TW202409077A - A conjugate consisting of or comprising at least a β-glucan or a mannan - Google Patents

Abstract

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

Description

A conjugate consisting of or containing at least one β-glucan or mannopolysaccharide

The present invention relates to polysaccharide adjuvants belonging to the class of C-type lectins (CLEC).

Vaccination is considered one of the most powerful means to save lives and reduce the burden of disease. With active immunization, vaccines are administered to induce the host's immune system to produce non-specific innate immune responses as well as specific antibodies, memory B cells and memory T cells that can act against the administered immunogen.

Polysaccharides constitute important virulence factors, especially for encapsulated bacteria that present complex carbohydrate structures on their surfaces. Bacterial as well as fungal or other polysaccharides are composed of repeating monosaccharide units linked by glycosidic bonds to form polymeric linear or branched structures. Antibody responses to various bacterial polysaccharides are known to be weak and, since they do not induce immune memory, are not enhanced by subsequent immunizations.

These properties are based on the nature 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. In contrast, 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 associated with major histocompatibility complex (MHC) class II molecules. Interaction with T cells induces B cells to differentiate into plasma cells and memory B cells. Unlike TI antigens, TD antigens are immunogenic and can be enhanced and potentiated 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, p. 1602-1609 (2011)).

The limitations of pure polysaccharide vaccines have been overcome by covalently conjugating the polysaccharide to a carrier protein that serves as a source of T-cell antigenic determinants. This concept has been successfully applied to several glycoconjugate vaccines currently on the market that have been developed against bacterial pathogens such as meningococci, Streptococcus pneumoniae, Haemophilus influenzae type b, and group B streptococci. All of these vaccines use pathogen-specific carbohydrates to induce carbohydrate-specific antibodies as the primary protective agent.

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

PRRs constitute a class of germline-encoded receptors that upon binding/activation are critical for the initiation of innate immunity, which plays a key role in the first line of defense until more specific adaptive immunity is developed. The innate immune response is the first line of defense against infectious diseases and tissue damage. Specialized cells, namely mainly APCs such as macrophages and DCs, but also some non-professional cells such as epithelial cells, endothelial cells and fibroblasts, are expressing these PRRs and play a role in pathogen recognition during the innate immune response important role. In addition, activation of APC by innate immune signaling is a key prerequisite for the generation of effective adaptive immunity, including antibody and memory responses.

The currently identified PRR family is divided into transmembrane receptors and receptors located in intracellular compartments. Transmembrane receptors include, for example, Tall-like receptors (TLRs 1-9) and C-type lectin receptors (CLR). Intracellular receptors include nucleotide-binding oligomerization domain-(NOD-)-like receptor (NLR), retinoic acid-inducible gene-(RIG-) I-like receptor (RLR) and AIM2-like receptor (ALR). ).

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

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 linked to a single terminal glucose. Inulin has no immunological activity in its natural soluble form, but when crystallized into different stable microcrystalline particles (inulin alpha-delta), it acquires potent protein-conjugated vaccine adjuvant activity. Delta inulin particles, marketed as Advax™ adjuvant, exhibit uniform spherulite discoidal particles with a diameter of 1-2 μm and are composed of a series of lamellae. δ- and γ-inulin are thought to act by surrogate complement activation, as the auxiliary mechanism of γ-inulin has been shown to involve increased C3 deposition on the macrophage surface, resulting in enhanced T cell activation (Kerekes et al. Man, J Leukoc Biol. 2001 Jan;69(1):69-74).

Another polysaccharide-based adjuvant candidate is chitosan-based adjuvants. Chitosan is a linear β-(1,4)-linked copolymer of D-glucosamine and N-acetyl-D-glucosamine (GlcNAc), prepared by partial alkaline deacetylation of chitin. Soluble chitosan itself is also poorly immunogenic. However, chitosan formulated as dry powder or solution has been widely used as an encapsulating agent for mucosal and systemic vaccine delivery and has also been 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.

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.).

Neimert-Andersson et al used ViscoGel, a soluble chitosan hydrogel. ViscoGel induced stronger humoral and cellular responses to antigens when used as an unadjuvanted vaccine with Haemophilus influenzae type b vaccine (Act-HIB). Serum IgG1 and IgG2a titers were significantly enhanced. The production of Th1, Th2, and Th17 interleukins was also increased. Unfortunately, Viscogel was not shown to be suitable for human use (Vaccine. 2014; 32:5967-5974).

Chitin and chitosan particles are readily phagocytosed, supporting a role for recognition via specific receptor-mediated phagocytosis. The receptors on bone marrow cells that bind chitin or chitosan and induce the phagocytic response have not been clearly identified. To date, several receptors have been shown to bind chitin or chitosan oligosaccharides, including: FIBCD1, a homotetrameric 55-kDa type II transmembrane protein expressed in the gastrointestinal tract; NKR-P1, an activation 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 GlcNac and is therefore a potential receptor for chitosan-based vaccine conjugates. ViscoGel reportedly triggers an immune response in a similar manner to chitin, as chitin molecules have been reported to act as PAMPs and recognize TLR-2 receptors on macrophages to induce innate immune responses.

Yu et al. (Mol. Pharmaceutics, 2016, DOI: 10.1021/acs.molpharmaceut.6b00138) also showed that the conjugate of inulin-chitosan and Mycobacterium tuberculosis CFP10-TB10.4 fusion protein (CT) had significantly increased hydrodynamic volume compared to non-adjuvant protein, and the vaccine induced high levels of Th1 (IFN-γ, TNF-α and IL-2) and Th2 interleukins (IL-4) as well as potent CT-specific antibody titers, mainly in the form of IgG1 and IgG2b. Pharmacokinetic studies showed that conjugation with inulin-chitosan prolonged the serum exposure of CT to the immune system. (Mol. Pharmaceutics 2016, 13, 11, 3626-3635).

One of the most prominent classes of polysaccharides used as adjuvants is the C-type lectins (CLECs), which interact with their own receptors, called C-type lectin receptors (CLRs). CLRs are considered ^Ca2+ -dependent carbohydrate recognition proteins. These receptors exhibit single or multiple carbohydrate recognition domains (CRDs) on their C-type lectin-like domain (CLECD), 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 polysaccharides and dextran.

CLR is divided into transmembrane CLR (TM-CLR) and soluble CLR (collagen lectin). TM-CLR is further divided into type I TM-CLR and type II TM-CLR. Type I TM-CLR includes mannose receptor (MR) and ENDO180 [mannose receptor type C2 (MRC2)] receptors, and binds to mannose, fucose, and N-acetylglucosamine. Type II TM-CLR includes dendritic cell-specific intracellular adhesion molecule-3-binding non-integrin (DC-SIGN), isletin, and macrophage galactose-type lectin (MGL) receptors. DC-SIGN is known to bind to N-linked glycans (branched trimannose structures), such as those present in the HIV-1 glycoprotein gp120 and other viruses such as hepatitis C virus, human cytomegalovirus, dengue or Ebola viruses )superior. DC-SIGN also recognizes lipoarabinomannan and mannan. Insulin is a Langerhans cell (LC)-associated CLR that binds mannose and fucose containing glycan residues. MGL has binding specificity for the terminal N-acetyl galactosamine (GalNAc) residue, and also shows affinity for Campylobacter jejuni and tumor-associated mucin MUC1, and is involved in controlling the adaptive immunity of effector T cells. In addition, the macrophage-inducible C-type lectin (Mincle, also known as CLEC4A) and the dectin-1/dectin-2 receptor family have been described. Dectin-1 plays an important role in mediating innate immunity against fungi and can interact with fungi (e.g., Saccharomyces cerevisiae β-glucan), lichens (e.g., Shitia polysaccharide, lichen polysaccharide), algae (e.g., Laminaria laminaria) polysaccharides) or beta-glucans found in grain species such as barley. Dectin-2 contains an EPN (Glu-Pro-Asn) amino acid motif that provides sensitivity to mannose ligands. Furthermore, dectin-2 interacts with Candida albicans.

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

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. Therefore, they link innate and adaptive immune responses.

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) demonstrated that oral administration of β-(1,3),β-(1,6) glucans in conjunction with rabies and canine parvovirus-2 vaccination in dogs resulted in earlier development of protective levels of antibodies against both viruses.

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 elicits a TH1 response, regardless of whether the antigen targets CLRDEC-205, islet proteins, Clec9A, or the Ig superfamily member Treml4.

In contrast, immunization under non-inflammatory (steady-state) conditions induces tolerance. Islet protein+ migratory DCs, but not lymph node-resident DCs, have been identified as the major Treg inducers, independent of the source of the dendritic cells (skin vs. lung) or the targeting receptor.

In particular, targeting mannose receptors (MRs) or other mannosaccharide-sensitive CLRs by using mannosaccharides as CLECs has been shown to be effective in inducing cellular and humoral immune responses; therefore, vaccines targeting MRs and other CLRs have received increasing attention in inducing specific tolerance in the treatment of cancer, infectious diseases, and autoimmune diseases.

Mannosan is a polysaccharide derived from yeast cell walls, consisting mainly of a β-(1,4)-linked mannose backbone and a small amount of α-(1,6)-linked glucose and galactose side chain residues. In addition, about 5% protein content has been detected in known mannosan preparations. As an important component of fungal cell walls, mannosan has been widely used as a component of carbohydrate-based Candida vaccines (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 mannosaccharide carrier-antigen complexes/conjugates have been developed, including mannosaccharide-mucin 1 (MUC1) fusion protein conjugates for tumor treatment or conjugates of mannosaccharide with model allergens such as ovalbumin (OVA), papain or Betv1.

Mucins are highly glycosylated proteins expressed on the cell surface. MUC1 is a prototype mucin that has been found to be overexpressed on a variety of tumor cells. Along these lines, a MUC1 fusion protein containing 5 tandem repeats of human MUC1 (containing the immunodominant epitope: APDTRPAPGSTAPPAHGVTS) and a peptide (Cpl3-32) was generated and combined with manna under oxidizing or reducing conditions. Polysaccharide binding causes distinct immune responses: oxidized mannan-MUC1 stimulates a Th1-type response mediated by CD8+ T cells, causing the secretion of IFN-γ and an antibody response mainly IgG2a, while reduced mannan-MUC1 It stimulates Th2 type response and produces IL-4 and high IgG1 antibody response. The fusion proteins used represent a single protein displaying T cell and B cell epitopes.

Protein-carbohydrate/mannosaccharide complexes of papain and OVA have also recently been generated to analyze their allergenic potential. It was found that coupling mannosaccharide to the protein surface reduced the binding and cross-linking of IgE antibodies against papain. Interestingly, in these experiments, coupling of mannosaccharide, dextran, or maltodextrin only reduced the allergenic potential of papain, but not that of 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 mannosaccharide conjugation resulted in increased IgG titers against OVA after intradermal immunization.

Similar to the neoglycoconjugate vaccine used for MUC1, Ghochikyan et al. (DNA AND CELL BIOLOGY, Volume 25, Issue 10, 2006, Pp. 571-580) and Petrushina et al. (Journal of Neuroinflammation 2008, 5:42) Amyloid beta (Aβ)28 is being used, a 28 amino acid residue peptide that carries the combined B-cell and T-cell epitopes of the human Aβ42 peptide, which can be conjugated to mannan in mice. Induces low-level anti-Aβ responses. These responses also demonstrated that amyloid deposition in the cortex and hippocampus of APP transgenic mice was attenuated after subcutaneous immunization. The immunization also resulted in an increase in induced anti-mannan titers in A[beta]28-mannan and BSA-mannan treated animals. However, this treatment was not developed further, most likely due to an increase in the occurrence of microbleeds in the brains of treated animals, which was attributed to the potentially deleterious role of mannan as a trigger of adverse vascular events, underscoring the importance of carbohydrate selection in designing effective treatments. and the importance of safe vaccines.

To date, there are no known conjugates using single B cell or T cell epitope peptides coupled to mannopolysaccharide or other related CLECs.

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

β-Glucans are a source of soluble, fermentable fiber (also known as prebiotic fiber) that provides substrates 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, daily intake of at least 3 grams of cereal β-glucan from oats can reduce total cholesterol and LDL cholesterol levels by 5% to 10% in people with normal or elevated blood cholesterol levels.

Typically, β-glucans form linear backbones with 1-3 β-glycosidic bonds, but vary in molecular weight, solubility, viscosity, branched structure, and gel properties. The β-glucans of yeast and fungi are usually built on the β-(1,3) main chain and contain β-(1,6) side branches, while the β-glucans of cereals contain both with and without β-(1,3) and β-(1,4) main chain bonds of side branches.

Beta-glucans are recognized by the innate immune system as pathogen-associated molecular patterns (PAMPs). PRR dectin-1 has emerged as the primary receptor for these carbohydrates, and binding of β-glucan to dectin-1 induces various cellular responses through the Syk/CARD9 signaling pathway, including phagocytosis, respiratory burst, and secretion of interleukins . In addition, complement receptor 3 (CR3, CD11b/CD18) is also considered to be a receptor for β-glucan. It has been reported that stimulation of dectin-1 triggers Th1, Th17 and cytotoxic T lymphocyte responses.

Members of the beta-glucan family include:

β-Glucan peptide (BGP) is a high molecular weight (approximately 100 kDa) branched polysaccharide extracted from the fungus Trametes versicolor . BGP is composed of a highly branched glucan moiety containing a β-(1,4) backbone and β-(1,3) side chains, and is rich in aspartic acid, glutamic acid, and other amines. The β-(1,6) side chain of the polypeptide portion of the amino acid is covalently attached.

Curdlan is a high molecular weight linear polymer from Agrobacterium spp., which consists of β-(1,3)-linked glucose residues.

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

Pustulan is a medium molecular weight (20 kDa), linear β-(1,6)-linked β-D-glucan from the lichen Lasallia pustulata, which can also Binds to dectin-1 as the main receptor and activates signaling through dectin-1.

Lichenan is a high molecular weight (about 22-245 kDa) linear β-(1,3) β-(1,4)-β-D glucan from the Icelandic lichen ( Cetraria islandica ). Its structure is similar to the β-glucans of barley and oats. Compared with the other two glucans, lichenan has a higher ratio of 1,3-β to 1,4-β-D bonds, with a β-(1,4)- to β-(1,3)-β-D bond ratio of about 2:1.

Beta-glucans from oats and barley are linear beta-(1,3) beta-(1,4)-beta-D glucans and are commercially available in different molecular weights (35.6 kDa, medium to up to products with a high molecular weight of 650 kDa).

Schizophyllan (SPG) is a gelling β-glucan derived from the fungus Schizophyllum commune . SPG is a high molecular weight (450 kDa) β-(1,3)-D-glucan with one β-(1) for every three β-(1,3)-glucosyl residues in the main chain. ,6) Monoglucosyl branch.

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

Whole glucan particles (WGP) are β-glucans that have attracted much attention for their ability to modulate immune responses. WGP Dispersible (WGP® Dispersible from Biothera) is a microparticulate preparation of brewing yeast ( Saccharomyces cerevisiae ) β-glucan composed of hollow yeast cell wall "ghosts" composed primarily of long polymers of β-(1,3) glucose obtained from the brewing yeast cell wall following a series of alkaline and acid extractions. Compared to other dectin-1 ligands such as zymosan, WGP Dispersible lacks TLR stimulatory activity. In contrast, soluble WGP can bind dectin-1 without activating this receptor and can significantly block WGP Dispersible's binding to macrophages and its immunostimulatory effects.

Zymosan is an insoluble preparation of yeast cells and activates macrophages via TLR2. TLR2 cooperates with TLR6 and CD14 in the response to zymosan. Zymosan can also be recognized by dectin-1, which acts as a phagocyte receptor expressed on macrophages and dendritic cells and cooperates with TLR2 and TLR6 to enhance the recognition of zymosan by each receptor. induced immune response.

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

Torosantucci et al. (2005) and Bromuro et al. (2010) revealed conjugates of branched β-glucan-laminarin and linear β-glucan-cadranan coupled with diphtheria toxoid CRM197. These conjugate vaccines induce high IgG titers against β-glucan and confer protection against fungal infections in mice. Alternatively, such conjugates can be used to detect high titers against CRM197 (Donadei et al., Mol Pharm. 2015 May 4;12(5):1662-72). The authors also generated β-glucan-CRM197 vaccines with synthetic linear β-(1,3)-oligosaccharides or β-(1,6)-branched β-oligosaccharides formulated with the human-acceptable adjuvant MF59. -(1,3)-oligosaccharides. All conjugates induced high titers of anti-β-(1,3)-glucan IgG and/or induced anti-β-(1,6) in addition to anti-β-(1,3)-glucan IgG. The high titers of )-dextran antibodies indicate the immunogenicity of different combinations of dextran and classic carrier proteins. Interestingly, Torosantucci et al. failed to demonstrate superior anti-CRM titers following immunization with CRM-dextran conjugates compared with unconjugated CRM alone.

Donadei et al. (2015) also analyzed conjugates of diphtheria toxoid CRM197 coupled with linear β-(1,3) glucan-cadranan or synthetic β-(1,3) oligosaccharides. Such conjugates are immunogenic and produce an antibody-like response to CRM197. Interestingly, the authors showed that CRM Cadran polysaccharide conjugates produced higher antibody titers when delivered intradermally compared to intramuscular (i.m.) immunization. However, intradermal administration of CRM-cadranan did not show different immunogenicity compared to subcutaneous administration. In addition, the in vivo effects of CRM-Cardran polysaccharide and non-Cardran polysaccharide coupled CRM using Alum as an adjuvant were comparable. Therefore, no additional benefit of CLEC coupling on the overall immune response could be detected in this system.

Liao et al. (2015) disclosed a series of linear β-(1,3)-β-glucan oligosaccharides (hexa-, octa-, deca-, and dodeca-β-glucans) that were coupled to KLH to produce glycoconjugates. These conjugates were shown to elicit robust 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 lethal pathogen Candida albicans ( C . albicans ). The article did not compare anti-KLH titers with unconjugated KLH, so no information on the potential benefits of β-glucans could be obtained in this experimental setting.

These findings are important for the suitability of glucan-based neoglycoconjugates as novel vaccines: latent anti-glucan antibodies induced after an initial glucan conjugate immunization could lead to rapid elimination of the same β-glucan vaccine in subsequent booster immunizations or could attenuate the immune response of the novel neoglycoconjugate vaccine for other indications, an effect that is well known for vector vaccines. As with the high levels of anti-glucan antibodies shown above for mannosaccharides and β-glucans (Petrushina et al. 2008, Torosantucci et al. 2005, Bromuro et al., 2010, Liao et al., 2015), the presence or even (re)stimulation of high levels of anti-glucan antibodies could therefore reduce or eliminate the latent immune response elicited by the conjugate vaccine. Therefore, for novel sustainable platforms using CLECs, especially β-glucan, as backbones for immunization, it will be crucial to ensure that the glucan antibody inducing capacity of the polysaccharides/oligosaccharides used is very low or absent.

Glucan particles (GP) are highly purified 2-4 µm hollow porous cell wall microspheres composed primarily of β-(1,3)-D-glucan with small amounts of β-(1,6)-D-glucan and chitin, typically isolated from brewing yeast using a series of hot alkaline, acid, and organic extractions. These particles interact with their receptors dectin-1 and CR3 (there is also evidence that interactions with ferulicityl receptors and CD5 are additional factors for GP function) and upregulate cell surface presentation of MHC molecules, causing changes in the expression of co-stimulatory molecules and inducing the production of pro-inflammatory interleukins. Due to their immunomodulatory properties, GP has been explored for vaccine delivery.

There are three general ways to use GP in vaccines: (i) as an adjuvant co-administered with one or more antigens to enhance T- and B-cell-mediated immune responses, (ii) chemical cross-linking with antigens and finally Commonly used (iii) serves as a physical delivery vehicle for antigen encapsulated within the GP cavity to provide targeted antigen delivery to APC.

(i): Antigen-specific adaptive immune responses can be enhanced by co-administering GP with the antigen. In this conventional adjuvant strategy, both the innate immune response and the adaptive immune response are activated to exert a protective response against the pathogen. For example, Williams et al. (Int J Immunopharmacol. 1989; 11(4): 403-10) added an adjuvant to a killed vaccine for Trypanosoma cruzi by co-administering GP. The immune response induced by this formulation resulted in an 85% survival rate in mice challenged with Trypanosoma cruzi. In contrast, the mortality rate in the control group that received dextrose, dextran, or the vaccine alone was 100%.

(ii): The carbohydrate surface of GP can also be covalently modified using _NaIO4 oxidation, carbodiimide cross-linking, or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate-mediated binding of antigen to the GP shell. Using this method, the coupling efficacy is extremely low (approximately 20%, as described in Pan et al. Sci Rep 5 , 10687 (2015)), which greatly limits the applicability and number of vaccine candidates compared to antigen encapsulation in GP or the platform technology provided in this application. Such covalently linked antigen-GP conjugates are used to study cancer immunotherapy and infectious diseases. For example, Pan et al. (2015) used OVA to cross-link to GP oxidized with periodate and used this vaccine to subcutaneously immunize mice. When mice were challenged with E.G7 lymphoma cells expressing OVA, a significant reduction in tumor size was observed. GP-OVA was detected in DCs (CD11c ⁺ MHC-II ⁺ ) in lymph nodes 12 and 36 hours after subcutaneous injection. Tumor protection was associated with increased total anti-Ova IgG titers, enhanced expression of MHC-II and co-stimulatory molecules (CD80, CD86), and elevated cytotoxic lymphocyte responses.

(iii): The most effective way to apply GP in vaccines is to use it to encapsulate vaccines/antigens in hollows. GP can encapsulate one or more antigens/DNA/RNA/adjuvants/drugs/combinations thereof with high payload efficiency, depending on the payload type and intended delivery mode.

Antigens can be encapsulated in the cavity of GP using polymer nanocomplexes, such as using bovine or mouse serum albumin and yeast RNA/tRNA to load the payload and complex or add calcium alginate or alginate-chitosan mixture. Using these strategies, for example, Huang et al. (Clin. Vaccine Immunol. 2013; 20:1585-91) reported that mice vaccinated with GP-OVA showed strong CD4+ T cell lymphoproliferation, Th1 and Th17-biased T cell-mediated immune responses, and high IgG1-specific and IgG2c-specific antibody responses to ovalbumin. Non-covalent encapsulation strategies induce stronger immune responses than GP co-administered with antigens.

An example of a GP-encapsulated subunit vaccine is GP coated with a soluble alkaline extract of a non-encapsulated strain of Cryptococcus neoformans (cap59), which induces antigen-specific CD4+ T cell responses (IFN- γ, IL-17A positive), reducing fungal colony-forming units (CFU) by more than 100-fold compared with the initial challenge dose, thereby protecting mice challenged with a lethal dose of highly virulent Cryptococcus neoformans (60% survival rate) (Specht CA et al. Mbio 2015; 6: e01905- e1915. and Specht CA et al., mBio 2017; 8: e01872- e1917.). In addition, inoculation of mice with GP-encapsulated antigens has been shown to be effective against Histoplasma capsulatum ( Deepe GS et al., Vaccine 2018; 36: 3359-67), Francisella tularensis ( F. tularensis ) (Whelan AO et al., PLOS ONE 2018; 13: e0200213), Blastomyces dermatitidis (Wuthrich M et al., Cell Host Microbe 2015; 17: 452-65) and Coccidioides posadasii ( C. posadasii ) (Hurtgen BJ et al., Infect. Immun . 2012; 80: 3960-74) is effective.

In addition to applications in cancer and infectious diseases, a limited number of studies using autologous antigens have been conducted using GP as an encapsulating agent for vaccine delivery. Along these lines, Rockenstein et al. (J. Neurosci., 2018 Jan 24 • 38(4):1000-1014) described the use of GP loaded with recombinant human α-synuclein (containing both B cell epitopes and T cell epitopes suitable for inducing anti-aSyn immune responses) and rapamycin, which is known to induce antigen-specific regulatory T cells (Tregs), in a mouse model of synucleinopathy. As expected from previous studies using full-length α-synuclein as an immunogen, administration of GP containing aSyn induced robust anti-α-synuclein antibody titers and reduced α-synuclein-triggered pathological changes in animals to a similar extent as previously published. The addition of rapamycin effectively induced the formation of iTreg (CD25 and FOXP3+) cells, as the number of these Treg cells increased significantly after rapamycin exposure. GP loaded with α-synuclein antigen and rapamycin thus triggered neuroprotective humoral and iTreg responses in a mouse model of synucleinopathy, with the combined vaccine (aSyn+rapamycin) being more effective than either humoral (GP aSyn) or cellular (GP rapamycin) vaccination alone. No information on the comparability of the effects of vaccination with known non-α-synuclein-containing GP has been reported in the literature.

β-glucan neoglycoconjugates efficiently target dendritic cells via the C-type lectin receptor dectin-1, enhancing their immunogenicity. Specifically, certain β-glucans are also used as 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 a potential vector for vaccination based on fusion proteins of MUC1 (Wang et al., Chem. Commun., 2019, 55, 253).

Xie et al. and Korotchenko et al. used branched β-glucan-laminarin as a backbone for OVA conjugation, and then administered these new glycoconjugates to mice epidermally or subcutaneously. Xie et al. showed that laminarin/OVA conjugates, but not the unconjugated mixture, induced increased anti-OVA CD4+ T cell responses compared to ovalbumin alone. Importantly, co-injection of unconjugated laminarin blocked this enhancement, supporting the function of laminarin-mediated APC targeting. As expected, native OVA and a mixture of OVA and laminarin stimulated low levels of anti-OVA antibody production. In contrast, the OVA/laminarin conjugate significantly enhanced the antibody response. Similarly, Korotchenko et al. demonstrated that the combination of laminarin with OVA significantly increased absorption and induced the activation of BMDC and the secretion of pro-inflammatory cytokines. These properties of the LamOVA conjugate also result in enhanced stimulation of OVA-specific naive T-cells co-cultured with BMDC. In prophylactic immunization experiments, the authors demonstrated that immunization with LamOVA reduced its sensitization and induced approximately three times higher IgG1 antibody titers than OVA after two immunizations. However, after the third immunization, in which all groups showed similar antibody titers, this effect disappeared in all treatment groups. Lam/OVA conjugates and OVA/alum conjugates have shown considerable therapeutic efficacy in murine models of allergic asthma. Therefore, these experiments cannot provide clear advantages of dextran-based conjugates over conventional vaccines.

Wang et al. (2019) analyzed the effects of β-glucan-based MUC1 cancer vaccine candidates. Again, the MUC1 tandem repeat sequence GVTSAPDTRPAPGSTPPAH, a well-studied cancer biomarker, was chosen as a peptide antigen to provide T cell and B cell antigenic determinants in the repeat sequence. β-glucan and MUC1 peptides were linked to yeast β-(1,3)-β-glucan polysaccharide using ethylene glycol (i.e., PEG) spacers using 1,1'-carbonyl-diimidazole (CDI)-mediated conditions. The size of the β-glucan-MUC1 nanoparticles was in the 150 nm range (actual average 162 nm), while the particles formed by unmodified β-glucan were approximately 540 nm. The β-glucan-MUC1 conjugate elicited high titers of anti-MUC1 IgG antibodies, significantly higher than the control group. Further analysis of the isotype and subtype of the antibodies produced showed IgG2b as the major subtype, indicating activation of a Th1-type response, as the ratio of IgG2b/IgG1 was >1. The large amounts of IgM antibodies observed indicated the involvement of the C3 component of the complement system, which often induces cytotoxicity, and the use of such backbones in vaccines where cytotoxicity should be avoided, such as vaccines for chronic or degenerative diseases, could be problematic.

