiopathic and exercise-induced allergic reactions, immunotherapy, eosinophil-related diseases, such as eosinophilic asthma, eosinophilic gastroenteritis, eosinophilic otitis media and eosinophilic esophagitis (see, for example, Holgate 2014 World Allergy Organ. J. 7: 17., US 8,741,294 B2). In addition, the vaccine or conjugate of the present invention is used to treat lymphoma or prevent the sensitizing side effects of antacid therapy (especially for gastric or duodenal ulcers or reflux). For purposes of the present invention, the term "IgE-related disease" includes or is used synonymously with the terms "IgE-dependent disease" or "IgE-mediated disease".

[0121] According to a preferred embodiment, the EMPD protein-derived polypeptide is derived from a native human IgE-BCR or is a mimetic with one or more aa exchanges to form a mimotope of the corresponding native sequence.

[0122] The development of dedicated antibodies that specifically target human or mouse EMPD IgE has allowed clinical and preclinical validation of this targeting strategy in vitro and in vivo (Liour et al., 2016 Pediatr Allergy Immunol August; 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 with 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) showed that mAbs specific for the N-terminal or middle segments of CemX could bind to B cells expressing mIgE and effectively induce their apoptosis and ADCC. CemX refers to the human membrane-bound e-chain. This isoform contains an additional domain of 52 aa residues located between the CH4 domain and the C-terminal membrane anchor peptide, called CemX or M1' peptide. This was particularly evident in 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 middle segment P2 (HSGQQQGLPRAAGGSVPHPR), while the C-terminal P3 (GAGRADWPGPP) was unsuccessful.

[0123] In addition, antibodies generated by active immunization against the human EMPD IgE region can mediate apoptosis and ADCC in vitro (Lin et al., Mol. Immunol., 52 (2012), pp. 190-199). Lin et al. disclosed the use of HBcAg immunogens carrying inserts of CemX or its P1, P2 and P1-P2 portions as anti-EMPD vaccines.

[0124] The first clinical anti-human EMPD IgE monoclonal antibody Quilizumab showed selective IgE inhibition in healthy volunteers in phase I and phase II studies, and showed clinical benefits in patients with allergic rhinitis and mild asthma (Scheerens et al., 2012 Asthma Therapy: Novel Approaches: p. A6791; Gauvreau et al., 2014 Sci. Transl. Med. 6, 243ra85.), but failed to improve the clinical outcomes of patients with severe bronchial asthma (Harris et al., 2016 Respir. Res. 17: 29.). The epitope of Quilizumab can also serve as a potential immunogen, located within an 11-residue segment of CemX, SAQSQRAPDRV.

[0125] WO 2017 / 005851 A1 and Vigl et al. (Journal of Immunological Methods 449 (2017) 28–36) disclose peptides as active anti-EMPD immunogens in combination with suitable protein carriers located in the membrane proximal domain of EMPD. The disclosed sequences include

[0126] AVSVNPGLAGGSAQSQRAPDRVLCHSGQQQGLPRAAGGSVP,QQQGLPRAAGG,QQLGLPRAAGG,QQQGLPRAAEG,QQLGLPRAAEG,QQQGLPRAAG,QQLGLPRAAG,QQQGL PRAAE, QQQLGLPRAAE, HSGQQQGLPRAAGG, HSGQQLGLPRAAGG, HSGQQQGLPRAAEG, HSGQQLGLPRAAEG, QSQRAPDRVLCHSG, GSAQSQRAPDRVL, and WPGPPELDV.

[0127] Although in principle, the present invention can improve all proposed IgE-related disease vaccine peptides, selected epitopes were specifically evaluated for their suitability for this platform. For example, SeqID43 / 44 (QQQGLPRAAGG) was shown to be superior to KLH-based vaccines.

[0128] In view of these favorable properties of the conjugates of the present invention, the conjugates and vaccines of the present invention are particularly suitable for active anti-human epidermal growth factor receptor 2 (anti-Her2) vaccination for the treatment and prevention of Her2-positive tumor diseases. Amplification or overexpression of Her2 occurs in approximately 15-30% of breast cancers and 10-30% of gastric / gastroesophageal cancers, and can be used as a prognostic and predictive biomarker. Her2 overexpression is also seen 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. According to a preferred embodiment, the polypeptide derived from the Her2 protein is derived from natural human Her2, or is a mimetic with one or more amino acid exchanges to form a mimetic epitope of the corresponding natural sequence.

[0129] Dakappagari et al. (JBC (2005) 280, 1, 54–63) disclosed that the conformational epitope aa626–649 was synthesized co-linearly with a promiscuous TH epitope (amino acids 288–302) from the measles virus fusion protein MVF and cyclized via a disulfide bond. The peptide was formulated with the muramyl dipeptide adjuvant nor-MDP (N-acetylglucosamine-3-yl-acetyl-L-alanyl-D-isoglutamine) and emulsified in Montanide ISA 720. The vaccine was immunogenic and immunization with the vaccine reduced tumor burden in tumor models.

[0130] EP 1 912 680 B1 and Allen et al. (J Immunol 2007; 179: 472-482) disclose immunogens using three conformational peptide constructs (aa266-296 (LHCPALVTYNTDTFESMPNPEGRYTFGASCV), aa298-333 (ACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEK) and aa315-333 (CPLHNQEVTAEDGTQRCEK) to mimic the region of the receptor dimerization loop. The candidate vaccine also contains the MVF T cell epitope (aa 288–302)KLLSLIKGVIVHRLEGVE and GPSL-linker. All peptides elicited a high anti-Her2 immune response, and the construct using peptide aa266–296 was equivalent to Herceptin. The aa266-296 peptide of the Her2 sequence (accession number P04626):

[0131]

[0132] As a vaccine, it significantly reduced tumor incidence in two transplantable tumor models and significantly reduced tumor development in two transgenic mouse tumor models.

[0133] Garret et al. (J Immunol 2007; 178: 7120-7131) disclosed that Her2 peptides aa563-598, aa585-598, aa597-626 and aa613-626 were synthesized in line with a promiscuous Th epitope from the measles virus fusion protein (aa 288-302) as immunogens and used in combination with Montanide ISA 720. The vaccine was immunogenic, and immunization with the vaccine carrying the aa597-626 epitope significantly reduced tumor burden in the tumor model.

[0134] Jasinska et al. (Int. J. Cancer: 107, 976-983 (2003)) disclosed 7 peptides of the Her2 extracellular domain as potential antigens for cancer immunotherapy: P1 aa115–132AVLDNGDPLNNTTPVTGA, P2aa149–162LKGGVLIQRNPQLC, P3 aa274–295YNTDTFESMPNPEGRYTFGAS, P4aa378–398PESFDGDPASNTAPLQPEQLQ, P5 aa489–504PHQALLHTANRPEDE, P6aa544–560CRVLQGLPREYVNARHC, P7 aa610–623YMPIWKFPDEEGAC, which were conjugated to tetanus toxoid and used Gerbu adjuvant. The induced humoral immune response had anti-tumor activity in animal models. Similarly, Wagner et al. (2007 Breast Cancer Res Treat. 2007; 106: 29-38) disclosed peptide immunogens for immunization studies using individual peptides P4 (aa378-394: PESFDGDPASNTAPLQPC), P6 (aa545-560: RVLQGLPREYVNARHC) and P7 (aa610-623: YMPIWKFPDEEGAC) coupled to tetanus toxoid and adjuvanted with Gerbu. Vaccination with or without IL12 resulted in anti-tumor efficacy in preclinical models. Tobias et al. 2017 (BMC Cancer (2017) 17:118) disclosed peptide immunogens for immunization studies, using single peptides P4 (aa378–394: PESFDGDPASNTAPLQP), P6 (aa545–560: RVLQGLPREYVNARHC) and P7 (aa610–623: YMPIWKFPDEEGAC) to combine into heteropeptides P467 (PESFDGDPASNTAPLQPRVLQGLPREYVNARHSLPYMPIWKFPDEEGAC) and P647 (RVLQGLPREYVNARHSPESFDGDPASNTAPLQPYMPIWKFPDEEGAC). The cysteine ​​(C) of P6 was replaced by "SLP" or "S", respectively. Both constructs were conjugated to either virions or diphtheria toxoid CRM197 (CRM) and combined with Montanide or sodium hydroxide (Alum) as adjuvants to induce antibodies that exhibited antitumor properties.

[0135] Riemer et al. (J Immunol 2004; 173: 394-401) reported the generation of peptide mimetics of the epitope recognized by trastuzumab on Her-2 / neu using a restricted 10-mer phage display library. The peptide mimetics were coupled to the immunogenic carrier tetanus toxoid (TT) and adjuvanted with aluminum hydroxide. The sequences contained: C-QMWAPQWGPD-C, C-KLYWADGELT-C, C-VDYHYEGTIT-C, C-QMWAPQWGPD-C, C-KLYWADGELT-C, C-KLYWADGEFT-C, C-VDYHYEGTIT-C, C-VDYHYEGAIT-C. Similarly, Singer et al. (ONCOIMMUNOLOGY 2016, 5(7), e1171446) disclosed mimotopes of trastuzumab epitopes derived from an AAV-mimotope library platform. The mimotope sequences tested included RLVPVGLERGTVDWV, TRWQKGLALGSGDMA, QVSHWVSGLAEGSFG, LSHTSGRVEGSVSLL, LDSTSLAGGPYEAIE, HVVMNWMREEFVEEF, SWASGMAVGSVSFEE. QVSHWVSGLAEGSFG and LSHTSGRVEGSVSLL were shown to be immunogenic and effective in tumor models.

[0136] Miyako et al. (ANTICANCER RESEARCH 31:3361-3368 (2011)) discloses peptides, inter alia, from the Her-2 / neu extracellular domain (aa167-175), in the form of Her-2 / neu-associated multiple antigenic peptides (MAPs). The Her-2 / neu peptide contains epitopes for CD4+ and CD8+ T cells, resulting in inhibition of the growth of tumor cells expressing Her-2 / neu. The disclosed sequences include:

[0137] Peptide sequence (B; tert-butyloxycarbonyl residue (Boc)).

[0138] N:143-162(RSLTEILKGGVLIQRNPQLC-BBB)8-K4K2KB

[0139] N:153-172(VLIQRNPQLCYQDTILWKDI-BBB)8-K4K2KB

[0140] N:163-182(YQDTILWKDIFHKNNQLALT-BBB)8-K4K2KB

[0141] N:173-192(FHKNNQLALTLIDTNRSRAC-BBB)8-K4K2KB

[0142] N:183-202(LIDTNRSRACHPCSMPCKGS-BBB)8-K4K2KB

[0143] N:193-212(HPCSMPCKGSRCWGESSEDC-BBB)8-K4K2KB

[0144] N:203-222(RCWGESSEDCQSLTRTVCAG-BBB)8-K4K2KB

[0145] N:213-232(QSLTRTVCAGGCARCKGPLP-BBB)8-K4K2KB

[0146] N:223-242(GCARCKGPLPTDCCHEQCAA-BBB)8-K4K2KB

[0147] N:233-252(TDCCHEQCAAGCTGPKHSDC-BBB)8-K4K2KB

[0148] N:243-263(GCTGPKHSDCLACLHFNHSG-BBB)8-K4K2KB

[0149] N:253-272(LACLHFNHSGICELHCPALV-BBB)8-K4K2KB

[0150] N:263-282(ICELHCPALVTYNTDTFESM-BBB)8-K4K2KB

[0151] N:273-292(TYNTDTFESMPNPEGRYTFG-BBB)8-K4K2KB

[0152] N:283-302(PNPEGRYTFGASCVTACPYN-BBB)8-K4K2KB

[0153] N:292-310(GASCVTACPYNYLSTDVGS-BBB)8-K4K2KB

[0154] N:300-321(PYNYLSTDVGSCTLVCPLHNQE-BBB)8-K4K2KB

[0155] N:312-330(TLVCPLHNQEVTAEDGTQR-BBB)8-K4K2KB

[0156] N:322-341(VTAEDGTQRCEKCSKPCARV-BBB)8-K4K2KB

[0157] N:332-351(EKCSKPCARVCYGLGMEHLR-BBB)8-K4K2KB

[0158] N:343-361(YGLGMEHLREVRAVTSANI-BBB)8-K4K2KB

[0159] N:352-370(EVRAVTSANIQEFAGCKKI-BBB)8-K4K2KB

[0160] Humoral immune responses were induced, tumor growth was inhibited in immunized mice, and tumor-infiltrating lymphocytes contained more CD8+ T cells and secreted large amounts of interleukin-2 after peptide restimulation.

[0161] Henle et al. (J Immunol. 2013 Jan 1; 190(1): 479-488) disclosed peptide epitopes derived from Her2 that generate cross-reactive T cells. For the HER-2 / neu HLA-A2 binding peptide aa369-377 (KIFGSLAFL), cytotoxic T lymphocytes (CTLs) specific for this epitope have been shown to 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. Among these sequences, sequence p373–382 (SLAFLPESFD) bound more strongly to HLA-A2 than p369–377 and was identified as a potential epitope for vaccination.

[0162] Kaumaya et al. (ONCOIMMUNOLOGY 2020, 9 (1) e1818437) disclosed a Her2 targeted vaccine (aa266-296 and aa597-626 combined with measles virus fusion peptide (MVF) amino acids 288-302 via four amino acid residues (GPSL) and emulsified in Montanide ISA 720VG) and a novel PD1 immune checkpoint targeted vaccine (PD-1 B cell peptide epitope (aa92-110; GAISLAPKAQIKESLRAEL) combined with viral fusion peptide (MVF) amino acids 288-302 via four amino acid residues (GPSL) and emulsified in Montanide ISA 720VG) for combined treatment of Her2-positive diseases. Therefore, providing a combination of anti-tumor disease vaccines, especially a combination of cancer target-specific vaccines and immune checkpoint targeted vaccines is also a preferred embodiment.

[0163] Although in principle, the present invention can improve all proposed Her2-related disease vaccine peptides, selected epitopes will be specifically evaluated for their suitability with this platform. For example, SeqID No47 / 48 (aa610-623: YMPIWKFPDEEGAC) was shown to be superior to CRM-based vaccines.

[0164] In view of these favorable properties of the conjugates of the present invention, the conjugates and vaccines of the present invention are particularly suitable for personalized neoantigen-specific therapy, preferably NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-C1, MAGE-C2, MAGE-C3, Survivin, gp100, tyrosinase, CT7, WT1, PSA, PSCA, PSMA, STEAP1, PAP, MUC1, 5T4, KRAS or Her2.

[0165] In view of these favorable properties of the conjugates of the present invention, the conjugates and vaccines of the present invention are particularly suitable for active anti-immune checkpoint vaccination to control the cancer microenvironment, treat and prevent tumor diseases, and treat and prevent T cell dysfunction in cancer / tumor diseases (e.g., avoiding exhaustion of CD8 T cells infiltrating cancer tissues) and chronic degenerative diseases, including diseases with reduced T cell activity, such as Parkinson's disease.

[0166] It is widely accepted that the T cell compartment is differentially altered in patients with Parkinson's disease compared to healthy controls (e.g., Bas et al., J Neuroimmunol 2001;113:146-52 or Gruden et al., J Neuroimmunol 2011;233:221-7). Such phenotypic changes in T cells in PD include: decreased absolute lymphocyte counts, decreased absolute and relative total T cell counts, decreased absolute and relative CD4+ lymphocyte counts, and sometimes CD8+ lymphocyte counts, increased Th1 / Th2 and Th17 / Treg ratios, and increased expression of inflammatory cytokines. However, most of these changes are also seen during healthy aging, making it difficult to discern the impact of a disease such as PD, which has a wide age range of onset (approximately 30-90 years) and a variable rate of progression. With regard to absolute cell counts, there seems to be consensus that there is a net decrease in CD3+CD4+ T cells in patients with PD. This CD4 decrease is supported by the altered CD4:CD8 ratio described above.

[0167] Along these lines, for example, Bhatia et al. (J Neuroinflammation (2021) 18:250) showed that PD patients have an overall reduction in the total number of CD3+ T cells, which correlates with disease severity (e.g., as measured by H+Y stage). This suggests that systemic T cell dysfunction continues to worsen as the disease progresses, which may reflect the combined effects of ongoing inflammation, drug treatment, and lifestyle changes. In addition, Lindestam Arlehamn et al. (2020) showed that the highest T cell activity can be detected in PD patients in the prodromal or early clinical stages (duration <10 years, H+Y stage 0-2).

[0168] Therefore, a preferred embodiment of the present invention is to provide for increasing or maintaining the number of PD patient T cells (especially the number of T effector cells) and the treatment of T cell function. This preferably includes a combination of multiple immune checkpoint inhibitors or multiple vaccines using anti-immune checkpoint inhibitor epitopes to induce the immune response of anti-checkpoint inhibitors and the target-specific vaccine combination of the present invention to increase or maintain the number of PD patient T cells (especially the number of T effector cells) and T cell function.

[0169] Patients receiving / suitable for treatment are characterized by an overall decrease in CD3+ cells, especially a decrease in CD3+CD4+ cells, which is a typical feature of PD patients at all stages of the disease. For this combination, the preferred disease stages defining the suitable patient groups are H+Y stages 1-4, preferably H+Y1-3, and most preferably H+Y 2-3, respectively.

[0170] Examples of such immune checkpoint targeted vaccines are vaccines that provide epitopes directed against: cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, accession number P16410) and programmed cell death protein 1 (PD-1, accession number Q15116) or their 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); having the following amino acid sequence:

[0171] Human CTLA4:>sp|P16410|CTLA4_Uniprot

[0172]

[0173] Human PD1:>sp|Q15116|PDCD1_Uniprot

[0174]

[0175] Human PD1-L1>sp|Q9NZQ7|PD1L1_Uniprot

[0176]

[0177] Human B7-H3–CD276>sp|Q5ZPR3|CD276_Uniprot

[0178]

[0179] Human B7-H4–VTCN1>sp|Q7Z7D3|VTCN1_Uniprot

[0180]

[0181] Human LAG3:>sp|P18627|LAG3_Uniprot

[0182]

[0183] Human Tim3:>sp|Q8TDQ0|HAVR2_Uniprot

[0184]

[0185] Antibodies targeting CTLA-4 inhibit immune responses in multiple ways, including hindering the activation of autoreactive T cells at the proximal steps of the immune response (usually in the lymph nodes). In contrast, the PD-1 pathway regulates T cells at the later stages of the immune response (usually in peripheral tissues). Therefore, there are currently two main intervention directions in the clinic, namely manipulating immune checkpoints by targeting CTLA-4 or PD-1 / PD-L1: anti-CTLA-4 is involved in the lymphocyte proliferation process after activation of antigen-specific T cell receptors, while anti-PD-1 / PD-L1 acts mainly on peripheral tissues during the effector step. However, CTLA-4 is also expressed on regulatory T lymphocytes and is therefore involved in the inhibition of peripheral T cell proliferation.

[0186] Nowadays, several immune checkpoint blockade antibodies such as ipilimumab (anti-CTLA-4 antibody), nivolumab and pembrolizumab (all anti-PD-1 antibodies), avelumab (anti-PD-L1 antibody) or atezolizumab and durvalumab (both anti-B7-H1 / PD-L1 antibodies) can induce high anti-cancer immunity with fewer side effects.

[0187] According to a preferred embodiment, the CTLA4 protein-derived polypeptide is derived from native human CTLA4, or is a mimetic having one or more amino acid exchanges forming a mimotope of the corresponding native sequence.

[0188] 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, forming a mimotope of the corresponding native sequence. The protein sequence corresponds to the extracellular domain of mouse PD1 (Q02242; Uniprot) and human PD1 (Q15116; Uniprot).

[0189] According to a preferred embodiment, the PD-L1 protein-derived polypeptide is derived from natural human PD-L1, or is a mimetic with one or more aa exchanges, thereby forming a mimotope of the corresponding natural sequence.

[0190] 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 to reduce tumor growth in a syngeneic BALB / c model with CT26 colon cancer cells. In addition, the combination of the disclosed PD1 epitope vaccine and HER-2 peptide vaccine showed an enhanced inhibitory effect on colon cancer tumor growth.

[0191] Tobias et al. (Front Immunol. 2020; 11: 895.) disclosed peptides / simulated epitopes from mouse and human PD-1 (= epitopes of anti-human PD1 mAb Nivolumab and anti-mouse mAb clone 29F.1A12). These peptides contain the human PD1-derived sequences PGWFLDSPDRPWNPP, FLDSPDRPWNPPTFS, and SPDRPWNPPTFSPA, corresponding to aa21-35, aa24-38, and aa27-41 positions of human PD1, respectively called JT-N1, JT-N2, and JT-N3. In addition, the simulated epitope of mouse PD1 contains ISLHPKAKIEESPGA (JT-mPD1), corresponding to amino acid residues aa126-140 of mPD1. Studies have shown that the anti-tumor effect of the simulated epitope JT-mPD1 is associated with a significant reduction in tumor proliferation and an increase in apoptosis rate in the Her-2 / neu-expressing homologous tumor mouse model used. Furthermore, the study showed that when the Her-2 / neu vaccine was combined with JT-mPD1, its anti-tumor effect was enhanced.

[0192] Chen et al. (Cancers 2019, 11, 1909) disclosed PDL1-Vax, a fusion protein composed of human PD-L1 (aa19-220 of human PD-L1) connected with T helper epitope sequence and human IgG1 Fc sequence as a novel PD-L1 targeted vaccine. Jorgensen et al. (Front Immunol. 2020; 11: 595035.) disclosed a 19-amino acid peptide (FMTYWHLLNAFTVTVPKDL) derived from the human PD-L1 signal peptide as a novel PD-L1 targeted vaccine. Tian et al. (Cancer Letters 476 (2020) 170-182) disclosed the fusion of a truncated mouse PDL1 extracellular domain (aa19-239) with the NitraTh epitope. hPDL1-NitraTh was also constructed as a novel PD-L1 targeted vaccine by fusing a truncated human PDL1 extracellular domain (aa19-238) with the NitraTh epitope.

[0193] These anti-immune checkpoint vaccines may be very effective when used alone or in combination with existing peptide vaccines. Therefore, a preferred embodiment is to provide a combination of anti-immune checkpoint vaccines and existing peptide vaccines to treat tumors or degenerative diseases (such as Parkinson's disease).

[0194] Although in principle the present invention can improve all proposed PD1 and PD-L1-related vaccine peptides, selected epitopes have been specifically evaluated for their suitability with this platform. For example, SeqID No 49 / 50 (GAISLAPKAQIKESLRAEL) was shown to be superior to KLH-based vaccines.

[0195] In view of these favorable properties of the conjugates of the present invention, the conjugates and vaccines of the present invention are particularly suitable for active anti-Aβ immunotherapy for the prevention, treatment and diagnosis of diseases associated with the formation and / or aggregation of β-amyloid protein. 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), Lewy body dementia and Down syndrome dementia, retinal ganglion cell degeneration in glaucoma, inclusion body myositis / myopathy.

[0196] Aβ peptides exist in multiple forms, including full-length Aβ1-42 and Aβ1-40, various modified forms of Aβ, including truncated, N-terminally truncated or C-terminally truncated, nitrated, acetylated and N-truncated species, pyroglutamate Aβ3-40 / 42 (i.e., AβpE3-40 and AβpE3-42) and Aβ4-42, which appear to play important roles in neurodegeneration.

[0197] According to a preferred embodiment, the Aβ peptide-derived polypeptide is selected from natural human Aβ1-40 and / or Aβ1-42 having the following amino acid sequence:

[0198] Aβ1-40:DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV

[0199] Aβ1-42:DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA

[0200] or a polypeptide comprising or consisting of amino acid residues of human Aβ1-40 and / or Aβ1-42, including truncated, particularly N-terminally truncated, C-terminally truncated, post-translationally modified, nitrated, glycosylated, acetylated, ubiquitinated peptide amino acids, or amino acids or peptides having a pyroglutamic acid residue at aa3 or aa11, including Aβaa1-6, 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 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, aa37-42.

[0201] According to a preferred embodiment, the polypeptide derived from Aβ1-40 or Aβ1-42 is selected from the mimetics of the above-mentioned Aβ-derived polypeptides, including mimetic epitopes and peptides containing amino acid substitutions that mimic pyroglutamylated amino acids. Schenk et al. (Nature. 1999 July 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 Aβ1-6 peptide epitopes coupled to CRM197 and adjuvanted with QS21, and Wiesner et al. (J Neurosci. 2011 June 22; 31(25): 9323-31) disclosed Aβ1-6 peptides coupled to Qβ virus-like particles as effective immunotherapeutic agents.

[0202] Wang et al. (Alzheimer's & Dementia: Translational Research & Clinical Interventions 3 (2017) 262-272) and US2018 / 0244739 A1 disclosed Aβ1-42 peptide immunogens, especially UB311, which contains two Aβ immunogens, namely cationic Aβ1-14-εK-KKK-MvF5Th[ISITEIKGVIVHRIETILF] and Aβ1-14-εK-HBsAg3 Th[KKKIITITRIITIITID] peptides, in equal molar ratios; they are mixed with polyanionic CpG oligodeoxynucleotides (ODN) to form a stable immunostimulatory complex of micron-sized particles, and aluminum mineral salts (Adju-Phos) are added to the final formulation.

[0203] Illouz et al. (Vaccine, Vol. 39, No. 34, August 9, 2021, pp. 4817-4829) disclosed Aβ1-11 fused to HBsAg as a vaccine for elderly mice.

[0204] 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) disclosed a vaccine comprising two foreign Th cell epitopes from tetanus toxin, P30 and P2, and three copies of the Aβ1-12 B cell epitope, adjuvanted with QuilA. Similarly, Davtyan H et al. (Alzheimer's & Dementia 10 (2014) 271–283) disclosed a DNA-based vaccine based on a protein coding region, consisting of an immunoglobulin (Ig) kappa chain signal sequence, 3 copies of Aβ1-11 B cell epitopes, 1 synthetic peptide (PADRE) and a string of 8 non-self promiscuous Th epitopes from tetanus toxin (TT) (P2, P21, P23, P30 and P32), hepatitis B virus (HBsAg, HBVnc) and influenza (MT), or also including 3 additional Th epitopes from TT (P7 (NYSLDKIIVDYNLQSKITLP); P17 (LINSTKIYSYFPSVISKVNQ); and P28 (LEYIPEITLPVIAALSIAES)).

[0205] Petrushina et al. (Journal of Neuroinflammation 2008, 5:42) disclosed Aβ1-28 with an N-terminal linker (n-CAGA) coupled to bromoacetylated Saccharomyces cerevisiae mannan as a potential vaccine, but with severe side effects.

[0206] US2011 / 0002949 A1 discloses a multivalent vaccine construct (Aβ3-10 / Aβ21-28) (MVC) and a monovalent vaccine construct Aβ1-8 (MoVC1-8) coupled to a carrier (KLH and administered with the saponin-based adjuvant ISCOMATRIX.

[0207] 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) disclosed Aβ1-15 as an array of Aβ1-15 sequences sandwiched between palmitoylated lysines at both ends, which are anchored on the surface of liposomes so that the peptide adopts an aggregated β-sheet structure, forming a conformational epitope.

[0208] Ding et al. (Neuroscience Letters, Vol. 634, November 10, 2016, pp. 1-6) disclosed peptides formed by coupling Aβ3-10 to the immunogenic carrier protein keyhole limpet hemocyanin (KLH) or by linearly concatenating five Aβ3-10 epitopes.

[0209] Bakrania et al. (Mol Psychiatry (2021). https: / / doi.org / 10.1038 / s41380-021- 01385-7 ) discloses 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 protein emulsified in incomplete Freund's adjuvant (IFA) as a suitable immunogen.

[0210] Lacosta et al. (Alzheimers Res Ther. 2018 Jan 29; 10(1): 12) disclosed an Aβ peptide immunogen comprising multiple repeats of a short C-terminal fragment of Aβ1-40. To generate an immune response, these repeats were coupled to a keyhole limpet hemocyanin (KHL) carrier protein and supplemented with an adjuvant, aluminum hydroxide.

[0211] Axelsen et al. (Vaccine, Vol. 29, No. 17, April 12, 2011, pp. 3260-3269) disclosed Aβ37–42 conjugated to keyhole limpet hemocyanin.

[0212] WO 2004 / 062556 A2, WO 2006 / 005707 A2, WO 2009 / 149486 A2 and WO 2009 / 149485 A2 disclose mimotopes of Aβ epitopes. The results show that these mimotopes can induce the formation of antibodies in vivo against untruncated Aβ1-40 / 42 and N-terminal truncated forms AβpE3-40 / 42, Aβ3-40 / 42, Aβ11-40 / 42, AβpE11-40 / 42 and Aβ14-40 / 42, respectively.

[0213] According to a preferred embodiment, the Aβ peptide-derived polypeptide is selected from:

[0214] Natural Aβ peptide

[0215]

[0216] Mimotope Aβ peptide

[0217]

[0218] These anti-Aβ vaccines are very effective when used alone or in combination with existing peptide vaccines against other pathological molecules involved in β-amyloidosis, tauopathy or synucleinopathy, especially mixed pathologies (i.e., the presence of Aβ pathology and Tau pathology and / or aSyn pathology). Therefore, a preferred embodiment is to provide a combination of an anti-Aβ vaccine with an anti-Tau and / or anti-ASyn peptide vaccine to treat degenerative diseases such as Alzheimer's disease, Down syndrome dementia, Lewy body dementia, Parkinson's disease dementia, Parkinson's disease or Tauopathies.

[0219] Although in principle, the present invention can improve all proposed Aβ and Aβ-related vaccine peptides, selected epitopes were specifically evaluated for their suitability with the present platform. For example, SeqID32 / 33 (AβpE3-8; pEFRHDS) were shown to be superior to KLH-based vaccines, while SeqID10 (Aβ1-6; DAEFRH) was shown to be immunogenic when coupled to different CLECs.

[0220] In view of these favorable properties of the conjugate of the present invention, the conjugate and vaccine of the present invention are particularly suitable for active anti-IL31 vaccination to treat and prevent IL31-related diseases and autoimmune inflammatory diseases.

[0221] IL31-associated diseases include pruritic allergic diseases, pruritic inflammatory diseases, and pruritic autoimmune diseases 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 origin (CPUO), primary localized cutaneous amyloidosis (PLCA), mastocytosis, chronic spontaneous urticaria, bullous pemphigoid, dermatitis herpetiformis, and other skin diseases including lichen planus, cutaneous amyloidosis, stasis dermatitis, scleroderma, pruritic and non-pruritic disorders associated with wound healing such as allergic asthma, allergic rhinitis, inflammatory bowel disease (IBD), osteoporosis, follicular lymphoma, Hodgkin lymphoma, and chronic myeloid leukemia.

[0222] According to a preferred embodiment, a single IL31 epitope can be used to trigger an immune response against different domains of IL31. In another preferred embodiment, a combination of multiple IL31 epitopes can be used to trigger an immune response against different domains of IL31 (particularly involving helix C or A, and also involving helix D), thereby preventing the binding of IL31 to IL31 receptor, interleukin 31 receptor alpha (IL-31RA) and oncostatin M receptor (OSMR).

[0223] The efficacy of anti-IL31 vaccine is very significant when used alone or in combination with peptide vaccines targeting other pathological molecules involved in allergic diseases, inflammatory diseases and autoimmune diseases that cause itching. Therefore, a preferred embodiment is to provide a combination of anti-IL31 vaccine and anti-IL4 and / or anti-IL13 peptide vaccines to treat allergic diseases, inflammatory diseases and autoimmune diseases that cause itching.

[0224] According to a preferred embodiment, the polypeptide derived from the IL31 protein is a fragment of the IL-31 protein, and / or is preferably selected from natural human IL31 (Genbank: AAS86448.1; MASHSGPSTSVLFLFCCLGGWLASHTLPVRLLRPSDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQ NYTLPCLSPDAQPPNNIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHE CKRFILTISQQFSECMDLALKSLTSGAQQATT); natural canine IL31 (Genbank: BAH97742.1; MLSHTGPSRFALFLLCSMETLLSSHMAPTHQLPPSDVRKIILELQPLSRGLLEDYQKKETGVPESNRTLLLCLTSDSQPPRLNSSAILPYFRAIRP LSDKNIIDKIIEQLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVFKSLNSGPQ); natural cat IL31(UNIPROT:A0A2I2UKP7; MLSHAGPARFALFLLCCMETLLPSHMAPAHRLQPSDVRKIILELRPMSKGLLQDYVSKEIGLPESN HSSLPCLSSDSQLPHINGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKFQREPEAKVSMPADNFER or natural horse IL31 (UNIPROT F7AHG9MVSHIGSTRFALFLLCCLGTLMFSHTGPIYQLQPKEIQAIIVELQNLSKKLLDDYVSALETSILSC FFKTDLPSCFTSDSQAPGNINSSAILPYFKAISPSLNNDKSLYIIEQLDKLNFQNAPETEVSMPTD NFERKRFILTILRWFSNCLEHRAQ) or any peptide sequence having at least 70, 75, 80, 85, 90 or 95% sequence identity to any of the foregoing, or any peptide sequence that differs from the naturally occurring sequence by multiple point mutations of surface exposed amino acids, wherein the number of point mutations is 1, 2 or 3.

[0225] According to a preferred embodiment, the polypeptide derived from IL31 protein is selected from the mimetics of the above-mentioned IL31 derived polypeptides, including mimotopes and peptides containing amino acid substitutions.

[0226] Further preferred target sequences include (presented as linear peptides or constrained peptides, such as cyclized peptides or peptides linked by suitable aa linkers such as ggsgg or the like):

[0227] For human IL31: peptides derived from sequences aa98-145, aa87-150, aa105-113, aa85-115, aa84-114, aa86-117, aa87-116; or fragments thereof and 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,I DEIIEHLDKLI,IIEHLDKLIFQD,HLDKLIFQDAPE,KLIFQDAPETNI,FQDAPETNISVP,APETNISVPTDT,TNISVPTDTHEC,SVPTDTHESKRF,TDTHECKRFILT,TDTHESKRFILT,TDTHERKRFILT,HECKRFILTISQ,HESKRFILTIS Q,HERKRFILTISQ,KRFILTISQQFS,ILTISQQFSECM,ILTISQQFSESM,ILTISQQFSERM,ISQQFSECMDLA,ISQQFSESMDLA,ISQQFSERMDLA,QFSECMDLALKS,QFSESMDLALKS,QFSERMDLALKS,SKMLLKDVEEEKG,EELQSLS K,KGVLVS,SPAIRAYLKTIRQLDNKSVIDEIIEHLDKLI,DEIIEHLDK,SVIDEIIEHLDKLI,SPAIRAYLKTIRQLDNKSVI,TDTHECKRF,HECKRFILT,HERKRFILT,HESKRFILT,SVPTDTHECKRF,SVPTDTHESKRF,and SVPTDTHERKRF

[0228] 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, aa 124-135 or fragments thereof and the peptides: SDVRKIILELQPLSRGLLEDYQKKETGV, DVRKIILELQPLSRGLLEDY ELQPLSRLSDKNIIDKIIEQLDKLKFQHE, LSDKNIIDKIIEQLDKLKFQ, KLKFQHE, LSDKNI, LDKL, LSDKN, ADTFECKSFILTILQQFSACLESVFKS and ADNFERKNF

[0229] For feline IL31: aa124-135 of the feline IL-31 sequence and peptides SDVRKIILELRPMSKGLLQDYVSKEIGL and DVRKIILELRPMSKGLLQDY, LSDKNTIDKIIEQLDKLKFQRE, ADNFERKNFILAVLQQFSACLEHVLQS and ADNFERKNF

[0230] For horse IL31: aa118-129 of horse IL-31 sequence and peptides: LQPKEIQAIIVELQNLSKKLLDDY, EIQAIIVELQNLSKKLLDDY, SLLNDKSLYIIEQLDKLNFQ, and TDNFERKRFILTILRWFSNCLEHRAQ

[0231] For mimotopes:

[0232] The canine IL-31 mimotope comprises the amino acid sequence SVPADTFERKSF, SVPADTFERKSF, NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF, APTHQLPPSDVRKIILELQPLSRG, TGVPES or variants thereof.

[0233] The feline IL-31 mimotope comprises the amino acid sequence SMPADNFERKNF, NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF, APAHRLQPSDIRKIILELRPMSKG, IGLPES or variants thereof.

[0234] The horse IL-31 mimotope comprises the amino acid sequence SMPTDNFERKRF, NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF, GPIYQLQPKEIQAIIVELQNLSKK, KGVQKF or variants thereof.

[0235] The human IL-31 mimetic epitope comprises the amino acid sequence SVPTDTHECKRF, SVPTDTHERKRF, HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF, LPVRLLRPSDDVQKIVEELQSLSKM, KGVLVS or variants thereof that retain anti-IL-31 binding activity.

[0236] According to a preferred embodiment, the IL31 epitope can be a conformational epitope, comprising at least two amino acids or amino acid sequences that are spatially distinct from each other but in close proximity to form respective paratopes. The paratope is usually bound to an anti-IL31 antibody, for example, a polyclonal anti-IL31 antibody that specifically recognizes natural IL31 obtained after vaccination of a mammal.

[0237] IL31 is a protein with a 4-helix bundle structure, such as that found in the gp30 / IL-6 cytokine family. The receptor for IL-31 is a heterodimer of interleukin 31 receptor α (IL-31RA, also known as GPL or gp130-like receptor) and oncostatin M receptor (OSMR). Both structures of this heterodimer are called IL-31 receptor or IL-31 co-receptor. Saux et al. described the putative interaction site between human IL-31 and its receptor (J Biol Chem 2010, 285, 3470-34). Targeting IL31 can be achieved by antibodies targeting IL-31 and / or its receptor. The development of dedicated monoclonal antibodies specifically targeting IL31 can allow clinical and preclinical validation of this targeting strategy in vitro and in vivo (Front Med (Lausanne. 2021-2-12; 8: 638325)).

[0238] BMS-981164 is an anti-IL-31 monoclonal antibody that targets circulating IL-31 and was developed by Bristol-Myers Squibb. A two-part Phase I single-dose, dose-escalation study was conducted between 2012 and 2015 to explore the safety and pharmacokinetic profile of BMS-981164 (NCT01614756). The study design was a randomized, double-blind, placebo-controlled, and the drug was administered in the form of 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 had to have at least moderate atopic dermatitis (rated as _3 on a scale of 0 to 5 by the physician's global assessment) and pruritus severity of at least 7 out of 10 on a visual analog scale. No results have been published so far from this study. As of 2016, BMS-981164 is no longer in Bristol-Myers Squibb's pipeline, and no new trials have been announced.

[0239] US 8,790,651 B2 describes monoclonal antibodies that bind to IL-31 for use in treating immune diseases such as atopic dermatitis. There is a monoclonal antibody (Lokivetmab, Zoetis) on the market that targets canine IL-31 for the treatment of canine atopic dermatitis. Lokivetmab is said to interfere with the binding of IL-31 to the co-receptor GPL.

[0240] EP 4 019 546 A1 discloses monospecific and multispecific antibodies whose variable regions block the binding of IL-31 to the IL-31 receptor alpha (IL-31RA) / oncostatin M receptor (OSMR) complex (IL-31RA / OS-MR complex).

[0241] Bachmann et al. disclosed a vaccine using whole canine IL-31 coupled with virus-like particles to immunize dogs for the treatment of atopic dermatitis. (Bachmann, MF; 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, US11,324,836B2, US11,207,390B2 and US10,556,003 and Fettelschloss et al. (doi: 10.1111 / eve.13408) disclose VLP-based immunogens for targeting IL31 and IL31-related diseases from different species (including human, canine, horse or porcine IL31). The characteristics of these VLP-based immunogens are anti-IL31 immunogens with full-length, natural and full-length modified IL31-derived sequences, respectively.

[0242] US2021 / 0079054A1 discloses a peptide-based immunogen targeting IL31 based on the UbiTh platform technology for treating and / or preventing pruritus or allergies, such as atopic dermatitis. Along these lines, B cell epitope-based immunogens derived from canine IL31 (Genbank: BAH97742.1; MLSHTGPSRFALFLLCSMETLLSSHMAPTHQLPPSDVRKIILELQPLSRGLLEDYQKKETGVPESNRTLLCLTSDSQPPRLNSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVFKSLNSGPQ) and human IL31 (Genbank: AAS86448.1; MASHSGPSTSVLFLFCCLGGWLASHTLPVRLLRPSDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQNYTLPCLSPDAQPPNNIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHE CKRFILTISQQFSECMDLALKSLTSGAQQATT) was presented, including:

[0243] 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 sequence aa98-145, aa87-150, aa105-113, aa85-115, aa84-114, aa86-117, aa87-116, with modifications where appropriate, e.g., serine and cysteine ​​substitutions. B cell epitopes are linear or constrained and fused to promiscuous T helper epitopes and formulated in the presence of adjuvants (eg, different CpG molecules, Alhydrogel, AdjuPhos, Montanides such as ISA50V2, ISA51, ISA720).

[0244] US2019 / 0282704A1 discloses a vaccine composition for immunizing and / or protecting a mammal from an IL-31-mediated disease, wherein the composition comprises a carrier polypeptide (e.g., CRM197) and at least one mimetic epitope of an IL31-derived epitope (selected from a cat IL-31 mimetic epitope, a canine IL-31 mimetic epitope, a horse IL-31 mimetic epitope, or a human IL-31 mimetic epitope); and an adjuvant. The mimetic epitope may be linear or constrained (e.g., cyclized).

[0245] The canine IL-31 mimotope comprises the amino acid sequence SVPADTFERKSF, SVPADTFERKSF, NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF, APTHQLPPSDVRKIILELQPLSRG, TGVPES or variants thereof.

[0246] The feline IL-31 mimotope comprises the amino acid sequence SMPADNFERKNF, NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF, APAHRLQPSDIRKIILELRPMSKG, IGLPES or variants thereof.

[0247] The horse IL-31 mimotope comprises the amino acid sequence SMPTDNFERKRF, NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF, GPIYQLQPKEIQAIIVELQNLS KK, KGVQKF or variants thereof.

[0248] The human IL-31 mimetic epitope comprises the amino acid sequence SVPTDTHECKRF, SVPTDTHERKRF, HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF, LPVRLLRPSDDVQKIVEELQSLSKM, KGVLVS or variants thereof that retain anti-IL-31 binding activity.

[0249] In addition, the region between approximately aa124-135 of the cat IL-31 sequence represented by (UNIPROT:A0A2I2UKP7); the region between approximately aa124-135 of the canine IL-31 sequence represented by (Genbank:BAH97742.1); and the region between approximately aa118-129 of the horse IL-31 sequence represented by (UNIPROT F7AHG9) are disclosed as suitable epitopes.

[0250] WO 2019 / 086694 A1 discloses a peptide-based immunogen targeting IL31, which is achieved by an IL31 antigen comprising an unpackaged IL31 helical peptide, or an epitope from canine, human, cat, horse, pig, cattle or camelid IL31. The antigen is coupled to a conventional carrier molecule (e.g., KLH) and adjuvanted with Imject Alum, or may be coupled to an anti-CD32 scFv construct that may contain a TLR9 agonist CpG or a TLR7 / 8 agonist imidazoquinoline. Specifically, the IL31 peptide comprises or consists of any of the following amino acid sequences helix A:

[0251] People: SDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQNYTL; and DVQKIVEELQSLSKMLLKDV,EELQSLSK and DVQK

[0252] Dog: SDVRKIILELQPLSRGLLEDYQKKETGV, and DVRKIILELQPLSRGLLEDY and ELQPLSR

[0253] Cat: SDVRKIILELRPMSKGLLQDYVSKEIGL and DVRKIILELRPMSKGLLQDY

[0254] Horse: LQPKEIQAIIVELQNLSKKLLDDY and EIQAIIVELQNLSKKLLDDY

[0255] Helix C

[0256] People:LDNKSVIDEIIEHLDKLIFQDA; andDEIIEH

[0257] Dog:LSDKNIIDKIIEQLDKLKFQHE,LSDKNIIDKIIEQLDKLKFQ,KLKFQHE,LSDKNI,LDKL,LSDKN,

[0258] Cat:LSDKNTIDKIIEQLDKLKFQRE

[0259] Horse:SLNNDKSLYIIEQLDKLNFQ

[0260] and / or Helix D:

[0261] People:TDTHECKRFILTISQQFSECMDALKS,TDTHESKRF and HESKRF

[0262] Dogs: ADTFECKSFILTILQQFSACLESVFKS and ADNFERKNF

[0263] Cats: ADNFERKNFILAVLQQFSACLEHVLQS and ADNFERKNF

[0264] Horse:TDNFERKRFILTILRWFSNCLEHRAQ

[0265] They can be used alone or in combination, or fused using the disclosed linker sequences.

[0266] WO 2022 / 131820 A1 discloses immunomodulatory or anti-inflammatory IL31-derived peptides as active ingredients in medicines or cosmetics for preventing or treating atopic dermatitis. The present invention also discloses a conjugate of an IL31 peptide or a fragment thereof coupled with a biocompatible polymer, wherein the biocompatible polymer is, for example, pullulan, chondroitin sulfate, hyaluronic acid (HA), glycol chitosan, starch, chitosan, dextran, pectin, curdlan gum, poly-L-lysine, polyaspartic acid (PAA), polylactic acid (PLA), polyethylene glycol lactide (polyglycolide, PGA), polycaprolactone (poly(ε-caprolactone), PCL), poly(caprolactone-lactide) random copolymer (PCLA), poly(caprolactone-glycolide) random copolymer (PCGA), poly(lactide-glycolide) random copolymer (PLGA), polyethylene glycol (PEG), pluronic F-68 and pluronic F-127 or a fatty acid, for example, hexanoic acid (hexanoic acid The peptides are conjugated to the peptides to increase their stability and skin permeability. The peptides or conjugates are not recommended as immunogens in this article.

[0267] Although in principle, the present invention enables improvement of all proposed IL31-related disease vaccine peptides, selected epitopes (see SeqIDs) have been specifically evaluated for their suitability for this platform compared to CRM197-based vaccines.

[0268] Selected Sequence

[0269] ·SeqID132 SKMLLKDVEEEKG-NHNH2 SeqID133 SKMLLKDVEEEKG-C

[0270] ·SeqID134 EELQSLSK-NHNH2; SeqID135 EELQSLSK-C;

[0271] ·SeqID136 KGVLVS-NHNH2; SeqID137 KGVLVS-C;

[0272] ·SeqID138 SVIDEIIEHLDKLI-NHNH2; SeqID139 SVIDEIIEHLDKLI-C;

[0273] ·SeqID140 SPAIRAYLKTIRQLDNKSVI-NHNH2; SeqID141

[0274] SPAIRAYLKTIRQLDNKSVI-C;

[0275] ·SeqID142 HERKRFILT-NHNH2; SeqID143 HERKRFILT-C;

[0276] ·SeqID144 HESKRFILT-NHNH2; SeqID145 HESKRFILT-C;

[0277] ·SeqID146 SVPTDTHERKRF-NHNH2,SeqID147 SVPTDTHERKRF-C

[0278] ·SeqID148 SVPTDTHESKRF-NHNH2,SeqID149 SVPTDTHESKRF-C

[0279] ·SeqID150 KRFILTISQQFS-NHNH2 SeqID151 KRFILTISQQFS-C

[0280] In view of these advantageous properties of the conjugate of the present invention, the conjugate and vaccine of the present invention are particularly suitable for active immunotherapy of calcitonin gene-related peptide (CGRP)-related diseases.

[0281] The CGRP-related disease is selected from episodic and chronic migraine and cluster headaches, hyperalgesia, hyperalgesia in functional pain states such as rheumatoid arthritis, osteoarthritis, visceral hyperalgesia syndrome, fibromyalgia, inflammatory bowel syndrome, neuropathic pain, chronic inflammatory pain and headache.

[0282] According to a preferred embodiment, the CGRP-derived polypeptide is derived from natural human CGRPα (ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF; a 37aa peptide fragment of aa83-119 of the calcitonin isomer α-CGRP precursor protein, accession number NP_001365879.1) or natural human CGRPβ aa82-228 (ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF; a 37aa peptide fragment of aa82-118 of the calcitonin gene-related peptide 2 precursor, accession number NP_000719.1) or its precursor molecule (NP_001365879.1 and NP_000719.1). The CGRP-derived polypeptide can also be a mimetic with one or more amino acid exchanges to form a mimetic epitope of the corresponding natural sequence.

[0283] According to a preferred embodiment, the CGRP-derived polypeptide is selected from the functional sites of natural human CGRP, including the central region of CGRP (e.g., aa8-35) or a fragment thereof, the C-terminal CGRP receptor binding region (e.g., aa11-37) or a fragment thereof, or the N-terminal region (e.g., aa1-20) that may also include the cyclic C2-C7 loop within CGRP or a fragment thereof consisting of amino acid residues derived from these sites or a mimic epitope.

[0284] Further preferred target sequences include ACDTATCVTH; ACDTATCVTHRLAGL; ACDTATCVTHRLAGLLSR; ACDTATCVTHRLAGLLSRSG; ACDTATCVTHRLAGLLSRSGGVVKN; TATCVTHRLAGLL; LLSRSGGVVKNNFVPTNVG; RLAGLLSRSGGVVKNNFVPTNVGSK; RLAGLLSRSGGVVKNNFVPTNVGSKAF; LLSRSGGVVKNNFVPTNVGSKA F; RSGGVVKNNFVPTNVGSKAF; GGVVKNNFVPTNVGSKAF; VVKNNFVPTNVGSKAF; NNFVPTNVGSKAF; VPTNVGSKAF; NVGSKAF; GSKAF

[0285] In US2022 / 0073582 A1, a polypeptide construct is disclosed containing a CGRP-derived peptide aa1-10ACDTATCVTH; aa 1-15ACDTATCVTHRLAGL; aa 1-18ACDTATCVTHRLAGLLSR; aa 1-20ACDTATCVTHRLAGLLSRSG; aa 1-25ACDTATCVTHRLAGLLSRSGGVVKN; aa4-16TATCVTHRLAGLL; aa 5-18ATCVTHRLAGLLSR; aa 11-18RLAGLLSR; aa11-25RLAGLLSRSGGVVKN; aa 11-30RLAGLLSRSGGVVKNNFVPT; aa 11-33RLAGLLSRSGGVVKNNFVPTNVG; aa 11-35RLAGLLSRSGGVVKNNFVPTNVGSK; aa 11-37RLAGLLSRSGGVVKNNFVPTNVGSKAF; aa 15-37LLSRSGGVVKNNFVPTNVGSKAF; aa18-37RSGGVVKNNFVPTNVGSKAF; aa 20-37GGVVKNNFVPTNVGSKAF; aa 22-37VVKNNFVPTNVGSKAF; aa 25-37NNFVPTNVGSKAF; aa 28-37VPTNVGSKAF; aa31-37NVGSKAF; Native human CGRP (Accession No.: NP_001365879.1) has the following amino acid sequence: MGFQKFSPFLALSILVLLQAGSLHAAPFRSALESSPADPATLSEDEARLLLAALVQDYVQMKASEL EQEQEREGSRIIAQKRACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAFGRRRRDLQA. The peptide immunogen construct disclosed in US 2022 / 0073582A1 requires that a CGRP-derived B cell epitope be coupled to one or more promiscuous T cell epitopes in order to function as a peptide immunogen construct targeting GCRP.

[0286] In addition to active immunotherapy, humanized anti-calcitonin gene-related peptide (CGRP) monoclonal antibodies have been proposed as an anti-CGRP targeting paradigm. Antibodies have been found to be effective in reducing the frequency of chronic migraine (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).