US 2013/171187 A1 discloses an immunogenic composition comprising a glucan and a pharmaceutically acceptable carrier to induce protective anti-glucan antibodies. Metwali et al. (Am. J. Respir. Crit. Care Med. 185 (2012), A4152; poster session C31 Regulation of Lung Inflammation) studied the immunomodulatory effects of glucan derivatives in pneumonia. WO 2021/236809 A2 discloses a multi-antigen determinant vaccine comprising amyloid-β and tau peptide for the treatment of Alzheimer's disease (AD). US 2017/369570 A1 discloses β-(1,6)-glucan linked to antibodies against cells present in the tumor microenvironment. US 2002/077288 A1 discloses synthetic immunogenic but non-amyloid peptides homologous to amyloid-β for the treatment of AD, either alone or in combination. US 2013/171187 A1 discloses anti-glucan antibodies for use as protective agents against fungal infections of Candida albicans. WO 2004/012657 A2 discloses microparticle β-glucan as a vaccine adjuvant. CN 113616799 A discloses a vaccine carrier composed of oxidized mannopolysaccharide and a cationic polymer. CN 111514286 A discloses a Zika virus E protein conjugate vaccine with glucan. US 4,590,181 A discloses a viral antigen mixed in a solution with agar polysaccharide or mold glucan. Lang et al. (Front. Chem. 8 (2020): 284) reviewed carbohydrate conjugates in vaccine development. Larsen et al. (Vaccines 8 (2020): 226) reported that Psoralea corylifolia polysaccharide activated dendritic cells derived from chicken bone marrow in vitro and promoted ex vivo CD4 ⁺ T cell recall responses to infectious bronchitis virus. US 2010/266626 A1 discloses glucans, preferably laminarin and calanin, as antigens bound to adjuvants for immunization against fungi. Mandler et al. (Alzh. Dement. 15 (2019), 1133-1148) reported the effects of single and combined immunotherapy approaches targeting amyloid-β and α-synuclein in a model of Lewy body dementia. Mandler et al. (Acta Neuropathol. 127 (2014), 861-879) reported a next-generation active immunization approach against synuclein pathologies that uses short immunogenic (B cell reactive) peptides that are too short to induce T cell responses (autoimmunity) and that do not carry native antigenic determinants but carry sequences that mimic the original antigenic determinants (e.g., oligomeric α-synuclein), and the impact of this approach on clinical trials in Parkinson's disease (PD). Mandler et al. (Mol. Neurodegen. 10 (2015), 10) reported that active immunization against α-synaptotagmin ameliorated degenerative lesions and prevented demyelination in a model of multiple systemic atrophy (MSA). Jin et al. (Vaccine 36 (2018), 5235-5244) reviewed β-glucan as a potential immune adjuvant, mainly focusing on adjuvant properties, structure-activity relationships and receptor recognition properties. WO 2022/060487 A1 discloses a vaccine comprising a specific α-synaptotagmin peptide for the treatment of neurodegenerative diseases. WO 2022/060488 A1 discloses a multi-antigen determinant vaccine comprising amyloid-β and α-synaptotagmin peptides for the treatment of AD. US 2009/169549 A1 discloses conformational isomers of modified forms of alpha-synuclein, which are produced by introducing cysteine into the alpha-synuclein polypeptide and disrupting the disulfide bonds to form stable and immunogenic isomers. WO 2009/103105 A2 discloses a vaccine having a mimetic antigenic determinant of an alpha-synuclein antigenic determinant extending from amino acid D115 to amino acid N122 in the native alpha-synuclein sequence.

To date, there has been no published report demonstrating the construction or use of individual B cell or T cell antigenic determinant peptides coupled to β-glucan, especially linear β-glucan and/or Psoralea corylifolia polysaccharide having high binding specificity/capacity for dectin-1, thereby forming the novel neoglycoconjugate proposed in the present application.

It is therefore an object of the present invention to provide improved vaccines in the form of conjugate vaccines made from a vaccination antigen in combination with a carbohydrate-based CLEC adjuvant, and in particular to provide vaccines that provide an improved immune response in vaccinated individuals compared to current state-of-the-art conjugate vaccines, in particular carbohydrate-based CLEC-peptide/protein conjugate vaccines.

Another object of the present invention is to provide vaccine compositions that use the CLEC backbone to confer short, easily interchangeable, highly specific B/T cell antigenic determinant immunity with efficacy, specificity and affinity that have not previously been met by conventional vaccines.

A specific aim of the present invention is to provide a vaccine for the dermal compartment with improved selectivity and/or specificity of CLEC-based vaccines.

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

Another object of the present invention is to provide vaccine compositions for the appropriate prevention and treatment of alpha-synuclein pathologies, which use a CLEC backbone to confer short, easily interchangeable, highly specific B/T cell antigenic determinant immunity to alpha-synuclein with efficacy, specificity and affinity that were previously unsatisfactory with conventional vaccines.

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

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

Another object of the present invention is to provide peptide immunogen constructs of alpha-synuclein (aSyn) and formulations thereof for use in treating synuclein pathologies.

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

The present invention successfully solves one or more of the above-listed objectives. This is surprising to those skilled in the art because, to date, no reports have been published in the art that demonstrate the structure and applicability or efficacy of compounds similar to the novel, small, modular new sugar conjugates according to the present invention.

Surprisingly, it has been demonstrated by the present invention that by conjugation (i.e., by covalent coupling; used synonymously herein) of a peptide/protein to a selected CLEC carrier according to the present invention, wherein said conjugation can be based on currently state-of-the-art chemistry, superior pharmaceutical formulations are obtained that achieve an immune response. In the art, a large number of different conjugation methods are available. During the development of the present invention, hydrazone formation or conjugation via a heterobifunctional linker has been identified as a particularly preferred method. In general, activation of the CLEC (e.g., formation of a reactive aldehyde on the vicinal OH group of the sugar moiety) and the presence of a reactive group on the selected peptide/protein (e.g., a hydrazide residue at the N-terminus or C-terminus, an SH group (e.g., via an N-terminal or C-terminal cysteine)) are required prior to conjugation. The reaction can be a single step reaction (e.g. mixing activated CLEC with hydrazide-peptide to cause hydrazone formation) or a multi-step process (e.g. reacting activated CLEC with hydrazide from a heterobifunctional linker and subsequent coupling of peptides/proteins via the respective reactive groups).

Thus, the components of the conjugates of the invention can be coupled directly to each other, for example by coupling B cell epitopes and/or T cell epitopes to β-glucan and/or carrier proteins or by coupling β- Dextran is coupled to the carrier protein (in all possible orientations). References herein to "B cell epitope polypeptide" or "T cell epitope polypeptide" default to the B cell or T cell epitope of "B cell epitope polypeptide" or "T cell epitope polypeptide" , and does not imply a B-cell or T-cell epitope of the carrier protein (if present), unless it is expressly referred to as a B-cell or T-cell epitope of the carrier protein. According to a preferred embodiment, the B cell epitope and/or the T cell epitope are preferably connected to β-glucan or mannan and/or carrier protein through a linker, more preferably a cysteine residue. or linkers containing cysteine or glycine residues; linkers produced by: hydrazine-mediated coupling, via heterobifunctional linkers such as N-β-maleic acid amide Aminopropionic acid hydrazine (BMPH), 4-[4-N-maleyl iminophenyl]butyric acid hydrazine (MPBH), N-[ε-maleyl iminohexyl Coupling of acid) hydrazine (EMCH) or N-[κ-maleimidodecanoic acid] hydrazine (KMUH)), imidazole-mediated coupling, reductive amination, carbodiimide coupling- -NH- _NH2 linker, -NRRA, NRRA-C or NRRA-NH- _NH2 linker, peptide linker, such as dimer, trimer, tetramer (or longer polymer) peptide group, Such as CG or CG, or a cleavage site, such as a cathepsin cleavage site, or a combination thereof, especially via a cysteine or NRRA-NH- _NH2 linker. Obviously, "linker resulting from coupling mediated by, for example, chelazine" refers to the chemical structure resulting in the conjugate after binding, ie, the chemical structure present in the resulting conjugate after binding. The amino acid linker may exist in a conjugated form via a peptide bond (eg, a linker containing glycine) or via a functional group of the amino acid (eg, a disulfide bond in a cysteine linker).

Novel classes of conjugates according to the present invention are demonstrated to confer immunity to short, easily interchangeable, highly specific B cell/T cell epitopes by using the CLEC backbone of the present invention, demonstrating previously unsatisfactory capabilities of conventional vaccines. Efficacy, specificity and affinity: In fact, the conjugates according to the invention are the first examples of the use of short B-cell/T-cell epitopes in CLEC-based vaccines, avoiding the need to present the epitopes in the form of fusion proteins. Requirements include tandem repeats forming epitopes or fusion of different tandem repeats to form a stable and potent immunogen (as required for the MUCl approach using mannans mentioned above).

By the present invention, the necessity of using full-length proteins for CLEC vaccines (i.e., the payload in glucan particles (GP)) can also be avoided. In addition, the problem of autoimmune reactions when using CLEC can be avoided, especially those caused by (undesirable) T cell epitopes (e.g., Syn, amyloid beta) present in immunogens (e.g., autologous proteins). Immune responses induced by T cell epitopes in T cell epitopes) or mixed autologous epitopes (e.g., MUCl-tandem repeats used as immunogens).

According to the present invention, short antigenic determinants (B cell and/or T cell antigenic determinants, mainly peptides, modified peptides) can be conjugated for the first time to a functional CLEC-based scaffold using covalent coupling based on recognized chemistry, wherein the possible conjugation methods can be adapted to the requirements of the specific antigenic determinant based on methods well known in the art.

The presentation of short peptides according to the present invention can be carried out in the form of a single binding moiety (in the form of a short peptide or a long protein) combined with individual foreign T cell antigen determinants, or in the form of a complex/conjugate with a larger carrier molecule to provide T cell antigen determinants to induce a sustainable immune response. The design of the vaccine according to the present invention enables the preparation of multivalent conjugates as a prerequisite for the efficient immune response induced by efficient B cell receptor (BCR) cross-linking.

Furthermore, with the present invention, a CLEC-based vaccine with excellent selectivity/specificity for the dermal compartment can be provided. In fact, the conjugate design according to the present invention is based on CLEC as a carrier of target-specific epitopes that show high binding specificity for PRRs on dermal APC/DC, especially dectin-1 ( or MR and DC-SIGN of mannan) to achieve skin selectivity/specificity and receptor-mediated absorption (=targeted vaccine delivery).

The CLEC polysaccharide used as a carrier according to the present invention serves to concentrate the carrier-peptide conjugate into optimal dermal/skin DC and initiate an immune response. This is particularly due to epidermal or dermal (not subcutaneous) specificity. The CLEC backbone and efficient dermal immune response priming according to the present invention also help to avoid the mandatory use of adjuvants that are typical for 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 eliminated, for example, when the addition of adjuvants is not indicated.

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

The CLEC according to the present invention has been selected to provide a novel solution to efficiently target skin-specific DCs and skin-specific immunization. The conjugate according to the present invention also exerts limited immunological activity in other classical vaccination tissues such as muscle or subcutaneous tissue, which is in contrast to the use of previously described CLEC-based vaccines/candidate vaccines in intramuscular or subcutaneous injections. As a result of experiments conducted during the course of the present invention, the vaccines according to the present invention, especially the vaccines using Psoralea corylifolia polysaccharide as CLEC, were identified as having unexpected selectivity for skin immunization.

The present invention relates to any V cell and/or T cell epitope polypeptide and any predominantly linear β-(1,6)-glucan having a ratio of (1,6)-coupled monosaccharide moieties to non-β-(1,6)-coupled monosaccharide moieties of at least 1:1. As also shown in the Examples section below, the present teachings enable and support any V cell and/or T cell epitope polypeptide and do not disclose any limitations with respect to such epitopes, particularly where the epitope is already part of a prior art and/or existing epitope. The specific V cell and/or T cell epitope polypeptides as shown and mentioned herein are preferred epitopes, but the present invention is not limited thereto. After the many epitopes tested so far in the course of the invention (see the group of epitopes with a very diverse function and structure investigated and experimentally confirmed in the Examples section (including a large number of model epitopes)), no restrictions on the nature and structure of the B-cell and/or T-cell epitopes appeared (linear polypeptides, autologous peptides, polypeptides with post-translational modifications, such as sugar structures or pyroglutamine, mimetic epitopes, allergens, structural epitopes, conformational epitopes, etc.), especially for Psoralea corylifolia as a β-(1,6)-glucan. In each case, the experiments showed that the β-glucan according to the invention and the covalent binding to the epitope polypeptide were responsible for the immunological efficacy, rather than the specific structural features of the individual epitopes.

As used herein, the term "B-cell and/or T-cell epitope" is a functional term recognized in the technical field of the present invention: T-cell epitopes are presented on the surface of antigen-presenting cells, where they bind to primary Histocompatibility complex (MHC) molecules. In humans, professional antigen-presenting cell lines exclusively present class II MHC peptides, whereas most nucleating somatic cells present class I MHC peptides. T cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, whereas MHC class II molecules present longer peptides (13 to 17 amino acids in length). And non-classical MHC molecules also present non-peptide epitopes, such as glycolipids. A B cell epitope is part of the antigen to which an immunoglobulin or antibody binds. B cell epitopes may be, for example, conformational or linear.

According to a preferred embodiment of the present invention, the conjugate according to the present invention comprises a polypeptide having at least one B cell antigenic determinant and at least one T cell antigenic determinant, preferably a B cell antigenic determinant + CRM197 conjugate covalently linked to β-glucan, in particular a peptide + CRM197 + linear β-(1,6)-glucan or a peptide + CRM197 + linear Pseudomonas aeruginosa polysaccharide conjugate.

The preferred ratio of glucan to peptide, especially the ratio of Shi fungus polysaccharide to peptide is 10 to 1 (w/w) to 0.1 to 1 (w/w), preferably 8 to 1 (w/w) to the range of 2 to 1 (w/w), especially 4 to 1 (w/w), with the proviso that if the conjugate includes a carrier protein, β-glucan or mannan and the B cell epitope - The preferred ratio of carrier polypeptide is 50:1 (w/w) to 0.1:1 (w/w), especially 10:1 to 0.1:1.

With the present invention, it is possible to focus on inducing a target-specific immune response while inducing no or only a very limited CLEC- or carrier protein-specific antibody response. Therefore, the conjugates according to the present invention solve the problems posed by classical conjugate vaccines, which must rely on the use of foreign carrier proteins to induce a sustainable immune response. The current development of advanced technology conjugate vaccines is largely based on carrier molecules such as KLH, CRM197, tetanus toxoid or other suitable proteins. These carrier molecules are complexed with target-specific short antigens to form immune responses against different target diseases. Delivery to substrates, such as infectious, degenerative or neoplastic diseases, including, for example, Her2-neu positive cancer; delivery of alpha synuclein for synucleinopathies (e.g., Parkinson's disease); delivery of amyloidosis (e.g., Deliver amyloid beta peptides for Alzheimer's disease); Deliver tau protein for the treatment of tauopathies, including Alzheimer's disease; Deliver PCSK9 for hypercholesterolemia; Deliver IL23 for psoriasis; Deliver frontotemporal lobar degeneration ( FTLD) and amyotrophic lateral sclerosis (ALS); deliver TDP43 and FUS for Huntington's disease, immunoglobulin light chain and heavy chain amyloidosis (AL, AH, AA) ( Mutant) huntingtin; delivery of islet amyloid polypeptide (IAPP) and amyloid for type 2 diabetes; delivery of (mutant) transthyretin for ATTR/transthyretin amyloidosis; and others.

In view of the guidance of the prior art, β-glucans, especially mainly linear β-(1,6)-glucans themselves, are mainly used as antigens to cause infection against fungi in which such β-glucans are present. The specific immune response, and therefore the immune potency and efficiency of the conjugates according to the invention and vaccines comprising such conjugates are also unexpected and unexpected (see, e.g., US 2013/171187 A1; Metwali et al., Am. J. Respir . Crit. Care Med. 185 (2012), A4152; poster session C31; US 2013/171187 A1; US 2010/266626 A1; Jin et al. (Vaccine 36 (2018), 5235-5244)). However, through the present invention, it was confirmed that the conjugate according to the present invention was unable to induce a significant immune response to β-glucan, but due to the structure of the conjugate of the present invention, the immune response was transferred to B covalently bound to β-glucan. Cellular and/or T cell epitope polypeptides. Combining these B cell and/or T cell epitope peptides with linear β-glucan seems to hide the immune response inducing ability of β-glucan, but exposes and greatly improves the covalently coupled B cells and /or presentation of T cell epitope peptides to the immune system. This teaching is neither disclosed in the prior art nor obvious from such prior art:

US 2017/369570 A1, which discloses beta-(1,6)-glucan linked to antibodies directed against cells present in the tumor microenvironment, is based on a completely different concept and mechanism (tumor therapy).

On the other hand, dextran is used as a component in vaccines (mostly in the form of "(liposome) dextran (nano) particles"), but without covalent coupling of B cell and/or T cell epitope peptides In dextran (for example, WO 2004/012657 A2; CN 113616799 A; US 4,590,181 A; Lang et al., Front. Chem. 8 (2020): 284; Larsen et al., Vaccines 8 (2020): 226).

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

In view of these advantageous properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention can be specifically used for active anti-Tau protein vaccination, also including those undergoing truncation, (hyper)phosphorylation, nitration, glycosylation and/or Or ubiquitinated variants for the treatment and prevention of tau protein disorders, especially Alzheimer's disease and Down syndrome (Down Syndrome), or other tau protein disorders, including Pick disease (Pick disease), progressive Supranuclear palsy (PSP), corticobasal degeneration, chromosome 17-related frontotemporal dementia (FTDP-17), and argyrophilic granulopathy. Other emerging disease entities and pathologies include glomerular glial tauopathies, primary age-related tauopathies (PART), which include neurofibrillary tangle dementia, chronic traumatic encephalopathy (CTE), and age-related tau astrocytosis. In addition, other disease entities are included, such as vacuolar tauopathies, gangliogliomas and gangliomas, and Guam amyotrophic lateral sclerosis (lytico-bodig) disease (Guam Parkinson-dementia syndrome). , meningeal angiomatosis, postencephalitic parkinsonism, and subacute sclerosing panencephalitis (SSPE).

Tauopathies often overlap with synucleinopathies, possibly due to interactions between synuclein and tau proteins. Therefore, the anti-Tau conjugates according to the invention can be specifically used as active anti-Tau against synucleinopathies, especially Parkinson's disease (PD), dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD). Protein vaccination.

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

According to a preferred embodiment, the Tau protein-derived polypeptide is selected from native human Tau (441 aa isoform; GenBank entry>AAC04279.1; Seq ID No or a polypeptide comprising or consisting of: amino acid residues derived from human Tau, including post-translationally modified, phosphorylated, diphosphorylated, hyperphosphorylated, 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, Tau260-264, Tau 267-273, Tau294-305, 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, Tau 418-426, Tau 420-426.

According to a preferred embodiment, the Tau protein-derived polypeptide is selected from the above-mentioned Tau-derived polypeptide mimics, including simulated epitopes and amino acid substitutions containing simulated phosphorylated amino acids (including D-substituted phosphorylated S and peptides with E substituted phosphorylated T), including Tau176-186, Tau200-207, Tau210-218, Tau213-221, Tau225-234, Tau379-408, Tau389-408, Tau391-408, Tau393-402, respectively. Tau393-406, Tau418-426, Tau420-426.

US 2008/050383 A1 and Asuni et al. (Journal of Neuroscience 34: 9115-9129) revealed that Tau379-408 with two phosphorylated aas: pS396 and pS404 is suitable for antibodies for immunotherapy against Tau pathology and Boutajangout et al. ( J. Neurosci., December 8, 2010 30(49):16559 -16566) revealed that the same epitope: dual phosphorylated peptide Tau379-408 with pSp396 and pS404 was combined with the adjuvant AdjuPhos in the htau/PS1 model In several tests, it was as effective as active immunotherapeutics in preventing cognitive decline, which is associated with pathological tau reduction in the brain. Bi et al. (2011, PLoS One 12: e26860.) also showed that using double-phosphorylated sequence Tau395-406 (with pS396) bound to KLH and adjuvanted with complete or incomplete Freund's adjuvant Tau-targeted immunization with the 10-mer peptide pS404 prevented the progression of neurofibrillary histopathology in aged P301L Tau transgenic mice.

Boimel M et al. (2010; Exp Neurol 2: 472-485) showed that the use of bis-phosphorylated peptides Tau195-213[pS202/pT205], Tau207-220[ pT212/pS214] and Tau224-238[pT231] can alleviate Tau-related pathologies in animals.

Troquier et al. (2012 Curr Alzheimer Res 4: 397-405) showed that targeting Tau by active Tau immunotherapy in the THYTau22 mouse model could be a suitable therapeutic approach as a reduction in insoluble Tau species (AT100 and pS422 immunoreactivity) could be detected that correlated with significant cognitive improvements using a Y-maze. The immunotherapy used an artificial peptide construct consisting of an N-terminal YGG linker fused to a 7-mer (Tau418-426) or 11-mer (Tau417-427) peptide derived from human Tau carrying pS422, coupled to KLH and with CFA as adjuvant.

US 2015/0232524 A1 and Davtyan H et al (Sci Rep. 2016;6:28912, Vaccine. 2017;35:2015-24 and Alzheimer's Research & Therapy (2019) 11:107) and Joly-Amado et al (Neurobiol Dis . February 2020; 134: 104636) revealed peptide immunogens and showed that the vaccines AV-1980R and AV-1980D induced strong immune responses and reduced tau pathology in different tau pathology models. The two vaccines are based on the MultiTEP platform, developed by It consists of three repeating sequences of Tau2-18 fused to several mixed T cell epitopes, and is used as a recombinant polypeptide or DNA vaccine respectively.

EP 3 097 925 B1 discloses a peptide immunogen composed of a phosphopeptide derived from human Tau441, and Theunis et al. (2013, PLoS ONE 8(8): e72301) showed that based on EP 3 097 925 B1, a liposome vaccine carrying Tau peptide - Tau 393-408 (carrying pS396 and pS402) can induce anti-phospho-Tau antibodies, accompanied by improvement of clinical symptoms and reduction of tau protein pathology indicators in the brain of Tau.P301L mice.

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 with pS202 and pT205 and Tau395-406 with pS396 and pS404) produced stable pTau antibodies and could significantly reduce tau pathology and improve behavioral deficits in Tau Tg animal models.

EP 2 758 433 B1 discloses peptide-based immunogens for interfering with Tauopathies. The present invention discloses the use as a peptide conjugate vaccine (eg, as a peptide KLH vaccine). Kontsekova et al. (Alzheimer's Research & Therapy 2014, 6:44) reveal that such a peptide vaccine (i.e., Axon peptide 108 (Tau294-305; KDNIKHVPGGGS), conjugated to KLH and adjuvanted with Alum; also known as AADvac1) induces Stable protective humoral immune response, antibodies distinguish pathological tau from physiological tau. Active immunotherapy reduces tau oligomer levels and neurofibrillary pathology in the brains of transgenic rats.

Although in principle the present invention is able to improve all proposed Tau vaccine peptides, the selected antigenic determinants were specifically evaluated for their suitability for the platform of the present invention. For example, Tau294-305, SeqID35+36 was shown to be superior to the KLH-based vaccines proposed by EP2 758 433 B1 and Kontsekova et al.

Other good target sequences include: Tau Location sequence 195-213 SGYSSPGSPGTPGSRSRTP 207-220 GSRSRTPSLPTPPT 224-238 KKVAVVRTPPKSPSS 393-408 VYKP SPVVSGDTP SPRHL 379 - 408[P-Ser396,404] RENAKAKTDHGAEIVYKpSPVVSGDTpSPRHL Tau260-264[P-Ser262] I G 294-305 KDNIKHVPGGGS 251-280 PDLKNVKSKIGSTENLKHQPGGGKVQIINK 256-285 VKSKIGSTENLKHQPGGGKVQIINKKLDLS 256-285-pS262 VKSKIGpSTENLKHQPGGGKVQIINKKLDLS 259-288 KIGSTENLKHQPGGGKVQIINKKLDLSNVQ 275-304 VQIINKKLDLSNVQSKCGSKDNIKHVPGGG 201-230-pT217 GSPGTPGSRSRTPSLPpTPPTREPKKVAVVR 379-408-pS396pS404 RENAKAKTDHGAEIVYKpSPVVSGDTpSPRHL 181-210-pS202pT205 TPPSSGEPPKSGDRSGYSSPGpSPGpTPGSRS 300-317 VPGGGSVQIVYKPVDLSK 267-273 QUR 298-304 KHVPGGG 329-335 HHVPGGG 361-367 THVPGGG 200-207 (pS202/pT205) PGpSPGpTPG 200-207 (pS202/pT205) phosphate mimetic PGDPGEPG 200-207 (pS202/pT205) phosphate mimetic SPGDPGEPG 210-218 (pT212/pS214) SRpTPpSLPTP 210-218 (pT212/pS214) phospho-mimetic SREPDLPTP 210-218 (pT212/pS214) phospho-mimetic SREPDLP 213-221 (pT217) PSLPpTPPTR 213-221 (pS214/pT217) PpSLPpTPPTR 213-221 (pT217) Phospho-mimetic PSLPEPPTR 213-221 (pS214/pT217) phospho-mimetic PDLPEPPTR 393-402 (pSer396) VxVd 393-402 (pSer396) phospho-mimetic VYKDPVVSG 393-406 (pSer396, pSer404) VYKP SPVVSGDTP SPR 393-406 (pSer396, pSer404) phospho-mimetic VYKDPVVSGDTDPR 176-186 (pT181) PPAPKpTPPSSG 176-186 (pT181) phosphate mimetic PPAPKEPPSSG 225-234 (pT231) KVAVVRTPPK 225-234 (pT231) Phospho-mimetic KVAVVREPPKS Tau379 - 408[P-Ser396,404] RENAKAKTDHGAEIVYKpSPVVSGDTpSPRHL Tau379 - 408[P-Ser396,404] phospho-mimetic RENAKAKTDHGAEIVYKDPVVSGDTDPRHL 389-408 GAEIVYKpSPVVSGDTpSPRHL 391-408 EIVYKpSPVVSGDTpSPRHL 389-408 Phosphoric acid analog GAEIVYKDPVVSGDTDPRHL 391-408 Phosphoric acid analog EIVYKDPVVSGDTDPRHL 418-426 DMVDpSPQLA 418-426 Phosphoric acid analog DMVDDPQLA 420-426 VxDV 420-426 Phosphoric acid analog VDDPQLA 362-366 HVPGG (LDNIT HVPGG GNKKIE) 235-246 SPSSAKSRLQTA

In view of these advantageous properties of the conjugate of the present invention, the conjugate and vaccine according to the present invention can be specifically used for active immunotherapy of IL12/IL23 related diseases and autoimmune inflammatory diseases. IL-23 related diseases are selected from the group consisting of: psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, diabetes (preferably type 1 diabetes), atherosclerosis, inflammatory bowel disease (IBD)/M. Crohn, multiple sclerosis, Behcet disease, ankylosing spondylitis, Vogt-Koyanagi-Harada disease ), chronic granulomatous disease, hidratenitis suppurativa, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, neurodegenerative disease (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. Additionally, IL-12/23-directed vaccines could be used together/in combination with vaccines targeting other targets, as recent data suggest that IL-23-driven inflammation can exacerbate other diseases, such as Alzheimer's disease or possibly diabetes.