[0287] Along these lines, US 8,597.649B2, EP 1957106 B1 and US 9.266,951B2 disclose clinically used monoclonal antibodies targeting aa25-37 and / or aa33-37 within human CGRP for the treatment of migraine, cluster headache and tension headache. US20120294797 A1 discloses a clinically used monoclonal antibody targeting CGRP, which is also specific for the C-terminal epitope aa26-37 (https: / / doi.org / 10.1080 / 21655979.2021.2006977) according to the co-crystallization results, indicating that the epitope is suitable for immunotherapy. US 9,505,838B2 also discloses a clinically used monoclonal antibody against CGRP, which binds to a C-terminal fragment having CGRP aa25-37 or to a C-terminal epitope within CGRP aa25-37.

[0288] Although in principle the present invention can improve all proposed CGRP-related disease vaccine peptides, specifically selected epitopes (see SeqID 152 to SeqID 162) were evaluated for suitability with the present platform by comparison with the CRM197-based vaccine.

[0289] Select the sequence for the experiment:

[0290] ·SeqID152 RLAGLLSR-NHNH2,SeqID153 RLAGLLSR-C

[0291] ·SeqID154 RLAGLLSRSGGVVKN-NHNH2,SeqID155 RLAGLLSRSGGVVKN-C

[0292] ·SeqID156 RSGGVVKN-NHNH2,SeqID157 RSGGVVKN-C

[0293] ·SeqID158 NNFVPTNVGSKAF-NHNH2,SeqID159 NNFVPTNVGSKAF-C

[0294] ·SeqID160 VPTNVGSKAF-NHNH2,SeqID161 VPTNVGSKAF-C

[0295] ·SeqID162 NVGSKAF-NHNH2,SeqID163 NVGSKAF-C

[0296] In view of these advantageous properties of the conjugates of the present invention, the CLEC-based conjugates and CLEC-based vaccines of the present invention are particularly suitable for specific allergen immunotherapy (AIT) for the treatment of IgE-mediated type I allergic diseases. Allergic diseases generally refer to a variety of diseases caused by hypersensitivity reactions of the immune system to substances in the environment that are usually harmless. These diseases include, but are not limited to, hay fever, seasonal-, food-, pollen-, mold spore-, poisonous plant-, medical / drug-, insect-, scorpion- or spider-venom, latex- or dust allergies, pet allergies, allergic bronchial asthma, allergic rhinitis and-conjunctivitis, atopic dermatitis, contact dermatitis to adhesives, antimicrobials, fragrances, hair dyes, metals, rubber components, topical medications, rosin, wax, polishes, cement and leather, chronic sinusitis, atopic eczema, autoimmune diseases in which IgE plays a role ("autoallergies"), chronic (idiopathic) and autoimmune urticaria, severe allergic reactions, especially idiopathic and exercise-induced severe allergic reactions.

[0297] To date, specific AIT is the only curative treatment for allergies, achieved through repeated injections of allergens containing extracts from different sources (e.g., food, pollen, animal dander, mites, or insect venom). However, the specific AIT modality currently used in clinical practice is characterized by a long treatment period, the need for frequent injections, and limited efficacy, which together lead to low patient compliance (Musa et al., Hum Vaccin Immunother. 2017 Mar;13(3):514–517. doi:10.1080 / 21645515.2016.1243632).

[0298] The main mechanism of AIT is the induction of so-called blocking antibodies, preferably of the IgG4 isotype, but other isotypes (e.g., IgG1 or IgA) are also possible. Studies have shown that naturally occurring IgA and IgG target epitopes on the surface of allergens that are different from the epitopes specifically recognized by IgE (the so-called IgE epitopes) (Shamji, Valenta et al. 2021; Allergy 76(12):3627-3641). The IgE epitope is responsible for the cross-linking of IgE that binds to mast cells through the high-affinity FcεRI receptor, thereby inducing an immediate allergic immune response.

[0299] In contrast, AIT-induced blocking antibodies (mainly IgG- and IgA-type) are directed against these IgE epitopes. Their binding to allergens interferes with the cross-linking of cell-bound IgE, thereby inhibiting the initiation of allergic reactions. IgG4 exhibits favorable properties as a blocking antibody, as it is unable to cross-link allergens and has a low affinity for activating IgG Fc receptors (FcγRs), while maintaining a high affinity for FcγRIIb. These properties make IgG4 an effective inhibitor of IgE-dependent reactions without causing the undesirable inflammation associated with IgG immune complex formation and complement activation (Shamji, Valenta et al., 2021). However, the blocking ability of IgG4 is not necessarily superior to that of other IgG subclasses {Ejrnaes et al., 2004; Molecular Immunology, Vol. 41, No. 5, 2004, pp. 471-478}, especially the early AIT blocking activity is also conferred by other IgG types (especially IgG1) (Strobl, Demir et al., 2023, Journal of Allergy and Clinical Immunology doi:10.1016 / j.jaci.2023.01.005).

[0300] According to a preferred embodiment, a single allergen epitope can be used to trigger an immune response against the corresponding allergen, such as the IgE epitopes mentioned in Tables A and B. In another preferred embodiment, a combination of multiple epitopes from one allergen can be used to trigger an immune response against different domains of the allergen.

[0301] These vaccines against single allergens are very effective when used alone or in combination with peptide vaccines against other allergen molecules involved in allergic diseases. Therefore, a preferred embodiment is to provide a combination of epitopes of different allergens to trigger immune responses against different allergens.

[0302] According to a preferred embodiment, the allergen derived polypeptide is a fragment of an allergen protein (especially as shown in Tables A and B) and / or is preferably selected from natural proteins (especially as listed in Tables A and B).

[0303] According to a preferred embodiment, the allergen derived polypeptides are linear fragments of allergen proteins, including those described in Tables A and B.

[0304] According to a preferred embodiment, the allergen derived polypeptide is selected from mimetics of the above allergen derived polypeptides, including mimotopes and peptides containing amino acid substitutions.

[0305] According to a preferred embodiment, the allergen-derived polypeptide is derived from a natural allergen, or is a mimetic having one or more amino acid exchanges, thereby forming a mimotope of the corresponding natural sequence.

[0306] According to a preferred embodiment, the allergen epitope may be a conformational 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. The paratope is usually bound by anti-allergen antibodies, for example, polyclonal anti-allergen antibodies that specifically recognize natural allergens obtained after vaccination of mammals with the vaccine.

[0307] According to a preferred embodiment, the corresponding conformational epitope or mimotope can be obtained from the literature or identified using a prediction algorithm (e.g., see: Dall'Antonia and Keller 2019, Nucleic Acids Research 47(W1):W496-W501) or a public database (e.g., https: / / www.iedb.org / ). Selected examples of potential target antigens and their corresponding epitopes / mimotopes for the present invention are shown in Tables A and B.

[0308] According to a preferred embodiment, further preferred target sequences include constrained peptides, such as cyclized peptides or peptides connected by suitable aa linkers known to those skilled in the art (e.g.: (G)n linker, (K)n linker, GGSGG or the like).

[0309] Table A – Preferred allergens used in CLEC vaccines

[0310]

[0311]

[0312]

[0313]

[0314]

[0315] Table B: Preferred allergen epitopes for CLEC-based vaccines

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322] 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 affinity of the induced blocking IgG does not increase further 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), which supports the view that AIT-induced inhibition of allergen binding to IgE can be mainly or only explained by the increased amount of induced specific IgG (Svenson et al., 2003, Molecular Immunology 39(10): 603-612; Jakobsen et al., 2005). Therefore, it is believed that the success rate of traditional AIT is quite limited, mainly due to the low immunogenicity of existing AIT compounds and the lack of further affinity maturation after long-term AIT application.

[0323] In contrast, the vaccines or conjugates of the present invention are particularly suitable for AIT and the desired induction of high-avidity IgG, since they induce an IgE-epitope-specific immune response with higher antibody levels (as conventional vaccines), which show prolonged affinity maturation after repeated immunizations (see e.g. Fig.13 and Fig.21 ). This results in immune sera with higher avidity than conventional vaccines including Alum-adjuvanted vaccines and conjugate vaccines (with and without adjuvant).

[0324] Currently, AIT uses only allergen extracts from natural sources, which represent a complex heterogeneous mixture of allergenic and non-allergenic proteins, glycoproteins and polysaccharides (Cox et al. 2005, Expert Review of Clinical Immunology 1(4):579-588.). The resulting product is difficult to standardize and may cause adverse side effects, including severe allergic reactions and late T cell-based responses (Mellerup, Hahn et al. 2000, Experimental Allergy 30(10):1423-1429).

[0325] Therefore, novel vaccine concepts in clinical development utilize platforms that provide universal T cell help (virus-like particles {Shamji, 2022#14} or carrier proteins such as KLH or hepatitis preS fusion protein (Marth et al. 2013, Journal of Immunology 190(7):3068-3078) and recombinant allergen proteins or peptides (containing allergen epitopes or their mimetic epitopes) to increase immunogenicity and affinity maturation (Bachmann et al., 2020, Trends in Molecular Medicine 26(4):357-368).

[0326] The latter approach, which uses peptide-carrier conjugates containing allergen epitopes or their mimotopes, is particularly advantageous for novel AIT modalities in patients because it focuses the immune response on the desired target epitope (i.e., IgE epitopes) and completely avoids both immediate side effects (i.e., cross-linking of the vaccine with cell-bound IgE) as well as late side effects (i.e., activation of allergen-specific T-cell responses).

[0327] Marth et al. (2013) disclosed AIT compounds based on fusion proteins of two non-allergic peptides PA and PB, which were derived from the IgE-reactive region of the major birch pollen allergen Bet v 1, fused to the hepatitis B surface protein PreS to form four recombinant fusion proteins containing different numbers and combinations of peptides. Similarly, the clinically tested AIT vaccine BM32 uses four fusion proteins composed of peptides from the four major timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5 and Phl p 6) fused to the hepatitis B PreS carrier protein. Weber et al. (2017; doi:10.1016 / j.jaci.2017.03.048) demonstrated that alum-adjuvanted BM32 and traditional extract-mediated AIT had similar immunogenicity in rabbits. However, despite encouraging initial clinical results (Eckl-Dorna, 2019EBioMedicine. 2019 Dec;50:421-432. doi:10.1016 / j.ebiom.2019.11.006.), further development of the BM32 approach was abandoned after a Phase IIb study. To date, no peptide-carrier conjugate or fusion protein AIT approaches, nor any other novel recombinant vaccines for AIT have been licensed (Pavón-Romero, 2022, Cells. 2022 Jan 8;11(2):212. doi:10.3390 / cells11020212).

[0328] Along these lines, a single injection of two monoclonal antibodies against two epitopes within the major cat allergen FeI d 1 has been shown to be as effective as years of traditional AIT in allergic patients (Orengo, Radin et al., 2018, Nature Communications 9(1):1421), suggesting that a small number of target epitopes within a given allergen may be sufficient to provide comprehensive protection from allergic immune responses. The vaccines or conjugates of the present invention are particularly suitable for combining universal T cell epitopes with such IgE epitopes or mimotopes on the CLEC backbone to treat allergies.

[0329] Although in principle the present invention can improve all proposed allergic disease vaccine polypeptides, selected epitopes (see Tables A and B and SeqID45 / 46) are particularly preferred. For example, SeqID45 / 46 was shown to be superior to KLH-based vaccines.

[0330] In view of these favorable properties of the conjugates of the present invention, the CLEC-based conjugates and CLEC-based vaccines of the present invention are particularly useful for enhancing the immunogenicity of commercially available peptide / sugar-conjugate vaccines, especially sugar-conjugate vaccines for the prevention of infectious diseases, such as microbial infections or viral infections, such as those caused by Haemophilus influenzae type b (Hib), Streptococcus pneumoniae, Neisseria meningitidis and Salmonella typhi or other infectious agents, including those causing hepatitis A or B, human papillomavirus infections, influenza, typhoid fever, measles, mumps and rubella. In addition, there are infections caused by group B meningococci, cytomegalovirus (CMV), respiratory syncytial virus (RSV), Clostridium difficile, extraintestinal pathogenic Escherichia coli (Expec), Klebsiella pneumoniae, Shigella, Staphylococcus aureus, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae, coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2), Ebola virus, Borrelia burgdorferi, HIV, etc.

[0331] To date, several carrier proteins have been used in licensed conjugate vaccines: genetically modified cross-reactive 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). Clinical trials have demonstrated the efficacy of these conjugate vaccines in preventing infectious diseases and modifying the transmission of Haemophilus influenzae type b, Streptococcus pneumoniae, Neisseria meningitidis, and typhoid fever. All carrier proteins are effective in improving the immunogenicity of vaccines, but they vary in the amount and avidity of antibodies they elicit, their ability to carry multiple polysaccharides in the same product, and their ability to be administered simultaneously with other vaccines.

[0332] According to a preferred embodiment, conjugate vaccines suitable for CLEC modification and enhanced immunogenicity include, but are not limited to, existing vaccines, including conjugate vaccines of Haemophilus type B (e.g. ), recombinant hepatitis B vaccines (e.g. Recombivax ), human papillomavirus vaccines (e.g. Gardasil ), meningococcal (groups A, C, Y and W-135) oligosaccharide diphtheria CRM197 conjugate vaccine (e.g. ), meningococcal (groups A, C, Y and W-135) polysaccharide diphtheria toxoid conjugate vaccines (for example: ), meningococcal (groups A, C, Y, W) TT-conjugate vaccines (e.g.: ), multivalent pneumococcal conjugate vaccines (e.g.: Prevnar ), anti-typhoid vaccine (e.g. Typhim Typhim Vi polysaccharide conjugated to avirulent recombinant Pseudomonas aeruginosa exotoxin A, or Vi-rEPA or polysaccharide tetanus toxoid conjugate vaccine ), varicella-zoster virus vaccine (e.g. ) and other anti-infective conjugate vaccines that carry a genetically modified cross-reactive substance of diphtheria toxin (CRM197), tetanus toxoid (TT), meningococcal outer membrane protein complex (OMPC), diphtheria toxoid (DT), Haemophilus influenzae protein D (HiD) or recombinant Pseudomonas aeruginosa exotoxin A (rEPA) as carrier molecules.

[0333] According to another aspect, the novel conjugate of the present invention can be used to prevent infectious diseases. Such diseases are, for example, microbial infections or viral infections, such as by Haemophilus influenzae type B (Hib), Streptococcus pneumoniae, Neisseria meningitidis and Salmonella typhi or other infectious agents, including infections caused by infectious agents that cause hepatitis A or B, human papillomavirus infection, influenza, typhoid fever, measles, mumps and rubella. Also there are infections caused by group B meningococci, cytomegalovirus (CMV), respiratory syncytial virus (RSV), Clostridium difficile, extraintestinal pathogenic Escherichia coli (Expec), Klebsiella pneumoniae, Shigella, Staphylococcus aureus, Plasmodium, coronavirus (SARS-CoV, MERS-CoV, SARS-CoV-2), Ebola virus, Borrelia burgdorferi, HIV, etc.

[0334] Although in theory, the present invention can improve all proposed anti-infective conjugate vaccines, selected vaccines were specifically analyzed. For example, the CLEC improved meningococcal (A, C, Y and W-135 groups) oligosaccharide diphtheria CRM197 conjugate vaccine (i.e. ) and Haemophilus influenzae type b conjugate vaccine Proven to be superior to commercially available and vaccine.

[0335] In view of these favorable properties of the conjugates of the present invention, the conjugates and vaccines of the present invention are particularly suitable for active immunotherapy of proprotein (proprotin) convertase subtilisin / kexin type 9 (PCSK9) related 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.

[0336] According to a preferred embodiment, the PCSK9 protein-derived polypeptide is derived from natural human PCSK9 (Accession No.: Q8NBP7) and has the following amino acid sequence:

[0337]

[0338] or a fragment thereof, or a mimetic with one or more amino acid exchanges, forming a mimetic epitope of the corresponding natural sequence. Further preferred target sequences include linear peptides or constrained (e.g. cyclized) peptides or peptides connected by suitable amino acid linkers (e.g. ggsgg or the like).

[0339] According to a preferred embodiment, the polypeptide derived from the PCSK9 protein is selected from the following regions: aa150-170, aa153-162, aa205-225, aa211-223, aa368-382, or a polypeptide comprising or consisting of amino acid residues or mimotopes derived from these subunits.

[0340] According to a preferred embodiment, the PCSK9 protein-derived polypeptide is selected from the PCSK9-derived sequences: NVPEEDGTRFHRQASK, NVPEEDGTRFHRQASKC, PEEDGTRFHRQASK, CPEEDGTRFHRQASK, PEEDGTRFHRQASKC, AEEDGTRFHRQASK, TEEDGTRFHRQASK, PQEDGTRFHRQASK, PEEDGTRFHRRASK, PEEDGTRFHRKASK, PEEDGTRFHRQASR, PEEDGTRFHRTASK, SIPWNLERITPPR, PEEDGTRFHRQASK, PEEDGTRFHRQA, EEDGTRFHRQASK, EEDGTRFHRQAS, SIPWNLERITP, SIPWNLERITP, SIPWNLERITP, SIPWNLERITP, SIPWNLERITP. IT,SIPWNLERITC,LRPRGQPNQC,SRHLAQASQ,SRHLAQASQC,SRSGKRRGER,SRSGKRRGERC,IIGASSDCSTCFVSQ,IIGASSDSSTSFVSQ,IIGASSDSSTSFVSQC,CIGASSDSSTSFVSC,IGASSDSSTSFVSC,CDGTRFHRQASK C,DGTRFHRQASKC,CDGTRFHRQASK,AGRDAGVAKGAC,RDAGVAKC,RDAGVAK,SRHLAQASQLEQC;SRHLAQASQLEQ,GDYEELVLALRC;GDYEELVLALR,LVLALRSEEDC;LVLALRSEED,AKDPWRLPC;AKDPWRLP,AARRGYLTKC, AARRGYLTK,FLVKMSGDLLELALKLPC;FLVKMSGDLLELALKLP,EEDSSVFAQC,EEDSSVFAQ,NVPEEDGTRFHRQASKC, NVPEEDGTRFHRQASK,CKSAQRHFRTGDEEPVN,KSAQRHFRTGDEEPVN,

[0341] According to a preferred embodiment, a single PCSK9-derived epitope can be used to trigger an immune response against different regions within the three different domains of PCSK9, namely the inhibitory prodomain (aa1-152), the catalytic domain (aa153-448), and the C-terminal domain (449-692). In another preferred embodiment, a combination of PCSK9-derived epitopes can be used to trigger an immune response against different epitopes within each domain of PCSK9, particularly involving the catalytic domain (aa153-449), further involving the inhibitory prodomain (aa1-152) and / or the C-terminal domain (449-692).

[0342] Vascular diseases such as hyperlipidemia, hypercholesterolemia, atherosclerosis, coronary heart disease and stroke are among the leading causes of death worldwide, and elevated LDL-C levels are a key factor in their pathogenesis. Therefore, LDL-C management is a very important factor in the successful treatment of hyperlipidemia, hypercholesterolemia and atherosclerosis. Accordingly, PCSK9 plays a vital role in LDL catabolism by directly acting on LDLR. Inhibition of PCSK9 is beneficial to LDL-C levels. Therefore, anti-PCSK9 therapy is a promising approach in beneficially regulating LDL-C levels and treating PCSK9-related diseases.

[0343] WO2015128287A1 and EP2570135A1 disclose PCSK9 mimetic epitope-carrier conjugate vaccines (for example: KLH or CRM197 as carriers), and disclose PEEDGTRFHRQASK, AEEDGTRFHRQASK, TEEDGTRFHRQASK, PQEDGTRFHRQASK, PEEDGTRFHRRASK, PEEDGTRFHRKASK, PEEDGTRFHRQASR, PEEDGTRFHRTASK and aa150-170 and / or aa205-225 sequences of PCSK9, in particular SIPWNLERITPPR, PEEDGTRFHRQASK, PEEDGTRFHRQA, EEDGTRFHRQASK, EEDGTRFHRQAS, SIPWNLERITP and SIPWNLERIT.

[0344] CN105085684A discloses a recombinant vaccine comprising a PCSK9 epitope and diphtheria toxin DTT. The epitope peptide is connected to the C-terminus of the transmembrane domain DTT of the carrier protein diphtheria toxin. CN106822881A discloses a protein vaccine characterized by a recombinant PCSK9 protein fragment polypeptide (catalytic domain and C-terminal domain).

[0345] WO2022150661A2 discloses viruses (including bacteriophage viruses or plant viruses) or virus-like particles for PCSK9 immunotherapy, especially comprising the PCSK9-derived sequence NVPEEDGTRFHRQASKC.

[0346] EP3434279A1 discloses an OSK-1-PCSK9 conjugate vaccine; using PCSK9 derived sequences LRPRGQPNQC, SRHLAQASQ and SRSGKRRGER. WO2021 / 154947A1 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, CDGTRFHRQASKC.

[0347] WO2011 / 027257A2 and WO 2012 / 131504 A1: Disclose PCSK9-derived peptide-VLP and PCSK9-derived peptide-carrier vaccines targeting PCSK9, including sequences SIPWNLERITPC, SIPWNLERITC, SIPWNLERITP, AGRDAGVAKGA, RDAGVAK; SRHLAQASQLEQ; GDYEELVLALR; LVLALRSEED; AKDPWRLP-; AARRGYLTK; FLVKMSGDLLELALKLP; EEDSSVFAQ. WO2015 / 123291A1: discloses a peptide-VLP (Qb) vaccine targeting PCSK9, comprising the sequences: NVPEEDGTRFHRQASKC and CKSAQRHFRTGDEEPVN, while WO2018 / 189705 discloses a peptide-carrier conjugate targeting PCSK9 based on the sequence SIPWNLERITPC and its modified derivatives.

[0348] Preferred polypeptide immunogen constructs of the present invention comprise B cell epitopes from α-synuclein and heterologous T helper cell (Th) epitopes coupled to CLEC. The present invention provides surprisingly superior new conjugates that surpass traditional vaccines in immunogenicity, cross-reactivity to α-synuclein, selectivity for α-synuclein species / aggregates, affinity, affinity maturation, and inhibitory capacity.

[0349] The covalent coupling of alpha synuclein polypeptides with beta-glucan or mannan according to the present invention unexpectedly enhances the immune response to such polypeptides. This is particularly impressive when directly compared with traditional vaccine formulations, such as those described by Rockenstein et al. (J. Neurosci., January 24, 2018, 38(4): 1000-1014), as also shown in the Examples section below.

[0350] Rockenstein et al. (2018) disclosed the application of yeast glucan whole particles (GP) with aSyn and rapamycin as non-covalent complexes as Parkinson's disease immunotherapeutics. These GPs are produced from Saccharomyces cerevisiae after a series of hot alkaline, organic and aqueous extraction steps. The final product consists of a highly purified yeast cell wall preparation with a diameter of 3-4 microns, free of cytoplasmic contents, and is coated with porous, insoluble β-glucan (mainly β1-3β-glucan).

[0351] Importantly, the vaccine composition disclosed by Rockenstein et al. (2018) consists of GP non-covalently complexed with ovalbumin and mouse serum albumin (MSA), human aSyn and MSA, or human aSyn, MSA and rapamycin. This complexing method relies on the co-incubation of different payloads with GP, followed by diffusion into the hollow GP cavity without covalent linkage, and is therefore similar to a group of vaccines in Example 28 of the present application, which only use a mixture of multiple components rather than covalent linkage to formulate the vaccine, and has been proven to be less efficient and unsuitable than the vaccine of the present invention.

[0352] 1) Rockenstein et al. showed that non-covalent mixing of aSyn and GP resulted in a detectable anti-aSyn immune response, 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 a significant increase in the function of such vaccines compared to controls. From this perspective, mixing of various adjuvants (GP and the mTOR inhibitor rapamycin) is required to provide a fully functional vaccine, such as the vaccine disclosed in the present invention.

[0353] 2) The vaccine disclosed by Rockenstein et al. is active in this aSyn overexpression model because it provides aSyn-specific T cell epitopes (as well as other T cell epitopes, such as MSA-derived epitopes) to exert their full functionality, i.e., inducing neuroprotective, anti-ASyn directed cellular (i.e.: T cell mediated) and humoral (i.e., antibody / B cell based) immune responses. This is in stark contrast to the teaching of the present invention, in which it is sufficient that only aSyn-specific B cell responses are elicited by the selected vaccine.

[0354] 3) The use of full-length aSyn also carries the risk of inducing / enhancing autoreactive aSyn-specific T cells, which could exacerbate the underlying neuropathology of PD and other synucleinopathies. Therefore, in this regard, the GP-aSyn-rapamycin vaccine proposed by Rockenstein et al. is also not suitable for human use.

[0355] 4) As shown in Example 5, similar to Rockenstein et al., non-covalent mixing of aSyn-derived peptides (e.g., SeqID2, i.e., B cell epitopes) and promiscuous T cell epitopes (e.g., SeqID7) with β-glucan particles (e.g., unoxidized pyruvic glycosides) can also induce low-level anti-aSyn antibody responses. However, the vaccines of the present invention based on the covalent attachment of such peptides to suitable glucans produce significantly different and superior immune responses (see also Figure 5 ).

[0356] In addition, Example 6 and Figure 7 It is also disclosed that such covalently linked vaccines also show a highly beneficial lack of anti-glucan antibody response compared to the non-covalent mixture vaccines based on glucan particles and peptides disclosed in the present invention.

[0357] Thus, the prior art of Rockenstein et al. does not suggest the claimed subject matter disclosed in the present invention.

[0358] Particularly preferred aSyn polypeptides to be coupled in the present invention are selected from natural α-synuclein, or polypeptides comprising or consisting of the following amino acid residues of natural human α-synuclein: 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:MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVHGVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDPDNEAYEMPSE EGYQDYEPEA (human aSyn(1-140aa): UNIPROT accession number P37840),

[0359] Preferably, the polypeptide comprises or consists of the following amino acid residues: 1-8, 91-100, 100-108, 103-135, 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 DQPVLPD, DQPVLPDN, DQPVLPDNE, DQPVLPDNEA, DQPVLPDNEAY, DQPVLPDNEAYE, DSPVLPDG, DHPVHPDS, DTPVLPDS, DAPVTPDT, DAPVRPDS, and YDRPVQPDR.

[0360] Existing CLEC vaccines are able to induce high titers against the carrier protein used (e.g., CRM197 or OVA). However, this high immunogenicity, as well as the structural complexity and heterogeneity of the carrier protein components, may lead to the induction of high levels of carrier / protein-specific antibodies at the expense of target-specific responses, which may therefore be underrepresented compared to the induced carrier responses.

[0361] In addition, affinity maturation of target-specific responses induced by repeated immunization with the carrier conjugate is also impaired due to overexpression of carrier-specific epitopes in the conjugate. As used and understood herein, immunological affinity maturation is the process by which T cells are attached to the target during an immune response. FH The process by which activated B cells produce antibodies with higher affinity for antigens. Through repeated exposure to the same antigen, the host will produce antibodies with gradually increasing affinity. The secondary response can elicit antibodies with affinity several times higher than the primary response. Affinity maturation occurs primarily on the surface immunoglobulins of germinal center B cells and is a process of somatic hypermutation (SHM) and T FH A direct result of cell selection (see also: https: / / en.wikipedia.org / wiki / Affinity_maturation). According to the Segen Medical Dictionary (https: / / medical-dictionary.thefreedictionary.com / affinity+maturation">affinity maturation), affinity maturation refers to the increase in the average affinity of antibodies for antigens after immunization. Affinity maturation is due to an increase in specific and more homogeneous IgG antibodies, following the less specific and more heterogeneous early response of IgM molecules.

[0362] Furthermore, high anti-vector responses also pose the risk of immune rejection and related safety issues.

[0363] Thus, according to the present invention, effective constructs with high immunogenicity, high target specificity and high tolerability / safety with low or absent carrier reactivity (i.e., against the protein carrier) were identified, successfully addressing this challenge through innovative solutions. Furthermore, it is critical for the novel vaccines of the present invention that the provided immunotherapeutics do not induce or induce very weak immune responses against the sugar backbone. This is particularly important because high anti-CLEC antibody levels induced by immunization can inhibit or reduce the efficacy of repeated immunizations using the same CLEC-based vaccine due to vaccine neutralization, or can also negatively affect the use of such vaccines for continuous immunization against various different targets.

[0364] The vaccine platform of the present invention also meets the need to combine various epitopes against one or more targets in one formulation without the risk of reduced efficacy due to unintended epitope diffusion as with traditional vaccines. The modular design of the platform of the present invention allows for easy exchange of B-cell epitopes and T-cell epitopes without the negative impact of vector-induced responses.

[0365] The present invention is based on a CLEC that has a highly specific binding to a cognate receptor. This binding is critical, and only strong binders can be effective as vaccine vectors / backbones.

[0366] According to the present invention, CLEC conjugation enables an effective immune response with novel characteristics. The conjugation of the present invention excludes the formation of anti-CLEC (especially pyralidin) antibodies, which can be impressively demonstrated in the process of the present invention. This lack of anti-CLEC antibody stimulation is very important for the reusability and re-boosting of a single vaccine designed using the platform of the present invention - whether the antigens are the same or different.

[0367] In contrast to the conjugated embodiments of the present invention, merely mixing the CLEC polysaccharide adjuvant with a B-cell or T-cell epitope peptide does not produce a similar effect in vivo. However, if conjugated, the orientation of the peptide does not significantly affect the performance of the compounds of the present invention; thus, CLEC conjugation is essentially independent of the orientation of the peptide in the construct. In the course of the present invention, it could be demonstrated that CLEC conjugation, especially with pyrifos, leads to improvements in new as well as existing peptide immunogens / antigens: this improvement is achieved by a higher, more target-specific and higher affinity antibody response (as shown by antibody selectivity and functionality). This effect is most pronounced for pyrenoids or similar β-glucans, which are primarily linear β-(1,6)-glucans in which the ratio of β-(1,6)-conjugated monosaccharide moiety to non-β-(1,6)-conjugated monosaccharide moiety is at least 1:1, preferably at least 2:1, more preferably at least 5:1, and especially at least 10:1, which perform surprisingly much better than even KLH or CRM in direct comparison, and even better than mannan or lichenan conjugates or conjugates comprising barley β-glucan.

[0368] As used herein, the term "predominantly linear" β-(1,6)-glucan refers to a β-(1,6)-D-glucan in which there are no or only a small amount of cross-linked sugar monomer entities, i.e., less than 1%, preferably less than 0.1%, and especially less than 0.01% of the monosaccharide moieties have more than two covalently linked monosaccharide moieties.

[0369] As mentioned above, pyrifos is the most preferred CLEC of the present invention. Pyrifos generally does not contain cross-linked sugar moieties and is mainly β-(1,6)-coupled, so that the common pyrifos preparations used to prepare the conjugates of the present invention have less than 1%, preferably less than 0.1%, and especially less than 0.01% of monosaccharide moieties with more than two covalently linked monosaccharide moieties, and have a maximum of 10% of impurities with β-(1,3)- or β-(1,4)-coupled monosaccharides.

[0370] The fact that dactylan was shown to be the most potent CLEC in the present invention was unexpected, as various references indicate that dactylan should be less effective in dectin-1 binding (e.g., Adams et al., J Pharmacol Exp Ther. 2008 Apr;325(1):115-23); in the literature, linear 1,3 and branched (1,3 backbone and 1,6 side chains) have been reported to be the most potent dectin-1 binders. For example, Adams et al. 2008 reported that murine recombinant dectin-1 only recognized and interacted with polymers containing a β-(1,3)-linked glucose backbone. Dectin-1 did not interact with glucans containing only a β-(1,6)-glucose backbone (dactylan), nor with non-glucan carbohydrate polymers such as mannan.

[0371] Therefore, according to a preferred embodiment of the present invention, the β-glucan of the present conjugate is a dectin-1 binding β-glucan. The ability of any compound (especially a glucan) to bind to dectin-1 can be easily determined by the methods disclosed herein (especially in the Examples section). In case of doubt, "dectin-1 binding β-glucan" refers to a β-glucan whose IC50 value for binding to the soluble mouse Fc-dectin-1a receptor is less than 10 mg / ml as determined by competitive ELISA (as disclosed in the Examples).

[0372] The Dectin-1 binding β-glucan (e.g., linear β-(1,6)-glucan) of the present invention has advantages over other glucans (e.g., DC-SIGN β-glucan (e.g., β-(1,2)-glucan)) in that such Dectin-1 binding glucan can reach a wider range of DCs (immature, mature, myeloid, plasmacytoid; and APC), which significantly increases the possibility of eliciting an effective immune response in vivo, compared with non-Dectin-1 binding glucans with limited applicability (immature DC, myeloid DC).

[0373] WO 2022 / 060487 A1 and WO 2022 / 060488 A1 disclose conjugates linking peptide immunogens to immunostimulatory polymer molecules such as β-(1,2) glucans. β-(1,2) glucans (including cyclic variants) have previously been considered 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, namely DC-SIGN (Zhang H et al., doi:10.1093 / glycob / cww041), specifically binding to N-linked high mannose oligosaccharides and branched fucose structures. Importantly, β-1,2 glucans do not bind to dectin (Zhang H et al., doi:10.1093 / glycob / cww041), thereby limiting their activity on DC-SIGN-positive cells.

[0374] DC-SIGN (CD209) is the first SIGN molecule to be identified and is highly expressed only on a limited subset of DCs, including immature (CD83 negative) DCs and specialized macrophages in the placenta and lungs (Soilleux EJ et al., doi: 10.1189 / jlb.71.3.445). In the periphery, such as in the skin or mucosal sites, expression and therefore the potential for biological activity as a receptor of the present invention is only detectable in immature DC subsets. 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).

[0375] In contrast, the target receptor of the β-glucan-based immunogen provided by the present invention is dectin-1. Dectin-1 is expressed on many different types of DCs, including not only immature DCs, myeloid DCs, but also plasmacytoid DCs that express dectin-1 at both 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).

[0376] Therefore, the biological activity of DC-SIGN targeting polymers (such as β-(1,2) glucan) is limited to a specific DC target cell population, while the dectin-1 targeting polymers used in the present invention can function in a variety of other DC types. Therefore, compared with other conjugates, these new conjugates can exert significantly different and superior immune responses. Therefore, the content disclosed in the prior art does not suggest the claimed subject matter disclosed in the present invention.

[0377] According to a particularly preferred embodiment, the conjugate of the present invention comprises a β-glucan that strongly binds to dectin-1, preferably a β-glucan that binds to a soluble mouse Fc-dectin-1a receptor, with an IC50 value of less than 10 mg / ml, more preferably an IC50 value of less than 1 mg / ml, even more preferably an IC50 value of less than 500 μg / ml, in particular an IC50 value of less than 200 μg / ml, as determined by competitive ELISA, for example, see the Examples. Specifically preferably, as determined by competitive ELISA, the conjugate binds to a soluble mouse Fc-dectin-1a receptor with an IC50 value of less than 1 mg / ml, more preferably an IC50 value of less than 500 μg / ml, even more preferably an IC50 value of less than 200 μg / ml, in particular an IC50 value of less than 100 μg / ml; and / or

[0378] - β-glucan which binds to soluble human Fc-dectin-1a receptor with an IC50 value of less than 10 mg / ml, more preferably with an IC50 value of less than 1 mg / ml, even more preferably with an IC50 value of less than 500 μg / ml, in particular with an IC50 value of less than 200 μg / ml, as determined by competitive ELISA; and / or

[0379] - The conjugate binds to soluble human Fc-dectin-1a receptor with an IC50 value of less than 1 mg / ml, more preferably an IC50 value of less than 500 μg / ml, even more preferably an IC50 value of less than 200 μg / ml, in particular an IC50 value of less than 100 μg / ml, as determined by competitive ELISA, for example see the Examples.

[0380] In addition, the conjugates of the present invention also show that the ratio of antibodies reacting with the target polypeptide to antibodies reacting with the carrier molecule is much higher than that of non-CLEC vaccines (especially vaccines without pyruvic polysaccharides). This significantly increases the specific focus of the antibody immune response on the target rather than the carrier, resulting in increased efficacy and specificity of the response.

[0381] The CLEC conjugates of the invention, especially the conjugates with pyruvic acid, also resulted in increased affinity maturation (AM) for the target protein (AM was strongly increased, whereas the KLH / CRM conjugate showed only limited AM after repeated immunizations).

[0382] In the field of vaccines, suitable vaccines having only B cell epitopes or only T cell epitopes have been disclosed. In certain cases, vaccines having only T cell epitopes or only B cell epitopes are suitable and preferred. However, most vaccines on the market contain two epitopes, i.e., T cell epitopes and B cell epitopes.

[0383] For example, vaccines containing only B cell epitopes are ineffective in most cases, even if they do result in a detectable antibody immune response. However, in most cases, this immune response is usually much less effective than vaccines containing both 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 can be detected.

[0384] On the other hand, vaccines containing only T cell epitopes (e.g., in vaccines in which a specific T cell response is the active component of the response) are of particular interest for certain applications, especially for cancer, where cancer-specific cytotoxic T lymphocyte and T helper cell epitopes or only CTL epitopes are combined with the vaccine platform of the present invention. In this case, the T cell epitopes with the CLEC polysaccharide adjuvant of the present invention are provided as only T cell epitopes. This is particularly preferred in cases where, for example, somatic mutations in cancer affect protein-coding genes (which can generate potential therapeutic new epitopes). These new epitopes can guide adoptive cell therapy and peptide-based (and RNA-based) new epitope vaccines to selectively target tumor cells using the patient's own cytotoxic T cells. This can be used according to the invention for universal antigens and personalized neoantigen-specific therapies (e.g. 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, 5T4, KRAS, Her2, etc.). For specific autoimmune diseases, the use of vaccines containing only T cell epitopes may also be preferred. The therapeutic effect of the corresponding conjugate containing only T cell epitopes is consistent with the reduction of effector T cells and regulatory T cells (T reg The development of autoimmune disease (such as multiple sclerosis or similar diseases) is associated with the inhibition of the corresponding autoimmune diseases (such as multiple sclerosis or similar diseases).

[0385] Since most commonly used vaccine combinations contain both B-cell and T-cell epitopes, the CLEC conjugates of the present invention also preferably contain both, separate B-cell and T-cell epitopes (minimum: at least one B-cell epitope and at least one T-cell epitope) to generate a sustained B-cell immune response. However, if desired, a weaker effect may demonstrate T-cell-independent immunity.

[0386] Therefore, the conjugate of the present invention is not limited by possible vaccine antigens. However, preferably, the vaccine antigen (i.e., B cell and / or T cell epitope polypeptide) has a length of 6-50 amino acid residues, preferably 7-40 amino acid residues, especially 8-30 amino acid residues.

[0387] Using the vaccine of the present invention, cross-linking of B cell receptors is also possible. According to a specific embodiment, the conjugate of the present invention is used for T cell-independent immunity. The T cell-independent response of polysaccharide vaccines is well known. These vaccines / polysaccharides produce immune responses by directly stimulating B cells without the assistance of T cells. T cell-independent antibody responses are short-lived. Antibody concentrations to pneumococcal capsular polysaccharides usually drop to baseline within 3-8 years, depending on the serotype. Typically, additional doses cannot be used to enhance vaccine responses because polysaccharide vaccines do not constitute immune memory. In children under two years old, polysaccharide vaccines are less immunogenic. The reason for direct stimulation here may be that B cells express a molecule called CR3 (complement receptor type 3). Macrophage-1 antigen or CR3 is a human cell surface receptor that can be found in B- and T-lymphocytes, polymorphonuclear leukocytes (mainly neutrophils), NK cells, and mononuclear phagocytes (such as macrophages). CR3 also recognizes iC3b when bound to foreign cells and β-glucan, meaning that direct uptake of the vaccine by B cells via the Pus-CR3 interaction could lead to cell stimulation and the generation of a low-level TI immune response.

[0388] The adjuvants, conjugates and vaccines of the present invention can fix complement and can be conditioned. The conditioned conjugates of the present invention can have enhanced B cell activation ability, which can lead to higher antibody titers and antibody avidity. This effect is known for C3d conjugates (Green et al., J. Virol. 77 (2003), 2046-2055) and can also be used in the process of the present invention unexpectedly.

[0389] Another unexpected advantage of the present invention is that the CLEC structure of the present invention allows for modular design of vaccines. For example, epitopes can be combined at will, and the platform does not rely on conventional carrier molecules. Although the main focus of the present invention is peptide-only vaccines, it is also applicable to proteins and peptides coupled independently to, and peptide-protein conjugates coupled to, the CLEC backbone of the present invention (Pyrgosaccharides). As shown in the Examples section involving Pyrgosaccharides, the present invention achieves significantly superior immune responses than traditional vaccines.

[0390] As mentioned above, the conjugate of the invention, if provided in the form of a pharmaceutical formulation (e.g. as a vaccine intended to be administered to a (human) subject in order to elicit an immune response to a specific polypeptide epitope coupled to a CLEC backbone, against which the immune response is to be directed), can be administered without the use (by co-administration) of a (additional) adjuvant in the formulation. According to a preferred embodiment, the pharmaceutical formulation comprising the conjugate of the invention is adjuvant-free.

[0391] A particularly preferred class of CLEC polysaccharide adjuvants of the present invention is β-glucan, especially chrysanthemum. Another preferred CLEC polysaccharide adjuvant is mannan. In contrast to the present invention, chrysanthemum has only been used in antifungal vaccines in the prior art (wherein chrysanthemum is used as an antigen, not as a carrier as in the present invention). Chrysanthemum also exhibits a different backbone, consisting only of β-(1,6) linked sugar moieties.

[0392] Pyran is a medium-sized linear β-(1,6) glucan. Pyran and synthetic forms of linear β-(1,6) glucans differ from all other glucans used as β-glucans, which usually consist of branched glucan chains (preferably β-(1,3) backbone with β-(1,6) side chains, such as yeast extract, GP, laminarin, schizophyllan, scleroglucan) or linear glucans that rely solely on β-(1,3) glucans, such as synthetic β-glucans, curdlan gum, Saccharomyces cerevisiae β-glucan (150 kDa) or linear β-(1,3:1,4) glucans, such as barley- and oat-β-glucans and lichenan.

[0393] As shown for the first time in the present invention, in vitro binding of dextran conjugates to the dectin-1 receptor is a proxy for subsequent in vivo efficacy: low-binding molecules can only exert a low level of immune response, medium binders are better, and high-efficiency binders induce a high-efficiency response (oat / barley BG < lichenan < pyracantha).

[0394] According to the present invention, CLEC is coupled to a single polypeptide (e.g., by standard techniques) to produce small nanoparticles with a low level of polydispersity (hydrodynamic radius (HDR) range: 5-15 nm), which are not cross-linked and do not 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, which are generally characterized by a size range of >100 nm (typical range (diameter; 150-500 nm, for example, Wang et al. (2019) provides particles with a diameter of 160 nm (DLS evaluation) and a size of about 150 nm (TEM evaluation); Jin et al. (Acta Biomater. 2018 Sep 15; 78: 211-223) provide β-glucan particles (nanoparticles of aminated β-glucan-ovalbumin) with a size of 180-215 nm (assessed by DLS and SEM, respectively).

[0395] By definition, the hydrodynamic radius measured by DLS is the radius of a hypothetical hard sphere that diffuses at the same rate as the particle being examined. The radius is calculated from the diffusion coefficient assuming a spherical shape of the molecule / particle and a given buffer viscosity. The HDR, also known as the Stokes radius, is a function of the Stokes-Einstein equation (see https: / / en.wikipedia.org / wiki / Stokes_radius ) calculated from the diffusion coefficient.

[0396] The preferred size range of the nanoparticles of the present invention can be those generally provided by the prior art, i.e. 1-5000 nm, preferably 1-200 nm, especially 2-160 nm, is the hydrodynamic radius (HDR) determined by dynamic light scattering (DLS). According to a preferred embodiment of the present invention, the particle size is smaller, such as 1-50 nm, preferably 1-25 nm, especially 2-15 nm, is the HDR determined by DLS. Therefore, these preferred particles are smaller, including peptide-only conjugates (about 5 nm average HDR) and CRM-pachysan conjugates (about 10-15 nm average HDR). Therefore, the preferred particles of the present invention are less than 100 nm, which is different from Wang et al.

[0397] Accordingly, the present invention also relates to a vaccine product designed for vaccination of an individual against a specific antigen, the product comprising a compound comprising β-glucan or mannan as a C-type lectin (CLEC) polysaccharide adjuvant covalently coupled to the specific antigen.

[0398] Preferably, the vaccine product of the invention comprises a conjugate as described herein or obtainable or obtained according to the method of the invention.

[0399] According to a preferred embodiment, the vaccine product of the present invention comprises an antigen containing 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.

[0400] According to a preferred embodiment, the covalently coupled antigen and CLEC polysaccharide adjuvant in the vaccine product of the present invention are present in the form of particles with a size of 1- to 5000 nm, preferably 1-200 nm, and especially 2-160 nm, the hydrodynamic radius (HDR) being determined by dynamic light scattering (DLS). As used herein, all particle sizes are median particle sizes, wherein the median separates the larger half of the particles from the smaller half. From the determined particle size, half of the particles are smaller than it, and the other half are larger than it.

[0401] According to a preferred embodiment, the covalently coupled antigen and CLEC polysaccharide adjuvant in the vaccine product of the present invention are present in the form of particles with a size of 1-50 nm, preferably 1-25 nm, especially 2-15 nm (HDR determined by DLS).

[0402] Preferably, the covalently coupled antigen and CLEC polysaccharide adjuvant in the vaccine product of the present invention are present as particles smaller than 100 nm, 50 nm, preferably smaller than 70 nm, especially smaller than 50 nm (HDR measured by DLS).

[0403] The vaccine product of the present invention has high storage stability. When stored in the form of liquid or frozen material (storage temperature: -80°C, -20°C, 2-8°C or stored at room temperature for a long time, at least 3 months), almost no aggregation occurs, as evidenced by the fact that the particle size does not increase significantly (i.e., more than 10%) during storage.

[0404] The extremely high potency of such small particles prepared using the medium molecular weight component dextrin of 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 (about 150 kDa) and branched glucans (containing a β(1,3) main chain and β(1,6) side chains), such as scleroglucan or glucan 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) suggest that dectin-1 has no or only weak interactions with chalcone, and also no interactions with non-glucan carbohydrate polymers such as mannan. In fact, various references report that chalcone is poorly effective in binding dectin-1. However, in general, linear 1,3 and branched (1,3 backbone and 1,6 side chains) are the most effective dectin-1 binders; Adams et al. (2008) showed that mouse recombinant dectin-1 only recognizes and interacts with polymers containing a β(1,3)-linked glucose backbone. Dectin-1 does not interact with glucans containing only a β(1,6)-glucose backbone (such as pyrenoids) nor with non-glucan carbohydrate polymers (such as mannans).

[0405] In contrast to these findings, it was discovered during the course of the present invention that dactylin-based conjugates can strongly bind to dectin-1 in vitro and elicit cellular responses.

[0406] According to a preferred embodiment of the present invention, β-(1,6)-glucan is used. The prior art generally reports that large particles are more effective at activating PRRs than small ("soluble") monomer preparations, so particles containing large glucans are superior (and therefore preferred), while small soluble glucans can be used to block the activation of DCs, thereby interfering with the desired effect. It is well known that granulated 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 remains controversial. Although there is a general consensus 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 fail 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.).

[0407] According to the present invention, it can be demonstrated that conjugates using high molecular weight glucans (10 times the size of schisan; for example: oat / barley 229 kDa / lichenan 245 kDa) perform less effectively than schisan particles (20 kDa). Korotchenko et al. showed that OVA / Lam conjugates are approximately 10 nm in diameter, bind dectin-1 and induce DC activation in vitro, but they are branched glucans, have no skin specificity, and are no more effective in vivo than OVA applied to the skin or OVA / alum applied subcutaneously. Wang et al. provided β-glucan particles >100 nm (average size: 160 nm). Jin et al. (2018) demonstrated aminated β-glucan-ovalbumin nanoparticles of 180-215 nm in size.

[0408] The present invention found that pyrenoid-based particles are potent dectin-1 binders, activate DC in vitro (with changes in surface marker expression) and elicit very strong immune responses, superior to a) other routes and b) comparable to KLH / CRM conjugate vaccines (which are also generally much larger particles) and c) larger glucans and mannans. This was true for Pep+Padre+pyrenoid (size 5nm) and Pep+CRM+pyrenoid (size 11nm).

[0409] In order to obtain an optimal immune response, the degree of activation of CLECs, especially pyrenoids, and the peptide / sugar ratio resulting from this degree of activation are crucial. Activation of the corresponding CLECs can be achieved by mild periodate oxidation. Thus, the degree of oxidation depends on the addition of periodate solution at a specified molar ratio: i.e. periodate:sugar subunit; 100% = 1 mole of periodate per mole of sugar monomer.

[0410] According to a preferred embodiment, the conjugate of the present invention comprises activated CLEC, wherein the ratio of periodate to β-glucan or mannan (monomer) part is 1 / 5 (i.e. 20% activation) to 2.6 / 1 (i.e. 260% activation), preferably 60% to 140%, especially 70% to 100%.

[0411] An optimal range of oxidation degrees between low / medium and high oxidation degrees (which will be proportional to the amount of epitope polypeptide in the final conjugate) can be defined as a reactivity with Schiff's fuchsin reagent similar to the reactivity of an equal amount of a given carbohydrate (e.g., pyrenoids) 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.

[0412] The preferred ratio of glucan to peptide is 10 to 1 (w / w) to 1 to 1 (w / w), preferably 8 to 1 (w / w) to 2 to 1 (w / w), especially 4 to 1 (w / w); that is, the molar ratio of sugar monomer to peptide is 24 to 1, but if the conjugate contains 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), especially 10:1 to 0.1:1; this ratio is lower than that of other reported effective vaccines (e.g. Liang et al., Bromuro et al.).

[0413] The degree of oxidation and the amount of reactive aldehydes available for sugar coupling were determined using existing methods such as: 1) gravimetric measurement, which allows determination of the total mass of the sample; 2) the anthrone method (according to Laurentin et al. 2003) - used to determine the concentration of intact, unoxidized sugars in the sample; in this case, dextran was quenched with concentrated H 2 SO 4 Dehydration forms furfural, which reacts with anthrone (H 2 SO 4 0.2% )Condensation forms a green complex, which can be measured colorimetrically at 620nm) or 3) Schiff analysis: The oxidation state of the carbohydrate used for coupling is assessed using the Schiff fuchsin-sulfite reagent. Briefly, the fuchsin dye is decolorized by sulfur dioxide. Reaction with fatty aldehydes (on dextran) restores the purple color of the fuchsin, which can then be measured at 570-600nm. The color reaction produced is proportional to the degree of oxidation of the carbohydrate (the number of aldehyde groups). Other suitable analytical methods are also possible. Peptide ratios can be assessed using suitable methods, including UV analysis (205nm / 280nm) and amino acid analysis (aa hydrolysis, derivatization, and RP-HPLC analysis).

[0414] The conjugates of the invention can also be used to induce a target-specific immune response while inducing no or only very limited CLEC- or carrier protein-specific antibody responses. As shown in the Examples section below, the present invention can also improve and focus the target-specific immune response, since it triggers an immune response away from reactions to carrier proteins or CLECs (e.g. in conventional peptide-carrier conjugates or in comparable settings without conjugation, especially also using non-oxidized CLECs, such as pyruvic acid).

[0415] Unless otherwise specified, "peptide" as used herein refers to a shorter polypeptide chain (2-50 amino acid residues), while "protein" refers to a longer polypeptide chain (more than 50 amino acid residues). Both are referred to as "polypeptides". According to the present invention, the B cell and / or T cell epitope polypeptide coupled to CLEC includes, in addition to polypeptides containing amino acid residues naturally used in normal gene expression and protein translation, all other forms of such polypeptide-based B cell and / or T cell epitopes, especially their natural or artificially modified forms, such as glycopeptides and all other post-translationally modified forms (e.g., the pyroglutamate form of Aβ disclosed in the Examples). In addition, the CLEC of the present invention is particularly suitable for presenting conformational epitopes, such as conformational epitopes that are part of a larger natural polypeptide, a mimetic epitope, a cyclic polypeptide, or a surface-bound construct.