According to a preferred embodiment, the IL12/IL23 protein-derived polypeptide is derived from native human IL12/IL23 or is a mimetic having one or more aa exchanges forming mimetic epitopes of the respective native sequences.

According to a preferred embodiment, the polypeptide derived from IL12/IL23 protein is selected from the subunit of heterodimeric protein IL23 - native human IL-23p19 or a polypeptide comprising or consisting of amino acid residues derived from the subunit or mimicking antigenic determinants. In WO 2005/108425 A1, peptides derived from IL-23p19, FYEKLLGSDIFTGE, FYEKLLGSDIFTGEPSLLPDSP, VAQLHASLLGLSQLLQP, GEPSLLPDSPVAQLHASLLGLSQLLQP, PEGHHWETQQIPSLSPSQP, PSLLPDSP, LPDSPVA, FYEKLLGSDIFTGEPSLLPDSPVAQLHASLLGLSQLLQP, LLPDSP, LLGSDIFTGEPSLLPDSPVAQLHASLLG, FYEKLLGSDIFTGEPSLLPDSPVAQLHASLLG, QPEGHHW, LPDSPVGQLHASLLGLSQLLQ and QCQQLSQKLCTLAWSAHPLV, were proposed as IL-23 vaccination peptides. In WO 03/084979 A2, GHMDLREEGDEETT, LLPDSPVGQLHASLLGLSQ and LLRFKILRSLQAFVAVAARV from IL-23p19 are mentioned as possible anti-interleukin vaccines. WO 2016/193405 A1 discloses a peptide immunogen derived from the IL12/23 p19 subunit (accession number: Q9NPF7) For a possible anti-interleukin vaccine, the subunit has the above amino acid sequence, especially aa136-145, aa136-143, aa 136-151, aa137-146, aa144-154, aa144-155 and others, especially the sequences: QPEGHHWETQQIPSLS, GHHWETQQIPSLSPSQPWQRL, QPEGHHWETQ, TQQIPSLSPSQ, QPEGHHWETQQIPSLSPSQ, QPEGHHWETQQIPSLSPS.

According to a preferred embodiment, the polypeptide derived from the IL12/IL23 protein is selected from the subunit of the heterodimeric protein IL23 - native human IL12/23p40 or a polypeptide comprising or consisting of: native human IL12/23p40 (Accession number: P29460 .1) Amino acid residues aa15-66, aa38-46, aa53-71, aa119-130, aa160-177, aa236-253, aa274-285, aa315-330, the IL12/23p40 has the following amino acids sequence:

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

Luo et al. J Mol Biol 2010 Oct 8;402(5):797-812. The conformational antigenic determinant aa15-66 of the anti-IL12/IL23p40 specific antibody Ustekinumab was revealed, which effectively reduced IL12/IL23-related diseases. Guan et al. (Vaccine 27 (2009) 7096-7104) disclosed immunogens aa38-46, aa53-71, aa119-130, aa160-177, aa236-253, aa274-285, aa315-330 of murine IL12/23 (accession numbers: P43432 (p40) and Q9EQ14 (p19)), wherein the IL12/23 has the following amino acid sequence: P43432 (p40): ; Connected to HBcAg in a recombinant manner.

Although in principle the invention is able to improve all proposed IL12/IL23 related disease vaccination peptides, the selected epitopes were specifically evaluated for their suitability for the platform of the invention. For example, SeqID37/38 and SeqID41/42 WISIT vaccines were shown to be superior to KLH based vaccines. The murine sequence SeqID39/40 showed similar efficacy to the KLH based conjugate in mice and was also active in IL12/23 recognition.

In view of these favorable properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention can be specifically used for active anti-EMPD (extracellular membrane proximal domain, as part of membrane IgE-BCR) vaccination to treat and prevent IgE-related diseases. Exclusive targeting and cross-linking of membrane IgE-BCR has been achieved by targeting the membrane anchoring region, i.e., the extracellular membrane proximal domain of IgE (EMPD IgE), which is only present on membrane-IgE and not on soluble serum IgE. IgE-related diseases include allergic diseases such as seasonal, food, pollen, mold spores, poisonous plants, drugs/medications, 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, waxes, polishes, cement, and leather; chronic sinusitis, atopic eczema; autoimmune diseases in which IgE plays a role ("autoallergy"); chronic (idiopathic) and autoimmune urticaria, choline-induced urticaria, mastocytosis, especially They are cutaneous mastocytosis, allergic bronchopulmonary aspergillosis, chronic or recurrent idiopathic angioedema, interstitial cystitis, systemic allergic reactions, especially idiopathic and exercise-induced systemic allergic reactions; immunotherapy, eosinophilic glomerulonephritis (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 according to the present invention is used to treat lymphoma or prevent allergic side effects of antacid therapy, especially for gastric or duodenal ulcers or reflux. For the present invention, the term "IgE-related disease" includes the term "IgE-dependent disease" or "IgE-mediated disease" or synonyms thereof.

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

The development of specialized antibodies that specifically target human or mouse EMPD IgE enables clinical and preclinical validation of this targeting strategy in vitro and in vivo (Liour et al., 2016 Pediatr Allergy Immunol Aug;27(5): 446-51). The concept of IgE-BCR cross-linking was initially developed 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.) was demonstrated in vivo by passive administration of anti-EMPD IgE antibodies. Chen et al. (2010 Journal of Immunology 184, 1748-1756) showed that mAbs specific to the N-terminal or central segment of CemX can bind to mIgE-expressing B cells and effectively induce their apoptosis and ADCC. CemX refers to 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 and is known as the CemX or M1' peptide. This antibody specifically targets 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). It shows antibodies against CemX, while the C-terminal P3 (GAGRADWPGPP ) without success.

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 immunogens carrying HBcAg inserts of CemX or its P1, P2 and P1-P2 portions as an anti-EMPD vaccine.

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

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 AVSVNPGLAGGSAQSQRAPDRVLCHSGQQQGLPRAAGGSVP, QQQGLPRAAGG, QQLGLPRAAGG, QQQGLPRAAEG, QQLGLPRAAEG, QQQGLPRAAG, QQLGLPRAAG, QQQGLPRAAE, QQLGLPRAAE, HSGQQQGLPRAAGG, HSGQQLGLPRAAGG, HSGQQQGLPRAAEG, HSGQQLGLPRAAEG, QSQRAPDRVLCHSG, GSAQSQRAPDRVL and WPGPPELDV.

Although, in principle, the present invention is capable of improving all proposed IgE-related disease vaccination polypeptides, selected epitopes are specifically assessed for their suitability for the platform of the present invention. For example, SeqID43/44 (QQQGLPRAAGG) was shown to be superior to KLH-based vaccines.

In view of these advantageous properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention can be specifically used for active anti-human epidermal growth factor receptor 2 (anti-Her2) vaccination to treat and prevent Her2-positive neoplastic diseases. Amplification or overexpression of Her2 occurs in approximately 15% to 30% of breast cancers and 10% to 30% of gastric/gastroesophageal cancers, and serves as a prognostic and predictive biomarker. Her2 overexpression is also seen in other cancers such as ovarian, endometrial and serous endometrial cancer, cervical cancer, bladder cancer, lung cancer, colorectal cancer, and head and neck cancer. According to a preferred embodiment, the polypeptide derived from the Her2 protein is derived from native human Her2 or is a mimetic having one or more aa exchanges to form a simulated epitope of the respective native sequence.

Dakappagari et al. (JBC (2005) 280, 1, 54-63) revealed a conformational antigenic determinant aa626-649 synthesized colinearly with a promiscuous TH antigenic determinant derived from the measles virus fusion protein MVF (amino acids 288-302) and cyclized via a disulfide bridge. The peptide was formulated with the muramyl dipeptide adjuvant nor-MDP (N-acetylglucosamin-3-yl-acetyl-L-propylaminoyl-D-isoglutamine) and emulsified in Montanide ISA 720. The vaccines have been immunogenic and immunization with these vaccines reduced tumor burden in tumor models.

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 simulate various regions of the dimerization loop of the receptor. The vaccine candidate also contains the T cell epitope of MVF (aa 288-302) KLLSLIKGVIVHRLEGVE and the GPSL linker. All peptides elicited high anti-Her2 immune responses and constructs using peptides aa266-296 were equally effective compared to Herceptin. aa266-296 peptide of Her2 sequence (accession number P04626): As a vaccine, it statistically reduced tumorigenesis in two transplantable tumor models and significantly reduced tumor development in two transgenic mouse tumor models.

Garret et al. (J Immunol 2007; 178:7120-7131) revealed Her2 peptides as immunogens aa563-598, aa585-598, aa597-626 and aa613-626 mixed with fusion proteins derived from measles virus (aa 288-302) Th epitopes are synthesized inline and administered in combination with Montanide ISA 720. The vaccines have become immunogenic and immunization with these vaccines carrying the aa597-626 epitope significantly reduced tumor burden in tumor models.

Jasinska et al. (Int. J. Cancer: 107, 976-983 (2003)) revealed 7 peptides from the extracellular domain of Her2 as potential antigens for cancer immunotherapy: P1 aa115-132 AVLDNGDPLNNTTPVTGA, P2 aa149-162 LKGGVLIQRNPQLC , P3 aa274-295 YNTDTFESMPNPEGRYTFGAS, P4 aa378-398 PESFDGDPASNTAPLQPEQLQ, P5 aa489-504 PHQALLHTANRPEDE, P6 aa544-560 CRVLQGLPREYVNARHC, P7 aa610-623 YMPIWKFPDEEGAC, these peptides are occasionally related to tetanus toxoid combined and used Gerbu as an adjuvant, and in animal models Induces humoral immune responses with anti-tumor activity. Similarly, Wagner et al. (2007 Breast Cancer Res Treat. 2007;106:29-38) disclosed peptide immunogens for vaccination studies, combining single peptides P4 (aa378-394: PESFDGDPASNTAPLQPC), P6 (aa545-560: RVLQGLPREYVNARHC) and P7 (aa610-623: YMPIWKFPDEEGAC) were administered coupled with tetanus toxoid using Gerbu as adjuvant. Vaccination was performed with or without the addition of IL12 and produced anti-tumor efficacy in preclinical models. Tobias et al. 2017 (BMC Cancer (2017) 17:118) revealed peptide immunogens for vaccination studies, combining single peptides P4 (aa378-394: PESFDGDPASNTAPLQP), P6 (aa545-560: RVLQGLPREYVNARHC) and P7 (aa610- 623: YMPIWKFPDEEGAC) was administered in combination with the following mixed peptides: P467 (PESFDGDPASNTAPLQPRVLQGLPREYVNARHSLPYMPIWKFPDEEGAC) and P647 (RVLQGLPREYVNARHSPESFDGDPASNTAPLQPYMPIWKFPDEEGAC). The cysteine (C) of P6 is replaced by "SLP" or "S" respectively. Both constructs were coupled to virions or diphtheria toxoid CRM197 (CRM), combined with Montanide or aluminum hydroxide (Alum) as adjuvants, and the induced antibodies exhibited antitumor properties.

Riemer et al. (J Immunol 2004; 173:394-401) reported the generation of peptide mimetics of the antigenic determinant recognized by Trastuzumab on Her-2/neu by using a defined 10-mer phage display library. The peptide mimetic antigenic determinant was coupled to the immunogenic carrier, tetanus toxoid (TT), and aluminum hydroxide was used as an adjuvant. The sequences included: 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, Vol. 5, No. 7, e1171446) disclosed mimetic epitopes of trastuzumab epitopes derived from an AAV-mimetic epitope library platform. The mimetic epitope sequences tested included RLVPVGLERGTVDWV, TRWQKGLALGSGDMA, QVSHWVSGLAEGSFG, LSHTSGRVEGSVSLL, LDSTSLAGGPYEAIE, HVVMNWMREEFVEEF, SWASGMAVGSVSFEE. QVSHWVSGLAEGSFG and LSHTSGRVEGSVSLL, which were shown to be immunogenic and effective in tumor models.

Miyako et al. (ANTICANCER RESEARCH 31: 3361-3368 (2011)) reveal, inter alia, peptides from the Her-2/neu extracellular domain (aa167-175) in the form of Her-2/neu-associated multiple antigenic peptides (MAP) exist. Her-2/neu peptide contains epitopes of CD4+ and CD8+ T cells, which contributes to the inhibitory effect on the growth of tumor cells expressing Her-2/neu. The sequence revealed includes: Peptide sequence (B; tert-butoxycarbonyl residue (Boc)). N:143-162 (RSLTEILKGGVLIQRNPQLC-BBB)8-K4K2KB N:153-172 (VLIQRNPQLCYQDTILWKDI-BBB)8-K4K2KB N:163-182 (YQDTILWKDIFHKNNQLALT-BBB)8-K4K2KB N:173-192 (FHKNNQLALTLIDTNRSRAC-BBB)8-K4K2KB N:183-202 (LIDTRNRSRACHPCSMPCKGS-BBB)8-K4K2KB N:193-212 (HPCSMPCKGSRCWGESSEDC-BBB)8-K4K2KB N:203-222 (RCWGESSEDCQSLTRTVCAG-BBB)8-K4K2KB N:213-232 (QSLTRTVCAGGCARCKGPLP-BBB)8-K4K2KB N:223-242 (GCARCKGPLPTDCCHEQCAA-BBB)8-K4K2KB N:233-252 (TDCCHEQCAAGCTGPKHSDC-BBB)8-K4K2KB N:243-263 (GCTGPKHSDCLACLHFNHSG-BBB)8-K4K2KB N:253-272 (LACLHFNHSGICELHCPALV-BBB)8-K4K2KB N:263-282 (ICELHCPALVTYNTDTFESM-BBB)8-K4K2KB N:273-292 (TYNTDTFESMPNPEGRYTFG-BBB)8-K4K2KB N:283-302 (PNPEGRYTFGASCVTACPYN-BBB)8-K4K2KB N:292-310 (GASCVTACPYNYLSTDVGS-BBB)8-K4K2KB N:300-321 (PYNYLSTDVGSCTLVCPLHNQE-BBB)8-K4K2KB N:312-330 (TLVCPLHNQEVTAEDGTQR-BBB)8-K4K2KB N:322-341 (VTAEDGTQRCEKCSKPCARV-BBB)8-K4K2KB N:332-351 (EKCSKPCARVCYGLGMEHLR-BBB)8-K4K2KB N:343-361 (YGLGMEHLREVRAVTSANI-BBB)8-K4K2KB N:352-370 (EVRAVTSANIQEFAGCKKI-BBB)8-K4K2KB

After immunization of mice, the humoral immune response was induced, tumor growth was inhibited, and tumor-infiltrating lymphocytes contained more CD8+ T cells, which secreted larger amounts of interleukin- 2.

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

Kaumaya et al. (ONCOIMMUNOLOGY 2020, Vol. 9, No. 1, e1818437) disclosed the following combination: 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), emulsified in Montanide ISA 720VG) and a novel PD1 immune checkpoint-targeted vaccine (PD-1 B cell peptide antigen determinant (aa92-110; GAISLAPKAQIKESLRAEL), combined with viral fusion peptide (MVF) amino acids 288-302 via four amino acid residues (GPSL), emulsified in Montanide ISA 720VG) for the treatment of Her2-positive disease. Therefore, providing a combination of anti-proliferative disease vaccines, especially a combination of cancer target-specific vaccines and immune checkpoint targeted vaccines is also a preferred embodiment.

Although in principle the invention is able to improve all proposed Her2-related disease vaccine peptides, the selected antigenic determinants were specifically evaluated for their suitability for the platform of the invention. For example, SeqID No 47/48 (aa610-623: YMPIWKFPDEEGAC) was shown to be superior to CRM-based vaccines.

In view of these favorable properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention can be specifically used for personalized neoantigen-specific therapy, preferably in the case of NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-C1, MAGE-C2, MAGE-C3, Survivin, gp100, tyrosinase, CT7, WT1, PSA, PSCA, PSMA, STEAP1, PAP, MUC1, 5 T4, KRAS or Her2.

In view of these favorable properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention can be specifically used for active anti-immune checkpoint vaccination to control the cancer microenvironment, for the treatment and prevention of proliferative diseases and for the treatment and prevention of T cell dysfunction in cancer/proliferative 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.

It is recognized in the field that compared with healthy controls, PD patients have different changes in the T cell compartment (for example: 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 are, for example, reduced absolute lymphocyte counts, reduced absolute and relative counts of total T cells, reduced absolute and relative counts of CD4+ and sometimes also reduced absolute and relative counts of CD8+ lymphocytes. , increased Th1/Th2 and Th17/Treg ratios, and increased expression of pro-inflammatory cytokines. However, most of these changes are also found during healthy aging, making it difficult to discern the impact of diseases such as PD, which exhibit a very wide range of onset (approximately 30 to 90 years) and variable progression rates. Regarding absolute cell numbers, there seems to be consensus that there is a net decrease in CD3+CD4+ T cells in PD. This CD4 reduction is supported by the change in CD4:CD8 ratio.

Along these lines, for example, Bhatia et al. (J Neuroinflammation (2021) 18:250) show an overall decrease in the total number of CD3+ T cells in PD that correlates with disease severity (e.g., as measured using H+Y phase) . This suggests that systemic T cell dysfunction progresses as the disease progresses, possibly reflecting the combined effects of ongoing inflammation, drug therapy, and lifestyle changes. Furthermore, Lindestam Arlehamn et al. (2020) showed that the highest T cell activity can be detected in PD patients during the prodromal or early clinical stage (<10 years duration and H+Y stage 0-2).

Therefore, providing treatment for enhancing or maintaining T cell numbers, especially T effector cell numbers and T cell functions in PD patients is a preferred embodiment of the present invention. This preferably includes combining a checkpoint inhibitor or a vaccine using anti-immune checkpoint inhibitor epitopes with the target-specific vaccine of the present invention to induce an anti-immune checkpoint inhibitor immune response to enhance or maintain immune response in PD patients. T cell number, especially T effector cell number and T cell function.

Patients suitable/applicable for treatment are characterized by an overall decrease in CD3+ cells, especially the overall decrease in CD3+CD4+ cells typical of PD patients at all disease stages. The preferred stages of disease to define the appropriate patient population for this panel are H+Y 1-4, preferably H+Y 1-3, and optimal H+Y 2-3.

Examples of such immune checkpoint targeted vaccines are vaccines that provide antigenic determinants to the following immune checkpoints: cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, accession number P16410) and planned cell death protein 1 (PD1, accession number Q15116) or its ligands - planned 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); these immune checkpoints have the following amino acid sequences: Human CTLA4: >sp|P16410|CTLA4_ Uniprot Human PD1:＞sp|Q15116|PDCD1_ Uniprot Human PD1-L1 ＞sp|Q9NZQ7|PD1L1_ Uniprot Human B7-H3 - CD276 ＞sp|Q5ZPR3|CD276_ Uniprot Human B7-H4 - VTCN1 ＞sp|Q7Z7D3|VTCN1_ Uniprot Human LAG3: >sp|P18627|LAG3_ Uniprot Human Tim3: >sp|Q8TDQ0|HAVR2_ Uniprot

Antibodies targeting CTLA-4 inhibit the immune response in several ways, including blocking autoreactive T cell activation at proximal steps of the immune response, typically in the lymph nodes. In contrast, the PD-1 pathway regulates T cells later in the immune response, usually in peripheral tissues. Therefore, there are currently two main clinical intervention directions to manipulate immune checkpoints by targeting CTLA-4 or PD-1/PD-L1: anti-CTLA-4 is involved in lymphocyte proliferation after activation of antigen-specific T cell receptors process, whereas anti-PD-1/PD-L1 mainly acts on peripheral tissues during the effector step. However, CTLA-4 is also expressed on regulatory T lymphocytes and is therefore involved in peripheral suppression of T cell proliferation.

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

According to a preferred embodiment, the polypeptide derived from the CTLA4 protein is derived from native human CTLA4 or is a mimetic having one or more aa exchanges to form a simulated epitope of the respective native sequence.

According to a preferred embodiment, the polypeptide derived from the PD1 protein is derived from native human PD1 or is a mimetic with one or more aa exchanges, forming a mimetic antigenic determinant of the respective native sequence. The protein sequence corresponds to the extracellular domain of mouse PD1 (Q02242; Uniprot) and human PD1 (Q15116; Uniprot).

According to a preferred embodiment, the polypeptide derived from the PD-L1 protein is derived from native human PD-L1 or is a mimetic having one or more aa exchanges to form a simulated epitope of the respective native sequence.

Guo et al. (Br J Cancer. 2021;125:152-154) and Kaumaya et al. (Oncoimmunology. 2020;9:1818437) revealed a PD1-derived peptide (aa92-110: GAISLAPKAQIKESLRAEL), which has the characteristics of CT26 colorectal cancer cells. Induction of antibodies that reduce tumor growth in the genotypic BALB/c model. Furthermore, the disclosed combination of PD1-epitope vaccine and HER-2 peptide vaccine exhibits enhanced tumor growth inhibition in colorectal cancer.

Tobias et al. (Front Immunol. 2020;11:895.) revealed peptides/mimetic epitopes from mouse and human PD-1 (= epitopes of anti-human PD1 mAb nivolumab and anti-mouse mAb pure line 29F.1A12). The peptides contain the human PD1-derived sequences PGWFLDSPDRPWNPP, FLDSPDRPWNPPTFS, and SPDRPWNPPTFSPA, corresponding to positions aa21-35, aa24-38, and aa27-41 on human PD1, respectively, denoted JT-N1, JT-N2, and JT-N3. In addition, the mimetic epitope for mouse PD1 includes ISLHPKAKIEESPGA (JT-mPD1) corresponding to amino acid residues aa126-140 of mPD1. The anti-tumor effect of the mimetic epitope JT-mPD1 was demonstrated to be associated with a significant reduction in tumor proliferation and an increase in apoptosis rate in the used syngeneic tumor mouse model expressing Her-2/neu. In addition, the anti-tumor effect of the Her2/neu vaccine was demonstrated to be enhanced when combined with JT-mPD1.

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

These anti-immune checkpoint vaccines can be highly effective when used alone or in combination with pre-existing peptide vaccines. Thus, a preferred embodiment provides a combination of an anti-immune checkpoint vaccine with a pre-existing peptide vaccine to treat a proliferative or degenerative disease, such as Parkinson's disease.

Although in principle the invention is able to improve all proposed PD1 and PD-L1 related vaccine peptides, the selected antigenic determinants were specifically assessed for their suitability for the platform of the invention. For example, SeqID No 49/50 (GAISLAPKAQIKESLRAEL) was shown to be superior to KLH based vaccines.

In view of these advantageous properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention can be specifically used for active anti-Aβ immunotherapy for prevention, treatment and diagnosis related to β-amyloid formation and/or aggregation. disease. The most prominent form of beta-amyloidosis is Alzheimer's disease (AD). Other examples include familial and sporadic AD, familial and sporadic Aβ cerebral amyloid vasculopathy, hereditary cerebral hemorrhage with amyloidosis (HCHWA), dementia with Lewy bodies and Down syndrome, glaucoma Retinal ganglion cell degeneration, inclusion body myositis/myopathy.

Aβ peptides exist in several 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, pyroglutamic acid Aβ3-40/42 (i.e., AβpE3-40 and AβpE3-42), and Aβ4-42, which appears to play a major role in neurodegeneration.

According to a preferred embodiment, the Aβ peptide-derived polypeptide is selected from native human Aβ1-40 and/or Aβ1-42, which has the following amino acid sequence: Aβ1-40: Aβ 1-42: Or polypeptides containing or consisting of amino acid residues derived from human Aβ1-40 and/or Aβ1-42, including truncation, especially N-terminal truncation, C-terminal truncation, post-translational modification, nitration , glycosylated, acetylated, ubiquitinated peptide amino acids or peptides carrying pyroglutamic acid residues at aa3 or a11, including Aβ aa1-6, aa1-7, aa1-8, aa1- 9. aa1-10, aa1-11, aa1-12, aa1-13, aa1-14, aa1-15, aa1-21, aa2-7, aa2-8, aa2-9, aa2-10, aa3-8, aa3-9, aa3-10, aa pE3-8, aa pE3-9, aa 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.

According to a preferred embodiment, the Aβ 1-40 or Aβ 1-42 derived polypeptide is selected from the mimics of the above-mentioned Aβ derived polypeptides, including simulated epitopes and amino acids containing simulated pyroglutamic acid amino acids. Substituted peptides. Schenk et al. (Nature. 1999 Jul 8;400(6740):173-7.) disclosed Aβ1-42 as an immunogen for anti-Aβ immunotherapy, and Pride et al. (Neurodegenerative Dis 2008;5:194-196) disclosed Aβ1-42 as an immunogen for anti-Aβ immunotherapy. CRM197 couples the peptide epitope of Aβ1-6 with QS21 as adjuvant, and Wiesner et al. (J Neurosci. 2011 Jun 22;31(25):9323-31) revealed that Qβ as potent immunotherapeutics Virus-like particle coupled Aβ1-6 peptide.

Wang et al. (Alzheimer's & Dementia: Translational Research & Clinical Interventions 3 (2017) 262-272) and US 2018/0244739 A1 disclose Aβ 1-42 peptide immunogens, especially UB311, which comprises two Aβ immunogens in an equimolar ratio, namely cationic Aβ1-14-εK-KKK-MvF5 Th [ISITEIKGVIVHRIETILF] and Aβ1-14-εK-HBsAg3 Th [KKKIITITRIITIITID] peptides, which are mixed with polyanionic CpG oligodeoxynucleotides (ODN) to form a stable immunostimulatory complex of micron-sized particles, to which aluminum mineral salt (Adju-Phos) is added to obtain the final formulation.

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

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 antigenic determinants from tetanus toxin - P30 and P2 and three copies of the B cell antigenic determinant of Aβ1-12, using QuilA as an adjuvant. Similarly, Davtyan H et al. (Alzheimer's & Dementia 10 (2014) 271-283) disclosed a DNA-based vaccine based on protein coding regions consisting of an immunoglobulin (Ig) k-chain signal sequence, three copies of the Aβ1-11 B cell epitope, a synthetic peptide (PADRE) and a string of eight non-self promiscuous Th epitopes from tetanus toxin (TT) (P2, P21, P23, P30 and P32), hepatitis B virus (HBsAg, HBVnc) and influenza (MT), or additionally comprising three other Th epitopes from TT (P7 (NYSLDKIIVDYNLQSKITLP); P17 (LINSTKIYSYFPSVISKVNQ); and P28 (LEYIPEITLPVIAALSIAES).