[0416] According to a preferred embodiment, the conjugate of the present invention comprises a CLEC polysaccharide backbone and a B cell epitope. A "B cell epitope" is a portion of an antigen that is bound by an immunoglobulin or antibody. B cell epitopes can be divided into two groups: conformational epitopes or linear epitopes. There are two main methods for epitope mapping: structural studies or functional studies. Methods for structural mapping of epitopes include X-ray crystallography, nuclear magnetic resonance, and electron microscopy. Methods for functional mapping of epitopes typically use binding assays such as western blots, dot blots, and / or ELISA to determine antibody binding. Competition methods determine whether two monoclonal antibodies (mAbs) can bind to an antigen simultaneously or compete with each other for binding at the same site. Another technique involves high-throughput mutagenesis, an epitope mapping strategy designed to improve rapid mapping of conformational epitopes of structurally complex proteins. Mutagenesis uses random / site-directed mutations at single residues to map epitopes. B cell epitope mapping can be used to develop antibody therapies, peptide-based vaccines, and immunodiagnostic tools (Sanchez-Trincado et al., J. Immunol. Res. 2017-2680160). For a variety of antigens, B cell epitopes are known and can be used in the current CLEC platform.

[0417] According to a particularly preferred embodiment, the conjugates of the invention comprise a CLEC polysaccharide backbone and one or more T cell epitopes, preferably promiscuous T cell epitopes and / or MHCII epitopes known to function with several / all MHC alleles of a given species as well as other species.

[0418] According to another aspect, the present invention also relates to improving known T cell epitopes using the present CLEC technology. Therefore, the present invention also includes β-glucan or mannan as a C-type lectin (CLEC) polysaccharide adjuvant for T cell epitope polypeptides, wherein β-glucan or mannan is covalently coupled to the T cell epitope polypeptide to form a conjugate of β-glucan or mannan and the T cell epitope polypeptide.

[0419] A single T-cell epitope that binds to more than one HLA allele is called a "promiscuous T-cell epitope". Preferred promiscuous T-cell epitopes bind to 5 or more, preferably 10 or more, and especially 15 or more HLA alleles. Promiscuous T-cell epitopes are applicable to different species, most importantly to several MHC / HLA haplotypes of a given species and other species (referring to MHC I and MHC II epitopes that are known to work with several / all MHC alleles). For example, the MHC II epitope PADRE (= non-natural pan-DR epitope (PADRE)) mentioned in the Examples section works in several human MHC alleles and mice (C57 / Bl6, but less effective in Balb / c). For example, the MHC II epitope PADRE (= non-natural pan-DR epitope (PADRE)) mentioned in the Examples section works in several human MHC alleles and mice (C57 / Bl6, but less effective in Balb / c). According to a preferred embodiment, the conjugate of the invention comprises a T cell epitope, preferably a T cell epitope comprising the amino acid sequence AKFVAAWTLKAAA ("PADRE (polypeptide)") or a PADRE (polypeptide) variant.

[0420] Preferred PADRE polypeptides or PADRE polypeptide variants include a linker (also preferred for other polypeptide epitopes used herein), such as a cysteine ​​residue or a linker comprising a cysteine ​​residue ("-C" or "C-"; particularly for maleimide coupling), NRRA, NRRA-C or NRRA-NH-NH2 linkers. Preferred PADRE polypeptide variants include variants disclosed in the prior art (e.g., Alexander et al., Immunity 1 (1994), 751-761; US ​​9,249,187 B2, or), preferably a shortened variant without a C-terminal A residue (AKFVAAWTLKAA), a variant in which the first residue alanine is substituted by an aliphatic amino acid residue (e.g., glycine, valine, isoleucine and leucine), a variant in which the third residue phenylalanine is substituted by L-cyclohexylalanine, a variant in which the thirteenth (last) amino acid residue alanine is substituted by an aliphatic amino acid residue (e.g., glycine, valine, isoleucine and leucine), a variant containing aminocaproic acid, preferably coupled to the C-terminus of the PADRE variant, or having the amino acid sequence AX 1 FVAAX 2 TLX 3 AX 4 A variant where X 1 Selected from W, F, Y, H, D, E, N, Q, I and K; X 2 Selected from F, N, Y and W, X 3 Selected from H and K, X 4Selected from A, D and E (provided that the oligopeptide sequence is not AKFVAAWTLKAAA; US 9,249,187 B2); in particular, the T cell epitope is selected from AKFVAAWTLKAAANRRA-(NH-NH2), AKFVAAWTLKAAAN-C, AKFVAAWTLKAAA-C, AKFVAAWTLKAAANRRA-C, aKXVAAWTLKAAaZC, aKXVAAWTLKAAaZCNRRA (SeqID7,8,87,88,89,90,91,92), aKXVAAWTLKAAa, aKXVAAWTLKAAaNRRA, aA(X)AAAK TAAAAa, aA(X)AAATLKAAa, aA(X)VAAATLKAAa, aA(X)IAAATLKAAa, aK(X)VAAWTLKAAa, and aKFVAAWTLKAAa (Sequences 760.5, 760.57, 906.09, 906.11, 965.10, 1024.03 of Alexander et al. 1994), 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.

[0421] T cell epitopes are presented on the surface of antigen presenting cells, where they bind to major histocompatibility complex (MHC) molecules. In humans, specialized antigen presenting cells exclusively 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 of 8–11 amino acids, while MHC class II molecules present longer peptides of 13–17 amino acids in length; nonclassical MHC molecules also present non-peptide epitopes, such as glycolipids. MHC class I and II epitopes can be reliably predicted by computational means alone, but not all T cell epitope in silico prediction algorithms are equally accurate. There are two main approaches to predict peptide-MHC binding: data-driven and structure-based. Structure-based approaches simulate peptide-MHC structures and require significant computational power. Data-driven approaches have higher prediction performance than structure-based approaches. Data-driven approaches predict peptide-MHC binding based on the sequence of the peptide that binds to the MHC molecule (Sanchez-Trincado et al., 2017). By identifying T cell epitopes, scientists can track, phenotype, and stimulate T cells. For many antigens, T cell epitopes are known and can be used in this CLEC platform.

[0422] Interestingly, recent breakthrough studies have shown that patients with Parkinson's disease (PD) have an increase in α-synuclein-specific T cells, which may be related to the risk haplotype of HLA, and also suggest that T cells are involved in autoimmunity in Parkinson's disease [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 confirmed by animal model studies [Williams et al., Brain. 2021;144:2047-2059]. A case study showed that the incidence of α-synuclein-reactive T cells increased several years before the onset of motor symptoms, and its frequency was highest around the time of motor symptoms and shortly thereafter in a large cross-sectional cohort of PD patients (Lindestam Arlehamn et al.). After the onset of motor symptoms, the T cell response to α-synuclein decreased with the duration of the disease. Thus, anti-aSyn T cell responses reach their highest levels before or shortly after PD diagnosis and gradually wane thereafter (i.e., maximal activity is detectable less than 10 years after diagnosis; Hoehn and Yahr (H+Y) stages 1 and 2 are preferred) (Lindestamn Arlehamn et al., 2020).

[0423] Thus, the human α-synuclein sequence contains common T cell epitopes. Examples are provided by Benner et al. (PLoS ONE 3(1):e1376.60), Sulzer et al. (2017), and Lindestam Arlehamn et al. (2020).

[0424] Benner et al. (Benner et al., (2008) PLoS ONE 3(1):e1376.) used a 60aa long, nitrated (at the Y residue) peptide containing the C-terminal portion of aSyn, which was emulsified in an equal volume of CFA containing 1 mg / ml Mycobacterium tuberculosis as an immunogen in a PD model. The article also disclosed the α-synuclein T cell epitope aa71-86 (VTGVTAVAQKTVEGAGNIAAATGFVK).

[0425] Sulzer et al. (Nature 2017; 546: 656-661) found two T cell antigenic regions in the N-terminal and C-terminal regions of α-synuclein in human PD patients. The first region is located near the N-terminus and contains MHCII epitopes aa31-45 (GKTKEGVLYVGSKTK) and aa32-46 (KTKEGVLYVGSKTKE), and also contains 9 peptides aa37-45 (VLYVGSKTK) as potential MHC class I epitopes. The second antigenic region disclosed by Sulzer et al. is located near the C-terminus (aa116–140) and requires phosphorylation of amino acid residue S129. Three phosphorylated aaS129 epitopes, aa116-130 (MPVDPDNEAYEMPSE), aa121-135 (DNEAYEMPSEEGYQD), and aa126-140 (EMPSEEGYQDYEPEA), generated significantly higher responses in PD patients than in healthy controls. The authors also demonstrated that the innate immune response to PD-associated α-synuclein has both MHC class I and class II restricted components.

[0426] In addition, Lindestam Arlehamn et al. (Nat Commun. 1875; 2020: 11) also disclosed α-synuclein peptide aa61-75 (EQVTNVGGAVVTGVT) as a T cell epitope (MHCII) for PD patients.

[0427] Accordingly, preferred T cell epitopes of the present invention include alpha synuclein polypeptide GKTKEGVLYVGSKTK

[0428] (aa31-45), KTKEGVLYVGSKTKE (aa32-46), EQVTNVGGAVVTGVT (aa61-75), VTGVTAVAQKTVEGAGNIAAATGFVK (aa71-86), DPDNEAYEMPSE (aa116-130), DNEAYEMPSEEGYQD (aa121-135), and EMPSEEGYQDYEPEA (aa126-140).

[0429] Regulatory T cells ("Treg cells" or "Tregs") are a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Treg cells have an immunosuppressive effect, usually inhibiting or downregulating the induction and proliferation of effector T cells. Tregs produced by the normal thymus are called "natural" Tregs. Selection of natural Tregs occurs on radioresistant hematopoietic-derived MHC class II expressing cells in the medulla or Hassal's corpuscles in the thymus. The process of Treg selection depends on the affinity of the interaction with the self-peptide-MHC complex. Selection to become a Treg is a "Goldilocks" process - not too high, not too low, but just right, T cells that receive very strong signals will undergo apoptosis; cells that receive weak signals will survive and be selected as effector cells. If a T cell receives an intermediate signal, it will become a regulatory cell. Due to the stochastic nature of the T cell activation process, all T cell populations with a given TCR will eventually become a mixture of Teffs and Tregs - the relative proportions are determined by the T cell's affinity for self-peptide-MHC. Tregs differentiated from immune naive T cells outside the thymus (ie, peripheral) or in cell culture are called "adaptive" or "induced" (ie, iTregs).

[0430] Natural Tregs are characterized by the expression of the CD4 T cell co-receptor and CD25, which is a component of the IL-2 receptor. Therefore, Tregs are CD4+CD25+. The expression of the nuclear transcription factor Forkhead box P3 (FoxP3) is a defining characteristic for the development and function of natural Tregs. Tregs inhibit the activation, proliferation, and cytokine production of CD4+ and CD8+ T cells and are thought to suppress B cells and dendritic cells, thereby inhibiting autoimmune responses.

[0431] Along these lines, several studies have shown that the number and function of Tregs in PD patients are reduced. 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 the ability of regulatory T cells (Tregs) in PD patients to inhibit effector T cell function is impaired, and the proportion of Th1 and Th17 cells increases, while the proportion of Th2 and Treg cells decreases. Thome et al. (npj Parkinson's Disease (2021) 7:41) showed that decreased PD Treg function is associated with increased activation of proinflammatory T cells, which can directly lead to subsequent increase in proinflammatory signals by other immune cell populations. Treg inhibition of T cell proliferation is significantly associated with peripheral proinflammatory immune cell phenotypes. Using the H&Y disease scale, the ability of PD Tregs to inhibit the proliferation of T effector cells (such as CD4+) decreases with increasing PD disease burden, with the highest activity in H+Y 1 and 2 stages. Importantly, Lindestam Arlehamn et al. (2020) showed that anti-aSyn T cell responses were highest before or shortly after diagnosis of motor PD and gradually declined thereafter (i.e., maximal activity was detectable less than 10 years after diagnosis; and Hoehn and Yahr (H+Y) stages 1 and 2 were preferred) (Lindestamn Arlehamn et al., 2020).

[0432] Therefore, the vaccine of the present invention is combined with

[0433] 1) a vaccine containing an alpha synuclein-specific Treg epitope (e.g., a CD4 epitope, 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

[0434] 2) Treg inducers, such as rapamycin, low-dose IL-2, TNF receptor 2 (TNFR2) agonists, anti-CD20 antibodies (such as rituximab), prednisolone, inosine pranobex, glatiramer acetate, sodium butyrate

[0435] It is preferred in the early stages of the disease (i.e., less than 10 years after diagnosis; and Hoehn and Yahr stages 1 and 2) to increase the number and activity of waning / declining Tregs, thereby reducing the autoimmune reactivity of aSyn-specific T effector cells and suppressing the autoimmune response in PD patients.

[0436] In addition, it has been found that Tregs are reduced and / or dysfunctional in a variety of diseases, especially chronic degenerative diseases or autoimmune diseases such as (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) as well as 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: Viglietta et 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)).

[0437] Therefore, it is also preferred to provide T cell epitopes suitable as Treg epitopes or Treg inducers in diseases where the Treg population is reduced or dysfunctional, as a combination with the vaccine of the present invention, so that the number and activity of reduced / decreased Tregs are increased, thereby reducing the autoimmune response of disease-specific T effector cells and inhibiting the patient's autoimmune response. Suitable Treg epitopes are defined as self-MHC epitopes (MHC II type), which are characterized by the ability to induce intermediate signals during T cell selection.

[0438] According to a preferred embodiment, the conjugate of the present invention comprises a polypeptide comprising or consisting of the amino acid sequence SeqID7, 8, 22-29, 87-131, GKTKEGVLYVGSKTK, KTKEGVLYVGSKTKE, ​​EQVTNVGGAVVTGVT, VTGVTAVAQKTVEGAGNIAAATGFVK, MPVDPDNEAYEMPSE), DNEAYEMPSEEGYQD, EMPSEEGYQDYEPEA or a combination thereof.

[0439] Therefore, preferred T cell epitopes are:

[0440]

[0441]

[0442] wherein X is L-cyclohexylalanine, Z is aminocaproic acid, and a is an aliphatic amino acid selected from the group consisting of alanine, glycine, valine, isoleucine and leucine.

[0443] According to another preferred embodiment, the conjugate of the present invention comprises B-cell epitopes and T-cell epitopes, preferably pan-specific / promiscuous T-cell epitopes, which are independently coupled to the CLEC polysaccharide backbone of the present invention, especially to Pseudomonas aeruginosa.

[0444] According to another preferred embodiment, the conjugate of the invention comprises a B-cell epitope coupled to a "classical" carrier protein, such as CRM197, the construct being further coupled to a CLEC carrier of the invention, in particular Pseudomonas aeruginosa.

[0445] For example, in the first step, a CRM conjugate can be formed by activating CRM such as GMBS or sulfo-GMBS; the maleimide group of the activated CRM then reacts 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. Subsequently, a one-pot reaction can be performed to mix the reduced CRM-conjugate with BMPH (N-β-maleimide-propionic acid hydrazide) and activated pyralidin (oxidized form) to generate a CLEC-based vaccine. The mechanism in the one-pot reaction may be that (in the case of pyralidin) oxidized pyralidin reacts with BMPH (having a hydrazide residue) and forms BMPH-hydrazone. The reduced CRM conjugate then reacts with the maleimide of BMPH-activated pyralidin via the SH group on the CRM-conjugate.

[0446] According to another preferred embodiment, the conjugate of the present invention comprises a "classical" carrier protein such as CRM197, which contains multiple T cell epitopes. The conjugate of the present invention also comprises a B cell epitope covalently coupled to a polysaccharide portion. In this embodiment, both polypeptides (B cell epitope and carrier molecule) are independently coupled to the CLEC carrier of the present invention (especially to Pseudomonas aeruginosa).

[0447] According to another preferred embodiment, the conjugate of the present invention further comprises a "classical" carrier protein such as CRM197, which contains multiple T cell epitopes. The conjugate of the present invention also comprises a B cell epitope covalently coupled to the "classical" carrier protein. The peptide-carrier conjugate of the present invention is also covalently coupled to the polysaccharide portion. In this embodiment, two polypeptides (B cell epitope and carrier molecule) are combined into a conjugate and coupled to the CLEC carrier of the present invention (especially to pyralidin). The carrier protein represents the connection between the β-glucan or mannan in the conjugate of the present invention and the B cell and / or T cell epitope polypeptide. The covalent coupling between the β-glucan or mannan and the B cell and / or T cell epitope polypeptide is then carried out by the carrier protein (as a functional linking part).

[0448] Preferred conjugates of the invention may comprise a B cell epitope coupled to CRM197, the construct further coupled to a CLEC polymer of the invention, in particular to a β-glucan, wherein the β-glucan is pyralidin, lichenan, laminarin, curdlan gum, β-glucan peptide (BGP), schizophyllan, scleroglucan, whole glucan particles (WGP), zymosan, or lentinan, preferably pyralidin, laminarin, lichenan, lentinan, schizophyllan or scleroglucan, in particular pyralidin.

[0449] The present invention found that the novel B cell epitope-CRM197 conjugate coupled to pyralidin is a potent dectin-1 binder, which can induce a very strong immune response and is superior to the traditional CRM conjugate vaccine.

[0450] The present invention has found that the conjugation of CLEC with a novel B cell epitope-CRM197 conjugate, in particular the generation of a B cell epitope-CRM197-dextran, more preferably a B cell epitope-CRM197-linear β-(1,6)-glucan or a B cell epitope-CRM197-chalcone conjugate, is essential for inducing the immunogenicity of various peptide-CRM197-CLEC (in particular, a peptide-CRM197-β-glucan, more preferably a peptide-CRM197-linear β-(1,6)-glucan or a peptide-CRM197-linear chalcone conjugate) that is superior to traditional CRM conjugate vaccines (with or without adjuvant, prepared by mixing with β-glucan / chalcone).

[0451] According to a preferred embodiment of the present invention, the CLEC conjugates of the present invention comprise oligosaccharides / polysaccharides as B cell epitopes coupled to carrier proteins as sources of T cell epitopes (e.g., CRM197, KLH, diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae protein D (HipD), 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)), the construct is further coupled to a CLEC polymer according to the invention, in particular to a β-glucan, which is truncatulan, lichenan, laminarin, curdlan gum, β-glucan peptide (BGP), schizophyllan, scleroglucan, whole glucan particles (WGP), zymosan or lentinan, preferably truncatulan, laminarin, lichenan, lentinan, schizophyllan or scleroglucan, in particular truncatulan. If the conjugate comprises a carrier protein, it is a preferred embodiment of the invention that the conjugate according to the invention comprises at least one additional, independently coupled T-cell or B-cell epitope. This preferred embodiment further clarifies that the present invention is not about eliciting specific antibodies against predominantly linear β-(1,6)-glucans, wherein the ratio of (1,6)-conjugated monosaccharide moieties to non-β-(1,6)-conjugated monosaccharide moieties is at least 1:1, such as pyruvic glycosides. Therefore, the present invention does not include conjugates containing predominantly linear β-(1,6)-glucans, wherein the ratio of (1,6)-conjugated monosaccharide moieties to non-β-(1,6)-conjugated monosaccharide moieties is at least 1:1, and which contain only carbohydrates as antigens and a carrier protein, because the conjugates of the present invention, if they contain additional T-cell or B-cell epitopes, significantly reduce or eliminate the induction of a strong de novo immune response against the glucan backbone in vivo (see, for example, Examples 7 and Figure 7 , below). In contrast, repeated application of uncoupled glucan (or glucan coupled only to a carrier protein) induced a strong anti-glucan immune response by increasing the level of antibodies against the glucan polysaccharide. This indicates that the conjugate of the present invention must have an additional T cell or B cell epitope polypeptide covalently bound to the conjugate of the predominantly linear β-(1,6)-glucan and the carrier protein. This also explains that the conjugate of the present invention does not include the prevention or treatment of diseases caused directly or indirectly by fungi (especially Candida albicans) by providing predominantly linear β-(1,6)-glucan (whose (1,6)-coupled monosaccharide portion to non-β-(1,6)-coupled monosaccharide portion ratio is at least 1:1) as an antigen (ultimately coupled to a carrier protein).

[0452] The present invention has found that such oligosaccharide / polysaccharide conjugate vaccines conjugated to Psoralea corylifolia are potent dectin-1 binders and can induce beneficial / effective immune responses if used in vivo.

[0453] Therefore, the present invention also relates to improving and / or optimizing carrier proteins by covalently coupling the carrier protein (already containing one or more T cell antigens (as part of its polypeptide sequence, optionally in a post-translationally modified form)) to the CLEC polysaccharide adjuvant of the present invention, i.e., β-glucan or mannan, preferably covalently coupled to pyralidin, lichenan, laminarin, curdlan gum, β-glucan peptide (BGP), schizophyllan, scleroglucan, whole glucan particles (WGP), zymosan or lentinan. Therefore, the present invention 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 coupled 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, wherein the carrier protein is covalently coupled to the β-glucan or mannan.

[0454] This improvement / optimization results in a significant reduction or elimination of B cell responses to CLEC and / or to the carrier protein, and / or an enhancement (or at least maintenance) of T cell responses to T cell epitopes of the carrier protein. This enables a reduction or elimination of antibody responses to CLEC and / or the carrier (the carrier then only transmits T cell responses), and a specific enhancement of antibody responses to the true target polypeptide coupled to the carrier and / or CLEC.

[0455] Therefore, a particularly preferred embodiment of the present invention is a conjugate consisting of or comprising

[0456] (a) β-Glucan

[0457] (b) at least one B-cell or T-cell epitope polypeptide, and

[0458] (c) a carrier protein,

[0459] The three components (a), (b) and (c) are covalently bound to each other.

[0460] This combination of three components can be provided in any orientation or order, i.e. in the order of (a)-(b)-(c), (a)-(c)-(b) or (b)-(a)-(c), wherein (b) and / or (c) can be covalently coupled from the N-terminus to the C-terminus or from the C-terminus to the N-terminus, or coupled via functional groups within the polypeptide (e.g. functional groups in lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, tyrosine, tryptophan or histidine residues, in particular the ε-amino group of lysine residues). Of course, the β-glucan can be coupled to one or more of each of components (b) and (c), preferably by the methods disclosed herein. Preferably, these components are coupled via a linker, in particular via a linker between all components (at least three). Preferred linkers are disclosed herein, such as cysteine ​​residues, or linkers comprising cysteine ​​or glycine residues, linkers resulting from hydrazide-mediated coupling, from coupling via heterobifunctional linkers (e.g., BMPH, MPBH, EMCH, or KMUH), from imidazole-mediated coupling, from reductive amination, from carbodiimide coupling -NH-NH 2 Connector; NRRA, NRRA-C or NRRA-NH-NH 2 Linkers, peptide linkers (e.g., dimers, trimers, tetramers (or longer polymers) peptide groups, such as CG or CG). For existing carrier proteins, especially CRM, CRM197 and KLH, the preferred order of the at least three components is (a)-(c)-(b), that is, β-glucan and at least one B cell or one T cell epitope polypeptide are coupled to the carrier protein.

[0461] According to another preferred embodiment, the conjugate of the invention comprises T cell epitopes and does not contain B cell epitopes, wherein the conjugate preferably comprises more than one T cell epitope, in particular two, three, four or five T cell epitopes. The construct is particularly suitable for cancer vaccines. The construct is also particularly suitable for autoantigens, in particular autoantigens associated with autoimmune diseases. The therapeutic effect of the corresponding conjugate is comparable to the reduction of effector T cells and regulatory T cells (T reg The presence of T reg cells in the brain is associated with the development of a population of T cells, which results in the suppression of the corresponding disease (e.g., autoimmune or allergic diseases), as shown in multiple sclerosis. Remarkably, these T reg cells perform powerful bystander immunosuppression, thereby ameliorating diseases caused by both cognate and noncognate self-antigens.

[0462] Preferred CLECs for use as the polysaccharide backbone of the present invention are pyracantha or other β-(1,6) glucans (including synthetic forms of such glucans); others that may be used are: mannan, members of the β-glucan family, especially linear β-(1,3) (Saccharomyces cerevisiae β-glucan (e.g., 150 kDa), curdlan gum) or glucans containing branched β-(1,3) and 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 gum (75-80 kDa or greater), β-(1,3) + β-(1,4) lichenan (22-250 kDa) β-(1 ,6) pyrenoids (20 kDa). Therefore, preferred CLECs according to the present invention are mannans and β-glucans, including linear and branched β-glucans characterized by having β-(1,3)-, β-(1,3)+β-(1,4)- and β(-1,6) main chains and attached side chains with β-(1,6) residues, more preferably linear β-glucans containing β-(1,3), β-(1,3)+β-(1,4) and β-(1,6) chains, more preferably linear β-(1,6) β-glucans, especially pyrenoids, their fragments or synthetic variants consisting of polymeric β-(1,6)-glucans (e.g. 4-mers, 5-mers, 6-mers, 8-mers, 10-mers, 12-mers, 15-mers, 17-mers or 25-mers).

[0463] Preferably, the minimum length of the CLEC of the present invention is 6-mer, since the oxidation reaction performed according to the present invention is problematic for smaller polysaccharides (ultimately other coupling mechanisms can be used for such smaller forms and / or end-linking by adding reactive forms). CLECs with 6 or more monomer units (i.e., 6-mers and larger) show good dectin binding. In general, the longer the CLEC, the better the dectin binding. A degree of polymerization (i.e., the number of individual glucose molecules within one glucan entity, DP) of 20-25 (i.e., DP20-25) certainly ensures good binding and in vivo efficacy (e.g., laminarin is a typical example of DP20-30).