Petrushina et al. (Journal of Neuroinflammation 2008, 5:42) revealed Aβ1-28 coupled to bromoacetylated brewing yeast mannosaccharide with an N-terminal linker (n-CAGA) as a potential vaccine, but it had serious side effects.

US 2011/0002949 A1 discloses a multivalent vaccine construct (Aβ3-10/Aβ21-28) (MVC) and a monovalent vaccine construct Aβ1-8 (MoVC1-8), which are combined with a carrier (KLH) and administered with a saponin-based adjuvant - ISCOMATRIX.

Muhs et al. (Proc Natl Acad Sci USA. 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) revealed that Aβ1-15 in an array of Aβ1-15 sequences, sandwiched between palmitoyl lysine at both ends, is anchored on the liposome surface so that the peptide adopts an aggregated β-sheet structure, forming a conformational antigenic determinant.

Ding et al. (Neuroscience Letters, Volume 634, November 10, 2016, Pages 1-6) revealed that by coupling Aβ3-10 to the immunogenic carrier protein keyhole hemocyanin (KLH) or borrowing A peptide composed of five Aβ3-10 epitopes linearly connected in series.

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

Lacosta et al. (Alzheimers Res Ther. 2018 Jan 29;10(1):12.) disclose Aβ peptide immunogens containing multiple repeat sequences of a short C-terminal fragment of Aβ1-40. To generate an immune response, the repeat sequences were conjugated to a keyhole limpet cyanine (KHL) carrier protein and formulated with the adjuvant aluminum hydroxide.

Axelsen et al. (Vaccine Vol. 29, No. 17, April 12, 2011, pp. 3260-3269) revealed Aβ37-42 coupled to keyhole limpet hemocyanin.

WO 2004/062556 A2, WO 2006/005707 A2, WO 2009/149486 A2 and WO 2009/149485 A2 disclose mimetic epitopes of the epitope of Aβ. They demonstrate that these mimetic epitopes can induce antibody formation in vivo against non-truncated 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.

According to a preferred embodiment, the Aβ peptide-derived polypeptide is selected from: Native A beta peptide A beta position sequence aa1-6 DAEFRH aa1-7 DAEFRHD aa1-8 DAEFRHDS aa1-9 DAEFRHDSG aa1-10 DAEFRHDSGY aa1-11 DAEFRHDSGYE aa1-12 DAEFRHDSGYEV aa1-13 DAEFRHDSGYEVH aa1-14 DAEFRHDSGYEVHH aa1-15 DAEFRHDSGYEVHHQ aa1-21 DAEFRHDSGYEVHHQKLVFFA aa2-7 AEFRHD aa2-8 AEFRHDS aa2-9 AEFRHSG aa2-10 AEFRHDSGY aa3-8 EFRHDS aa3-9 EFRHDSG aa3-10 EFRHDSGY aa pE3-8 p(E)FRHDS aa pE3-9 p(E)FRHDSG aa pE3-10 p(E)FRHDSGY aa11-16 EVHHQK aa11-17 EVHHQKL aa11-18 EVHHQKLV aa11-19 EVHHQKLVF aa12-19 VHHQKLVF aa13-19 HHQKLVF aa14-19 HQKLVF aa14-20 HQKLVFF aa14-21 HQKLVFFA aa14-22 HQKLVFFAE aa14-23 HQKLVFFAED aa30-40 AIIGLMVGGVV aa31-40 IIGLMVGGVV aa32-40 IGLMVGGVV aa33-40 GLMVGGVV aa34-40 LMVGGVV aa30-42 AIIGLMVGGVVIA Mimic epitope A beta peptide A beta position sequence aa1-6 DKELRI aa1-7 DKELRID aa1-8 DKELRIDS aa1-9 DKELRIDSG aa1-10 DKELRIDSGY aa1-6 SWEFRT aa1-7 SWEFRTD aa1-8 SWEFRTDS aa1-9 SWEFRTDSG aa1-10 SWEFRTDSGY aa1-7 TLHEFRH aa1-7 TLHEFKH aa1-7 THTDFRH aa1-7 THTDFKH aa2-7 AEFKHD aa2-7 AEFKHG aa2-7 SEFRHD aa2-7 SEFRHG aa2-7 SEFKHD aa2-7 SEFKHG aa2-7 ILFRHG aa2-7 ILFRHD aa2-7 ILFKHG aa2-7 ILFKHD aa pE3-8 IRWDTP aa pE3-8 IRYDAPL aa pE3-8 IRYDMAG

These anti-Aβ vaccines are very effective when used alone or in combination with existing peptide vaccines targeting other pathological molecules involved in β-amyloidosis, tauopathy or synucleinopathy, especially in mixed pathological conditions (i.e., the presence of Aβ pathology and Tau pathology and/or aSyn pathology). Therefore, a preferred embodiment provides a combination of anti-Aβ vaccines and anti-Tau and/or anti-Syn peptide vaccines to treat degenerative diseases such as Alzheimer's disease, dementia in Down syndrome, dementia with Lewy bodies, Parkinson's disease dementia, Parkinson's disease or tauopathy.

Although, in principle, the present invention is capable of improving all proposed Aβ and Aβ-related vaccination polypeptides, selected epitopes are specifically assessed for their suitability for the platform of the invention. For example, SeqID32/33 (AβpE3-8; pEFRHDS) was shown to be superior to KLH-based vaccines and SeqID10 (Aβ1-6; DAEFRH) was shown to be immunogenic in combination with different CLECs.

In view of these favorable properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention can be specifically used for active anti-IL31 vaccination to treat and prevent IL31-related diseases and autoimmune inflammatory diseases.

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 conditions, such as uremic pruritus, cholestatic pruritus, pemphigoid and chronic urticaria, allergic contact dermatitis (ACD), dermatomyositis, chronic pruritus of unknown origin (CPUO), primary localized cutaneous amyloidosis (PLCA), hypertrophic Cystic eczema, chronic spontaneous urticaria, pemphigoid, dermatitis herpetiformis and other skin conditions including lichen planus, eczematous dermatosis, stasis dermatitis, scleroderma, pruritus associated with wound healing, and non-pruritic diseases such as allergic asthma, allergic rhinitis, inflammatory bowel disease (IBD), osteoporosis, follicular lymphoma, Hodgkin lymphoma and chronic myeloid leukemia.

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 IL31 epitopes can be used to trigger an immune response against different domains of IL31, particularly involving spirochetes C or A and further involving spirochetes D, thereby preventing IL31 from binding to two IL31 receptors, interleukin 31 receptor α (IL-31RA) and oncostatin M receptor (OSMR).

Anti-IL31 vaccines can be very effective when used alone or in combination with peptide vaccines targeting other pathological molecules involved in pruritic allergic diseases, pruritic inflammatory diseases, and pruritic autoimmune diseases. Therefore, a preferred embodiment provides a combination of anti-IL31 vaccines with anti-IL4 and/or anti-IL13 peptide vaccines to treat pruritic allergic diseases, pruritic inflammatory diseases, and pruritic autoimmune diseases.

According to a preferred embodiment, the IL31 protein-derived polypeptide is a fragment of the IL31 protein, and/or is preferably selected from native human IL31 (Genbank: AAS86448.1; MASHSGPSTSVLFLFCCLGGWLASHTLPVRLLRPSDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQNYTLPCLSPDAQPPNNIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHECKRFILTISQQFSECMDLALKSLTSGAQQATT); native canine IL31 (Genbank: BAH97742.1; MLSHTGPSRFALFLLCSMETLLSSHMAPTHQLPPSDVRKIILELQPLSRGLLEDYQKKETGVPESNRTLLLCLTSDSQPPRLNSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVFKSLNSGPQ); native cat IL31 (UNIPROT: A0A2I2UKP7 MLSHAGPARFALFLLCCMETLLPSHMAPAHRLQPSDVRKIILELRPMSKGLLQDYVSKEIGLPESNHSSLPCLSSDSQLPHINGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKFQREPEAKVSMPADNFERKNFILAVLQQFSACLEHVLQSLNSGPQ); or native horse IL31 (UNIPROT F7AHG9 MVSHIGSTRFALFLLCCLGTLMFSHTGPIYQLQPKEIQAIIVELQNLSKKLLDDYVSALETSILSCFFKTDLPSCFTSDSQAPGNINSSAILPYFKAISPSLNNDKSLYIIEQLDKLNFQNAPETEVSMPTDNFERKRFILTILRWFSNCLEHRAQ) or any peptide sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to any of the foregoing, or a plurality of point mutations in surface-exposed amino acids that differ from the naturally occurring sequence, wherein the number of point mutations is 1, 2 or 3.

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

Other preferred target sequences include (presented as linear or constrained peptides, such as cyclized peptides or peptides linked by suitable aa linkers (e.g., ggsgg or similar)):

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, IDEIIEHLDKLI, 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,EELQSLSK,KGVLVS,SPAIRAYLKTIRQLDNKSVIDEIIEHLDKLI,DEIIEHLDK,SVIDEIIEHLDKLI,SPAIRAYLKTIRQLDNKSVI,TDTHECKRF,HECKRFILT,HERKRFILT,HESKRFILT,SVPTDTHECKRF,SVPTDTHESKRF,SVPTDTHERKRF

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 peptides consisting of SDVRKIILELQPLSRGLLEDYQKKETGV, DVRKIILELQPLSRGLLEDY ELQPLSR LSDKNIIDKIIEQLDKLKFQHE, LSDKNIIDKIIEQLDKLKFQ, KLKFQHE, LSDKNI, LDKL, LSDKN, ADTFECKSFILTILQQFSACLESVFKS, and ADNFERKNF

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

For equine IL31: aa118-129 and peptides of equine IL-31 sequence: LQPKEIQAIIVELQNLSKKLLDDY, EIQAIIVELQNLSKKLLDDY, SLNNDKSLYIIEQLDKLNFQ and TDNFERKRFILTILRWFSNCLEHRAQ

For the mimetic antigenic determinant: The canine IL-31 mimetic antigenic determinant comprises the amino acid sequence SVPADTFERKSF, SVPADTFERKSF, NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF, APTHQLPPSDVRKIILELQPLSRG, TGVPES or its variants. The cat IL-31 mimetic antigenic determinant comprises the amino acid sequence SMPADNFERKNF, NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF, APAHRLQPSDIRKIILELRPMSKG, IGLPES or its variants. The horse IL-31 mimetic antigenic determinant comprises the amino acid sequence SMPTDNFERKRF, NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF, GPIYQLQPKEIQAIIVELQNLSKK, KGVQKF or its variants. 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.

According to a preferred embodiment, the IL31 antigenic determinant may be a conformational antigenic determinant comprising at least two amino acids or amino acid sequences that are spatially distinct from each other but closely adjacent to each other so as to form respective complementary sites. Complementary sites are usually bound by anti-IL31 antibodies (e.g., polyclonal anti-IL31 antibodies obtained after vaccination of mammals) and specifically recognize naturally occurring IL31.

IL31 is a protein with a 4-helical bundle structure, as seen in the gp 30/IL6 interleukin family. The receptor for IL31 is a heterodimer of interleukin 31 receptor α (IL-31 RA, also known as GPL or gp130-like receptor) and oncostatin M receptor (OSMR). Both structures of the heterodimer are called IL-31 receptors or IL-31 co-receptors. The putative interaction sites between human IL31 and its receptor have been described by Saux et al. (J Biol Chem 2010, 285, 3470-34). Targeting of IL31 can be achieved by antibodies targeting IL-31 and/or its receptor. The development of a dedicated monoclonal antibody that specifically targets IL31 has enabled clinical and preclinical validation of this targeting strategy in vitro and in vivo (Front Med (Lausanne. 2021 Feb 12;8:638325)).

BMS-981164 is an anti-IL-31 monoclonal antibody targeting circulating IL-31, which 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 study with subcutaneous (SC) and intravenous (IV) formulations (0.01 to 3 mg/kg) administered to healthy volunteers (Part 1) and patients with atopic Medication for adults with dermatitis (Part 2). Adults in Part 2 were required to have at least moderate atopic dermatitis (a score of 3 on a physician global scale of 0 to 5) and a visual analog scale (10 points) of at least itch severity of 7. To date, the results of this study have not been published. As of 2016, BMS-981164 is no longer in Bristol-Myers Squibb's development pipeline, and no new trials have been announced.

US 8,790,651 B2 describes monoclonal antibodies that bind to IL-31 for the treatment of immune disorders, such as atopic dermatitis. A monoclonal antibody against canine IL-31 (Lokivetmab, Zoetis) is commercially available for the treatment of canine atopic dermatitis. Logivimab is hypothesized to interfere with the binding of IL-31 to the co-receptor GPL.

EP 4 019 546 A1 discloses monospecific and multispecific antibodies in which the antibody variable domain blocks IL-31 and the interleukin 31 receptor alpha (IL-31RA)/oncostatin M receptor (OSMR) complex (IL-31RA/OS-MR complex).

Bachmann et al. disclose a vaccine utilizing intact canine IL-31 conjugated to virus-like particles for immunization of dogs for the treatment of atopic dermatitis. (Bachmann, M. F.; Zeltins, A.; Kalnins, G.; Balke, I.; Fischer, N.; Rostaher, A.; Tars, K.; Favrot, C. Vaccination against IL-31 for the Treatment of Atopic Dermatitis in Dogs. J. Allergy Clin. Immunol. 2018, 142, 279-281. e1). Similarly, US11,324,836 B2, US11,207,390 B2 and US10,556,003 and Fettelschloss et al. (doi: 10.1111/eve.13408) revealed that IL31 and IL31 target IL31 from different species (including human, canine, horse or porcine) VLP-based immunogens for relevant diseases. These VLP-based immunogens were characterized by anti-IL31 immunogens having full-length, native, and full-length modified IL31-derived sequences, respectively.

US2021/0079054A1 discloses peptide-based immunogens based on UbiTh platform technology targeting IL31 for the treatment and/or prevention of pruritic or allergic symptoms, such as atopic dermatitis. Along these lines, derived from canine IL31 (Genbank: BAH97742.1; MLSHTGPSRFALFLLCSMETLLSSHMAPTHQLPPSDVRKIILELQPLSRGLLEDYQKKETGVPESNRTLLLCLTSDSQPPRLNSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVFKSLNSGPQ) and human IL31 (Genbank: AAS86448.1; MASHSGPSTSVLFLFCCLGGWLASHTLPVRLLRPSDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQNYTLPCLSPDAQPPNNIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHECKRFILTISQQFSECMDLALKSLTSGAQQATT) based on B cell antigen determinant immunogen presentation includes: For canine IL31: peptides consisting of aa97-144, aa97-133, aa97-122, aa97-114, aa90-110, aa90-144, aa86-144, aa97-149, aa90-149, aa86-149; for human IL31: peptides derived from the sequences aa98-145, aa87-150, aa105-113, aa85-115, aa84-114, aa86-117, aa87-116 with modifications (e.g., serine and cysteine substitutions) when applicable. B cell epitopes are linear or restricted and fused to promiscuous helper T epitopes and formulated in the presence of adjuvants (e.g. different CpG molecules, Alhydrogel, AdjuPhos, Montanides, such as ISA50V2, ISA51, ISA720).

US2019/0282704 A1 discloses a vaccine composition for immunizing mammals and/or protecting them from IL-31-mediated diseases, wherein the composition comprises a combination of: a carrier polypeptide (e.g., CRM197); and at least one mimetic epitope selected from the following IL31-derived epitopes: 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).

The canine IL-31 mimic epitope includes the amino acid sequences SVPADTFECKSF, SVPADTFERKSF, NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF, APTHQLPPSDVRKIILELQPLSRG, TGVPES or variants thereof.

The feline IL-31 mimetic epitope comprises the amino acid sequence SMPADNFERKNF, NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF, APAHRLQPSDIRKIILELRPMSKG, IGLPES or variants thereof.

The equine IL-31 mimic epitope includes the amino acid sequence SMPTDNFERKRF, NS SAILPYFKAISPSLNNDKSLYIIEQLDKLNF, GPIYQLQPKEIQAIIVELQNLS KK, KGVQKF or variants thereof.

Human IL-31 mimetic epitopes include the amino acid sequences SVPTDTHECKRF, SVPTDTHERKRF, HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF, LPVRLLRPSDDVQKIVEELQSLSKM, KGVLVS or variants thereof that retain anti-IL-31 binding.

In addition, a region between approximately amino acid residues 124 and 135 of the cat IL-31 sequence represented by (UNIPROT: A0A2I2UKP7); a region between approximately amino acid residues 124 and 135 of the canine IL-31 sequence represented by (Genbank: BAH97742.1); and a region between approximately amino acid residues 118 and 129 of the horse IL-31 sequence represented by (UNIPROT F7AHG9) were revealed as suitable antigenic determinants.

WO 2019/086694 A1 discloses a peptide-based immunogen targeting IL31, which is achieved by an IL31 antigen from canine, human, cat, horse, pig, cow or camel IL31, which comprises an unassembled IL31 helical peptide, or an antigenic determinant contained in the above animals. The antigen is coupled to a known carrier molecule (e.g., KLH) and Imject Alum is used as an adjuvant, or it 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 an amino acid sequence identified as any of the following: Treponema A: Human: SDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQNYTL; and DVQKIVEELQSLSKMLLKDV, EELQSLSK, and DVQK Canine: SDVRKIILELQPLSRGLLEDYQKKETGV, and DVRKIILELQPLSRGLLEDY and ELQPLSR Cat: SDVRKIILELRPMSKGLLQDYVSKEIGL and DVRKIILELRPMSKGLLQDY Horse: LQPKEIQAIIVELQNLSKKLLDDY and EIQAIIVELQNLSKKLLDDY Treponema C Human: LDNKSVIDEIIEHLDKLIFQDA; and DEIIEH Canine: LSDKNIIDKIIEQLDKLKFQHE, LSDKNIIDKIIEQLDKLKFQ, KLKFQHE, LSDKNI, LDKL, LSDKN, Cat: LSDKNTIDKIIEQLDKLKFQRE Horse: SNNNDKSLYIIEQLDKLNFQ and/or spirochete D: Human: TDTHECKRFILTISQQFSECMDLALKS, TDTHESKRF, and HESKRF Canine: ADTFECKSFILTILQQFSACLESVFKS and ADNFERKNF Cat: ADNFERKNFILAVLQQFSACLEHVLQS and ADNFERKNF Horse: TDNFERKRFILTILRWFSNCLEHRAQ The spirochetes are present alone or in combination, and are also fused using the linker sequences disclosed.

WO 2022/131820 A1 discloses immunomodulatory or anti-inflammatory IL31-derived peptides as active ingredients for preventing or treating atopic dermatitis in the form of pharmaceutical or cosmetic preparations. It also discloses conjugates in which the IL31 peptide or fragment thereof is conjugated to a biocompatible polymer, such as pluronic acid, chondroitin sulfate, hyaluronic acid (HA), glycol chitosan, starch, chitosan, dextran, pectin, curdlan, poly-L-lysine, polyaspartic acid (PAA), polylactic acid (PLA), polyglycolic acid (polyglycol Ride) (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 (pluronic F-127) or fatty acids, such as hexanoic acid (hexanoic acid). Acid), caprylic acid (caprylic acid, C8), capric acid (capric acid, C10), lauric acid (lauric acid, C12), myristic acid (myristic acid, C14), palmitic acid (C16), stearic acid (C18) and cholesterol are added to increase the stability and skin permeability of the peptide. In the invention, neither the peptide nor the conjugate is suggested as an immunogen.

Although in principle the present invention enables the improvement of all proposed IL31-related disease vaccination polypeptides, the selected antigenic determinants (see SeqID) were specifically assessed for their suitability for the platform of the present invention compared to CRM197-based vaccines. Selected sequences • SeqID132 SKMLLKDVEEEKG-NHNH2 SeqID133 SKMLLKDVEEEKG-C • SeqID134 EELQSLSK-NHNH2; SeqID135 EELQSLSK-C; • SeqID136 KGVLVS-NHNH2; SeqID137 KGVLVS-C; • SeqID138 SVIDEIIEHLDKLI-NHNH2; SeqID139 SVIDEIIEHLDKLI-C; • SeqID140 SPAIRAYLKTIRQLDNKSVI-NHNH2; SeqID141 SPAIRAYLKTIRQLDNKSVI-C; • SeqID142 HE R KRFILT-NHNH2; SeqID143 HE R KRFILT-C; • SeqID144 HE S KRFILT-NHNH2; SeqID145 HE S KRFILT-C; • SeqID146 SVPTDTHE R KRF-NHNH2, SeqID147 SVPTDTHE R KRF-C • SeqID148 SVPTDTHE S KRF-NHNH2, SeqID149 SVPTDTHE S KRF-C • SeqID150 KRFILTISQQFS-NHNH2 SeqID151 KRFILTISQQFS-C

In view of these advantageous properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention can be specifically used for active immunotherapy against calcitonin gene-related peptide (CGRP)-related diseases.

CGRP-related disorders are selected from the group consisting of: intermittent and chronic migraine and cluster headache, hyperalgesia, hyperalgesia in dysfunctional pain states such as rheumatoid arthritis, osteoarthritis, visceral pain hypersensitivity syndrome, Fibromyalgia, inflammatory bowel disease syndrome, neuropathic pain, chronic inflammatory pain and headaches.

According to a preferred embodiment, the CGRP-derived polypeptide is derived from native human CGRP α (ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF; a 37 aa peptide fragment of aa83-119 of the calcitonin isoform α-CGRP preproprotein, accession number NP_001365879.1) or native human CGRP β (ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF); aa82-228 of the calcitonin gene-related peptide 2 proprotein, accession number NP_000719.1) or its pro-promoter molecules (NP_001365879.1 and NP_000719.1). CGRP-derived polypeptides may also be mimetics with one or more aa exchanges forming mimetic epitopes of the respective native sequences.

According to a preferred embodiment, the CGRP-derived polypeptide is selected from the functional sites of native 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 or a fragment thereof that may also contain the C2-C7 loop (e.g., aa1-20) within CGRP, which is composed of amino acid residues derived from these sites that mimic antigenic determinants.

Other preferred target sequences include ACDTATCVTH; ACDTATCVTHRLAGL; ACDTATCVTHRLAGLLSR; ACDTATCVTHRLAGLLSRSG; ACDTATCVTHRLAGLLSRSGGVVKN; TATCVTHRLAGLL; ATCVTHRLAGLLSR; RLAGLLSR; RLAGLLSRSGGVVKN; RSGGVVKN; RLAGLLSRSGGVVKNNFVPT; RLAGLLSRSGGVVKNNFVPTNVG; RLAGLLSRSGGVVKNNFVPTNVGSK; RLAGLLSRSGGVVKNNFVPTNVGSKAF; LLSRSGGVVKNNFVPTNVGSKAF; RSGGVVKNNFVPTNVGSKAF; GGVVKNNFVPTNVGSKAF; VVKNNFVPTNVGSKAF; NNFVPTNVGSKAF; VPTNVGSKAF; NVGSKAF; GSKAF

In US 2022/0073582 A1, a polypeptide construct of native human CGRP (Accession No.: NP_001365879.1) is disclosed, which contains a CGRP-derived peptide aa1-10 ACDTATCVTH；aa 1-15 ACDTATCVTHRLAGL；aa 1-18 ACDTATCVTHRLAGLLSR；aa 1-20 ACDTATCVTHRLAGLLSRSG；aa 1-25 ACDTATCVTHRLAGLLSRSGGVVKN；aa 4-16 TATCVTHRLAGLL；aa 5-18 ATCVTHRLAGLLSR；aa 11-18 RLAGLLSR；aa 11-25 RLAGLLSRSGGVVKN；aa 11-30 RLAGLLSRSGGVVKNNFVPT；aa 11-33 RLAGLLSRSGGVVKNNFVPTNVG;aa 11-35 RLAGLLSRSGGVVKNNFVPTNVGSK;aa 11-37 RLAGLLSRSGGVVKNNFVPTNVGSKAF;aa 15-37 LLSRSGGVVKNNFVPTNVGSKAF;aa 18-37 RSGGVVKNNFVPTNVGSKAF;aa 20-37 GGVVKNNFVPTNVGSKAF;aa 22-37 VVKNNFVPTNVGSKAF;aa 25-37 NNFVPTNVGSKAF;aa 28-37 VPTNVGSKAF;aa 31-37 NVGSKAF; having the following amino acid sequence: MGFQKFSPFLALSILVLLQAGSLHAAPFRSALESSPADPATLSEDEARLLLAALVQDYVQMKASELEQEQEREGSRIIAQKRACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAFGRRRRDLQA. The peptide immunogen construct disclosed in US 2022/0073582 A1 requires a CGRP-derived B cell antigenic determinant coupled to one or more promiscuous T cell antigenic determinants as a peptide immunogen construct targeting GCRP.

In addition to active immunotherapeutics, humanized anti-calcitonin gene-related peptide (CGRP) monoclonal antibodies have been shown as an anti-CGRP targeting paradigm. Several studies have found that antibodies are effective in reducing the frequency of chronic migraines (Dodick DW et al. (2014) Lancet Neurol. 13: 1100-1107; Dodick DW et al. (2014) Lancet Neurol. 13: 885-892; Bigal ME et al. (2015) Lancet Neurol. 14: 1081-1090; Bigal ME et al. (2015) Lancet Neurol. 14: 1091-1100; and Sun H et al. (2016) Lancet Neurol. 15: 382-390).

Along these lines, US 8,597.649 B2, EP 1957106 B1 and US 9.266,951 B2 disclose clinically used monoclonal antibodies targeting aa25-37 and/or aa33-37 in human CGRP for the treatment of migraine, cluster headache and tension headache. US20120294797 A1 discloses clinically used CGRP-targeting monoclonal antibodies, and according to the co-crystallization results, this antigenic determinant is also specific for the C-terminal antigenic determinant aa26-37 (https://doi.org/10.1080/21655979.2021.2006977), and the antigenic determinant is suitable for immunotherapy. US 9,505,838 B2 also discloses a monoclonal antibody against CGRP for clinical use, which binds to a C-terminal fragment having amino acids 25-37 of CGRP or a C-terminal epitope within amino acids 25-37 of CGRP.

Although, in principle, the present invention is capable of improving all proposed CGRP-related disease vaccination polypeptides, selected epitopes (see SeqID 152 to SeqID 162) were specified based on their suitability for the platform of the present invention compared to CRM197-based vaccines. assessment. Selected sequence for experiment: • SeqID152 RLAGLLSR-NHNH2, SeqID153 RLAGLLSR-C • SeqID154 RLAGLLSRSGGVVKN-NHNH2, SeqID155 RLAGLLSRSGGVVKN-C • SeqID156 RSGGVVKN-NHNH2, SeqID157 RSGGVVKN-C • SeqID158 NNFVPTNVGSKAF-NHNH2, SeqID159 NNFVPTNVGSKAF-C • SeqID160 VPTNVGSKAF-NHNH2, SeqID161 VPTNVGSKAF-C •SeqID162 NVGSKAF-NHNH2, SeqID163 NVGSKAF-C

In view of these favorable properties of the conjugates of the present invention, the CLEC-based conjugates and CLEC-based vaccines according to the present invention are particularly useful for specific allergen immunotherapy (AIT) to treat IgE-mediated type I allergic diseases. Allergic diseases generally refer to a variety of conditions caused by the immune system's sensitivity to normally harmless substances in the environment. Such diseases include (but are not limited to) hay fever, seasonal, food, pollen, mold spore, poisonous plant, agent/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 ("autoallergy"); chronic (idiopathic) and autoimmune urticaria; systemic allergic reactions, especially idiopathic and exercise-induced systemic allergic reactions.

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

The main mechanism of AIT is the induction of so-called blocking antibodies, preferably of the IgG4 isotype, as well as other isotypes (e.g., IgG1 or IgA). It has been shown that the antigenic determinants targeted by naturally occurring IgA and IgG on the surface of allergens are different from the antigenic determinants specifically recognized by IgE (the so-called IgE antigenic determinants) (Shamji, Valenta et al. 2021; Allergy 76(12): 3627-3641). However, the latter antigenic determinant is responsible for cross-linking to IgE on mast cells via the high-affinity cγRI receptor and is therefore responsible for inducing immediate allergic immune responses.

In contrast, AIT-induced blocking antibodies (mainly IgGa and IgA types) are directed against the IgE antigenic determinants. Their binding to allergens interferes with the crosslinking of cell-bound IgE, thereby inhibiting the initiation of allergic reactions. IgG4 exhibits advantageous characteristics as a blocking antibody, as it is unable to crosslink allergens and shows low affinity for the Fc receptors of activating IgG (FcγR), while maintaining a high affinity for FcγRIIb. These characteristics enable IgG4 to become an effective inhibitor of IgE-dependent reactions without causing 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 other IgG subclasses {Ejrnaes et al. 2004; Molecular Immunology Vol. 41, No. 5, .2004, P. 471-478}, especially in the early stages of 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).

According to a preferred embodiment, a single allergen antigenic determinant can be used to trigger an immune response against a respective allergen (e.g., the IgE antigenic determinants mentioned in Tables A and B). In another preferred embodiment, a combination of antigenic determinants from one allergen can be used to trigger an immune response against different domains of the allergen.

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 antigenic determinants of different allergens to trigger an immune response against different allergens.

According to a preferred embodiment, the allergen-derived polypeptide is a fragment of an allergen protein, especially one of those described in Table A and Table B, and/or is preferably selected from native proteins, especially those listed in Table A and Table B.

According to a preferred embodiment, the allergen-derived polypeptide is a linear fragment of an allergen protein, including those described in Table A and Table B.

According to a preferred embodiment, the allergen-derived polypeptide is selected from the mimetics of the allergen-derived polypeptides mentioned above, including mimetic antigenic determinants and peptides containing amino acid substitutions.

According to a preferred embodiment, the allergen-derived polypeptide is derived from a native allergen or is a mimetic having one or more aa exchanges to form a simulated epitope of the respective native sequence.

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 different but closely related to each other. adjacent to form respective complementary bits. The paratope typically binds to an anti-allergen antibody (eg, multiple anti-allergen antibodies obtained after vaccination of a mammal) and specifically recognizes a naturally occurring allergen.

According to a preferred embodiment, the respective conformational epitopes or simulated epitopes can be obtained from the literature or using predictive algorithms (such as Dall'Antonia and Keller 2019, Nucleic Acids Research 47(W1): W496-W501 disclosed) or publicly available databases (e.g.: https://www.iedb.org/). Selected examples of potential target antigens and their respective epitopes/mimetic epitopes to be used with the present invention are summarized in Table A and Table B.

According to a preferred embodiment, other preferred target sequences include restricted peptides, such as cyclized peptides or suitable aa linkers known to those skilled in the art, such as: (G)n linkers, (K) Peptides connected by n-linker, GGSGG or similar. Table A - Preferred allergens for use in CLEC-based vaccines Allergen source allergens UNIPROT Common alder ( Alnus glutinosa ) Aln g 1 P38948 Alternaria alternata Alt a 1 P79085 Alt a 3 P78983 Alt a 4 Q00002 Alt a 5 P42037 Alt a 6 Q9HDT3 Alt a 7 P42058 Alt a 8 P0C0Y4 Alt a 10 P42041 Alt a 12 P49148 Alt a 13 Q6R4B4 Ragweed ( Ambrosia artemisiifolia ) Amb a 1 P27761 Amb a 1.05 P27762 Amb a 3 P00304 Amb a 5 P02878 Amb a 6 O04004 Amb a 7 No complete sequence available Amb a 8 Q2KN24 Amb a 9 Q2KN27 Amb a 10 Q2KN25 European honey bee ( Apis mellifera ) Api m 1 P00630 Api m 2 Q08169 Api m 3 Q4TUB9 Api m 4 01501 Api m 5 B2D0J4 Api m 6 Q27SJ8 Api m 7 Q8MQS8 Api m 8 B2D0J5 Api m 9 C9WMM5 Api m 10 Q1HHN7 Api m 11 B3GM11 Api m 12 Q868N5 Celery ( Apium graveolens ) Api g 1 P49372 Api g 2 E6Y8S8 Api g 3 P92919 Api g 4 Q9XF37 Api g 5 P81943 Peanut ( Arachis hypogae ) Ara h 1 P43238 Ara h 2 Q6PSU2-1 Ara h 3 O82580 Ara h 3.02 Q9SQH7 Ara h 5 Q9SQI9 Ara h 6 Q647G9 Ara h 7 Q9SQH1 Ara h 8 Q6VT83 Betula verrucosa Bet v 1 P15494 Bet v 2 P25816 Bet v 3 P43187 Bet v 4 Q39419 Bet v 6 O65002 Bet v 7 P81531 Domestic dog ( Canis familiaris ) Can f 1 O18873 Can f 2 O18874 Can f 3 P49822 Can f 4 D7PBH4 Can f 5 P09582 Can f 6 H2B3G5 Can f 7 Q28895 Can f 8 F1PHB6 European hornbeam ( Carpinus betulus ) car b 1 P38949 European chestnut ( Castanea sativa ) Cas s 1 B7TWE3 Cas s 5 Q42428 Cas s 8 Unknown sequence Cladosporium herbarum Cla h 2 No complete sequence available i h 5 P42039 i h 6 P42040 i h 7 P42059 i h 8 P0C0Y5 ci h 9 B7ZK61 CI h 10 P40108 CI h 12 P50344 Corylus avellana Cor a 1 Q08407 Cor a 2 Q9AXH5 Cor a 6 A0A0U1VZC8 Cor a 8 Q9ATH2 Cor a 9 Q8W1C2 Cor a 10 Q9FSY7 Cor a 11 Q8S4P9 Japanese cedar ( Cryptomeria japonica ) Cry j 1 P18632 Cry j 2 P43212 European carp ( Cyprinus carpio ) Cyp c 1 Q8UUS3 Wild carrot ( Daucus carota ) Dau c 1 O04298 Dau c 4 Q8SAE6 European house dust mite ( Dermatophagoides pteronyssinus ) Der p 1 P08176 (variant) Der p 2 P49278 Der p 3 P39675 Der p 4 Q9Y197 Der p 5 P14004 Der p 6 P49277 Der p 7 P49273 Der p 8 P46419 Der p 9 Q7Z163 Der p 10 O18416 Der p 11 Q6Y2F9 Der p 14 Q8N0N0 Der p 20 B2ZSY4 Der p 21 Q2L7C5 Der p39 XP_027203190.1 European beech ( Fagus sylvatica ) Fags 1 B7TWE6 Domestic cat ( Felis domesticus ) Fel d 1 P30438 (chain 1), P30440 (chain 2) Fel d 2 P49064 Fel d 3 Q8WNR9 Fel d 4 Q5VFH6 Fel d 5 No complete sequence available Fel d 6 No complete sequence available Fel d 7 E5D2Z5 Fel d 8 F6K0R4 Rubber tree ( Hevea brasiliensis ) Hev b 1 P15252 Hev b 2 P52407 Hev b 3 O82803 Hev b 4 Q6T4P0 Hev b 5 Q39967 Hev b 6 P02877 Hev b 7.01 O04008 Hev b 7.02 O65811 Hev b 8 O65812 Hev b 9 Q9LEJ0 Hev b 10 P35017 Hev b 11 Q949H3 Hev b 12 Q8RYA8 Hev b 13 Q7Y1X1 American cedar ( Juniperus ashei ) Jun a 1 P81294 Jun a 2 Q9FY19 Jun a 3 P81295 Apple ( Malus domestica ) Mal d 1 P43211 Mal d 2 Q9FSG7 Mald 3.0101w Q5J026 Mal d 4 Q9XF42 White oak ( Quercus alba ) Que a 1 B6RQS1 Phleum pratense PhIp 1 Q40967 PhI p 2 P43214 PhI p 4 Q5ZQK5 PhI p 5 Q40960 PhI p 6 P43215 PhI p 7 O82040 PhI p 11 Q8H6L7 PhI p 12 P35079 PhI p 13 Q9XG86 Polistes annularis Pol a 1 Q9U6W0 Pol a 5 Q05109 Paper Wasp ( Polistes dominulus ) Pol d 1 Q6Q252 Pol d 4 Q7Z269 Pol d 5 Q68KJ8 Guinea Paper Wasp ( Polistes exclamans ) Pol e 1 No complete sequence available Pol e 4 No complete sequence available Pol e 5 Q68KJ9 Northern paper wasp ( Polistes fuscatus ) Pol f 5 P35780 Polistes gallicus Pol g 1 P83542 Pol g 5 P83377 Polistes metricus Pol m 5 P35780 Table B: Preferred allergen epitopes for use in CLEC-based vaccines allergens UNIPROT Epitope / Mock epitope Amb a 1 P27761 GMLATVAFNTFTDNVDQR (Mol Immunol. 1988 Apr;25(4):355-65), AFNKFTDNVDQR, MPRCRFGF, WRTQNDVLENG (Mol Immunol. 2009 Feb;46(5):873-83.), TFTDNVDQRMPRCRH (Peptides. 2021 Nov; 2019, 66, 37-44) Amb a 3 P00304 16 3):229-35) Alt a1 P79085 MISTSRK, QKRNTIT, (Li et al. J Immunol (2019) 203 (1): 31-38.), KISEFYGRKP, VATATLPNYC, YSCGENSFMD, YYNSLGFNIK (Kurup et al., Peptides. 2003 Feb;24(2):179- 85.) Ara h 1 P43238 REREREEDWRQPREDWRRPS, RTRGRQPGDYDDDRRQPRRE, DDDRRQPRREEGGRWGPAGP, TTNQRSPPGERTRGRQPGDY, QPREDWRRPSHQQPRKIRPE, GREGEQEWGTPGSHVREETS, PVNTPGQFEDFFPASSRDQS, AGGEQEERGQRRWSTRSSEN, REGEPDLSNNFGKLFEVKPD, NASSELHLLGFGINAENNHR, IDQ IEKQAKDLAFPGSGEQV, LAFPGSGEQVEKLIKNQKES (Cell Rep Med 2(10): 100410.), AKSSPYQKKT, QEPDDLKQKA, LEYDPRLUYD, GERTRGRQPG, PGDYDDDRRQ, PRREEGGRWG, REREEDWRQP, EDWRRPSHQQ, QPKKIRPEGR, TPGQFEDFFP, SYLQEFSRNT, FNAEFNEIRR, EQEERGQRRW, DITNPINLRE, NNFGKLFEVK, GTGNLELVAV, RRYTARLKEG, ELHLLGFGIN, HRIFLAGDKD, IDOIEKOAKD, KDLAFPGSGE, KESHFVSARP, PEKESPEKED (Eur J Biochem 245 (2): 334-339) Ara h 2 Q6PSU2-1 HASARQQWEL, QWELQGDRRC, DRRCQSQLER, LRPCEQHLMQ, KIQRDEDSYE, YERDPYSPSQ, SQDPYSPSPY, DRLQGRQQEQ, KRELRNLPQQ, QRCDLDVESG (Arch Biochem Biophys 342 (2): 244-253), HASARQQWEL, ERDPYSPSQDPYSPS, RRCQSQLER, CDLEVESGGRDRY, ERDPYSPSQDPYSPS, NLRPCEQHLMQKIQRD, PQRCDLE, RQQEQQFKRELRNLPQQ, SDRLQGRQQ (Deak, Vrabel et al. 2017), RRCQSQLERANLRPCEQHLMQKIQR DEDSYGRDPYSPSQDPY (Mol Immunol 85: 81-88) Ara h 3 O82580 IETWNPNNQEFECAG, GNIFSGFTPEFLEQA, VTVRGGLRILSPDRK, DEDEYEYDEEDRG (J Clin Invest 103(4): 535-542), EDEYEYDEEDRRRGRGSRGR, NIFSGFTPEFLEQAFQVDDR, ESEEEGAIVTVRGGLRILSP, SGFTPEFLEQAFQVDDRQIV, EEGAIVTVRGGLRILSPDRK, TYE EPAQQGRRYQSQRPPRR, RRADEEEEYDEDEYEYDEED, NHEQEFLRYQQQSRQSRRRS, QEFLRYQQQSRQSRRRSLPY, QEEREFSPRGQHSRRERAGQ (Cell Rep Med 2(10): 100410 ) Ara h 6 Q647G9 CDELNEMENTQR, CEALQQIMENQCD, CNFRAPQRCDLDV, KPCEQHIMQRI, YDSYDIR, KRELRMLPQQ, MRRERGRGGDSSSS (Sci Rep 7(1): 3981) Bet v 1 P15494 KAEQVKASKEMGETLLRAVESYLLAHSDAYN, GPGTIKKISFPEGFPFKYVKDRVDEVDHTN, DGGSILKISNKYHTKGDHEVKAEQVKASKE, LFPKVAPQAISSVENIEGNGGPGTIKKISF, MGETLLRAVESYLL, MGVFNYETETTSVIPAARLFKAFI, VDHTNFKYNYSVIEGGPIGDTLEKISNEIK (Clin Exp Allergy 34 (10): 1525-1533), ISFPEGFPFK, FILDGDNLFPKVAPQAISSVE, NIEGNGGPGTIKK (Anal Chem 90(19): 11315-11323), DGDNLFPKVA ( Int Arch Allergy Appl immunol 89: 410-415), ILDGDNLFPKVAPQAI int Arch Allergy Immunol 101 (1): 89-94), Skemgetllravesyllahsd (J Allergy Clinol 116 (2): 370-3 76). Immunol 186 (9): 5333-5344), EQVKASKEMGETLLLLLLA (Allergy 67 (12): 1530-1537), GDNLFPKVAPQAIS, ISFPEGFPFKD (Clin Exp Alyeralgy 4 4 (2): 288-299), AA 32-37, AA42- 50, aa138-153 (BIOLOGICS AND IMMUNOTHERAPY, Vol. 149, Issue 1, p200-211, January 2022), Mimic epitopes: CQQFLSVRALC, QQFLSVRALC, CQQFLSVRAL Cry j 1 P18632 DALTLRTATNIW, DGDALTLRTATN, DGRGAQVYIGNG, NATPQLTKNAGV, NGGPCVFIKRVS, NGNATPQLTKNA, NSDDDPVNPAPG, VENGNATPQLTK (Biol Pharm Bull 28(8): 1496-1499), NAGVLTCSLSK (Mol Immunol 30(2): 183-189) Cry j 2 P43212 KWVNGREI, GQCKWVNGREICNDRDRPTA, GRENSRAEVSYVHVNGAKFI, PGNKKFVVNNLFFNGPCQPH, SHIIYENVEMINSENPILINQFYCT, YCTSASACQNQRSAVQIQDV (Clin Exp Allergy 33(2): 211-217), TYKNIRGTSATAAAIQLKCS (J Clin Immunol 33( 5): 977-983), AEVSYVHVNGAK (Eur J Immunol 32(6): 1631-1639) Der p 1 P08176 (variant) KGIPNTKAP, DMFQIGKYG, GIREVWPAG, SSMGAYWGG, KGTTGVRNT (Molecular Immunology 45(5): 1308-1317), CQIYPPNANKIREALAQ, GYSNAQGVDYWI, NQSLDLAEQELVDCASQHGC, VRNSWDTNWGDNGY (Mol Immunol 29(6): 739-749) , EQSYPCWLSGTPSTP, TPLCDYAAARVGACG, KEQLPTSYPPERAGW, NCLSSDEPLHIRWCQ, EALSEEEWPRYTSHP, SCDATQRAQGRCS, SCDSSQKKQGRCS, SCDESRRRQGRCS (Clin Exp Allergy 29(11): 1563-1571), simulated epitopes: KGIPNTKAP, DMFQIGKYG, GIREVWPAG, SSMGAYWGG, KGTTGVRNT, CKGIPNTKAP,CDMFQIGKYG, CGIREVWPAG, CSSMG AYWGG, CKGTTGVRNT, CKGIPNTKAPC, CDMFQIGKYGC, CGIREVWPAGC, CSSMGAYWGGC, KGTTGVRNTC, KGIPNTKAPC, DMFQIGKYGC, GIREVWPAGC, SSMGAYWGGC, KGTTGVRNTC, Der p 2 P49278 FVVEYTKKW, SWWNLPQIG, KGITTKWMA, AGISYTKTW (Molecular Immunology 45(5): 1308-1317), DQVDVKDCANHEIKK, VPGIDPNACHYMKCK (Mol Immunol 28(11): 1225-1232), APKSENVVVTVKVMGDNGVLACAIATHAKIRD, CH GSEPCIIHRGKPFQLEAVFEANQNSKTAK, DQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGK, EANQNSKTAKIEIKASIEGLEVDVPGIDPNAC, EVDVPGIDPNACHYMKCPLVKGQQYDIKYTWIVPKIAPKSEN (Allergy 67(5 ; , CKGITTKWMA, CAGISYTKTW Der p39 XP_027203190.1 QEELREAFRMY, TSALREILRAL, NDELDEMIAEI (World Allergy Organization Journal (2022) 15:100651) Fel d 1 P30438 (chain 1), P30440 (chain 2) KALPVVLENARILKNCVDAKMTEEDKE, KRDVDLFLTGTPDEYVEQVAQYKALPV (Proc Natl Acad Sci USA 90(16): 7608-7612), DAKMTEEDKENALS, EICPAVKRDVDLFLTG, EPERTAMKKIQDCY, FAVANGNELLLDLS, KKIQDCYVENGLIS, LLDKIYTSPLC, VAQYKALPVVLENA , VKMAETCPIFYDVF, VQNTVEDLKLNTLGR (J Allergy Clin Immunol 93(1 Pt 1): 34- 43), EICPAVKRDVDLFLTGTPDEYVEQVAQYK, SSKDCMGEAVQNTVEDLKLNTLGR, VKMAITCPIFYDVFFAVANGNELLLDLSLTKVNA (Clin Exp Allergy 44(6): 882-894), CPAVKRDVDLFLT, EQVAQYKALPVVLENA KALPVVLENARILNCV RILKNCVDAKMTEEDKE KEN ALSLLDKIYTSPL TAMKKIQDCY VENGLI SRVLDGLVMTTISSSK (J ALLERGY CLIN IMMUNOL Vol. 131, Issue 1, 109.e4), ENLAYTKALLTG ; NNISVDGLLTS; VIRNELLLTYA; VINDLLLVLA; GDFYPL; NDFYPE; LDLNP (Molecular Immunology 71 (2016) 176-183), FAVANGNELL; LKLNTLGREICPAVKRGVDL; January 2013, 109.e1) PhIp 1 Q40967 VRYTTEGGTKTEAEDVIPEGWKADTSYESK, APYHFDLSGHAFGAM, HITDDNEEPIAPYHFDLSGHA, NEEPIAPYHFDLSGHAFG, CFEIKCTKPEACSGEPVVV, CTKPEACSGEPVVVHITDDNEEPIAPYHFDLSGH, DNEEPIAPYHF, EPIAPYHFDLSGH, EQKLRSAGELELQFRRVKC, GEPVVVHITDDNEEPIA PYHFDLSGHAFGAMAKKG, GYKDVDKPPFSGMTGCGNTPIFKSGRGCGSCFEIKCTKPEACS, HITDDNEEPIAPYHF, HITDDNEEPIAPYHFDLSGHAFGA, HVEKGSNPNYLALLVKYVNGDGDV VAV, KCTKPEACSGEPVVVHITDDNEEPIAPYHFDLS, KPPFSGMTGCGNT, KTEAEDVIPEGWKADTSYESK, LTGPFTVRYTTEGGTKTEAEDVIPEGWKADTSYESK, PIAPYHFD, PIAPYHFDLSGHAFG (Faseb j 13( 11): 1277-1290) PhI p 2 P43214 VPKVTFTVEKGSNEKHLAVLVKYEGDTMAEVEL, VEKGSNEKHLAVLVKYEGDTMAEVELREHGSD, REHGSDEWVAMTKGEGGVWTTFDSEEPLQGPFN, FRFLTEKGMKNVFDDVVPEKYTIGATYAPEE (J Allergy Clin Immunol 135(5): 1207-1207.e1201-1211), CFRFLTEKGM KNVFDDVVPEKYTIGATYAPEE (EBioMedicine 11 (2016) 43-57), FRFLTEKGMKNVFDDVVPEKYTIGATYAPEEC PhIp5 Q40960 AEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDK, ATTEEQKLIEKINAGFKAALAAAAGVQPADKYR, FVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSK, ADLGYGPATPAAPAAGYTPATPAAPAEAAPAGK, AYKLAYKTAEGATTPEAKYDAYVATLSEALRI, EAAFNDAIKASTGGAYESYKFIPALEAAVK, TVATAPEV KYTVFETALKKAITAMSEAQKAAK (J Allergy Clin Immunol 133(3): 836-845.e811), SRLGRSSAWV, THWQLGERPD, PSTPGERVRH, RGGPDDLTAL, PFWVRGTTW, PSTPGSRQNM, PSTPGDNPLV, KFVVNGRWID, KFLVNGRWID, RLTENTEPLL, FTWGGLRDKS, ERAGAMERAN, RSVSKEEPGM, KLGKFGAARV, VQDLMKSSGV (J Allergy Clin Immunol 114(6): 1294-1300), simulated epitopes: KLGKFGAARV, CKLGKFGAARVC, CKLGKFGAARV, KLGKFGAARVC, PhI p 6 P43215 GKATTEEQKLIEDVNASFRAAMATTANVPPAD, YKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYN, DAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKY, AADHAAPEDKYEAFVLHFSEALRIIAGTPEVHA (J Allergy Clin Immunol 135(5): 1207-1207.e1201-1211)

A positive result of AIT is 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 further increase over time (Strobl et al. 2023; Jakobsen CG et al., 2005, Clinical & Experimental Allergy, 35: 193-198. doi: 10.1111/j.1365-2222.2005.02160.x), which supports the view that AIT-induced inhibition of allergen binding to IgE can be mainly or exclusively explained by the induction of increased amounts of specific IgG (Svenson et al., 2003, Molecular Immunology 39 (10): 603-612; Jakobsen et al., 2005). It is therefore believed that the rather limited success of AIT can be primarily attributed to the low immunogenicity of existing AIT compounds and the lack of further affinity maturation upon prolonged AIT administration.

In contrast, the vaccine or conjugate according to the invention is particularly suitable for the induction of AIT and the required high-affinity IgG, since it induces IgE- with higher antibody levels (compared to conventional vaccines) after repeated immunizations. Epitope-specific immune responses and exhibit prolonged affinity maturation (see, eg, Figure 13 and Figure 21). Compared with classic vaccines, including vaccines using Alum as adjuvant and conjugate vaccines (with or without adjuvant), the present invention can produce immune serum with higher affinity.

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

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 proteins (Marth et al. 2013, The Journal of Immunology 190 (7): 3068-3078) and recombinant allergenic proteins or peptides (containing allergenic epitopes or mimetic epitopes thereof) to increase immunogenicity and affinity maturation (Bachmann et al., 2020, Trends in Molecular Medicine 26 (4): 357-368).

The latter approach of administering peptide-carrier conjugates comprising allergenic epitopes or mimetic epitopes thereof will be particularly advantageous for the novel AIT paradigm in patients, as it focuses the immune response on the desired target epitope (i.e., IgE epitope) and completely avoids both immediate (i.e., cross-linking of vaccine with cell-bound IgE) and late side effects (i.e., activation of allergen-specific T cell responses).

Marth et al. (2013) disclosed an AIT compound based on a fusion protein of two non-allergenic peptides, PA and PB, derived from the IgE-reactive region of the major birch pollen allergen Bet v 1, fused to the hepatitis B surface protein PreS in four recombinant fusion proteins containing different numbers and combinations of peptides. Similarly, the clinically tested AIT vaccine BM32 uses four fusion proteins consisting of peptides from four Timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5 and Phl p 6) fused to the PreS carrier protein from hepatitis B. Weber et al. (2017; doi: 10.1016/j.jaci.2017.03.048) could demonstrate similar immunogenicity in rabbits of BM32 and known extract-mediated AIT with Alum as adjuvant. However, despite initial promising clinical results (Eckl-Dorna, 2019 EBioMedicine. 2019 Dec;50:421-432. doi: 10.1016/j.ebiom.2019.11.006.), further development of the BM32 approach was 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).

Along these lines, it has been demonstrated that a single injection of two monoclonal antibodies against two epitopes of the major cat allergen Fel d 1 in allergic patients is equally effective as AIT, which has been known for many years. (Orengo, Radin et al. 2018, Nature Communications 9 (1): 1421), which shows that a small number of target epitopes within a given allergen can be sufficient to provide comprehensive protection against allergic immune responses. Vaccines or conjugates according to the invention are particularly suitable for combining universal T cell epitopes with such IgE epitopes or mimic epitopes on the CLEC backbone for the treatment of allergies.

Although in principle, the present invention enables the improvement of all proposed polypeptides for vaccination against allergic diseases, the 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.

In view of these advantageous properties of the conjugates of the present invention, the CLEC-based conjugates and CLEC-based vaccines according to the present invention can be specifically used to enhance commercially available peptide/glycoconjugate vaccines, especially for the prevention of infectious diseases. Immunogenicity of glycoconjugate vaccines. Such diseases are, for example, microbial or viral infections, caused for example by Haemophilus influenzae type B (Hib), Streptococcus pneumoniae, Neisseria meningitidis and Salmonella Typhi or other infectious agents. agents, including those causing hepatitis A or B, human papillomavirus infection, influenza, typhoid, measles, mumps and rubella. In addition, there are serogroup B meningococci, cytomegalovirus (CMV), respiratory syncytial virus (RSV), Clostridioides Difficile , extraintestinal pathogenic Escherichia coli (Expec), Klebsiella pneumoniae ( Klebsiella Pneumoniae , Shigella , Staphylococcus Aureus , Plasmodium falciparum , P. vivax , P. ovale And infections caused by Plasmodium malariae ( P. malariae ), coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2), Ebola virus, Borrelia burgdorferi , HIV and other diseases.

To date, several carrier proteins have been used in licensed conjugate vaccines, including: 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), and recombinant Pseudomonas aeruginosa exotoxin (rEPA). Clinical trials have demonstrated the efficacy of these conjugate vaccines in preventing infectious diseases and altering the transmission of Haemophilus influenzae type b, Streptococcus pneumoniae, and meningococci and typhoid fever. All carrier proteins are effective in increasing vaccine immunogenicity, but they vary in the amount and affinity of antibodies elicited, their ability to carry multiple polysaccharides in the same product, and their ability to be administered simultaneously with other vaccines.