[0464] The molecular weight of synthetic CLEC can also be smaller, for example, as low as 1-2 kDa, while the preferred molecular weight range of glucan and its fragments can be 1-250 kDa (e.g., laminarin, lichenan, Saccharomyces cerevisiae β-glucan, pyruvate, curdlan gum and barley glucan, etc.), preferably 4.5-80 kDa (e.g., laminarin, pyruvate, curdlan gum, low molecular weight lichenan, etc.), especially 4.5-30 kDa (e.g., laminarin, pyruvate, low molecular weight lichenan, etc.). Mannans are linear polymers of mannose. Plant mannans have β-(1,4) bonds. They are a storage sugar form. Mannan cell wall polysaccharides found in yeast have an α-(1,6) linked backbone and α-(1,2) and α-(1,3) linked branches. It is serologically similar to the structure on mammalian glycoproteins.

[0465] In order to produce the conjugates of the invention, CLECs, especially pyralidin, must be activated (e.g., by using mild periodate-mediated oxidation), the degree of oxidation being important for the immune response. As described above, a practical range of oxidation (particularly for pyralidin) is about 20-260% oxidation. In many cases, the optimal range of oxidation is between low / moderate oxidation (i.e., 20-60% oxidation) and high oxidation (i.e., 140-260% oxidation), i.e., in the range of 60-140% oxidation. Optimization of other CLECs can be readily adapted by those skilled in the art, e.g., for lichenan, more than 200% is required to obtain a similar amount of aldehyde groups.

[0466] Therefore, the range can also be alternatively defined as the reactivity with Schiff's red reagent, using agarose as an example, as follows: low / medium oxidation at a molar ratio (sugar monomer: periodate) of 0.2-0.6, the optimal range 0.6-1.4, and high oxidation at 1.4-2.6.

[0467] In any case, the degree of oxidation should be defined to meet the optimal range for each specific CLEC. Preferably, the linear β-glucan, more preferably β-(1,6)β-glucan, in particular chrysanthemum, chrysanthemum fragments or synthetic variants thereof consisting of polymeric β(1,6)-glucan (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, resulting in cleavage of the ortho-OH groups, thereby generating the active aldehyde. Mild periodate oxidation refers to the use of sodium periodate (NaIO 4), a well-known mild reagent, effectively oxidizes vicinal diols in carbohydrate sugars to generate reactive aldehyde groups. The carbon-carbon bonds between adjacent hydroxyl groups are cleaved. By varying the amount of periodate, aldehydes can be introduced stoichiometrically into a smaller or larger number of sugar moieties of a given polysaccharide.

[0468] Other exemplary methods for activating carbohydrates are well known in the art and include cyanation of hydroxyl groups (e.g., by using organic cyanation reagents such as 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP) or N-cyanotriethylammonium tetrafluoroborate (CTEA)), reductive amination of carbohydrates, or activation and coupling using carboxylic acid reactive chemical groups such as carbodiimides.

[0469] The activated carbohydrate then reacts with the polypeptide to be conjugated to the activated CLEC to form a conjugate of CLEC and the B cell or T cell epitope polypeptide.

[0470] Therefore, the present invention also relates to a method for producing the conjugate of the present invention, wherein β-glucan or mannan is activated by oxidation, and the activated β-glucan or mannan is contacted with a B cell and / or T cell epitope polypeptide to obtain a conjugate of β-glucan or mannan and a B cell and / or T cell epitope polypeptide.

[0471] Preferably, the β-glucan or mannan is obtained by periodate oxidation, reductive amination or cyanation of hydroxyl groups at vicinal positions.

[0472] According to a preferred embodiment, the β-glucan or mannan is oxidized to a degree of oxidation, defined as a reactivity with Schiff's fuchsin reagent, corresponding to the degree of oxidation of an equivalent amount of pyrenoids oxidized by periodate, in a molar ratio of 0.2-2.6, preferably 0.6-1.4, especially 0.7-1.

[0473] Preferably, the conjugate is produced by coupling a hydrazide to a carbonyl (aldehyde) via a hydrazone-based coupling, or by coupling a sulfhydryl (e.g., cysteine) to a carbonyl (aldehyde) via a heterobifunctional maleimide-and-hydrazide linker (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).

[0474] The polypeptide to be coupled to the CLEC of the present invention is or comprises at least one B cell or at least one T cell epitope. Preferably, the polypeptide coupled to the CLEC comprises a single B cell or T cell epitope (even in embodiments where more than one polypeptide is coupled to the CLEC polysaccharide backbone). As shown in the Examples section, the preferred length of the polypeptide is 5-29 amino acid residues, preferably 5-25, more preferably 7-20, even more preferably 7-15, especially 7-13 amino acid residues. In this regard, it is important to note that these length ranges are only for epitope sequences and do not include linkers, including peptide linkers, such as cysteine ​​or glycine or di-, tri-, tetra-(or longer) polypeptide groups, such as CG or CG, or cleavage sites, such as cathepsin cleavage sites; or combinations thereof (e.g., -NRRAC). Examples of epitopes have been tested in the Examples section; it can be seen from these results that the platform of the present invention is not limited to any particular polypeptide. Therefore, almost all possible epitopes are suitable for the present invention, including those known in the art, especially those that have been described as being integrable into presentation platforms (e.g., with "classical" carrier molecules or adjuvants).

[0475] It is particularly preferred if the epitope can be coupled to the activated β-glucan based on existing coupling methods, including hydrazide-mediated coupling, coupling via heterobifunctional linkers (e.g., BMPH, MPBH, EMCH, KMUH, etc.), imidazole-mediated coupling, reductive amination, carbodiimide coupling, etc. (more to be added). The epitope used comprises a single peptide, which can be contained in a peptide or protein, or can be presented as a peptide-protein conjugate and then coupled to CLEC.

[0476] Thus, preferred coupling methods for providing conjugates of the invention are hydrazide coupling or coupling using thioester formation (e.g., maleimide coupling using BMPH (N-β-maleimidopropionic acid hydrazide), MPBH, EMCH, KMUH), especially wherein the pyruvic polysaccharide is coupled to BMPH via hydrazone formation and the polypeptide is coupled via thioester.

[0477] In this embodiment, it is preferred to provide a polypeptide with two preferred linkers, such as a hydrazide polypeptide / epitope for hydrazone coupling:

[0478] N-terminal coupling of peptide: H 2 N-NH-CO-CH 2 -CH 2 -CO-polypeptide-COOH; preferably in combination with succinic acid or an alternative suitable linker, such as other suitable dicarboxylic acids, in particular also glutaric acid as a spacer / linker;

[0479] C-terminal coupling (preferred coupling direction of the present invention): NH 2-Peptide-NH-NH 2 .

[0480] Alternatively, unmodified polypeptides / epitopes may be used in the present invention, for example, polypeptides containing (additional) cysteine ​​residues or another source of SH groups at the C or N termini for heterobifunctional linker-mediated coupling (particularly BMPH, MPBH, EMCH, KMUH): NH 2 -Cys-Pep-COOH or NH 2 -Pep-Cys-COOH.

[0481] The length of the B cell polypeptide preferably used in the present invention is 5-19 amino acid residues, preferably 6-18, especially 7-15 amino acid residues. The B cell epitope is preferably a short linear polypeptide, a glycopeptide, a lipopeptide, other post-translationally modified polypeptides (e.g., phosphorylated, acetylated, nitrated, containing pyroglutamic acid residues, glycosylated, etc.), a cyclic polypeptide, etc.

[0482] Preferred B cell epitopes are B cell epitopes representing autoantigens, B cell epitopes representing antigens present in tumor 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 conformational epitopes, B cell epitopes representing carbohydrate epitopes, immobilized / coupled to polypeptides or proteins to form multivalent B cell epitope-protein / polypeptide conjugates suitable for CLEC coupling, including carrier molecules such as B cell epitopes of 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); non-peptide antigens (including linear polypeptides, polypeptides representing conformational epitopes, mimetic epitopes or polypeptide variants from natural epitopes / sequences, glycopeptides, lipopeptides, other post-translationally modified peptides (e.g., phosphorylated, acetylated, containing pyroglutamic acid residues, etc.), cyclic polypeptides, etc.) that can be coupled to the active aldehydes present on pyrocatechol / CLEC.

[0483] The T cell polypeptide preferably used in the present invention has a length of 8-30 amino acid residues, preferably 13-29 amino acid residues, and more preferably 13-28 amino acid residues.

[0484] The preferred specificity of T cell epitopes used in the present invention is a linear short peptide suitable or known to be suitable for presentation through MHC I and II (as known to those skilled in the art), especially MHCII epitopes for CD4 effector T cells and CD4 Treg cells, MHC I epitopes for cytotoxic T cells (CD8+) and CD8 Treg cells, for example, can be used for cancer, autoimmunity or infectious diseases, and are known to be effective in humans or animals; a linear short peptide suitable for presentation through MHC I and II (as known to those skilled in the art), which adds a lysosomal protease cleavage site at the N-terminus or C-terminus, specifically a cathepsin family member-specific site, more specifically a site for cysteine ​​cathepsins, such as cathepsins B, C, F, H, K, L, O, S, V, X and W, especially cathepsin S- or L-, most preferably a cathepsin L cleavage site, promoting effective endo / lysosomal release of the peptide for MHC presentation, especially MHCII, with known efficacy in humans or animals. Cathepsin cleavage sites in various proteins have been identified and are well known in the art. This includes literature on sequences or methods for identifying such sequences: for example: Biniosek 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, etc. Specifically, the adaptation of the peptide sequence with an artificial protease cleavage site as shown in the present invention is based on the surprising effect of these sequence extensions: when the antigen is coupled to CLEC, the CLEC vaccine of the present invention induces a more effective immune response after skin administration. The vaccine of the present invention is taken up by dendritic cells, and then the peptide antigen is processed by lysosomes and presented to MHC.

[0485] Lysosomes are membrane-bound organelles within cells that are characterized by an acidic interior and carry a variety of hydrolases, including lipases, proteases, and glycosidases, which are involved in cellular catabolism. Among the various enzymes carried by lysosomes, cathepsins are a family of lysosomal proteases with a broad spectrum of functions. All cathepsins are divided into three different protease families: serine proteases (cathepsins A and G), aspartic proteases (cathepsins D and E), and 11 cysteine ​​cathepsins. In humans, 11 cysteine ​​cathepsins are known, which also have structures similar to papain: cathepsins B, C (J, dipeptidyl peptidase I or DPPI), F, H, K (O2), L, O, S, V (L2), X (P, Y, Z), and W (lymphoplasmin).

[0486] The various cathepsins show similarities in cellular localization and biosynthesis, but there are some differences in expression patterns. Among all lysosomal proteases, cathepsins L, B, and D are the most abundant, and their lysosomal concentrations are equivalent 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 mainly expressed in immune cells.

[0487] In addition to playing a major role in lysosomal protein recycling, cathepsins also play an important role in various physiological processes. Cathepsin S is the main protease involved in MHC II antigen processing and presentation. Mice without cathepsin S show significant differences in the generation of MHC II-bound li fragments and presentation, because where cathepsin S is abundantly expressed, li degradation in professional APCs is significantly reduced. In addition, endocytosis selectively targets exogenous substances to cathepsin S in human dendritic cells. Enrichment of MHC II molecules in late endocytic structures is also persistently seen in splenic DCs of mice lacking cathepsin S. Recent studies have shown that cathepsins B and D are both involved, but are not essential for MHC II-mediated antigen presentation. Cathepsin L also plays a role in a wide variety of cellular processes, including antigen processing, tumor invasion and metastasis, bone resorption, and turnover of intracellular and secreted proteins involved in growth regulation. Although cathepsin L is generally considered a lysosomal protease, it is also a secreted enzyme. This broad-spectrum protease can effectively degrade a variety of extracellular proteins (laminin, fibronectin, collagen I and IV, elastin, and other structural proteins of the basement membrane) as well as serum proteins, cytoplasmic proteins, and nuclear proteins.

[0488] As a novel means to enhance the potency of T cell epitopes in vaccines, especially CLEC-based vaccines, the addition of a lysosomal protease cleavage site at the N-terminus or C-terminus is a preferred embodiment of the present invention.

[0489] Such cleavage sites according to the invention may have the following characteristics:

[0490] Cathepsin L-like cleavage site:

[0491] The expected cathepsin L-like cleavage site is defined according to the protease cleavage site sequences known in the art, particularly 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. The preferred consensus sequence of the C-terminal cathepsin L site consists of the following formula:

[0492] X n -X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8

[0493] X n : 3-27 amino acids from an immunogenic peptide

[0494] X 1 : Any amino acid

[0495] X 2 : Any amino acid

[0496] X 3 : Any amino acid

[0497] X 4 : N / D / A / Q / S / R / G / L; preferably N / D, more preferably N

[0498] X 5 : F / R / A / K / T / S / E; preferably F or R, more preferably R

[0499] X 6 : F / R / A / K / V / S / Y; preferably F or R, more preferably R

[0500] X 7 : Any amino acid, preferably A / G / P / F, more preferably A

[0501] X 8: Cysteine ​​or linker like NHNH 2

[0502] The most preferred sequence: X n -X 1 X 2 X 3 NRRA-Connector

[0503] Cathepsin S-like cleavage site:

[0504] The expected cathepsin S cleavage site is based on protease cleavage site sequences known in the art, in particular those disclosed in Biniossek et al. (J. Proteome Res. 2011, 10, 5363-5373) and https: / / en.wikipedia.org / wiki / Cathepsin_S, characterized by the consensus sequence:

[0505] X n -X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8

[0506] The characteristics of X are

[0507] X n : 3-27 amino acids from an immunogenic peptide

[0508] X 1 : Any amino acid

[0509] X 2 : Any amino acid

[0510] X 3 : any amino acid, preferably V, L, I, F, W, Y, H, more preferably V

[0511] X 4 : any amino acid, preferably V, L, I, F, W, Y, H, more preferably V

[0512] X 5 : K, R, E, D, Q, N, preferably K, R, more preferably R

[0513] X 6 : Any amino acid

[0514] X 7 : Any amino acid, preferably A

[0515] X 8:Preferred A

[0516] X 8 : Cysteine ​​or linker like NHNH 2

[0517] The most preferred sequence: X n -X 1 X 2 VVRAA-Connector

[0518] T cell epitopes contained in proteins suitable for coupling to CLECs include carrier proteins, particularly non-toxic cross-reactive substances of diphtheria toxin (CRM), particularly CRM 197 , KLH, diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae protein D (HipD), group B meningococcal outer membrane protein complex (OMPC), recombinant atoxic form of Pseudomonas aeruginosa exotoxin A (rEPA), flagellin, Escherichia coli heat-labile enterotoxin (LT), cholera toxin (CT), mutant toxins (such as LTK63 and LTR72), virus-like particles, albumin binding protein, bovine serum albumin, ovalbumin, synthetic peptide dendrimers, such as multiple antigenic peptides (MAP) or other commercially available carrier proteins, preferably CRM197 and KLH, most preferably CRM197, preferably 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.

[0519] According to a preferred embodiment of the present invention, the CLEC conjugate of the present invention comprises (a) CLEC coupled to a single B- and / or T-cell epitope, including a mixture of multiple B- or T-cell epitopes, especially these epitopes coupled to pyralidin; (b) CLEC coupled to a polypeptide-carrier protein conjugate, preferably a polypeptide-KLH or polypeptide CRM197 conjugate coupled to pyralidin, most preferably a polypeptide-CRM197 conjugate coupled to pyralidin; (c) CLEC coupled to a single B- and T-cell epitope from a self protein (cancer) or pathogen (infectious disease), not a promiscuous MHC / HLA specific but a known disease-specific T-cell epitope; coupled to CLEC, most preferably coupled to pyralidin; d) CLEC is singly ("singly" herein means that the polypeptide chains are not present in the form of fusion proteins, tandem repeat polypeptides or peptide-protein conjugates, but exist as independent entities; i.e., an independent polypeptide containing B cell epitopes and an independent polypeptide containing T cell epitopes) coupled to B cell epitopes and T cell epitopes contained in polypeptides or proteins, such as carrier proteins, self proteins, foreign proteins from pathogens, allergens, etc.; (e) CLEC is singly ("single", also has the same meaning as (d)) coupled to T cell epitopes representing linear MHC I and MHC II epitopes or T cell epitopes contained in proteins, such as carrier proteins or target proteins, for example, for the treatment of tumor diseases or autoimmune diseases.

[0520] In view of these favorable properties of the conjugate of the present invention, the conjugate and vaccine of the present invention are particularly suitable for active anti-Aβ, anti-Tau and / or anti-α-synuclein vaccines for the treatment and prevention of β-amyloidosis, Tau disease or synuclein disease, preferably Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), Parkinson's disease dementia (PDD), axonal dystrophy, Alzheimer's disease (AD), amygdala-restricted Lewy body dementia (AD / ALB), Down syndrome dementia, Pick's disease, progressive supranuclear palsy (PSP), cortical basal degeneration, frontotemporal dementia and Parkinson's disease associated with chromosome 17 (FTDP-17) and argyrophilic grain disease.

[0521] Therefore, the conjugate of the present invention is particularly suitable for preventing or treating diseases, for example for humans, mammals or birds, in particular for treating and preventing human diseases. Therefore, one aspect of the present invention is the use of the conjugate in the medical field as a medical indication. The present invention relates to the use of the conjugate of the present invention for treating or preventing diseases. The present invention therefore also relates to the use of the conjugate of the present invention for preparing a medicament for preventing or treating a disease, preferably for preventing or treating an infectious disease, a chronic disease, an allergy or an autoimmune disease. Accordingly, the present invention also relates to a method for preventing or treating a disease, preferably for preventing or treating an infectious disease, a chronic disease, an allergy or an autoimmune disease, wherein an effective amount of the conjugate of the present invention is administered to a patient in need.

[0522] According to another aspect, the novel glycoconjugates of the invention can be used for the prevention of infectious diseases, preferably with the proviso that the prevention or treatment of diseases caused directly or indirectly by fungi, in particular Candida albicans, is excluded by providing as antigens mainly linear β-(1,6)-glucans with a ratio of (1,6)-conjugated monosaccharide moieties to non-β-(1,6)-conjugated monosaccharide moieties of at least 1:1, ultimately conjugated to a carrier protein. Such diseases are, for example, microbial infections, such as those caused by Haemophilus influenzae type b (Hib), Streptococcus pneumoniae, Neisseria meningitidis and Salmonella typhi or other infectious agents.

[0523] According to another aspect, the present invention also relates to a pharmaceutical composition comprising the above-mentioned conjugate or vaccine and a pharmaceutically acceptable carrier.

[0524] Preferably, the pharmaceutically acceptable carrier is a buffer, preferably a phosphate or TRIS-based buffer.

[0525] According to a preferred embodiment of the present invention, the pharmaceutical composition is contained in a needle-based delivery system, preferably a syringe, a microneedle system, a hollow needle system, a solid microneedle system, or a system containing 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 soluble MAP (D-MAP); an electrophoretic system, an iontophoretic system, a laser-based system, especially an erbium YAG laser system; or a gene gun system. The conjugate of the present invention is not limited to any form of production, storage or delivery state. Therefore, all traditional and typical forms are suitable for the present invention. Preferably, the composition of the present invention can contain the conjugate or vaccine of the present invention as a solution or suspension, a deep-frozen solution or suspension; a lyophilizate, a powder or granules.

[0526] The present invention is further illustrated by the following examples and drawings, but is not limited thereto. BRIEF DESCRIPTION OF THE DRAWINGS

[0527] Figure 1 : Binding activity of CLEC conjugates to ConA and DC receptor (i.e., dectin-1) in vitro A) Pus binds dectin-1 more efficiently than Lichenan (Lich); B) Oat β-glucan (oat_BG265, oat_BG391) and barley β-glucan (Barley_BG229) have limited binding efficacy compared to Pus. C) Different types of glucans (i.e., Pus, mannan, and barley glucan (229 kd)) retain high or moderate receptor binding activity after glucan oxidation, as assessed by competitive binding assays. "20% and 40% oxidation" indicates the oxidation state of the glucan moieties used for conjugation. % inhibition represents the inhibition of binding of soluble dectin-1 receptor (Pseudo-glucan and Barley_BG229) or ConA (mannan) to plate-bound β-glucan or mannan in the presence of the indicated concentrations of the tested CLECs. D) Pseudo-glucan conjugates and E) lichenan conjugates retain approximately 50% of dectin-1 binding capacity compared to unconjugated β-glucan as assessed by competition binding assays. F) Pseudo-glucan conjugates generated via heterobifunctional linkers retain high binding potency for dectin-1. Data are presented as relative light units (RLU) of a luminol-doped ELISA. Pus70 conjugates 1-3 refer to three different CLEC peptide conjugates (SeqID2, SeqID10, and SeqID16), respectively. Pus 70% and Lich 200% refer to Pseudo-glucan and Lichenan in their respective oxidized states. BMPH Pus refers to activated Pseudo-glucan. BMPH conjugate 2 refers to the CLEC-SeqID10 conjugate.

[0528] Figure 2 : Flow cytometric analysis of dendritic cells activated by lipopolysaccharide (LPS) and different pyrin preparations.

[0529] Immature, bone marrow-derived mouse dendritic cells (BMDC) were generated in vitro with granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF-BMDC were stimulated for 24 hours with LPS (equivalent to the dose contained in oxidized chrysanthemum and in chrysanthemum conjugate preparations), SeqID2+SeqID7+chrysanthemum conjugate, or oxidized chrysanthemum alone. Dose escalation (up to 500 μg / mL) was started for chrysanthemum conjugate and chrysanthemum alone, each starting at 62.5 μg / mL. DCs were identified based on CD11c / CD11b expression, and surface expression of CD80 and major histocompatibility complex (MHC) class II was measured by flow cytometry by A) and C) SeqID2+SeqID7+chrysanthemum conjugate or B) and D) oxidized chrysanthemum alone. For DCs treated with the pyrenoid preparation (= measured values) and DCs treated with an equal amount of LPS (= expected values), the expression of activation markers was analyzed using the CytExpert software.

[0530] Figure 3 : The particle size of CLEC conjugates was determined by dynamic light scattering (DLS).

[0531] Particle size was determined by measuring the random variation in the intensity of light scattered from a suspension or solution using DLS. Regularisation analysis and the corresponding 24 h cumulative radius analysis are shown for A) SeqID5+SeqID7+Pseudomonas aeruginosa (80% oxidized state) conjugate, B) SeqID6+CRM+Pseudomonas aeruginosa conjugate and C) unmodified Pseudomonas aeruginosa, respectively.

[0532] Figure 4 : Comparison of the immunogenicity of different CLEC-based vaccines.

[0533] Female BALB / c mice aged 8-12 weeks were vaccinated intradermally for a total of 3 times at 2-week intervals. Blood samples were collected at the start of vaccination and after each vaccination to obtain information on the kinetics of the ensuing immune response. Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide (SeqID3) responses to mannan, barley and pyralidin-based vaccines (SeqID2+SeqID7+CLEC) and B) anti-peptide (SeqID3 and SeqID11) responses to pyralidin-based and lichenin-based vaccines (SeqID2+SeqID7+CLEC and SeqID10+SeqID7+CLEC).

[0534] Figure 5 : Comparative analysis of the immunogenicity of peptide-pastelan conjugates and vaccines consisting of unconjugated peptide and CLEC.

[0535] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Samples were collected 2 weeks after the third vaccination and analyzed for anti-peptide responses (SeqID3). Vaccines used: SeqID2+SeqID7+CLEC or a mixture of unconjugated SeqID2, SeqID7, and CLEC.

[0536] Figure 6 : Comparative analysis of the immunogenicity of pyrifos polysaccharide conjugates containing both B-cell epitopes and T-cell epitopes and conjugates containing only B-cell epitopes or only T-cell epitopes.

[0537] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Vaccines used: SeqID5+SeqID7+CLEC or SeqID5+CLEC, and SeqID7+CLEC. Samples were collected 2 weeks after the third vaccination and analyzed for anti-peptide responses (SeqID6).

[0538] Figure 7 :Comparative analysis of anti-pastosan antibody responses in mice after repeated immunization with peptide-pastosan conjugates or vaccines containing the respective unconjugated components

[0539] Female BALB / c mice, 8-12 weeks old, received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Pre-plasma and t1-t3 represent the immune response detectable before vaccination (pre-plasma) or after the first vaccination (t1), the second vaccination (t2), or the third vaccination (t3). Samples were collected 2 weeks after the third vaccination and analyzed for anti-pasteuran responses. A) Analysis of anti-pasteuran responses induced by different vaccines. B) Kinetics of immune responses. C) Inhibition ELISA demonstrates the specificity of the ELISA system. Vaccines used: SeqID2+SeqID7+CLEC or a mixture of unconjugated SeqID2, SeqID7, and CLEC

[0540] Figure 8 : Comparative analysis of immune responses elicited by CLEC-based vaccines using differentiated peptide conjugation orientations.

[0541] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Four different CLEC-based prototype vaccine candidates were tested (two different peptides, conjugated to pyralidin via their C-terminus or N-terminus). Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and B) anti-aSyn protein responses. Vaccines used: SeqID1 / 2 / 4 / 5+SeqID7+CLEC

[0542] Fig. 9 : Comparative analysis of the immunogenicity of CLEC-based vaccines using different promiscuous T helper cell epitopes.

[0543] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Nine different CLEC-based vaccines (vaccines 1-9) containing the same B cell epitope and different T helper epitopes (i.e., SeqID7, SeqID22-29) were evaluated for immune responses against the respective peptide-KLH conjugates (vaccine 10). Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and B) anti-aSyn protein responses.

[0544] Fig.10 : Comparative analysis of target-specific and carrier protein-specific immunogenicity induced by CLEC-based vaccines using carrier protein KLH as a source of T helper cell epitopes and traditional peptide-protein conjugate vaccines.

[0545] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal or subcutaneous (sc) vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. The immune responses elicited by two peptide-protein conjugate vaccines using KLH as a source of T helper epitopes in combination with CLEC modification (SeqID3+KLH+Alumin and SeqID6+KLH+Alumin, respectively) were evaluated and compared with conventional peptide-KLH conjugates (i.e., SeqID3+KLH and SeqID6+KLH) in combination with Alum / Responses elicited by subcutaneous vaccination (sc) or intradermal vaccination (id) without additional adjuvant were compared. Samples were collected 2 weeks after the third vaccination and analyzed by ELISA for A) anti-peptide and anti-aSyn protein responses and B) anti-KLH responses.

[0546] Fig.11:Comparative analysis of target-specific and carrier protein-specific immunogenicity induced by CLEC-based vaccines with carrier protein CRM197 as the source of T helper cell epitopes and traditional peptide-protein conjugate vaccines

[0547] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal or subcutaneous vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Two different CRM-based vaccine types were used in this study. SeqID6+CRM+Pus represents a peptide-CRM conjugate that was subsequently coupled to pyruvic acid, while SeqID5+CRM+Pus represents a conjugate in which the peptide component and the carrier molecule were coupled to CLEC, respectively. The immune response induced by both types was evaluated against the respective conventional peptide-CRM conjugates (i.e., SeqID6+CRM adjuvanted with Alum / Alhydrogel, administered subcutaneously). Samples were collected 2 weeks after the third vaccination and analyzed by ELISA for A) anti-peptide and anti-aSyn protein responses and B) anti-CRM responses.

[0548] Fig.12 : Comparative analysis of the selectivity of immune responses elicited by CLEC-based vaccines against two different aSyn forms in vivo.

[0549] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal or subcutaneous vaccinations at 2-week intervals. CLEC-based vaccines (SeqID2+SeqID7+Pus and SeqID5+SeqID7+Pus; intradermal) and alternative CLEC-based vaccines (SeqID3+KLH+Pus and SeqID6+CRM+Pus; intradermal) were evaluated compared to traditional peptide-component vaccines (SeqID3+KLH+Alum and SeqID6+CRM+Alum, subcutaneous). Samples were collected 2 weeks after the third vaccination for aSyn selectivity assay (inhibition ELISA). Black line: monomeric aSyn for inhibition; dashed line: filamentous aSyn for inhibition.

[0550] Fig.13 : Comparative analysis of the avidity of immune responses elicited by CLEC-based vaccines.

[0551] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal or subcutaneous vaccinations at 2-week intervals. CLEC-based vaccines (SeqID2+SeqID7+Pus and SeqID5+SeqID7+Pus, intradermal) and alternative CLEC-based vaccines (SeqID3+KLH+Pus and SeqID6+CRM+Pus, intradermal) were evaluated compared to traditional peptide-component vaccines (SeqID3+KLH+Alum and SeqID6+CRM+Alum, subcutaneous). Samples were collected 2 weeks after the second immunization (T2) or 2 weeks after the third immunization (T3) to assess antibody affinity for aSyn by ELISA-based affinity assays.

[0552] Fig.14 : Comparative analysis of the affinity of immune responses elicited by CLEC-based vaccines.

[0553] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal or subcutaneous vaccinations at 2-week intervals. CLEC-based vaccines (SeqID2+SeqID7+Pus and SeqID5+SeqID7+Pus, intradermal vaccination) and alternative CLEC-based vaccines (SeqID3+KLH+Pus and SeqID6+CRM+Pus, intradermal vaccination) were evaluated compared to traditional peptide-component vaccines (SeqID3+KLH+Alum and SeqID6+CRM+Alum, subcutaneous vaccination). Samples were collected 2 weeks after the third vaccination and the antibody equilibrium dissociation constant (Kd) against aSyn was assessed by aSyn displacement ELISA assay.

[0554] Fig.15 : Comparative analysis of the in vitro functionality of immune responses elicited by CLEC-based vaccines.

[0555] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal and subcutaneous vaccinations at 2-week intervals. Samples were collected 2 weeks after the third vaccination and modulation of aSyn aggregation in the presence of aSyn-specific antibodies was assessed by ThT fluorescence assay. A) Aggregation of aSyn for 0-72 hours in the presence of CLEC vaccine-induced antibodies (SeqID2+SeqID7+Pus; intradermal vaccination), traditional peptide-component-induced antibodies (SeqID3+KLH+Alum, subcutaneous vaccination), or mouse plasma. B) Aggregation of aSyn or aSyn with preformed fibrils for 0-92 hours in the presence of CLEC vaccine-induced antibodies (SeqID5+SeqID7+Pus and SeqID6+CRM+Pus, both intradermal vaccination), traditional peptide-component-induced antibodies (SeqID6+CRM+Alum, subcutaneous vaccination), or mouse plasma. Kinetic curves were calculated by normalizing to the ThT fluorescence at t0, and the percentage inhibition of aSyn aggregation was calculated using the slope values ​​extracted from the linear regression analysis of the exponential growth phase of ThT kinetics.

[0556] Fig.16 : Comparative analysis of the effects of immunization route on immune responses elicited by CLEC-based vaccines.

[0557] Female BALB / c mice aged 8-12 weeks were vaccinated 3 times at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Two alternative routes, including subcutaneous (sc) and intramuscular (im), were compared with intradermal (id) vaccination of CLEC vaccines. Three doses of CLEC-based vaccine (SeqID2+SeqID7+Pus) were administered per route. Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and B) anti-aSyn protein responses.

[0558] Fig.17 : Comparative analysis of immune responses elicited by CLEC vaccines containing B-cell epitopes characterized by post-translationally modified peptide Aβ.

[0559] Female BALB / c mice, 8-12 weeks old, were vaccinated a total of 3 times (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. CLEC-based vaccines (SeqID33+SeqID7+Pus, id) were evaluated with conventional peptide-conjugate-based vaccines (SeqID32+KLH+Alum, sc). Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide, anti-AβpE3-40, anti-AβpE3-42, and anti-Aβ1-42 responses. B) Antibody affinity for AβpE3-42 was assessed by ELISA-based affinity testing.

[0560] Fig.18 : Comparative analysis of immune responses elicited by CLEC vaccines containing B-cell epitopes derived from intracellular proteins and the autoantigen Tau.

[0561] Female BALB / c mice aged 8-12 weeks were vaccinated 3 times (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. CLEC-based vaccines (SeqID36+SeqID7+Pus, id) were evaluated with traditional peptide-component based vaccines (SeqID35+KLH+Alum, sc). Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and anti-recombinant Tau 441 protein responses. B) Antibody avidity to SeqID35 was assessed by ELISA-based avidity testing.

[0562] Fig.19 : Comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes derived from secreted proteins, autoantigens, and the conformational epitope IL23.

[0563] Female BALB / c mice aged 8-12 weeks were vaccinated 3 times (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Three CLEC-based vaccines (SeqID38 / SeqID40 / SeqID42 all conjugated to SeqID7 and Psoralen, id) were evaluated in comparison to conventional peptide-conjugate-based vaccines (SeqID37 / SeqID39 / SeqID41 conjugated to KLH and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third vaccination and analyzed for anti-peptide and anti-IL23 protein responses.

[0564] Fig. 20: Comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes derived from self-epitopes present in the transmembrane protein: extracellular membrane-membrane-bound IgE proximal domain (EMPD).

[0565] Female BALB / c mice aged 8-12 weeks were vaccinated 3 times (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. A CLEC-based vaccine (SeqID44+SeqID7+Pus, id) was evaluated compared to a traditional peptide-component based vaccine (SeqID43+KLH+Alum, sc). Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-injected peptide and anti-EMPD peptide responses. B) Antibody avidity to the EMPD peptide was assessed by ELISA-based avidity assay.

[0566] Fig.21 : Comparative analysis of immune responses elicited by CLEC vaccines containing B-cell epitopes derived from allergens, mimotopes and conformational epitopes Bet v 1.

[0567] Female BALB / c mice aged 8-12 weeks were vaccinated 3 times (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. A CLEC-based vaccine (SeqID46+SeqID7+Pus, id) was evaluated compared to a traditional peptide-component based vaccine (SeqID45+KLH+Alum, sc). Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and anti-Bet v 1 protein responses. B) Antibody avidity to Bet v 1 was assessed by ELISA-based avidity assay.

[0568] Fig. 22 : Comparative analysis of the immune responses elicited by CLEC vaccines containing a B cell epitope (ie, oncogene) present in different forms of cancer / tumor diseases: Her2.

[0569] Female BALB / c mice aged 8-12 weeks were vaccinated 3 times (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. A CLEC-based vaccine (SeqID48+SeqID7+Pus, id) was evaluated compared to a traditional peptide-component based vaccine (SeqID47+KLH+Alum, sc). Samples were collected 2 weeks after the third vaccination and analyzed for anti-peptide and anti-Her2 protein responses.

[0570] Fig.23: Comparative analysis of the immune responses elicited by CLEC vaccines containing the B cell epitope (i.e. oncogene) PD1 present in different forms of neoplastic diseases / cancers.

[0571] Female BALB / c mice aged 8-12 weeks were vaccinated 3 times (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. CLEC-based vaccines (SeqID50+SeqID7+Pus, id) were evaluated with traditional peptide-component based vaccines (SeqID49+KLH+Alum, sc). Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and anti-PD1 protein responses. B) Antibody avidity to SeqID49 was assessed by ELISA-based avidity assay.

[0572] Fig.24 :Comparative analysis of target protein-specific immunogenicity induced by CLEC-based peptide-CRM197 conjugate vaccines with different peptide-CRM197 / CLEC ratios

[0573] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Five different peptide-CRM-based vaccines were used in this study, which employed different peptide-CRM / Pus ratios (w / w). All 5 groups were immunized with SeqID6+CRM+Pus conjugates. 1:1, 1:2,5, 1:5, 1:10 and 1:20 represent conjugates, where the weight ratio (w / w) of peptide-CRM conjugate / CLEC was 1 / 1, 1 / 2,5, 1 / 5, 1 / 10 and 1 / 20. The induced immune response was evaluated using samples collected 2 weeks after the third vaccination, and the response against aSyn protein was analyzed by ELISA. Titer determination was based on OD 最大 Calculation of / 2.

[0574] Fig.25 : Comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes derived from aSyn (aa1-8).

[0575] Female BALB / c mice aged 8-12 weeks were vaccinated 3 times (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. CLEC-based vaccines (SeqID12+SeqID7+Asterin, id) were evaluated in comparison to conventional peptide-component conjugate-based vaccines (SeqID13 conjugated with KLH and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and anti-ASyn protein responses and A) aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn for inhibition; dashed line: filamentous aSyn for inhibition.

[0576] Fig.26 Shown is a comparative analysis of the immune responses elicited by CLEC vaccines containing B cell epitopes derived from aSyn (aa100-108).

[0577] Female BALB / c mice aged 8-12 weeks were vaccinated 3 times (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. CLEC-based vaccines (SeqID16+SeqID7 and pyruvic acid, id) were evaluated in comparison to conventional peptide-component conjugate-based vaccines (SeqID17 conjugated with KLH and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and anti-aSyn protein responses and B) aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn for inhibition; dashed line: filamentous aSyn for inhibition.

[0578] Fig. 27 Comparative analysis of immune responses elicited by CLEC vaccines containing B-cell epitopes derived from aSyn (aa91-97) is shown. Female BALB / c mice aged 8-12 weeks were vaccinated 3 times (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. CLEC-based vaccines (SeqID14+SeqID7 and Pseudomonas polysaccharide, id) were evaluated in comparison to vaccines based on traditional peptide-component conjugates (SeqID15 conjugated with KLH and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third vaccination and analyzed for anti-peptide and anti-aSyn protein responses.

[0579] Fig.28Comparative analysis of immune responses elicited by CLEC vaccines containing B-cell epitopes derived from aSyn (aa130-140) is shown. Female BALB / c mice aged 8-12 weeks were vaccinated a total of 3 times (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. CLEC-based vaccines (SeqID20+SeqID7 and pyruvic acid, id) were evaluated in comparison to vaccines based on traditional peptide-component conjugates (SeqID21 conjugate with KLH and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and anti-aSyn protein responses and B) aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn for inhibition; dashed line: filamentous aSyn for inhibition.

[0580] Fig.29 Comparative analysis of immune responses elicited by CLEC vaccines containing B-cell epitopes derived from aSyn (aa115-122) is shown. Female BALB / c mice aged 8-12 weeks were vaccinated a total of 3 times (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. CLEC-based vaccines (SeqID51+SeqID7 and pyrenoidin, id) and vaccines based on traditional peptide-component conjugates (SeqID52 conjugated with CRM and Alhydrogel (Alum), sc) were evaluated. Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and anti-aSyn filamentous responses and B) aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn for inhibition; dashed line: filamentous aSyn for inhibition.

[0581] Fig.30 Comparative analysis of immune responses elicited by CLEC vaccines containing B-cell epitopes derived from aSyn (aa115-124) is shown. Female BALB / c mice aged 8-12 weeks received a total of 3 vaccinations (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. CLEC-based vaccines (SeqID67+SeqID7 and pyrenoidin, id) were evaluated compared to vaccines based on traditional peptide-component conjugates (SeqID68 conjugated with CRM and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and anti-aSyn filamentous responses and B) aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn for inhibition; dashed line: filamentous aSyn for inhibition.

[0582] Fig.31 Comparative analysis of immune responses elicited by CLEC vaccines containing B-cell epitopes derived from aSyn (aa107-113) is shown. Female BALB / c mice aged 8-12 weeks were vaccinated a total of 3 times (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. CLEC-based vaccines (SeqID73+SeqID7 and pyrenoidin, id) were evaluated compared to vaccines based on traditional peptide-component conjugates (SeqID74 conjugated with CRM and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and anti-aSyn filamentous responses and B) aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn for inhibition; dashed line: filamentous aSyn for inhibition.

[0583] Fig.32 A comparative analysis of the in vitro functionality of the immune responses elicited by CLEC-based vaccines is shown.

[0584] Female BALB / c mice aged 8-12 weeks were vaccinated 3 times (id and sc) at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Samples were collected 2 weeks after the third vaccination and evaluated for ThT kinetics (i.e., the fibrillar fraction of aSyn) in the presence of A) CLEC vaccine-induced antibodies (SeqID67 / 71 / 73 + SeqID7 and pyrenoidin, id) or conventional peptide-component-induced antibodies (SeqID68 / 72 / 74 coupled to CRM and Alhydrogel (Alum), sc) or D) aSyn-specific monoclonal antibody LB09 or untreated mouse plasma.

[0585] Fig.33 The in vitro binding activity of the CRM197-CLEC conjugate to the murine DC receptor (ie, dectin-1) is shown.

[0586] The figure shows a comparative analysis of dectin-1 binding capacity as determined by ELISA. A) Pus refers to unmodified pus oxi and pus oxi refers to activated pus oxi. CRM-pus conjugate 1 refers to SeqID6+CRM197+pus oxi conjugate and CRM conjugate 1 refers to CRM197+SeqID6 conjugate without β-glucan modification. Negative control refers to samples without inhibitors. B) SeqID52 / 66 / 68 / 70 / 72 refer to CRM197-pus oxi conjugates with the indicated B cell epitopes. C) Lich oxi refers to activated lichen oxi and CRM-Lich conjugate 1 refers to SeqID6+CRM197+lichen oxi conjugate. D) Lam oxi refers to activated laminarin and CRM-Lam conjugate 1 refers to SeqID6+CRM197+laminarin conjugate.

[0587] Fig.34 The in vitro binding activity of the CRM197-CLEC-conjugate to the human DC receptor (ie, dectin-1) is shown.

[0588] The figure shows a comparative analysis of dectin-1 binding capacity determined by ELISA. Lich conjugate refers to SeqID6+CRM197+lichenin conjugate, Pus conjugate refers to SeqID6+CRM197+pastosan conjugate, Lam conjugate refers to SeqID6+CRM197+laminarin conjugate, and negative control refers to a sample without inhibitor added.

[0589] Fig.35 A comparison of the immunogenicity of different CRM-chalcone based vaccines is shown.

[0590] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at the start of vaccination and after each vaccination to obtain information on the kinetics of the ensuing immune response. Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide responses and B) responses against aggregated aSyn filaments.

[0591] Fig.36 A comparative analysis of the selectivity of the immune responses elicited in vivo by vaccines based on peptide+CRM+pastosan for aSyn filaments is shown.

[0592] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal or subcutaneous vaccinations at 2-week intervals. CRM-chalcone-based vaccines were evaluated compared to conventional CRM vaccines. Samples were collected 2 weeks after the third vaccination and assayed for aSyn selectivity (inhibition ELISA). IC50 values ​​of antibodies inhibited with increasing doses of aSyn filaments are shown.

[0593] Fig.37 The avidity of antibodies induced by the peptide+CRM197+Aglabracan vaccine is shown.

[0594] Shown are the stability of aSyn-antibody complexes induced by peptide+CRM197+pastosan or peptide+CRM197 vaccines after challenge with different concentrations of the chaotropic agent sodium thiocyanate (NaSCN) and the determined avidity index.

[0595] Fig.38 A comparison of the immunogenicity of different CLEC-based vaccines is shown.

[0596] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Samples were collected 2 weeks after the third vaccination and analyzed for responses against SeqID6 peptide (A) and aSyn filaments (B) induced by vaccines based on peptide+carrier+dextran or non-CLEC-modified, alum-adjuvanted vaccines; dose: 20 μg peptide equivalent / injection; pustulan refers to SeqID6+CRM+pustulan, lichenan refers to SeqID6+CRM+lichenan, laminarin refers to SeqID6+CRM+laminarin, sc+Alum refers to non-CLEC-modified, alum-adjuvanted vaccine SeqID6+CRM.

[0597] Fig.39 The binding activity of peptide-CLEC-conjugates to the murine (A) and human (B) DC receptor (ie dectin-1) in vitro is shown.

[0598] Comparative analysis of dectin-1 binding capacity determined by ELISA is shown. Lich conjugate refers to SeqID5+SeqID7+lichenin conjugate, Pus conjugate refers to SeqID5+SeqID7+pastosan conjugate, Lam conjugate refers to SeqID5+SeqID7+laminarin conjugate, and negative control refers to samples without inhibitors.

[0599] Fig.40Comparison of the immunogenicity of different CLEC-based vaccines is shown. Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Samples were collected 2 weeks after the third vaccination and analyzed for anti-peptide responses (SeqID6, shown as peptides) and anti-aSyn responses (shown as proteins) induced by peptide-glucan-based vaccines (i.e., SeqID5+SeqID7+CLEC, doses: 5 μg and 20 μg / injection; lichenan refers to SeqID5+SeqID7+lichenan; laminarin refers to SeqID5+SeqID7+laminarin, and pustulan refers to SeqID5+SeqID7+pustulan).

[0600] Fig.41 The results show that the glycoconjugate-Aglais sutchuenensis conjugate has in vitro DC receptor (ie, dectin-1) binding activity.

[0601] The two CLEC-modified vaccines, oligosaccharide + CRM197 + pyrifos and polysaccharide + TT + pyrifos, both maintained high dectin-1 binding potency. Comparative analysis of dectin-1 binding capacity determined by ELISA is shown in the figure. Act-Pus refers to a conjugate of tetanus toxoid (TT) with pyrifos modified capsular polysaccharide (polyribosyl-ribitol-phosphate, PRP) of Haemophilus influenzae type b. Act refers to the unmodified β-glucan Conjugate vaccine, Men refers to the CRM197 conjugate vaccine containing Neisseria meningitidis oligosaccharides (A, C, W135 and Y) without β-glucan modification Men-Pus refers to the polysaccharide modified with pyruvate Vaccine, pus oxi refers to activated pus oxilan for modification.

[0602] Fig.42 A comparison of the immunogenicity of different CLEC-based glycoconjugate vaccines is shown.

[0603] Female BALB / c mice aged 8-12 weeks were vaccinated 3 times subcutaneously / intramuscularly at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Blood samples were collected 2 weeks after the third vaccination to analyze the anti-vaccine response induced by the oligo / polysaccharide-carrier-dextran or non-dextran-modified conjugate vaccines. A) shows the conjugated pyruvate ( + pyruvate) induced responses: Neisseria meningitidis (A, C, W135, Y) + CRM197 + pyruvate (80%), or unmodified Neisseria meningitidis (A, C, W135, Y) + CRM197, (dose: 5 μg); B) shows the Coupling with pyrifos polysaccharide ( + pyruvate) induced responses: Haemophilus influenzae (b) PRP + TT + pyruvate (80%), or unmodified Haemophilus influenzae (b) PRP + TT (dose: 2 μg)

[0604] Fig.43 Shown: Comparative analysis of the immunogenicity of CLEC-based vaccines using different IL31 peptide epitopes.

[0605] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Eight different CLEC-based vaccines (SeqID132+SeqID7+pastelan; SeqID134+SeqID7+pastelan; SeqID136+SeqID7+pastelan; SeqID138+SeqID7+pastelan; SeqID140+SeqID7+pastelan; SeqID142+SeqID7+pastelan; SeqID144+SeqID7+pastelan; and SeqID146+SeqID7+pastelan) were evaluated. Figure 2 shows the immune responses elicited by SeqID132+SeqID7+Pseudomonas aeruginosa) compared to the corresponding peptide-CRM197 conjugates adjuvanted with Alum (i.e., SeqID133+CRM197; SeqID135+CRM197; SeqID137+CRM197; SeqID139+CRM197; SeqID141+CRM197; SeqID143+CRM197; SeqID145+CRM197; and SeqID147+CRM197). Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and B) anti-IL31 protein responses. C) shows the avidity of antibodies induced by SeqID132+SeqID7+Pseudomonas aeruginosa or SeqID133+CRM vaccines determined by challenge with different concentrations of the chaotropic agent sodium thiocyanate (NaSCN).

[0606] Fig.44 Comparative analysis of the immunogenicity of CLEC-based vaccines using different IL31 peptide epitopes

[0607] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. The 10 different CLEC-based vaccines (i.e., SeqID133+CRM197+pastelan; SeqID135+CRM197+pastelan; SeqID137+CRM197+pastelan; SeqID139+CRM197+pastelan; SeqID141+CRM197+pastelan; SeqID143+CRM197+pastelan; SeqID145+CRM197+pastelan; SeqID147+CRM197+pastelan; SeqID149+CRM197+pastelan; and SeqID1 The immune responses elicited by the peptide-CRM197 conjugates (SeqID133+CRM197; SeqID135+CRM197; SeqID137+CRM197; SeqID139+CRM197; SeqID141+CRM197; SeqID143+CRM197; SeqID145+CRM197; SeqID147+CRM197; SeqID149+CRM197; and SeqID151+CRM197) were compared with the respective unmodified peptide-CRM197 conjugates (i.e., SeqID133+CRM197; SeqID135+CRM197; SeqID137+CRM197; SeqID139+CRM197; SeqID141+CRM197; SeqID143+CRM197; SeqID145+CRM197; SeqID147+CRM197; SeqID149+CRM197; and SeqID151+CRM197) adjuvanted with Alum. Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and B) anti-IL31 protein responses. C) shows the avidity of antibodies induced by SeqID133+CRM197+Pseudomonas aeruginosa or SeqID133+CRM vaccines determined by challenge with different concentrations of the chaotropic agent sodium thiocyanate (NaSCN).

[0608] Fig.45 Demonstration of inhibition of IL31 signaling by anti-IL31 antibodies induced by IL31 peptide-CLEC vaccine

[0609] The inhibitory effect of vaccine-induced antibodies on human IL-31 signaling was evaluated in human A549 cells (ATCC, Virginia, USA). The vaccine-induced antibodies used were from animals repeatedly immunized with IL31 peptide + SeqID7 + cleavage polysaccharide conjugate (CLEC; IL31 peptides: SeqID132, SeqID134, SeqID136, SeqID138, SeqID140, SeqID142, SeqID144, SeqID146) and traditional IL31-peptide + CRM conjugate with Alum adjuvant (CRM-Alum; IL31 peptides: SeqID133, SeqID135, SeqID137, SeqID139, SeqID141, SeqID143, SeqID145, SeqID147). Positive control: commercially available anti-IL31 blocking antibody; w / o inhibitor: IL31 stimulation only, bg: background, no IL31 stimulation.

[0610] Fig.46 Demonstration of inhibition of IL31 signaling by anti-IL31 antibodies induced by IL31 peptide-vector-CLEC vaccine

[0611] The inhibitory effect of vaccine-induced antibodies on human IL-31 signaling was evaluated in human A549 cells (ATCC, Virginia, USA). The vaccine-induced antibodies used were from animals repeatedly immunized with IL31 peptide + CRM197 + cleavage sugar conjugate (CRM-CLEC; IL31 peptide: SeqID133, SeqID135, SeqID137, SeqID139, SeqID141, SeqID143, SeqID145, SeqID147, SeqID149, SeqID 151) and traditional IL31-peptide + CRM conjugate with Alum as adjuvant (CRM-Alum; IL31 peptide: SeqID133, SeqID135, SeqID137, SeqID139, SeqID141, SeqID143, SeqID145, SeqID147, SeqID149, SeqID 151). Positive control: commercially available anti-IL31 blocking antibody; no inhibitor: IL31 stimulation only, bg: background, no IL31 stimulation.

[0612] Fig.47 Comparative analysis of the immunogenicity of CLEC-based vaccines using different CGRP peptide epitopes

[0613] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. The immune responses elicited by 6 different CLEC-based vaccines (SeqID152+SeqID7+Pyridoxine; SeqID154+SeqID7+Pyridoxine; SeqID156+SeqID7+Pyridoxine; SeqID158+SeqID7+Pyridoxine; SeqID160+SeqID7+Pyridoxine; and SeqID162+SeqID7+Pyridoxine) were evaluated, compared to the respective peptide-CRM197 conjugates adjuvanted with Alum (i.e., SeqID153+CRM197; SeqID155+CRM197; SeqID157+CRM197; SeqID159+CRM197; SeqID161+CRM197; and SeqID163+CRM197). Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and B) anti-CGRP protein responses. C) shows the avidity of antibodies induced by SeqID152+SeqID7+Pseudomonas aeruginosa or SeqID153+CRM vaccines, determined upon challenge with different concentrations of the chaotropic agent sodium thiocyanate (NaSCN).