According to a preferred embodiment, conjugate vaccines suitable for CLEC modification and immunogenicity enhancement include (but are not limited to) currently available vaccines, including Haemophilus type b conjugate vaccines (for example: PedvaxHIB®, ActHIB® , Hiberix®), recombinant hepatitis B vaccine (for example: Recombivax HB®, PREHEVBRIO®, Engerix-B, HEPLISAV-B®), human papillomavirus vaccine (for example: Gardasil®, Gardasil 9®, Cervarix®), meningeal Pneumococcal (groups A, C, Y and W-135) oligosaccharide diphtheria CRM197 conjugate vaccine (such as Menveo®), meningococci (groups A, C, Y and W-135) polysaccharide diphtheria toxoid conjugate vaccine ( For example: Menactra®), meningococcal (groups A, C, Y and W-135) TT conjugate vaccine (for example: MenQuadfi®), multivalent pneumococcal conjugate vaccine (for example: Prevnar-13®, Prevnar 20 ®, Pneumovax-23®, Vaxneuvance®), anti-typhoid vaccines (e.g. Typhim V®, Typhim VI®, Typherix®, Vi polysaccharide or Vi-rEPA conjugated to non-toxic recombinant Pseudomonas aeruginosa exotoxin A or polysaccharide tetanus Toxoid conjugate vaccines (Typbar-TCV®), varicella-zoster virus vaccines (e.g., Shingrix®) and other anti-infectious conjugate vaccines that carry as carrier molecules: genetically modified cross-reactive substances of diphtheria toxin ( CRM197), or tetanus toxoid (TT), or meningococcal outer membrane protein complex (OMPC), or diphtheria toxoid (DT), or Haemophilus influenzae protein D (HiD) or recombinant Pseudomonas aeruginosa outer membrane protein complex toxin (rEPA).

According to another aspect, the novel conjugates according to the present invention can be used to prevent infectious diseases. Such diseases are, for example, microbial infections or viral infections, which are caused, for example, 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 infection, influenza, typhoid, measles, mumps and rubella. In addition, there are infections caused by serogroup B meningococci, cytomegalovirus (CMV), respiratory syncytial virus (RSV), Clostridium difficile, extraenteric pathogenic Escherichia coli (Expec), Klebsiella pneumoniae, Shigella spp., Staphylococcus aureus, Plasmodium spp., coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2), Ebola virus, Borrelia burgdorferi, HIV, and others.

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

In view of these favorable properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention can be specifically used for active immunotherapy against proprotein convertase subtilisin/kexin type 9 (PCSK9)-related diseases, including (but not limited to) hyperlipidemia, hypercholesterolemia, atherosclerosis, increased serum levels of low-density lipoprotein cholesterol (LDL-C) and cardiovascular events, stroke or various forms of cancer.

According to a preferred embodiment, the PCSK9 protein-derived polypeptide is derived from native human PCSK9 (accession number: Q8NBP7), which has an amino acid sequence: or a fragment thereof, or a mimetic having one or more aa exchanges to form a mimetic epitope of the respective native sequence. Other preferred target sequences include linear or restricted peptides (eg cyclized peptides) or peptides linked by a suitable aa linker (eg ggsgg or similar).

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

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

According to a preferred embodiment, a single PCSK9-derived epitope can be used to trigger targeting of 3 different domains of PCSK9 (i.e., the inhibitory prodomain (aa1-152), the catalytic domain (aa153-448), and the C-terminal domain (449 -692)) immune response in different regions. In another preferred embodiment, combinations of PCSK9-derived epitopes can be used to trigger immune responses against different epitopes within each domain of PCSK9, particularly involving the catalytic domain (aa153-449), and further involving the inhibitory prodomain (aa1-152) and/or the C-terminal domain (449-692).

Vascular diseases such as hyperlipidemia, hypercholesterolemia, atherosclerosis, coronary heart disease and stroke are one of the leading causes of death worldwide, and elevated LDL-C levels play a key role in their pathogenesis. Therefore, LDL-C management is a very important factor in the successful treatment of hyperlipidemia, hypercholesterolemia, and atherosclerosis. Therefore, PCSK9 plays a key role in LDL catabolism via direct action on LDLR. Inhibiting PCSK9 has been shown to benefit LDL-C levels. Therefore, anti-PCSK9 therapy is a promising approach in the beneficial regulation of LDLC levels and the treatment of PCSK9-related diseases.

WO2015128287A1 and EP2570135A1 disclose PCSK9 mimicking antigen determinant-carrier conjugate vaccines (e.g., KLH or CRM197 as carriers) and disclose sequences PEEDGTRFHRQASK, AEEDGTRFHRQASK, TEEDGTRFHRQASK, PQEDGTRFHRQASK, PEEDGTRFHRRASK, PEEDGTRFHRKASK, PEEDGTRFHRQASR, PEEDGTRFHRTASK and aaa150 to 170 and/or aa205 to 225 of PCSK9, in particular SIPWNLERITPPR, PEEDGTRFHRQASK, PEEDGTRFHRQA, EEDGTRFHRQASK, EEDGTRFHRQAS, SIPWNLERITP and SIPWNLERIT.

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

WO2022150661A2 discloses a virus (including bacteriophage virus or plant virus) or virus-like particle for PCSK9 immunotherapy, which particularly contains the PCSK9 derived sequence NVPEEDGTRFHRQASKC.

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

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

Preferred polypeptide immunogen constructs according to the present invention contain B cell epitopes from alpha synuclein and allogeneic helper T cell (Th) epitopes coupled to CLEC. The present invention provides novel conjugates that are unexpectedly superior in terms of immunogenicity, cross-reactivity against alpha-synuclein, selectivity for alpha-synuclein species/aggregates, affinity, affinity maturity and inhibitory capacity. All surpassed conventional vaccines.

The covalent binding of alpha-synuclein polypeptides according to the present invention to beta-glucan or mannan enables unexpected and unexpected enhancement of the immune response of such polypeptides. This is particularly impressive when compared to traditional vaccine formulations such as those described by Rockenstein et al. Confirmed in part.

Rockenstein et al. (2018) revealed the application of yeast whole glucan particles (GP) non-covalently complexed with aSyn and rapamycin as immunotherapeutic agents for Parkinson's disease. These GPs are produced after a series of thermal alkaline, organic and aqueous extraction steps with Saccharomyces cerevisiae, resulting in a product that contains no cytoplasmic content and consists of beta-glucans (mainly ß1-3 beta-glucans). The final product consists of a highly purified 3 to 4 μm diameter yeast cell wall preparation surrounded by a porous insoluble shell.

Importantly, the vaccine compositions disclosed by Rockenstein et al. (2018) consist of GP non-covalently complexed with ovalbumin and mouse serum albumin (MSA), human aSyn and MSA, or human aSyn, MSA and rapamycin. This complexation method relies on the co-incubation of different payloads with GP and subsequent diffusion into the GP cavity without covalent attachment, and is therefore similar to a set of vaccines disclosed in Example 28 provided in this application, which formulates vaccines by merely mixing the components without covalent attachment, and has been shown to be less effective and unsuitable than vaccines according to the present invention.

1) Rockenstein et al. demonstrated that non-covalent mixing of aSyn and GP elicited a detectable immune response against aSyn, thus demonstrating that GP can serve as an adjuvant. However, Rockenstein et al. also demonstrated that non-covalent addition/co-complexation of rapamycin was required to induce significantly enhanced functionality of such vaccines compared to controls. From this perspective, a mixture 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.

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) in order to exert its full functionality, that is, the induction of neuroprotective, anti-aSyn directed cellular (i.e., T cell mediated) and humoral (i.e., antibody/B cell based) immune responses. This is in direct contrast to the teachings of the present invention, where it is sufficient if the selected vaccine elicits only aSyn-specific B cell responses.

3) The use of full-length aSyn also carries the risk of inducing/enhancing autoreactive Syn-specific T cells, which have the potential to exacerbate the underlying neuropathology in PD and other synuclein pathologies. Therefore, the GP-aSyn-rapamycin vaccine proposed by Rockenstein et al. is not suitable for human use with respect to this issue.

4) As shown in Example 5, similar to Rockenstein et al., non-covalent mixing of Syn-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 Psoralea corylifolia) was also able to induce low-level antibody responses against aSyn. However, the vaccines according to the present invention based on the covalent linkage of such peptides to suitable glucans exerted significantly different and superior immune responses (see also FIG. 5 ).

In addition, and also disclosed in Example 6 and Figure 7, such covalently linked vaccines also show extremely beneficial deficiencies compared to non-covalent mixed vaccines based on dextran particles and peptides as disclosed in the present invention. Anti-glucan antibody response.

Therefore, the prior art disclosure of Rockenstein et al. does not indicate the claimed subject matter of the present invention.

The specific preferred aSyn polypeptide to be bound in the present invention is selected from native α-synaptic nucleoprotein or a polypeptide consisting of the following amino acid residues of the amino acid sequence of native human α-synaptic nucleoprotein: 1 to 5, 1 to 8, 1 to 10, 60 to 100, 70 to 140, 85 to 99, 91 to 100, 100 to 108, 102 to 108, 102 to 109, 103 to 129, 103 to 135, 107 to 130, 109 to 126, 110 to 130, 111 to 121, 111 to 135, 115 to 121, 115 to 122, 115 to 123, 115 to 124, 115 to 125, 115 to 126, 118 to 126, 121 to 127, 121 to 140 or 126 to 135, the amino acid sequence is: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA (Human aSyn (1-140 aa): UNIPROT Accession No. P37840), Preferably, the polypeptide comprises or consists of the following amino acid residues: 1 to 8, 91 to 100, 100 to 108, 103 to 135, 107 to 130, 110 to 130, 115 to 121, 115 to 122, 115 to 123, 115 to 124, 115 to 125, 115 to 126, 118 to 126, 121 to 127 or 121 to 140; or a mimetic antigenic determinant selected from the following group: DQPVLPD, DQPVLPDN, DQPVLPDNE, DQPVLPDNEA, DQPVLPDNEAY, DQPVLPDNEAYE, DSPVLPDG, DHPVHPDS, DTPVLPDS, DAPVTPDT, DAPVRPDS and YDRPVQPDR.

Current state-of-the-art CLEC vaccines all 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 component may result in the induction of high levels of carrier/protein-specific antibodies at the expense of target-specific responses, so that target-specific responses may be insufficient compared to the induced carrier responses.

Furthermore, affinity maturation of target-specific responses induced after repeated immunizations with the carrier conjugate is also impaired due to over-representation of carrier-specific epitopes in the conjugate. As used and understood herein, affinity maturation in immunology is the process by which _TFH cell-activated B cells produce antibodies with increased affinity for an antigen during the course of an immune response. With repeated exposure to the same antigen, the host will produce antibodies with progressively increasing affinity. The secondary reaction can elicit antibodies with several times higher affinity than the primary reaction. Affinity maturation occurs primarily on surface immunoglobulins of germinal center B cells and is a direct result of somatic hypermutation (SHM) and T _FH cell selection (see also: https://en.wikipedia.org/wiki/Affinity_maturation) . According to Segen's Medical Dictionary (https://medical-dictionary.thefreedictionary.com/affinity+maturation''＞affinity maturation＜/a＞), affinity maturation is the increase in the average affinity of the antibody for the antigen after immunization. . Affinity maturation results from an increase in specific and more homogeneous IgG antibodies and is followed by an early response of less specific and more heterogeneous IgM molecules.

In addition, high anti-carrier reactions also pose risks of immune rejection and related safety issues.

Therefore, effective constructs according to the present invention with high immunogenicity, high target specificity and high tolerability/safety with low or non-existent carrier reactivity (i.e. against protein carriers) are achieved through the innovative The workaround successfully addresses this challenge. Additionally, for novel vaccines according to the present invention, it is crucial to provide immunotherapeutic agents that induce no or only a very weak immune response against the glycoskeleton. This is particularly important because high anti-CLEC antibody levels induced after immunization may inhibit or reduce the efficacy of repeated immunizations with the same CLEC-based vaccine through vaccine neutralization, or may also compromise the effectiveness of this type of vaccine. Have adverse effects on continuous immunization against different targets.

The vaccine platform according to the invention also meets the need to combine various epitopes for one or several targets in one formulation without incurring the risk of reduced efficacy due to unintended epitope diffusion as reported for classical vaccines . The modular design of the platform according to the present invention allows for easy exchange of B cell epitopes and T cell epitopes without negative effects of vector-induced responses.

The present invention is based on CLECs that exert highly specific binding to cognate receptors. This binding is critical and only the stronger binders are effective as vaccine vectors/backbones.

According to the present invention, CLEC conjugation enables an effective immune response with novel characteristics. The conjugation according to the present invention prevents the formation of anti-CLEC antibodies, especially for Psoralea corylifolia, and such prevention can be impressively demonstrated in the process of the present invention. This lack of induction of anti-CLEC antibodies is very important for the reusability and boosting of individual vaccines designed with the platform according to the present invention - whether using the same or different antigens.

In contrast to the conjugation examples of the present invention, merely mixing the CLEC polysaccharide adjuvant with a B-cell or T-cell antigenic determinant peptide does not produce a comparable effect in vivo. However, if the two are conjugated, the orientation of the peptide does not significantly affect the potency of the compounds according to the present invention; thus, CLEC binding is essentially independent of the orientation of the peptide in the construct. In the course of the present invention, it was shown that CLEC conjugation (particularly with Psoralea corylifolia polysaccharide) results in improvements of novel as well as existing peptide immunogens/antigens: this improvement is achieved by higher, more target-specific and more affinity antibody responses (e.g., as can be demonstrated by antibody selectivity and functional display). This effect is most pronounced in Pyricularia polysaccharides or similar β-glucans, which are predominantly linear β-(1,6)-glucans in which the ratio of β-(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, especially at least 10:1, which surprisingly outperform even significantly KLH or CRM in direct comparisons, and even outperform mannosaccharide or lichen conjugates or conjugates comprising barley β-glucan.

As used herein, the term "predominantly linear" β-(1,6)-glucan refers to 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.

As has been stated above, Lycoris polysaccharide is the best CLEC according to the present invention. The fungus polysaccharide usually does not contain cross-linking sugar moieties and is mainly β-(1,6) coupled, so that the common fungus polysaccharide preparations used to prepare the conjugates according to the invention contain less than 1%, preferably less than 0.1%, Especially less than 0.01% of the monosaccharide moieties and having more than two covalently linked monosaccharide moieties, and the fungus polysaccharide contains up to 10% of the monosaccharide moieties having β-(1,3) or β-(1,4) coupling. Monosaccharide impurities.

The fact that Pseudomonas aeruginosa was shown to be the most effective CLEC in the process of the present invention is surprising, since various references have shown that Pseudomonas aeruginosa should be less effective in dectin-1 binding (e.g. Adams et al., J Pharmacol Exp Ther. 2008 Apr;325(1):115-23); in which linear 1,3 and branched (1,3 backbone and 1,6 side branches) have been reported as the most effective dectin-1 binders. For example, Adams et al., 2008 have reported that murine recombinant dectin-1 only recognizes and interacts with polymers containing a β-(1,3) linked glucose backbone. Dectin-1 does not interact with glucans consisting solely of a β-(1,6)-glucose backbone (pyrifos), nor does it interact with non-glucan carbohydrate polymers such as mannan.

Therefore, according to a preferred embodiment of the present invention, the β-glucan of the conjugate of the present invention is a dectin-1-conjugated β-glucan. The ability of any compound, especially dextran, to bind to C dectin-1 can be readily determined by methods as disclosed herein, particularly in the Examples section. For the avoidance of doubt, "dectin-1 binding beta-glucan" is beta-glucan that binds to the soluble murine Fc-dectin-1a receptor with an IC50 value below 10 mg/ml, as determined by competitive ELISA. Glycans, for example, are disclosed in the Examples.

Compared to other glucans, such as DC-SIGN β-glucans (such as β-(1,2)-glucans), the dectin-1-conjugated β-glucans (such as linear β-(1,6)-glucans) according to the present invention are more advantageous because a wider range of DCs (immature, mature, myeloid, plasmacytoid; in addition: APC) can be covered by such dectin-1-conjugated glucans, which significantly increase the potential to induce an effective immune response in vivo compared to non-dectin-1-conjugated glucans of limited applicability (immature DCs, myeloid DCs).

WO 2022/060487 A1 and WO 2022/060488 A1 disclose conjugates that link peptide immunogens to immunostimulatory polymer molecules such as β-(1,2) glucans. β-(1,2) glucans, including cyclic variants, have previously been suggested as potential adjuvants (Martirosyan A et al., doi:10.1371/journal.ppat.1002983), which are a type of glucan that primarily binds to specific PRRs (i.e., DC-SIGN, Zhang H et al. doi:10.1093/glycob/cww041) and specifically binds to N-terminally linked high mannose oligosaccharides and branched fucosylated structures. Importantly, β-1,2 glucan is unable to bind to dectin-1 (Zhang H et al., doi:10.1093/glycob/cww041), thereby limiting its activity on DC-SIGN positive cells.

DC-SIGN (CD209) was the first SIGN molecule to be identified and was found to be highly expressed only in a limited subset of DCs, including immature (CD83 negative) DCs and special macrophages in the placenta and lungs (Soilleux EJ et al., doi: 10.1189/jlb.71.3.445). In the periphery, for example in the skin or at mucosal sites, expression and thus potential for biological activity as a receptor according to the present invention can only be detected 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).

In contrast, the target receptor of the glucan-based immunogens provided in the present invention is dectin-1. Dectin-1 is expressed on a variety of different DC types, including not only immature DC, bone marrow DC, but also plasmacytoid DC, which express dectin-1 at the mRNA and protein levels, as well as DC-like Langerhans cells in the skin. (Patente et al., doi: 10.3389/fimmu.2018.03176; Joo et al. doi: 10.4049/jimmunol.1402276).

Therefore, the biological activity of DC-SIGN targeting polymers (such as β-(1,2) glucan) is limited to specific DC target cell populations, while dectin-1 targeting polymers such as those used in the present invention can be used in It functions in many different other DC types. Therefore, these novel conjugates can exert significantly different and superior immune responses compared with other conjugates. Therefore, the prior art disclosure is not indicative of the claimed subject matter of the present disclosure.

According to a particularly preferred embodiment, the conjugate of the present invention comprises a strong dectin-1 binding β-glucan, preferably a β-glucan that binds to a soluble murine 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, for example as disclosed in the examples. Particularly preferred are the following binders, as determined by competitive ELISA, which bind to soluble murine Fc-dectin-1a receptor with an IC50 value of less than 1 mg/ml, more preferably with an IC50 value of less than 500 µg/ml, even more preferably with an IC50 value of less than 200 µg/ml, in particular with an IC50 value of less than 100 µg/ml; and/or -the following β-glucan, as determined by competitive ELISA, 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; and/or - wherein the conjugate binds to soluble human Fc-dectin-1a receptor with an IC50 value of less than 1 mg/ml, more preferably less than 500 µg/ml, even more preferably less than 200 µg/ml, in particular less than 100 µg/ml as determined by competitive ELISA, for example as disclosed in the examples.

In addition, compared with vaccines without CLEC, especially without Psoralea corylifolia polysaccharide, the conjugate according to the present invention also shows a highly proportional increase in the ratio of antibodies reacting with the target polypeptide and antibodies reacting with the carrier molecule. This significantly increases the specific focus of the antibody immune response on the target rather than the carrier, thereby increasing the efficacy and specificity of the immune response.

The conjugation of CLEC according to the present invention, especially CLEC conjugated to Pseudomonas aeruginosa, also resulted in increased affinity maturation (AM) for the target protein (AM was greatly increased, while KLH/CRM conjugate showed only limited AM after repeated immunization).

In the field of vaccines, suitable vaccines having only B cell epitopes or only T cell epitopes have been disclosed. In certain cases, it may be suitable and preferred to use a vaccine having only T cell epitopes or only B cell epitopes. However, most vaccines on the market contain two epitopes, namely T cell epitopes and B cell epitopes.

For example, vaccines containing only B-cell epitopes are not very effective in most cases, even if they do elicit a detectable antibody immune response. However, in most cases, this immune response is usually much less effective compared to vaccines containing B-cell and T-cell epitopes. This is also consistent with the examples given in the Examples section of the present invention, where lower levels of response could be detected.

On the other hand, vaccines containing only T cell epitopes (e.g. in vaccines where a specific T cell response is an active component of the response) are particularly interesting for certain applications, especially cancer, where cancer-specific cytotoxicity T lymphocyte and helper T cell epitopes or only CTL epitopes are combined with the vaccine platform according to the invention. In this case, the T cell epitopes with CLEC polysaccharide adjuvant according to the invention are provided with only T cell epitopes. This is particularly preferred in certain circumstances, such as where somatic mutations in cancer affect protein-coding genes, which may generate potentially therapeutic neo-epitope(s). These neo-epitopes can guide adoptive cell therapies and peptide-based (and RNA-based) neo-epitope vaccines to selectively target tumor cells using the patient's own cytotoxic T cells. According to the present invention, this can be used for general antigen and individualized neoantigen-specific treatments (for example, in NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-C1, MAGE-C2, MAGE-C3, Survivin, gp100 , tyrosinase, CT7, WT1, PSA, PSCA, PSMA, STEAP1, PAP, MUC1, 5 T4, KRAS, Her2 and others). For certain autoimmune diseases, it may also be preferable to use vaccines containing only T cell epitopes. The therapeutic effect of each conjugate containing only T cell epitopes is associated with the reduction of effector T cells and the formation of a regulatory T cell (T _reg cell) population, thereby enabling the suppression of the respective autoimmune disease (e.g., multiple sclerosis or similar diseases).

Since most commonly used vaccine designs contain both B cell antigenic determinants and T cell antigenic determinants, the CLEC conjugates according to the present invention preferably also contain respective B cell antigenic determinants and T cell antigenic determinants (at least: at least one B cell antigenic determinant and at least one T cell antigenic determinant) to achieve a sustained B cell immune response. However, if desired, a weak effect may demonstrate T cell-independent immunity.

Therefore, the conjugates according to the invention are not limited in terms of possible vaccine antigens. However, the vaccine antigen (ie, B cell and/or T cell epitope polypeptide) preferably has 6 to 50 amino acid residues, more preferably 7 to 40 amino acid residues, especially 8 to 30 amino acid residues in length.

Cross-linking of B cell receptors is also possible using the vaccine according to the invention. According to a specific embodiment, the conjugate according to the invention is used for T cell-independent immunity. T cell-independent responses are well known for polysaccharide vaccines. These vaccines/polysaccharides produce an immune response by directly stimulating B cells without the help of T cells. T cell-independent antibody responses are short-lived. Antibody concentrations to S. pneumoniae capsular polysaccharide typically decrease to baseline within 3-8 years, depending on the serotype. It is generally not possible to enhance the vaccine response by using additional doses because polysaccharide vaccines do not constitute immune memory. In children under two years of age, polysaccharide vaccines are poorly immunogenic. The reason for the direct stimulation here may be that the B cells express a molecule called CR3 (complement receptor type 3). Macrophage-1 antigen, or CR3, is a human cell surface found on B and T lymphocytes, polymorphonuclear leukocytes (mainly neutrophils), NK cells, and mononuclear phagocytes (such as macrophages). receptor. CR3 also recognizes iC3b when bound to the surface of foreign cells and β-glucan, which means that direct uptake of the vaccine by B cells through the Pus-CR3 interaction may cause stimulation of the cells and produce a low-level TI immune response.

Adjuvants, conjugates and vaccines according to the invention fix complement and can be opsonized. The opsonized conjugates according to the present invention may have increased B cell activation capacity, which may result in higher antibody titers and antibody affinity. Such effects are known for C3d conjugates (Green et al., J. Virol. 77 (2003), 2046-2055) and unexpectedly may also be used in the present process.

Another unexpected advantage of the present invention is that the CLEC framework of the present invention enables modular design of vaccines. For example, antigenic determinants can be combined at will, and the platform is independent of conventional carrier molecules. Although the main focus of the present invention is vaccines containing only peptides, it is also applicable to independent coupling of proteins and peptides, as well as coupling of peptide-protein conjugates with the CLEC backbone according to the present invention, especially with Pyricularia polysaccharide. As shown in the example section using Pyricularia polysaccharide, the vaccine according to the present invention obtains an immune response significantly higher than that of classical vaccines.

As mentioned above, the conjugate according to the invention, if provided in the form of a pharmaceutical preparation (for example, as a vaccine), is intended to be administered to a (human) individual to elicit an immune response to a specific polypeptide epitope bound to the CLEC backbone. , the epitope is an epitope that should elicit an immune response), can be administered in this formulation without the use (by co-administration) of (other) adjuvants. According to a preferred embodiment, the pharmaceutical preparation comprising the conjugate according to the invention does not contain adjuvants.

One particularly preferred class of CLEC polysaccharide adjuvants according to the present invention are β-glucans, especially Schizophora polysaccharides. Another preferred CLEC polysaccharide adjuvant is mannan. In contrast to the present invention, fungus polysaccharides have only been used in antifungal vaccines in the prior art (wherein the fungus polysaccharides were used as antigens and not as carriers as in the present invention). The fungus polysaccharide also exhibits a different backbone, as it consists only of β-(1,6)-linked sugar moieties.

Schizophora polysaccharide is a medium-sized linear β-(1,6) glucan. The synthetic form of polysaccharides and linear β-(1,6) glucans differs from that of all other glucans, which are usually composed of branched glucan chains (preferably β-(1,6)). 1,3) Main chain and β-(1,6) side chain, such as yeast extract, GP, laminarin, schizophyllan, scleroglucan) β-glucan may only rely on β-(1 ,3) Linear glucan of glucan, such as synthetic β-glucan, Cardranan, Saccharomyces cerevisiae β-glucan (150kDa) or linear β-(1,3:1,4) glucan Sugars (such as barley and oat beta-glucan and lichenin).

As shown here for the first time, binding of dextran conjugates to the dectin-1 receptor in vitro is a surrogate for subsequent in vivo efficacy: low-binding molecules exert only a low immune response, and medium-binding conjugates are more The best, and the high-efficiency conjugate induces a high-efficiency reaction (oat/barley BG<lichenin<lichen polysaccharide).

According to the present invention, CLEC are coupled (e.g., by standard techniques) to individual polypeptides to produce small nanoparticles with low polydispersity (hydrodynamic radius (HDR) range: 5-15 nm) that are not cross-linked with each other , and will not aggregate to form larger particles similar to those of conventional CLEC vaccines, such as glucan particles (2-4µm) or β-glucan particles disclosed in the literature. These particles are usually characterized by a size range >100 nm (typical range) (diameter; 150-500 nm), e.g. Wang et al. (2019) provide particles with a diameter of 160 nm (evaluated by DLS) and a size of about 150 nm (evaluated by TEM); Jin (Acta Biomater. 2018 Sep 15;78:211-223) provided β-glucan particles (aminated β-glucan) with a size of 180-215 nm (evaluated by DLS and SEM, respectively). -Ovalbumin nanoparticles).