[0614] Fig.48 Comparative analysis of the immunogenicity of CLEC-based vaccines using different CGRP peptide epitopes

[0615] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. The immune responses elicited by six different CLEC-based vaccines (SeqID153+CRM197+pastelan; SeqID155+CRM197+pastelan; SeqID157+CRM197+pastelan; SeqID159+CRM197+pastelan; SeqID161+CRM197+pastelan; and SeqID163+CRM197+pastelan) were evaluated and compared with the respective unmodified peptide+CRM197 conjugates (i.e., SeqID153+CRM197; SeqID155+CRM197; SeqID157+CRM197; SeqID159+CRM197; SeqID161+CRM197; and SeqID163+CRM197) adjuvanted with Alum. Samples were collected 2 weeks after the third vaccination and analyzed for A) anti-peptide and B) anti-CGRP protein responses. C) shows the avidity of antibodies induced by SeqID 153 + CRM197 + pyrifos or SeqID 153 + CRM vaccines after challenge with different concentrations of the chaotropic agent sodium thiocyanate (NaSCN).

[0616] Fig.49 It was shown that antibodies induced by SeqID5+SeqID7+Agar vaccine inhibited aSyn aggregation in the in vivo PFF model.

[0617] C57BL / 6 mice were injected with recombinant aSyn PFFs into the right substantia nigra under stereotaxic conditions and then immunized four times with SeqID5+SeqID7+Pseudomonas vaccine (vaccine) or unconjugated CLEC (vector) as control starting on the day of PFF inoculation. Plasma was collected after the third immunization. Brain, plasma and cerebrospinal fluid (CSF) were collected 126 days after PFF inoculation. (A) Titers of specific antibodies against the peptides used for vaccination in plasma collected two weeks after the third immunization (day 126). (B) Comparison of titers of specific antibodies against the B cell peptides of the vaccine in CSF and plasma at day 126. (C) Analysis of phosphorylated S129 aSyn positive aggregates in all brain regions of SeqID5+SeqID7+Pseudomonas vaccine-vaccinated and CLEC-treated mice. (D) Correlation between antibody response and synucleinopathy level in vaccine recipients (r = -0.9391; CI (95%) -0.9961 to -0.3318, p = 0.0179, R2 = 0.882). (EH) Representative pSer129aSyn staining of injected hemispheres at the level of (E, F) substantia nigra and (G, H) striatum. (E, G) Vehicle-treated mice and (F, H) vaccine-treated mice after injection of PFFs. Error bars represent mean ± SEM of n = 5-9 animals per group. Statistical differences were assessed by unpaired t-test; **p < 0.01; *p < 0.05.

[0618] Fig.50 Analysis of carrier-specific immunogenicity of peptide+CLEC and peptide+CRM+CLEC conjugates

[0619] Female BALB / c mice aged 8-12 weeks received intradermal / subcutaneous vaccinations for a total of three times at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. The immune responses elicited by four different CLEC-based vaccines (CRM-CRM197; i.e., SeqID6+CRM197+CRM197; SeqID133+CRM197+CRM197; SeqID135+CRM197+CRM197; and SeqID137+CRM197+CRM197) were evaluated, respectively, compared to the respective peptide-CRM197 conjugates adjuvanted with Alum (CRM-Alum; i.e., SeqID6+CRM197; SeqID133+CRM197; SeqID135+CRM197; and SeqID137+CRM197). Samples were collected 2 weeks after the third inoculation and analyzed for in vivo anti-CRM responses induced by A) SeqID6+CRM197+Pseudomonas and B) SeqID133+CRM197+Pseudomonas; SeqID135+CRM197+Pseudomonas; and SeqID137+CRM197+Pseudomonas.

[0620] Fig.51 Analysis showing CLEC-specific immunogenicity of peptide+CLEC and peptide+CRM+CLEC conjugates

[0621] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. The immune responses elicited by 14 different CLEC-based vaccines were evaluated. Samples were collected 2 weeks after the third vaccination and analyzed for in vivo responses against laminarin; A) Samples: SeqID6+CRM197+laminarin, SeqID6+CRM197+laminarin; B) Samples SeqID6+CRM197+laminarin; laminarin conjugated at the indicated conjugate / laminarin ratios (w / w); C) Samples: SeqID133+CRM19 7+chalcone; SeqID135+CRM197+chalcone; and SeqID137+CRM197+chalcone; D) SeqID132+SeqID7+chalcone; SeqID134+SeqID7+chalcone; and SeqID136+SeqID7+chalcone; pre-serum: sample before vaccination; positive control: sample from an animal immunized with non-oxidized chalcone only.

[0622] Fig.52: Analysis of the immunogenicity of peptide-carrier-dextran conjugates and vaccines consisting of peptide-carrier conjugates and unconjugated dextran.

[0623] Female BALB / c mice aged 8-12 weeks received a total of 3 intradermal vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to obtain information on the kinetics of the ensuing immune response. Samples were collected 2 weeks after the third vaccination and analyzed for responses against SeqID6 peptide (A) and against ASyn monomer (B). Vaccines used: SeqID6+CRM197+pastosan, a mixture of SeqID6+CRM197 and non-oxidized pastosan, and non-CLEC-modified, unadjuvanted SeqID6+CRM197. Example

[0624] Materials and Methods

[0625] 1) Oxidation of CLEC / dextran backbone

[0626] In order to form vaccine conjugates, polysaccharides, and in particular CLEC / β-glucans, need to be chemically modified to produce reactive groups that can be used for attachment to proteins / peptides. Two commonly used methods for polysaccharide activation are periodate oxidation of vicinal hydroxyls and cyanation of hydroxyls. Other methods for polysaccharide activation are possible and are well known in the art. The examples shown in this Examples section rely on mild periodate oxidation.

[0627] Depending on their solubility, CLEC and β-glucans (eg, mannan, lichenan, pyrifos or β-glucan from barley) can be oxidized using the periodate oxidation method in aqueous solution or DMSO.

[0628] The degree of oxidation was predetermined by adding periodate solution in a molar ratio (periodate:sugar subunit; 100% = 1 Mol periodate per Mol sugar monomer) ranging from 1:5 (ie 20% oxidation degree) to 2,6:1 (260% oxidation degree).

[0629] Briefly, sodium periodate was added at a molar ratio of 1:5 to 2,6:1 (periodate:sugar subunit, corresponding to oxidation degrees of 20% and 260%) to open the furanose ring between the vicinal diols of β-glucan, leaving two aldehyde groups as substrates for subsequent coupling reactions. 10% (v / v) 2-propanol was added as a free radical scavenger. The reactants were incubated on an orbital shaker (1000 rpm) at room temperature in the dark for 4 h. Subsequently, the reaction was performed using a Slide-A-Lyzer TM (Thermo Scientific) or Pur-A-Lyzer TMThe oxidized dextran was dialyzed 3 times against water to remove sodium (perio)iodate and low molecular weight dextran impurities using a Sigma Aldrich (Sigma Aldrich) dark box (cutoff 20 kDa). The dialyzed dextran can be directly used for peptide coupling reactions or stored at -20°C or lyophilized and stored at 4°C for further use.

[0630] 2) Conjugated WISIT vaccine

[0631] 2a. Formation via hydrazone

[0632] The polypeptide contains a hydrazide group at the N-terminus or C-terminus for aldehyde coupling. If the coupling direction is through the N-terminus of the selected peptide to the aldehyde group of the dextran moiety, the peptide is designed to contain a suitable linker / spacer arm, such as succinic acid. Alternatively, the dextran coupling is performed with the intact protein (e.g., CRM197).

[0633] Typical example of such peptides: N-terminal coupling of peptide: H 2 N-NH-CO-CH 2 -CH 2 -CO-peptide-COOH; C-terminal coupling: NH 2- Peptide-NH-NH 2 .

[0634] For coupling, a solution of active dextran (i.e. oxidized pyruvic acid) is stirred with dissolved hydrazide-modified peptides or intact proteins (e.g. CRM197) in coupling buffer (sodium acetate buffer at pH 5.4 or DMEDA at neutral pH (6.8) depending on the isoelectric point of the peptide). The free hydrazide groups in these peptides react with aldehyde groups to form hydrazone bonds leading to the final conjugate. For proteins, coupling with active dextran is based on the reaction of the amino groups of lysine residues with the active aldehydes on the dextran in the presence of sodium cyanoborohydride.

[0635] Subsequently, the conjugate is reduced by adding sodium borohydride in borate buffer (pH 8.5). This step reduces the hydrazine bond to a stable primary amine and converts the unreacted aldehyde group in the sugar backbone to a primary alcohol. The carbohydrate concentration in the conjugate is estimated by the anthrone method, and the peptide concentration is estimated by UV spectroscopy or determined by amino acid analysis.

[0636] 2b. Coupling using heterobifunctional linkers

[0637] The second coupling technique used relies on heterobifunctional 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), maleimide-and-hydrazide cross-linkers are used to couple sulfhydryls (cysteine) to carbonyls (aldehydes)).

[0638] The peptide contains a cysteine ​​(Cys) at the N-terminus or C-terminus for maleimide coupling. Typical examples of such peptides: N-terminal coupling of peptide: Cys-peptide-COOH; C-terminal coupling: NH 2 -Peptide-Cys-COOH.

[0639] For coupling, the active dextran solution (i.e., oxidized pyralidin) was reacted with BMPH overnight (the ratio used was BMPH:pyralidin 1:1 (w / w) to 2:1), and then dialyzed three times with PBS. The BMPH-activated dextran was then mixed with the dissolved Cys-modified peptide in a coupling buffer (e.g., phosphate-buffered saline, PBS). The maleimido group reacts with the sulfhydryl group in the peptide to form a stable thioether bond, and together with the hydrazone formed between the linker and the active aldehyde, leads to a stable conjugate. The carbohydrate concentration in the conjugate was estimated by the anthrone method, and the peptide concentration was determined by amino acid analysis or the Ellmann assay using Ellman's reagent (5,5'-dithio-bis-(2-nitrobenzoic acid), DTNB). DTNB reacts with thiol groups to form colored products, which provides a reliable method for measuring reduced cysteine ​​and other free thiol groups in solution by spectrophotometry (λmax = 412 nm; ε = 14,150 / M·cm).

[0640] 2c) Peptide KLH / CRM coupling

[0641] Peptides (containing N-terminal or C-terminal Cys residues, see above) were coupled to the carriers CRM-197 (e.g.: EcoCRM, Fina Biosolutions) or KLH (Sigma Aldrich) using the heterobifunctional crosslinkers GMBS or SMCC (ThermoFisher). Briefly, CRM-197 / KLH was mixed with excess GMBS or SMCC (according to the manufacturer's protocol) at room temperature to allow activation, and then excess GMBS was removed by centrifugation through a desalting column. Excess peptide was then added to the activated carrier for coupling (buffer: 200 mM sodium phosphate (pH = 6,8)), followed by dialysis 3 times against PBS. The coupling efficiency / peptide content was assessed using the Ellmann assay for quantification of free thiol groups in the solution (Ellmann reagent: 5,5'-dithio-bis-(2-nitrobenzoic acid). The peptide CRM-197 / KLH conjugate was further reacted with Alum ( When the CRM-197 / KLH vaccine was compared with other vaccines of the present invention, each mouse was injected with the same amount of the coupled polypeptide.

[0642] 2d) Formation of sugar-neocomplexes using peptides, KLH / CRM197 and dextran

[0643] Peptide-KLH and peptide-CRM197 conjugates prepared as described in 2c) were also coupled to activated dextran at different ratios of peptide-KLH and peptide-CRM197 to dextran (i.e., 1 / 1 (w / w), 1 / 2 (w / w), 1 / 5 (w / w), 1 / 10 (w / w), and 1 / 20 (w / w), respectively). After formation of the peptide conjugates, the Pep-KLH or Pep-CRM conjugates were reduced with dithiothreitol (DTT). The reduced carrier conjugates were coupled to activated dextran in the presence of an excess of the heterobifunctional linker BMPH. The coupling was achieved via the formation of a stable thioether bond between the maleimido group of BMPH and the sulfhydryl residue of the reduced KLH or CRM197 conjugate, and the coupling of the aldehyde group in the sugar to the hydrazide group of BMPH. After 2 hours at room temperature, the generated hydrazone was reduced to a stable primary amine by incubation with sodium cyanoborohydride overnight. Subsequently, glucose-neoconjugate was lysed using Slide-A-Lyzer TM (ThermoScientific) or Pur-A-Lyzer TM (Sigma Aldrich) cassettes were dialyzed three times against PBS or water to remove low molecular weight impurities (see also: Example 23).

[0644] 3) Determination of in vitro bioactivity of CLEC-conjugates

[0645] The in vitro bioactivity of mannan and glucan conjugates was analyzed by ELISA with soluble murine Fc-dectin-1a receptor (InvivoGen) or ConA as described by Korotchenko et al. 2020. Briefly, ELISA plates were coated with reference glucans (CLR agonists, CLECs), such as chalcone, lichenan, or mannan, and reacted with fluorescently labeled ConA (for mannan) or soluble murine Fc-dectin-1a receptor (for chalcone and other β-D-glucans), which could be detected by HRP-labeled secondary antibodies. Oxidized carbohydrates as well as glycoconjugates were tested in a competitive ELISA (increasing concentrations of CLEC or conjugates were added to the soluble receptor used in the assay to reduce receptor binding to the coated CLEC) to demonstrate their functionality. IC 50 The value determines the biological activity (ie, the potency of binding to the soluble receptor compared to the non-oxidized, unconjugated ligand).

[0646] 4) Activation analysis using bone marrow-derived dendritic cells

[0647] Bone marrow-derived dendritic cells (BMDCs) were harvested from mouse femurs and tibias and incubated with 20 ng / mL murine GM-CSF (Immunotools) as described by Korotchenko et al. 2020 with slight modifications. + MHCII + CD11b int GM-CSF-derived dendritic cells (GM-DC) were subjected to FACS analysis to evaluate the effect of various conjugates as well as a positive control (=LPS) on CD80 and MHCII expression.

[0648] 5) Determine the hydrodynamic radius

[0649] The hydrodynamic radius of the conjugate is analyzed by dynamic light scattering (DLS). In brief, the sample (i.e., the conjugate) is centrifuged at 10,000g for 15 minutes (Merck Millipore, Ultrafree-MC-VV Durapore PVDF). All sample wells are sealed with silicone oil to prevent evaporation, and data for about 24 hours are continuously collected. All measurements are carried out at 25°C in 1536-well plates (1536W SensoPlate, Greiner Bio-One) using WYATT DynaProPlateReader-II. Sample measurements are performed in triplicate. All measurements are filtered by a baseline value of 1.00 ± 0.005 so that only curves with values ​​returned between 0.995 and 1.005 are considered for further analysis (e.g., cumulants radius and regularization analysis). The sample analysis was performed according to https: / / www.wyatt.com / library / application-notes / by-technique / dls.html and the DYNAMICS User Guide (M1406 Rev. C, Version 7.6.0), Technical Notes TN2004 and TN2005 (both available at: www.wyatt.com).

[0650] 6) Animal experiments

[0651] Female BALB / c mice, n=5 per group, were immunized with different CLEC conjugates (id, im, sc), peptide-CRM-197 / KLH conjugates (id) or peptide-CRM-197 / KLH conjugates adsorbed to Alum (sc) and respective controls (e.g., unconjugated CLEC, mixture of CLEC and peptide, etc.) Animals were vaccinated 3 times every two weeks, and blood was collected regularly one day before each vaccination and two weeks after the last vaccination unless otherwise stated.

[0652] 7) Quantification of vaccine-induced antibodies in mouse plasma by ELISA

[0653] Whole blood was collected from mice using heparin as an anticoagulant and centrifuged to obtain plasma. Plasma samples were stored at -80°C. To detect anti-target specific antibodies, peptide-BSA conjugates or recombinant proteins / fragments (usually at a concentration of 1 μg / ml) were coated on ELISA plates (Nunc Maxisorb) with 50 mM sodium carbonate buffer at 4°C overnight. All anti-peptide ELISAs used in the examples were performed with Pep-BSA conjugates (e.g., SeqID3 (sequence: DQPVLPD) has a C-terminal C for coupling with maleimide-activated BSA; named: Pep1c (DQPVLPD-C, SeqID 3), which was used as bait for the detection of Pep1b (SeqID2; DQPVLPD-(NH-NH 2) )- and Pep1c-conjugate vaccines induce anti-Pep1-specific responses. The plates were blocked with 1% bovine serum albumin (BSA) and the plasma samples were serially diluted in the plates. The target-specific antibodies were detected with biotinylated anti-mouse IgG (Southern Biotech) and then developed with streptavidin-POD (Roche) and TMB. EC 50 The values ​​were calculated by nonlinear regression analysis (four-parameter logistic fit function) using GraphPad Prism software (Graph Pad Prism www.graphpad.com / scientific-software / prism / ).

[0654]

[0655]

[0656] 8) Characterization of the binding preference of aSyn-specific antibodies by inhibition ELISA

[0657] ELISA plates (Nunc Maxisorb) were coated with aSyn monomer (Abcam) or aSyn filaments (Abcam) and blocked with 1% bovine serum albumin (BSA). Control antibodies and plasma samples were incubated with serial dilutions of aSyn monomer or aSyn filaments in low binding ELISA plates. Next, the pre-incubated antibody / plasma samples were added to the monomer / filament coated plates and binding was detected with biotinylated anti-mouse IgG (Southern Biotech), followed by color development with streptavidin-POD (Roche) and TMB. The logIC50 value was calculated as the concentration of monomer or filamentous aSyn required to quench half of the ELISA signal and used as an estimate of the selectivity of the Abs for the antigen being studied. logIC 50The values ​​were calculated using GraphPad Prism software (GraphPad Prism www.graphpad.com / scientific-software / prism / ) according to nonlinear regression analysis (four-parameter logistic fit function).

[0658] 9) Quantification of aSyn aggregation

[0659] Automated protein aggregation assays were performed in a GENIOS microplate reader (Tecan, Austria) with continuous orbital shaking in black flat-bottom 96-well plates (0.1 ml reaction volume). Kinetics were monitored by reading the peak fluorescence intensity every 20 minutes using filters with 450 nm excitation and 505 nm emission. The absence and presence of antibodies (antibody / protein molar ratios from 6×10 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 -5 to 3×10 -3 unequal) when the fibril is formed.

[0660] Furthermore, fibril formation in the absence and presence of antibodies was also initiated by the presence of preformed fibrils. Briefly, aSyn preformed fibrils (1 μM) were aggregated for 0-24 hours in the presence of activated aSyn monomers (10 μM) and 10 μM ThT in 100 μl PBS.

[0661] For data analysis, the mean value of negative control samples (i.e., ThT background fluorescence) was calculated in, for example, Microsoft Excel, and each sample at a given time point was divided by the mean value. In order to compare different conditions / inhibitors in the aggregation assay, the fluorescence reading determined at the beginning of the assay was set to 1 (t0=1), and each sample was standardized against it.

[0662] To evaluate the kinetic curves, the Michaelis-Menten kinetic model was applied: Km (substrate concentration yielding half-maximal rate) and Vmax (maximum rate) values ​​under each condition were calculated by enzyme kinetic analysis (Michaelis-Menten) using GraphPad Prism software.

[0663] To compare different conditions / inhibitors in the aggregation assay, the slope values ​​of the exponential growth phase of ThT kinetics were calculated by linear regression using GraphPad Prism software.

[0664] 10) Determination of affinity and avidity

[0665] To determine Ab (antibody) affinity, a modification of the standard ELISA test was used in which replicate wells containing antibodies binding to different antigens for each example were exposed to increasing concentrations of chaotropic thiocyanate ions. Resistance to thiocyanate elution was used to measure affinity, and the index representing 50% effective antibody binding (avidity index) was used to compare different sera. Briefly, plasma was diluted to 1 / 500 in PBS and spread on coated and blocked ELISA plates (Nunc Maxisorb). After incubation for 1 hour, sodium thiocyanate (NaSCN, SIGMA; in PBS) was added to the samples at concentrations ranging from 0.25 to 3 M. The plates were incubated at room temperature for 15 minutes, then washed, detected, and developed with streptavidin-POD (Roche) and TMB. Subsequent absorbance readings in the presence of increasing concentrations of NaSCN were converted to appropriate percentages of total bound antibody, assuming that the absorbance reading in the absence of NaSCN represented effective total binding of the specific antibody (100% binding). The data were fitted to a graph of (% binding) versus NaSCN (log) concentration and an affinity index representing the concentration of NaSCN required to reduce the initial optical density by 50% was estimated by linear regression analysis. Data were rejected if the correlation coefficient of the line fit was less than 0.88.

[0666] To determine the k D The value (binding affinity) was determined using a displacement ELISA, which allows a simple determination of the k value of the complex formed by the antibody and its competing ligand. D Briefly, equal concentrations of antibodies were incubated with increasing concentrations of free aSyn filaments on plates immobilized with aSyn filaments, and free antibody titers were measured. Relative binding of antibodies was expressed as a percentage of the maximum binding observed for each sample in the assay; in displacement curves, competition with aSyn filaments (5 μg / ml) was defined as representing 0% binding (nonspecific binding), while the reaction without competition represented 100% (maximum) binding.

[0667] Competition binding curves were analyzed according to the one-site model using GraphPad computer-assisted curve fitting software.

[0668] 11) Inducing synuclein disease in mice

[0669] To induce synucleinopathy, preformed polymorphic fibrils (PFFs; i.e., preformed sonicated τ-polymorphic aSyn fibrils 1B) were stereotaxically injected into the right substantia nigra of nine-week-old male C57BL / 6 mice. The preparation and validation of PFFs are described in Sci. Adv. 2020, 6, eabc4364, doi: 10.1126 / sciadv.abc4364; DOI: 10.1126 / sciadv.abc4364. Briefly, each animal was injected unilaterally with 2 μL of PFFs 1B solution (concentration: 2.5 mg / ml) into the area just above the right substantia nigra (coordinates from bregma: -2.9AP, ±1.3L, and -4.5DV) at a flow rate of 0.4 μL / min [Sci. Adv. 2020, 6, eabc4364, doi:10.1126 / sciadv.abc4364; DOI:10.1126 / sciadv.abc4364], and the needle was left in place for 5 minutes and then slowly withdrawn from the brain.

[0670] Animals received three id immunizations (i.e., weeks 0, 2, and 4) biweekly, starting from the day of vaccination, with either the CLEC-based vaccine (n=5) or unconjugated CLEC (n=10) as a control, followed by a booster immunization at week 10. At the end of the study (day 126), cerebrospinal fluid (CSF) was collected by cisterna magna puncture, and the brain was carefully removed and fixed with paraformaldehyde (PFA; 4%). Coronal serial sections of the whole brain (from the rostral forebrain cortex to the medulla oblongata - i.e., bregma - 6.72 mm) were collected at 50 μm intervals using a cryostat and processed for immunohistochemical analysis.

[0671] 12) Immunohistochemistry (IHC)

[0672] IHC staining of phosphor-S129 aSyn (pS129 aSyn) on coronal serial sections was performed as described in [Sci.Adv.2020,6,eabc4364,doi:10.1126 / sciadv.abc4364;DOI:10.1126 / sciadv.abc4364]. Monoclonal rabbit anti-pS129 aSyn antibody EP1536Y (ab51253, Abcam) was used, followed by incubation with labeled polymer-HRP anti-rabbit (Dako EnVision+TM Kit, K4011). pS129 aSyn staining was visualized with DakoDAB (K3468), and sections were counterstained with Nissl staining. The actual number of pS129 aSyn aggregates in each structure (cerebral cortex, striatum, thalamus, substantia nigra, and brainstem) and the total numb...

Claims

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

1. The conjugate according to claim 1 , wherein the β-glucan is pyruvate.

3. A conjugate according to claim 1 or 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 10:1 (w / w) to 1:1 (w / w), preferably 8:1 (w / w) to 2:1 (w / w), especially 4:1 (w / w).

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

6. The conjugate according to any one of claims 1 to 5, wherein the length of the B cell epitope polypeptide is 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 length of the T cell epitope polypeptide is 8 to 30 amino acid residues, preferably 13 to 29 amino acid residues, in particular 13 to 28 amino acid residues.

8. The conjugate according to any one of claims 1 to 7, wherein the conjugate further comprises a carrier protein, preferably a non-toxic cross-reactive substance of diphtheria toxin (CRM) (especially CRM 197) , KLH, diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae protein D (HipD), outer membrane protein complex of meningococcal serogroup B (OMPC), recombinant avirulent 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 protein, bovine serum albumin, ovalbumin, synthetic peptide dendrimers such as multiple antigenic peptides (MAP), provided that when the conjugate comprises a carrier protein, the conjugate comprises at least another, independently conjugated T cell or B cell epitope polypeptide, especially 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 from 1 / 0.1 to 1 / 10.

9. The conjugate according to any one of claims 1 to 8, wherein the polypeptide is or comprises a B cell epitope polypeptide or a T cell epitope polypeptide, preferably 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 T-cell epitopes and does not contain B-cell epitopes, wherein the conjugate preferably comprises more than one T-cell epitope, in particular two, three, four or five T-cell epitopes.

11. Use of the conjugate according to any one of claims 1 to 10 for preventing or treating a disease, preferably for preventing or treating an infectious disease, a chronic disease, an allergy or an autoimmune disease.

12. Use of the conjugate according to any one of claims 1 to 11 as an active anti-Aβ, anti-Tau and / or anti-α-synuclein vaccine for the treatment and prevention of β-amyloidosis, tauopathy, or synucleinopathy, preferably Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), Parkinson's disease dementia (PDD), axonal dystrophy, Alzheimer's disease (AD), amygdala restricted Lewy body AD (AD / ALB), Down syndrome dementia, Pick's disease, progressive supranuclear palsy (PSP), cortical basal degeneration, frontotemporal dementia and Parkinson's disease associated with chromosome 17 (FTDP-17) and argyrophilic grain disease.

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

14. The method according to claim 13, wherein the β-glucan is obtained by periodate oxidation on the ortho-hydroxyl groups, as reductive amination, or as cyanation of the hydroxyl groups.

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

Images