By definition, the hydrodynamic radius measured by DLS is the radius of an assumed hard sphere with the same diffusion velocity as the measured particle. The radius is calculated from the diffusion coefficient assuming that the molecule/particle is spherical and that the buffer has a given viscosity. HDR is also called Stokes radius and is calculated from the diffusion coefficient using the Stokes-Einstein equation (see https://en.wikipedia.org/wiki /Stokes_radius).

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

Therefore, the present invention also relates to a vaccine product designed for vaccinating an individual against a specific antigen, wherein the product comprises a compound, preferably β-glucan or mannosaccharide, as a C-type lectin (CLEC) polysaccharide adjuvant covalently coupled to the specific antigen.

Preferably, the vaccine product according to the invention comprises a conjugate as disclosed herein or obtainable or obtainable by a method according to the invention.

According to a preferred embodiment, the vaccine product according to the present invention comprises an antigen comprising at least one B cell antigenic determinant and at least one T cell antigenic determinant, preferably, wherein the antigen is a polypeptide comprising one or more B cell antigenic determinants and T cell antigenic determinants.

According to a preferred embodiment, the covalently coupled antigen and CLEC polysaccharide adjuvant in the vaccine product according to the present invention are present in particles with a size of 1 to 5000 nm, preferably 1 to 200 nm, especially 2 to 160 nm, Determined by dynamic light scattering (DLS) as hydrodynamic radius (HDR). As used herein, all particle sizes are mid-size, where "mid" is the value that distinguishes half of the particles with larger sizes from half of the particles with smaller sizes. It is a defined size according to which half of the particles are smaller and the other half are larger.

According to a preferred embodiment, the covalently coupled antigen and CLEC polysaccharide adjuvant in the vaccine product according to the present invention are present in particles with a size of 1 to 50 nm, preferably 1 to 25 nm, especially 2 to 15 nm, Measured by DLS in HDR format.

Preferably, the covalently coupled antigen and CLEC polysaccharide adjuvant in the vaccine product according to the present invention are present as particles with a size of less than 100 nm, preferably less than 70 nm, especially less than 50 nm, and are obtained by DLS in the form of HDR. Determination.

The vaccine product according to the invention shows high storage stability. Virtually no aggregation occurs when stored as liquid or frozen material (storage temperature: -80°C, -20°C, 2-8°C, or at room temperature for an extended period of at least 3 months), transparent particle size It is determined that it will not increase significantly (i.e. more than 10%) during storage.

The extremely high efficacy of such small particles produced by using the medium molecular weight component Trichophyton polysaccharide according to the present invention is unexpected: for example, according to Adams et al. (J Pharmacol Exp Ther. 2008 Apr;325(1):115 -23), the best dectin-1 substrates are linear phosphate β(1,3) glucan (about 150kda) and branched glucan (which contains β(1,3) backbone and β(1 , 6) side chain), such as scleroglucan or glucan or lichen from Candida albicans. In addition, the data of 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 does not interact or interacts only weakly with agaric polysaccharides, nor with non-glucan carbohydrate polymers such as mannan. In fact, there are multiple references reporting that Shigu polysaccharide is less effective in binding to dectin-1. However, in general, linear 1,3 and branched (1,3 main chain and 1,6 side branches) are the most effective dectin-1 conjugates; Adams et al. (2008) demonstrated that murine recombinant dectin- 1 only recognizes and interacts with polymers containing a β(1,3)-linked glucose backbone. Dectin-1 does not interact with glucans composed entirely of β(1,6)-glucose backbones (e.g., polysaccharides), nor with non-glucan carbohydrate polymers (e.g., mannans) .

In contrast to these findings, it was shown in the course of the present invention that Pseudomonas aeruginosa-based conjugates are able to bind strongly to dectin-1 and elicit cellular responses in vitro.

According to a preferred embodiment of the present invention, β-(1,6)-glucan is used. It is generally reported in the prior art that large particles are more effective at activating PRRs than small ("soluble") monomeric formulations, so particles containing large glucans are more advantageous (and therefore preferred), while small soluble glucans can be used to block DC activation, thereby interfering with the intended effect. It is generally believed that particulate β-glucans (such as the widely used yeast cell wall component zymosan) bind to and activate dectin-1, thereby inducing cellular responses. In contrast, the interaction of soluble β-glucans with dectin-1 is controversial. However, 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), can bind to dectin-1 but cannot 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.).

According to the present invention, it can be shown that conjugates using high molecular weight glucans (10 times the size of Psoralen; for example: oat/barley 229kDa/lichenin 245kDa) are not as effective as Psoralen particles (20kDa). Korotchenko et al. demonstrated that OVA/Lam conjugates have a diameter of about 10 nm and bind to dectin-1 in vitro and induce DC activation, but they are branched glucans, not skin-specific, and their effects in vivo are not superior to OVA applied to the skin or OVA/alum applied subcutaneously. Wang et al. provided β-glucan particles with a size of >100 nm (average size: 160 nm). Jin et al. (2018) demonstrated aminated β-glucan-ovalbumin nanoparticles with a size of 180-215 nm.

According to the present invention, it was shown that Pep-based particles are potent dectin-1 binders that can activate DC in vitro (changes in surface marker expression) and induce very strong immune responses, superior to a) other routes and b) KLH/CRM conjugate vaccines (usually also larger particles) and c) larger glucans and mannoses. This was true for both Pep+Padre+Pep-based (5 nm size) and Pep+CRM+Pep-based (11 nm size).

In order to obtain the best immune response, the degree of activation of CLEC, especially the degree of activation of Shigu polysaccharide, and the peptide/sugar ratio generated by this degree of activation are decisive. Activation of individual CLECs is achieved by mild periodate oxidation. The degree of oxidation is therefore determined based on the addition of a periodate solution at a defined molar ratio: periodate:sugar subunits; 100% = 1 mole of periodate per mole of sugar monomer.

According to a preferred embodiment, the combination according to the present invention contains 1/5 (i.e. 20% activation) to 2.6/1 (i.e. 260% activation), preferably 60% to 140%, especially 70% to 70% 100% ratio of periodate to beta-glucan or mannan (monomeric) moieties activated CLEC.

The optimal range of oxidation degrees between low/medium and high oxidation degrees (which will be directly proportional to the amount of antigenic determinant polypeptide in the final conjugate) can be defined as the degree of reaction with Schiff's fuchsin-reagent, which is similar to the degree of oxidation of an equivalent amount of a given carbohydrate (e.g., Pyrrolizidine) with periodate at a molar ratio (sugar monomer:periodate salt) of 0.2-2.6 (low/medium), 0.6-1.4 (optimal range), and 1.4-2.6 (high), respectively.

The preferred ratio of glucan to peptide is in the range of 10 to 1 (w/w) to 1 to 1 (w/w), preferably 8 to 1 (w/w) to 2 to 1 (w/w), and especially 4 to 1 (w/w), i.e. the molar ratio of sugar monomer to peptide is 24 to 1, with the proviso that if the conjugate comprises a carrier protein, the preferred ratio of β-glucan or mannopolysaccharide to B cell-antigen determinant-carrier polypeptide is 50:1 (w/w) to 0.1:1 (w/w), especially 10:1 to 0.1:1; which is lower than that of other reported effective vaccines (e.g. Liang et al.; Bromuro et al.).

The degree of oxidation and the amount of reactive aldehyde available for sugar coupling are determined using state-of-the-art methods such as: 1) gravimetric analysis, which can be used to determine the total mass of the sample; 2) the anthrone method (according to Laurentin et al., 2003), which is used for the concentration of intact non-oxidized sugars in the sample; in this case, _dextran is dehydrated with concentrated _H2SO4 to form furfural, which condenses with anthrone (0.2% in _H2SO4 ) to form a green complex that can be measured colorimetrically at a wavelength of 620 nm); or 3) Schiff analysis: Schiff's fuchsin sulfite reagent is used to assess the oxidation state of the carbohydrate available for coupling. Briefly, the _fuchsin dye is decolorized by sulfur dioxide and reaction with aliphatic aldehydes (on the dextran) restores the purple color of the fuchsin, which can then be measured at a wavelength of 570-600 nm. The color reaction produced is proportional to the degree of oxidation (number of aldehyde groups) of the carbohydrate. Other suitable analytical methods are also possible. Suitable methods including UV analysis (205 nm/280 nm) and amino acid analysis (aa hydrolysis, derivatization and RP-HPLC analysis) can be used to assess the peptide ratio.

The conjugates according to the invention can further be used to induce target-specific immune responses while inducing no or only a very limited CLEC- or carrier protein-specific antibody response. As also shown in the Examples section below, the present invention is also able to improve and focus target-specific immune responses because it directs the triggered immune response away from responses to carrier proteins or CLECs (e.g., in conventional peptide-carrier conjugates or non- In the comparative setting of binding properties, it is especially applicable to non-oxidized CLEC, such as auricularia polysaccharides).

Unless otherwise specified, "peptide" as used herein refers to a shorter polypeptide chain (2 to 50 amino acid residues), while "protein" refers to a longer polypeptide chain (more than 50 amino acid residues). Both are referred to as "polypeptides". In addition to polypeptides with natural amino acid residues of normal gene expression and protein translation, the B-cell and/or T-cell antigenic determinant polypeptides that bind to CLEC according to the present invention also include all other forms of such polypeptide-based B-cell and/or T-cell antigenic determinants, especially their natural or artificially modified forms, such as glycosyl polypeptides and all other post-translationally modified forms thereof (e.g., the Aβ pyroglutamine form disclosed in the Examples). Furthermore, CLECs according to the present invention are particularly suitable for use with conformational epitopes, such as those that are part of a larger native polypeptide, a mimetic epitope, a cyclic polypeptide or a surface-bound construct.

According to a preferred embodiment, the conjugate according to the present invention includes a CLEC polysaccharide backbone and a B cell epitope. A "B cell epitope" is the part of an antigen to which immunoglobulins or antibodies bind. B cell epitopes can be divided into two groups: conformational or linear. There are two main approaches to epitope mapping: structural studies or functional studies. Methods for structurally localizing epitopes include X-ray crystallography, nuclear magnetic resonance and electron microscopy. Methods of functionally localizing epitopes typically use binding assays such as Western blotting, disk blotting, and/or ELISA to measure antibody binding. Competition methods are used to determine whether two monoclonal antibodies (mAbs) can bind to an antigen simultaneously or compete with each other to bind at the same site. Another technique involves high-throughput mutagenesis, an epitope mapping strategy designed to improve the rapid localization of conformational epitopes on structurally complex proteins. Mutagenesis uses random/site-directed mutagenesis on individual residues to localize epitopes. B cell epitope mapping can be used to develop antibody therapies, peptide-based vaccines, and immunodiagnostic tools (Sanchez-Trinchado et al., J. Immunol. Res. 2017-2680160). For many antigens, B cell epitopes are known and can be used in the CLEC platform of the invention.

According to a particularly preferred embodiment, the conjugate according to the invention comprises a CLEC polysaccharide backbone and one or more T cell epitopes, preferably comprising hybrid T cell epitopes and/or known to be associated with a given species and MHCII epitopes in which several/all MHC alleles of other species act together.

According to another aspect, the invention also relates to the use of the present CLEC technology to improve known T cell epitopes. Accordingly, the present invention also includes beta-glucans or mannans for use as adjuvants for C-type lectin (CLEC) polysaccharides of T cell epitope polypeptides, wherein the beta-glucans or mannans are covalently bound to the T cells The epitope polypeptide is used to form a conjugate of β-glucan or mannan and T cell epitope polypeptide.

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

Preferred PADRE polypeptides or PADRE polypeptide variants include a linker (preferred for other polypeptide antigenic determinants used herein), such as a cysteine residue or a linker comprising a cysteine residue ("-C" or "C-"; particularly useful for maleimide coupling), a NRRA, NRRA-C or NRRA-NH-NH2 linker. Preferred PADRE polypeptide variants include variants disclosed in the prior art (e.g., Alexander et al., Immunity 1 (1994), 751-761; US9,249,187B2) or preferably a shortened variant without a C-terminal A residue (AKFVAAWTLKAA), wherein the first residue alanine is replaced by an aliphatic amino acid residue (e.g., glycine, valine, isothiocyanate, thiazolidine ... variants in which the third amino acid residue phenylalanine is substituted with L-cyclohexylalanine, variants in which the thirteenth (last) amino acid residue alanine is substituted with an aliphatic amino acid residue (e.g., glycine, valine, isoleucine and leucine), variants comprising aminocaproic acid, which are preferably coupled to the C-terminus of the PADRE variant, or variants having the amino acid sequence AX1FVAAX2 Variant _{AX4A of TLX3} , wherein _X1 is selected from W, F, Y, H, D, E, N, Q, I and K; _X2 is selected from the group consisting of F, N, Y and W, _X3 is selected from the group consisting of H and K, and _X4 is selected from the group consisting of A, D and E (the prerequisite is that the oligopeptide sequence is not AKFVAAWTLKAAA; US9,249,187B2); in particular, wherein the T cell antigen determinant is selected from AKFVAAWTLKAAANRRA-(NH-NH2), AKFVAAWTLKAAAN-C, AKFVAAWTLKAAA-C, AKFVAAWTLKAAANRRA-C, aKXVAAWTLKAAaZC, aKXVAAWTLKAAaZCNRRA (Seq ID 7, 8, 87, 88, 89, 90, 91, 92), aKXVAAWTLKAAa, aKXVAAWTLKAAaNRRA, aA(X)AAAKTAAAAa, aA(X)AAATLKAAa, aA(X)VAAATLKAAa, aA(X)IAAATLKAAa, aK(X)VAAWTLKAAa and aKFVAAWTLKAAa (Sequences 760.5, 760.57, 906.09, 906.11, 965.10, 1024.03 according to Alexander et al., 1994), wherein X is L-cyclohexylalanine, Z is aminohexanoic acid, and a is an aliphatic amino acid residue selected from alanine, glycine, valine, isoleucine and leucine.

T cell epitopes are present on the surface of antigen presenting cells where they are bound to major histocompatibility complex (MHC) molecules. In humans, specialized antigen presenting cells are specialized for presenting class II MHC peptides, while most nucleosome cells present class I MHC peptides. T cell epitopes presented by class I MHC molecules are typically peptides between 8 and 11 amino acids in length, while class II MHC molecules present longer peptides (13 to 17 amino acids in length); and atypical MHC molecules also present non-peptide epitopes such as glycolipids. Class I and class II MHC epitopes can be reliably predicted by computational methods alone, but not all in silico T cell epitope prediction algorithms are equal in accuracy. There are two main approaches to predict peptide-MHC binding: data-driven and structure-based. Structure-based approaches model the peptide-MHC structure 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, type, and stimulate T cells. For many antigens, the T cell epitopes are known and can be used with the CLEC platform of the present invention.

Interestingly, recent breakthrough studies have shown that α-synuclein-specific T cells are increased in PD patients, which may be related to the risk haplotype of HLA, and suggest that T cells are involved in autoimmunity in PD [Sulzer et al., Nature 2017;546:656-661 and Lindestamn Arlehamn et al., Nat Commun. 1875;2020:11]. A recent animal model study also further confirmed the causal role of α-synuclein-reactive T cells [Williams et al., Brain. 2021;144:2047-2059). In one case study, the presence of α-synuclein-reactive T cells began to increase several years before the onset of motor disease and was highest in frequency shortly before or after the onset of motor disease in a large cross-sectional group of PD patients (Lindestam Arlehamn et al., 2018). After the onset of motor disease, T cell responses to α-synuclein decreased with increasing disease duration. Thus, anti-aSyn T cell responses were highest before or shortly after the diagnosis of motor PD and then gradually weakened (i.e., maximal activity was detectable less than 10 years after diagnosis; and preferably Hoehn and Yahr (H+Y) stages 1 and 2) (Lindestamn Arlehamn et al., 2020).

Thus, the human α-synuclein sequence contains well-known T cell antigenic determinants. Examples are provided by Benner et al. (PLoS ONE 3(1): e1376.60); Sulzer et al., (2017) and Lindestam Arlehamn et al. (2020).

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

Sulzer et al. (Nature 2017;546:656-661) identified two T cell antigenic regions in the N-terminal and C-terminal regions of α-synuclein from human PD patients. The first region is located near the N-terminus and is composed of MHCII antigenic determinants aa31-45 (GKTKEGVLYVGSKTK) and aa32-46 (KTKEGVLYVGSKTKE), and also contains a 9-mer peptide aa37-45 (VLYVGSKTK) as a potential MHC I class antigenic determinant. The second antigenic region revealed by Sulzer et al. is close to 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 naturally occurring immune response to α-synuclein associated with PD has both MHC class I and MHC class II restricted components.

In addition, Lindestam Arlehamn et al. (Nat Commun. 1875;2020:11) also revealed that alpha-synuclein peptide aa61-75 (EQVTNVGGAVVTGVT) serves as a T cell epitope (MHCII) in PD patients.

Therefore, preferred T cell epitopes according to the present invention include alpha synuclein polypeptides GKTKEGVLYVGSKTK (aa31-45), KTKEGVLYVGSKTKE (aa32-46), EQVTNVGGAVVTGVT (aa61-75), VTGVTAVAQKTVEGAGNIAAATGFVK (aa71-86), DPDNEAYEMPSE ( aa116-130), DNEAYEMPSEEGYQD (aa121-135) and EMPSEEGYQDYEPEA (aa126-140).

Regulatory T cells ("Treg cells" or "Treg") are a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Treg cells have immunosuppressive effects and usually inhibit or downregulate the induction and proliferation of effector T cells. Tregs produced by the normal thymus are called "natural". Selection of natural Tregs occurs on radioresistant hematopoietic-derived MHC class II expressing cells in the medulla or on Hassal's corpuscles in the thymus. The process of Treg selection depends on the affinity of interaction with the autologous peptide MHC complex. Choosing to become a Treg is a "Goldilocks" process. It is neither too high nor too low, as long as it is just right. T cells that receive a very strong signal will undergo apoptotic death; Cells with weak signals will survive and be selected to become effector cells. If a T cell receives a moderate signal, it becomes 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 Teff and Tregs, with the relative ratio of the two determined by the T cell's affinity for self-peptide-MHC. Tregs formed by differentiation of naive T cells outside the thymus (i.e., in the periphery) or in cell culture are called "resilient" or "inducible" (i.e., iTregs).

Natural Tregs are characterized by expressing both the CD4 T cell co-receptor and CD25, which is a component of the IL-2 receptor. Therefore Tregs are CD4+ CD25+. The nuclear transcription factor forkhead box P3 (FoxP3) appears to be a key property that determines the formation and function of natural Treg. Tregs inhibit the activation, proliferation and interleukin production of CD4+ T cells and CD8+ T cells, and are thought to inhibit B cells and dendritic cells, thereby inhibiting autoimmune responses.

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 regulatory T cells (Treg) in PD patients inhibit effector T cells The 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) demonstrated that decreased Treg function in PD patients is associated with increased pro-inflammatory T cell activation, which can directly lead to subsequent increased pro-inflammatory signaling in other immune cell populations. The inhibition of T cell proliferation by Tregs is significantly related to the peripheral pro-inflammatory immune cell phenotype. When using the H&Y disease scale, the ability of PD Tregs to inhibit the proliferation of T effector cells (e.g., CD4+) decreases with the increase in PD disease burden, with the highest activity occurring in H+Y stages 1 and 2. Importantly, Lindestam Arlehamn et al. (2020) demonstrated that anti-aSyn T cell responses are highest before or shortly after diagnosis of motor PD and diminish thereafter (i.e., maximal activity is detectable less than 10 years after diagnosis ; and preferably Hoehn and Yahr (H+Y) 1 and 2) (Lindestamn Arlehamn et al., 2020).

Therefore, the vaccine according to the present invention is combined with the following: 1) a vaccine containing α-synaptic protein specific Treg antigenic determinants (e.g. CD4 antigenic determinants, such as those disclosed by Brenner et al., Sulzer et al. and Lindestam Arlehamn et al. (aa31-45 (GKTKEGVLYVGSKTK), aa32-46 (KTKEGVLYVGSKTKE), aa61-75 (EQVTNVGGAVVTGVT), aa71-86 (VTGVTAVAQKTVEGAGNIAAATGFVK), aa116-130 (MPVDPDNEAYEMPSE), aa121-135 (DNEAYEMPSEEGYQD) and aa126-140 (EMPSEEGYQDYEPEA)); and/or 2) Treg inducers, such as rapamycin, low-dose IL-2, TNF receptor 2 (TNFR2) agonists, anti-CD20 antibodies (e.g., rituximab), prednisolone, inosine pranobex, glatiramer acetate, sodium butyrate Preferably in the early stages of the disease (i.e., less than 10 years after diagnosis; and preferably Hoehn and Yahr (H+Y) stages 1 and 2) to increase the number and activity of weakened/diminished Tregs, and thereby reduce the autoimmune reactivity of aSyn-specific T effector cells and inhibit the autoimmune response of PD patients.

In addition, Tregs have been found to be reduced and/or dysfunctional in many diseases, especially chronic degenerative 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: Putnam et 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)).

Therefore, it is also preferred to provide a T cell antigenic determinant suitable as a Treg antigenic determinant or Treg inducer in diseases with a reduced or dysfunctional Treg population, which in combination with a vaccine according to the present invention increases the number and activity of weakened/reduced Tregs, and thereby reduces the autoimmune reactivity of disease-specific T effector cells and inhibits the patient's autoimmune response. Suitable Treg antigenic determinants are defined as autologous MHC antigenic determinants (MHC class II), which are characterized by the ability to induce a medium signal in the selection process of T cells.

According to a preferred embodiment, the conjugate according to the present invention includes a polypeptide that includes or consists of the following amino acid sequences: SeqID7, 8, 22-29, 87-131, GKTKEGVLYVGSKTK, KTKEGVLYVGSKTKE, EQVTNVGGAVVTGVT, VTGVTAVAQKTVEGAGNIAAATGFVK, MPVDPDNEAYEMPSE ), DNEAYEMPSEEGYQD, EMPSEEGYQDYEPEA or combinations thereof.

Therefore, the preferred T cell epitope is: SeqID7 AKFVAAWTLKAAANRRA-(NH-NH2) PADRE SeqID8 AKFVAAWTLKAAAN-C PADRE SeqID22 AKFVAAWTLKAAA-(NH-NH2) PADRE-Original SeqID23 KAAAVKAAFWTAL-NRRA-(NH-NH2) Alternative synthetic peptides SeqID24 DSETADNLEKTVAALSILPGHGC-(NH-NH2) Diphtheria toxin (TV-TT exchange) SeqID25 DSETADNLEKTVAALSILPGHGCNRRA-(NH-NH2) Diphtheria toxin (TV-TT exchange) SeqID26 ISITEIKGVIVHRIETILF-(NH-NH2) MvF5 Th (UBITh®1) SeqID27 ISITEIKGVIVHRIETILFNRRA-(NH-NH2) MvF5 Th (UBITh®1) SeqID28 ISQAVHAAHAEINEAGR-(NH-NH2) Chicken Ova (323-339) SeqID29 ISQAVHAAHAEINEAGNRRA-(NH-NH2) Chicken Ova (323-339) SeqID87 AKFVAAWTLKAAA-C Padre of maleimide coupling (original) SeqID88 AKFVAAWTLKAAANRRA-C Maleimide Coupling Tridem' Padre (Original) SeqID89 aKXVAAWTLKAAaZC PADRE, alternative aas SeqID90 aKXVAAWTLKAAaZCNRRA PADRE, alternative aas SeqID91 aKXVAAWTLKAAa PADRE, alternative aas SeqID92 aKXVAAWTLKAAaNRRA PADRE, alternative aas SeqID93 DSETADNLEKTTAALSILPG diphtheria SeqID94 DSETADNLEKTTAALSILPGNRRA diphtheria SeqID95 LSEIKGVIVHRLEGV f SeqID96 LSEIKGVIVHRLEGVNRRA f SeqID97 KLLSLIKGVIVHRLEGVE f SeqID98 KLLSLIKGVIVHRLEGVENRRA f SeqID99 VSIDKFRIFCKANPK P23-TT SeqID100 LKFIIKRYTPNNEIDS P32-TT SeqID101 IREDNNTLKLDRCNN P21-TT SeqID102 FNNFTVSFWLRVPKVSASHLE P30-TT SeqID103 QYIKANSKFIGITE P2-TT SeqID104 LEYIPEITLPVIAALSIAES TT SeqID105 LINSTKIYSYFPSVISKVNQ TT SeqID106 NYSLDKIIVDYNLQSKITLP TT SeqID107 PHHTALRQAILCWGELMTLA HBV nucleocapsid SeqID108 FFLLTRILTIPQSLD HBV surface AG SeqID109 YSGPLKAEIAQRLEDV MT influenza matrix epitope SeqID110 FFLLTRILTIPQSL HBsAg SeqID111 GAYARCPNGTRALTVAELRGNAEL Bordetella pertussis SeqID112 ALNIWDRFDVFCTLGATTGYLKGNS cholera toxin SeqID113 QYIKANSKFIGITEL Clostridium tetani TT1 SeqID114 FNNFTVSFWLRVPKVSASHLE Clostridium tetani TT2 SeqID115 KFIIKRYTPNNEIDSF Clostridium tetani TT3 SeqID116 VSIDKFRIFCKALNPK Clostridium tetani TT4 SeqID117 WVRDIIDDFTNESSQKT Clostridium tetani 2 SeqID118 AGLTLSLLVICSYLFISRG EBV BHRF1 SeqID119 PGPLRESIVCYFMVFLQTHI EBV EBNA-1 SeqID120 VPGLYSPCRAFFNKEELL EBV CP SeqID121 TGHGARTSTEPTTDY EBV GP340 SeqID122 KELKRQYEKKLRQ EBVBPLF1 SeqID123 TVFYNIPPPMPL EBV EBNA-2 SeqID124 DKREMWMACIKELH HCMV IE1 SeqID125 FVFTLTVPSER Influenza MP1-1 SeqID126 PKYVKQNTLKLAT influenza hemagglutinin SeqID127 EKKIAKMEKASSVFNV Malaria CS:T3 epitope SeqID128 FFLLTRILTI Hepatitis B surface antigen SeqID129 DQSIGDLIAEAMDKVGNEG heat shock protein 65 SeqID130 QVHFQPLPPAVVKL Bacillus Calmette-Guérin SeqID131 KQIINMWQEVGKAMYA HIV gp120 Wherein X is L-cyclohexyl alanine, Z is aminocaproic acid and a is an aliphatic amino acid selected from alanine, glycine, valine, isoleucine and leucine.

According to another preferred embodiment, the conjugate according to the present invention comprises a B cell antigenic determinant and a T cell antigenic determinant, preferably a pan-specific/promiscuous T cell antigenic determinant, and the B cell and T cell antigenic determinants are independently coupled to the CLEC polysaccharide backbone according to the present invention, in particular to Psoralea corylifolia polysaccharide.

According to another preferred embodiment, the conjugate according to the invention comprises a B cell antigenic determinant coupled to a "classical" carrier protein, such as CRM197, wherein said construct is further coupled to a CLEC carrier according to the invention, in particular to Psoralea corylifolia.

For example, in the first step, the formation of the CRM conjugate can be carried out by activating the CRM with GMBS or sulfo-GMBS, etc., and then activating the maleimide group of the CRM and the SH group of the peptide (cysteine) The CRM conjugate is then treated with DTT to reduce the disulfide bonds and generate SH groups on the cysteine. Subsequently, a one-pot reaction of mixed reduced CRM-conjugates with BMPH (N-β-maleimide-propionic acid hydrazine) and activated Schizophora polysaccharide (which has been oxidized) can be produced based on Vaccine for CLEC. The mechanism of the one-pot reaction may be that (compared to the phylloxera polysaccharide) the oxidized phylloxera polysaccharide reacts with BMPH (having a hydrazine residue) and forms BMPH-hydrazone, and the reduced CRM conjugate is then taken up by the CRM conjugate The SH group reacts with the maleimide of BMPH-activated Schizophora polysaccharide.

According to another preferred embodiment, the conjugate according to the invention comprises a "classical" carrier protein, such as CRM197, which contains multiple T cell epitopes. Conjugates according to the invention also comprise B cell epitopes covalently coupled to the polysaccharide moiety. In this example, the two polypeptides (B cell epitope and carrier molecule) are independently coupled to the CLEC carrier according to the invention, in particular to the fungus polysaccharide.

According to another preferred embodiment, the conjugate according to the invention also comprises a "classical" carrier protein, such as CRM197, which contains multiple T cell antigenic determinants. The conjugate according to the invention also comprises a B cell antigenic determinant covalently coupled to a "classical" carrier protein. The peptide-carrier conjugate according to the invention is also covalently coupled to a polysaccharide portion. In this embodiment, two polypeptides (B cell antigenic determinant and carrier molecule) are coupled to the CLEC carrier according to the invention in the form of a conjugate, in particular to Psoralea corylifolia. The carrier protein graft serves as a link between the β-glucan or mannosaccharide and the B cell and/or T cell antigenic determinant polypeptides in the conjugate according to the invention. The covalent binding between the β-glucan or mannan and the B cell and/or T cell antigenic determinant polypeptide is then carried out by the carrier protein (as a functional linker).

Preferred conjugates according to the present invention may comprise a B cell epitope coupled to CRM197, wherein the construct is further coupled to a CLEC polymer according to the present invention, especially β-glucan, wherein the β-glucan is Pyrrolidone, Lichenin, Laminaria, Curdlan, β-glucan peptide (BGP), Schizophyllan, Scleroglucan, Whole Glucan Particles (WGP), Yeast Polysaccharide or Mushroom Polysaccharide, preferably Pyrrolidone, Laminaria, Lichenin, Mushroom Polysaccharide, Schizophyllan or Scleroglucan, especially Pyrrolidone.

According to the present invention, it is shown that the novel B cell antigen determinant-CRM197 conjugate coupled with Psoralea corylifolia polysaccharide is a potent dectin-1 conjugate and induces a very strong immune response, which is superior to the conventional CRM conjugate vaccine.

According to the present invention, it is shown that the binding of CLEC to novel B cell antigen determinant-CRM197 conjugates, in particular the production of B cell antigen determinant-CRM197-glucan, more preferably B cell antigen determinant-CRM197-linear β-(1,6)-glucan or B cell antigen determinant-CRM197-Pseudomonas aeruginosa conjugates, is essential for inducing the superior immunogenicity described for various peptide-CRM197-CLEC, in particular peptide-CRM197-β-glucan, more preferably peptide-CRM197-linear β-(1,6)-glucan or peptide-CRM197-linear Pseudomonas aeruginosa conjugates compared to conventional CRM conjugate vaccines with or without the addition of adjuvants by mixing with β-glucan/Pseudomonas aeruginosa.

According to a preferred embodiment of the present invention, the CLEC conjugate according to the present invention comprises an oligosaccharide/polysaccharide as a B cell antigenic determinant coupled to a carrier protein as a source of T cell antigenic determinant (e.g., CRM197, KLH, diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae protein D (HipD) and outer membrane protein complex of meningococcal serogroup B (OMPC), recombinant non-toxic form of Pseudomonas aeruginosa exotoxin A ( r EPA), flagellin, Escherichia coli heat-labile enterotoxin (LT), cholera toxin (CT), mutant toxins (such as LTK63 and LTR72), wherein the construct is further coupled to a CLEC polymer according to the present invention, especially β-glucan, wherein the β-glucan is auricularia polysaccharide, lichen polysaccharide, laminarin polysaccharide, curdlan polysaccharide, β-glucan peptide (BGP), schizophyllan, scleroglucan, whole glucan particles (WGP), yeast polysaccharide or mushroom polysaccharide, Preferred are Pyricularia polysaccharides, Laminaria polysaccharides, Lichen polysaccharides, Mushroom polysaccharides, Schizophyllan polysaccharides or Scleroglucan, especially Pyricularia polysaccharides. If the conjugate comprises a carrier protein, a preferred embodiment of the present invention is that the conjugate according to the present invention comprises at least one additional, independently bound T cell or B cell antigenic determinant. The preferred embodiment further illustrates that the present invention is not intended to induce antibodies specific for predominantly linear β-(1,6)-glucan, the (1,6)-coupled monosaccharide portion of which is bound to The ratio of non-β-(1,6)-coupled monosaccharide moieties is at least 1:1, such as Psoralen. Therefore, predominantly linear β-(1,6)-glucan conjugates containing only carbohydrates as antigens and carrier proteins and containing a ratio of (1,6)-coupled monosaccharide moieties to non-β-(1,6)-coupled monosaccharide moieties of at least 1:1 are excluded from the present invention, because if the conjugate contains additional T-cell or B-cell antigenic determinants (see Example 7 and Figure 7 below), then according to the present invention The conjugate significantly reduced or eliminated the induction of a strong neonatal immune response in vivo against the glucan backbone. In contrast, repeated administration of unconjugated glucan (or glucan conjugated 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 according to the present invention must have an additional T cell or B cell antigenic determinant polypeptide covalently bound to the conjugate of the predominantly linear β-(1,6)-glucan and the carrier protein.

This also explains that the conjugates according to the present invention do not cover the prevention or treatment of diseases caused directly or indirectly by fungi, especially Candida albicans, by providing mainly linear β-(1,6)-glucans (ultimately coupled to a carrier protein) with a ratio of at least 1:1 of β-(1,6)-coupled monosaccharide moieties to non-β-(1,6)-coupled monosaccharide moieties as antigens.

According to the present invention, such oligosaccharide/polysaccharide conjugate vaccines coupled with Psoralea corylifolia polysaccharide are demonstrated to be potent dectin-1 conjugates and to induce beneficial/effective immune responses if administered in vivo.

Therefore, the present invention also relates to improving and/or optimizing the carrier protein by covalently coupling the carrier protein (already comprising one or more T cell antigens (as part of its polypeptide sequence, optionally in a post-translationally modified form)) with a CLEC polysaccharide adjuvant according to the present invention, i.e., β-glucan or mannosaccharide, preferably Pyricularia polysaccharide, Lichen polysaccharide, Laminaria polysaccharide, Curdlan polysaccharide, β-glucan peptide (BGP), Schizophyllan, Scleroglucan, Whole Glucan Particles (WGP), Yeast Polysaccharide or Mushroom Polysaccharide. Therefore, the present invention relates to β-glucan or mannopolysaccharide used as a C-type lectin (CLEC) polysaccharide adjuvant for B cell and/or T cell antigenic determinant polypeptides, wherein the β-glucan or mannopolysaccharide is covalently bound to the B cell and/or T cell antigenic determinant polypeptide to form a conjugate of β-glucan or mannopolysaccharide and B cell and/or T cell antigenic determinant polypeptide, wherein a carrier protein is covalently coupled to the β-glucan or mannopolysaccharide.

Such modifications and/or optimization result in a significant reduction or elimination of B cell responses to CLEC and/or the carrier protein and/or enhancement (or at least maintenance) of T cell responses to T cell epitopes of the carrier protein. This can reduce or eliminate the antibody response to CLEC and/or the vector (subsequently delivering only the T cell response), and specifically enhance the antibody response to the actual target polypeptide bound to the vector and/or CLEC.

Therefore, a specific preferred embodiment of the present invention is composed of or includes a combination of the following (a)-β-glucan (b) at least one B cell or T cell epitope polypeptide, and (c) a carrier protein, The three components (a), (b) and (c) are covalently bonded to each other.

The combination of these three ingredients can be provided in any direction or order, i.e., in the order (a)-(b)-(c), (a)-(c)-(b) or (b)-(a)-(c) , wherein (b) and/or (c) can be covalently bonded from the N-terminus to the C-terminus or from the C-terminus to the N-terminus, or through functional groups in the polypeptide (for example, through lysine, arginine, Tianmen Functional groups in asparagine, glutamate, asparagine, glutamine, serine, threonine, tyrosine, tryptophan or histidine residues, especially through ionized amines The ε-ammonium group of the acid residue) is bound. Of course, beta-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 bound by linkers, in particular by linkers between all at least three components. Preferred linkers are disclosed herein, for example, cysteine residues or linkers containing cysteine or glycine residues, linkers produced by: hydrazine-mediated coupling, via heterobis Functional linkers, such as coupling of BMPH, MPBH, EMCH or KMUH, imidazole-mediated coupling, reductive amination, carbodiimide coupling - -NH- _NH linker, -NRRA, NRRA-C or NRRA-NH- _NH2 linker, peptide linker such as dimeric, trimeric, tetrameric- (or longer) peptidyl groups such as CG or CG. In the case of existing carrier proteins, especially CRM, CRM197 and KLH, the preferred sequence of the at least three components is (a)-(c)-(b), that is, the β-glucan and the at least one B Cells or a T cell epitope polypeptide are coupled to a carrier protein.

According to another preferred embodiment, the conjugate according to the invention contains a T cell epitope and does not contain a B cell epitope, wherein the conjugate preferably contains more than one T cell epitope, especially two , three, four or five T cell epitopes. This construct is particularly suitable for use in cancer vaccines. The construct is also particularly suitable for use with self-antigens, especially those associated with autoimmune diseases. The therapeutic effects of the respective conjugates are related to the reduction of effector T cells and the formation of regulatory T cell (T _reg cell) populations. These two phenomena enable the suppression of corresponding diseases, such as autoimmune diseases or allergic diseases, such as multiple Sexual sclerosis is shown. Notably, these Treg cells perform strong bystander immunosuppression and thus ameliorate diseases induced by homologous and non-homologous autologous antigens.

Preferred CLECs used as the polysaccharide backbone according to the present invention are Shigu polysaccharides or other β-(1,6) glucans (also including synthetic forms of such glucans); others used: mannan polysaccharides, β -Members of the glucan family, especially linear β-(1,3) (Saccharomyces cerevisiae β-glucan (e.g. 150kDa), Cardranan) or containing branched β-(1,3) and β- Glucans of (1,6), such as: laminarin (4,5-7kDa), scleroglucan, schizophyllan, preferably linear glucan, (for example: β (1,3): brewing Yeast β-glucan (150kD), Cadranan (75-80kDa or larger), β-(1,3)+β-(1,4) lichenan (22-250kDa) β-(1, 6) Fungi polysaccharide (20 kDa). Therefore, preferred CLECs according to the present invention are mannan polysaccharides and β-glucans, including linear and branched β-glucans, characterized by the presence of β-(1,3 )-, β-(1,3)+β-(1,4)- and β(-1,6) backbones and additional side chain residues with β-(1,6), preferably containing Linear β-glucans of β-(1,3), β-(1,3)+β-(1,4) and β-(1,6) chains, preferably linear β-(1,6 ) β-glucan, especially agaric polysaccharide or polysaccharide β-(1,6)-glucan monosaccharide (such as 4-mer, 5-mer, 6-mer, 8-mer, 10-mer , 12-mer, 15-mer, 17-mer or 25-mer) fragments or synthetic variants thereof.

Preferably, the minimum length of the CLEC according to 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 polysaccharide forms and/or end-linking by increasing the reaction form). 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 amount of a single glucose molecule in a glucan mass, DP) of 20-25 (i.e. DP20-25) can ensure good binding and in vivo efficacy (e.g. laminarin is a typical example with a DP of 20-30).

The molecular weight of synthetic CLEC may also be smaller, correspondingly as low as 1-2kDa, while the preferred molecular weight range of glucans and their fragments may be 1-250kDa (e.g. laminarin, lichenin, Saccharomyces cerevisiae β-glucan saccharide, Lycoris polysaccharide, Calderan polysaccharide, barley glucan, etc.), preferably 4.5 to 80 kDa (such as laminarin, Lycoris polysaccharide, Calderan polysaccharide, low molecular weight lichenan, etc.), especially 4.5 to 30 kDa (For example, laminarin, Shigu polysaccharide, low molecular weight lichenin, etc.).

Mannan is a linear polymer polysaccharide composed of mannose. Plant mannans have beta-(1,4) linkages and are a form of storage polysaccharide. The mannan cell wall found in yeast has an α-(1,6)-linked backbone and α-(1,2) and α-(1,3)-linked branches. It is serologically similar to structures found on mammalian glycoproteins.

In order to produce the conjugates according to the present invention, the CLEC, especially Pseudomonas polysaccharide, must be activated (e.g. by using mild periodate-mediated oxidation), and the degree of oxidation is very important for the immune response. As disclosed above, the practical oxidation range, especially for Pseudomonas polysaccharide, is about 20% to 260% oxidation. In many cases, the optimal oxidation range is between low/medium oxidation (i.e. 20-60% oxidation) and high oxidation (i.e. 140-260% oxidation), i.e. in the range of 60-140% oxidation. The optimization for other CLECs can be easily adjusted by those skilled in the art, for example, for lichen polysaccharide, an oxidation degree of more than 200% is necessary to obtain a similar amount of aldehyde groups.

Therefore, the range can also be alternatively defined as the degree of reaction with Schiff's red reagent. For the example of Pyrrolidone, it can be defined as follows: low/medium oxidation degree at a molar ratio (sugar monomer:periodate salt) of 0.2-0.6, an optimal range of 0.6-1.4, and a high oxidation degree of 1.4-2.6.

In any case, the degree of oxidation should be defined to satisfy the optimal range for each specific CLEC. Preferably, linear β-glucan, more preferably β-(1,6)-glucan, especially Pyrrolidone, Pyrrolidone fragments or synthetic variants thereof consisting of poly β(1,6)-glucan monosaccharides (e.g., 4-mer, 5-mer, 6-mer, 8-mer, 10-mer, 12-mer, 15-mer, 17-mer or 25-mer) are activated by mild periodate oxidation, resulting in cleavage of vicinal hydroxyl groups and thereby generating reactive aldehydes. Mild periodate oxidation refers to the use of sodium periodate (NaIO ₄ ), which is a well-known mild reagent that can effectively oxidize vicinal diols in carbohydrate sugars to generate reactive aldehyde groups. Carbon-carbon bonds are cleaved between adjacent hydroxyl groups. By varying the amount of periodate salt used, aldehydes can be stoichiometrically introduced into a smaller or larger number of sugar moieties of a given polysaccharide.

Other exemplary methods for activating carbohydrates are well known in the art and include cyanation of hydroxyl groups (e.g., by using an organic cyanation reagent 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.

Next, the activated carbohydrate reacts with the polypeptide to couple to the activated CLEC and form a conjugate of CLEC and the B cell or T cell epitope polypeptide.

Therefore, the present invention also relates to a method for producing a conjugate according to the present invention, wherein β-glucan or mannopolysaccharide is activated by oxidation and wherein the activated β-glucan or mannopolysaccharide is contacted with a B cell and/or T cell antigenic determinant polypeptide, thereby obtaining a conjugate of β-glucan or mannopolysaccharide and a B cell and/or T cell antigenic determinant polypeptide.

Preferably, the β-glucan or mannopolysaccharide is obtained by periodate oxidation of adjacent hydroxyl groups, reductive amination or cyanation of hydroxyl groups.

According to a preferred embodiment, the β-glucan or mannopolysaccharide is oxidized to a degree of oxidation, which is defined as the degree of reaction with Schiff's fuchsin reagent, which is equivalent to the degree of oxidation of an equivalent amount of Pyrrolidone polysaccharide oxidized with periodate at a molar ratio of 0.2-2.6, preferably 0.6-1.4, and especially 0.7-1.

Preferably, the conjugate is produced by coupling a hydrazide to a carbonyl group (aldehyde) via a hydrazone-based coupling, or by coupling a hydrazide (e.g., cysteine) to a carbonyl group (aldehyde) via coupling using an isobifunctional cis-butenediimide and a hydrazide linker (e.g., BMPH (N-β-cis-butenediimidopropionic acid hydrazide, MPBH (4-[4-N-cis-butenediimido-phenyl]butyric acid hydrazide), EMCH (N-[ε-cis-butenediimidohexanoic acid) hydrazide), or KMUH (N-[κ-cis-butenediimidoundecanoic acid] hydrazide).

The polypeptide coupled to the CLEC according to the present invention is or comprises at least one B cell antigenic determinant or at least one T cell antigenic determinant. Preferably, the polypeptide coupled to the CLEC comprises a single B cell or T cell antigenic determinant (even in embodiments where more than one polypeptide is coupled to the CLEC polysaccharide backbone). Also as shown in the Examples section, the preferred length of the polypeptide is 5 to 29 amino acid residues, preferably 5 to 25 amino acid residues, more preferably 7 to 20 amino acid residues, even more preferably 7 to 15 amino acid residues, especially 7 to 13 amino acid residues. In this regard, it is important to note that these length ranges are for the epitope sequence only, but do not include linkers, including peptide linkers, such as cysteine or glycine or dimer, trimer, tetramer (or longer) peptide groups, such as CG or CG, or cleavage sites, such as a tissue protease cleavage site; or combinations thereof (e.g. -NRRAC). Examples of epitopes have been tested in the Examples section; as can be seen from these results, the platform according to the present invention is not limited to any particular polypeptide. Therefore, virtually all possible epitopes are eligible for the present invention, including those epitopes known in the art, in particular epitopes that have been described as being incorporable into display platforms (e.g. in conjunction with "classical" carrier molecules or adjuvants).

If the epitope can be coupled to activated β-glucan by coupling methods based on current advanced technology, including hydrazine-mediated coupling, through heterobifunctional linkers (for example: BMPH, MPBH, EMCH, KMUH, etc.) Coupling, imidazole-mediated coupling, reductive amination, carbodiimide coupling, etc. (more to be added), then this epitope is particularly preferred. The epitopes used comprise individual peptides, may be contained within a peptide or protein, or may be presented as a peptide-protein conjugate prior to coupling to CLEC.

Therefore, preferred coupling methods for providing conjugates according to the present invention are hydrazine couplings or couplings using thioester formation (for example using BMPH (N-β-maleiminopropionic acid hydrazide), Maleimide coupling of MPBH, EMCH, KMUH), in particular, in which the polysaccharide is coupled to BMPH through the formation of hydrazone, and the polypeptide is coupled through the thioester.

In this embodiment, it is preferred to provide a polypeptide having two preferred linkers, such as hydrazide polypeptide/antigenic determinant for hydrazone coupling: N-terminal coupling of polypeptide: _H2N -NH-CO- _CH2 - _CH2 -CO-polypeptide-COOH; preferably in combination with succinic acid or an alternative suitable linker, such as other suitable dicarboxylic acids, especially glutaric acid used as a spacer/linker; C-terminal coupling (this is the preferred coupling direction according to the present invention): _NH2 -polypeptide-NH- _NH2 .

Alternatively, unmodified polypeptides/epitopes may be used in the present invention, e.g. polypeptides containing an (additional) cysteine residue at the C or N terminus or an alternative source of SH group for heterobifunctional linker-mediated coupling (especially BMPH, MPBH, EMCH, KMUH): _NH2 -Cys-Pep-COOH or _NH2 -Pep-Cys-COOH).

The length of the B cell polypeptide used according to the present invention is preferably 5 to 19 amino acid residues, more preferably 6 to 18 amino acid residues, and especially 7 to 15 amino acid residues. The B cell antigen determinant 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.

Preferred B cell epitopes are B cell epitopes representing self-antigens, B cell epitopes representing antigens present in neoplastic diseases, B cell epitopes representing antigens present in allergic IgE-mediated diseases, B cell epitopes representing antigens present in autoimmune diseases, B cell epitopes representing antigens present in infectious diseases, B cell epitopes representing conformational epitopes, B cell epitopes representing carbohydrate epitopes, polypeptides or proteins immobilized/coupled to form multivalent B cell epitope protein/polypeptide conjugates suitable for CLEC coupling B cell antigenic determinants, such conjugates include carrier molecules, such as CRM197, KLH, tetanus toxoid or other commercially available carrier proteins or carriers known to those skilled in the art, preferably CRM197 and KLH, and most preferably CRM197; non-peptide-derived antigens suitable for coupling with reactive aldehyde groups present on Pyricularia auriculariae polysaccharide/CLEC (including linear polypeptides, polypeptides representing conformational antigenic determinants, polypeptide variants mimicking antigenic determinants or derived from natural antigenic determinants/sequences, glycopeptides, lipopeptides, other post-translationally modified peptides (e.g., phosphorylated, acetylated, containing pyroglutamic acid residues, etc.), cyclic polypeptides, etc.).

The length of the T cell polypeptide used according to the present invention is preferably 8 to 30 amino acid residues, more preferably 13 to 29 amino acid residues, especially 13 to 28 amino acid residues.

Preferred specificities for T cell epitopes for use in the present invention are short linear peptides that are suitable or known to be suitable for presentation by MHC I and II (as known to those skilled in the art), especially CD4 effector T cells and MHCII epitopes of CD4 Treg cells, MHCII epitopes of cytotoxic T cells (CD8+) and CD8 Treg cells, which may, for example, have known efficacy in cancer, autoimmune or infectious diseases in humans or animals; suitable Short linear peptides presented by MHC I and II (as known to those skilled in the art), which have a lysosomal protease cleavage site attached to the N or C terminus, especially a site specific for members of the cathepsin family, and more Especially cysteine cathepsins, such as cathepsins B, C, F, H, K, L, O, S, V, X and W, especially cathepsin S or L site, preferably cathepsin L Cleavage sites that promote efficient endo/lysosomal release of MHC-presenting peptides, 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 methods for revealing such sequences or identifying such sequences: for example: Biniossek et al., J. Proteome Res. 2011, 10, 12, 5363-5373; Adams-Cioaba et al., Nature Comm. 2011, 2:197; Ferrall-Fairbanks PROTEIN SCIENCE 2018 VOL 27:714—724; Kleine-Weber et al., Scientific Reports (2018) 8:1659, https://en.wikipedia.org/wiki/Cathepsin_S and others. Specifically, the adjustment of the peptide sequence using artificial protease cleavage sites as shown in the present invention is based on the fact that when the antigen is coupled to CLEC, these sequence extensions trigger more effective immunity after cutaneous application of the CLEC vaccine according to the present invention. Unexpected effects of reactions. The vaccine according to the invention is taken up by DC and the peptide antigen is subsequently processed by lysosomes and presented on the MHC.

Lysosomes are organelles bound to the cell membrane within cells. Their interior is acidic and contains a variety of hydrolases, including lipases, proteases and glycosidases that participate in cell catabolism. Among the various enzymes contained in lysosomes, cathepsins are a family of lysosomal proteases with a wide range of functions. All cathepsins belong to three different protease families: serine proteases (cathepsins A and G), aspartic proteases (cathepsins D and E), and eleven cysteine cathepsins. In humans, 11 cysteine cathepsins are known to 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 (lymphase).

Cathepsins show similarities in their cellular localization and biosynthesis, but there are some differences in their expression patterns. Of all the lysosomal proteases, cathepsins L, B, and D are the most abundant, with lysosomal concentrations equivalent to 1 mM. Cathepsins B, H, L, C, X, V, and O are expressed in all cells, 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.

In addition to its main function in lysosomal protein recycling, cathepsins also play an important role in various physiological processes. Cathepsin S is the main protease involved in MHC II Ag processing and presentation. Cathepsin S-deficient mice show significant differences in the production and presentation of MHC II-bound Li fragments, due to significantly reduced Li degradation in professional APCs where cathepsin S is abundantly expressed. Furthermore, endocytosis selectively targets exogenous substances to cathepsin S in human DCs. Enrichment of MHC II molecules in late endocytic structures has also been consistently noted in splenic DCs from cathepsin S-deficient mice. Recent studies have shown that both cathepsins B and D are involved but are not required for MHC II-mediated Ag presentation. Cathepsin L also plays a role in a variety of cellular processes, including antigen processing, tumor invasion and metastasis, bone resorption, and the turnover of intracellular and secreted proteins involved in growth regulation. Although generally considered a lysosomal protease, cathepsin L is also secreted. 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 and cytoplasmic and nuclear proteins.

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

Characteristics of such cleavage sites according to the present invention may be as follows: Cathepsin L-like cleavage site: A given cathepsin L-like cleavage site is defined based on protease cleavage site sequences known to those skilled in the art, in particular, Also as 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 is composed of the following formula: X _n -X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇ -X ₈ X _n : from immunogenicity 3-27 amino acids of the peptide X ₁ : any amino acid X ₂ : any amino acid X ₃ : any amino acid X ₄ : N/D/A/Q/S/R/G/L; more Preferably N/D, _preferably N X ₅ : F/R/A/K/T/S/E; preferably F or R, preferably R /Y; preferably F or R, more preferably R X ₇ : Any amino acid, preferably A/G/P/F, more preferably A X ₈ : Cysteine or linker, such as NHNH _{2 Optimal} sequence: X _{n -X 1} Also as the sequence disclosed in Biniossek et al. (J. Proteome Res. 2011, 10, 5363-5373) and https://en.wikipedia.org/wiki/Cathepsin_S, and is characterized by the following common sequence: X _n -X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇ -X ₈ where X is characterized by X _n : 3-27 amino acids from the immunogenic peptide X ₁ : any amino acid X ₂ : Any amino acid X ₃ : Any amino acid, preferably V, L, I, F, W, Y, H, more preferably V X ₄ : Any amino acid, preferably V, L, I , F, W, Y, H, preferably V X ₅ : K, R, E, D, Q, N, preferably K, R, more preferably R X ₆ : any amino acid X ₇ : any amino group _{Acid , preferably A ​ ​}

The T cell antigen determinant is contained in a protein suitable for coupling to CLEC, including a carrier protein, in particular a non-toxic cross-reactive material (CRM) of diphtheria toxin, in particular CRM197, KLH, diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae protein D (HipD) and the outer membrane protein complex of meningococcal serogroup B (OMPC), a recombinant non-toxic form of Pseudomonas aeruginosa exotoxin A ( r EPA), 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, e.g., multiple antigenic peptides (MAP) or other commercially available carrier proteins, preferably CRM197 and KLH, most preferably CRM197, preferably, wherein the ratio of the carrier protein to the 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.

According to a preferred embodiment of the present invention, the CLEC conjugate according to the present invention comprises (a) CLEC conjugated to individual B cell and/or T cell antigenic determinants, including a mixture of B cell or T cell antigenic determinants, especially such antigenic determinants coupled to Psoralea corylifolia polysaccharide; (b) CLEC conjugated to a polypeptide-carrier protein conjugate, preferably a polypeptide-KLH or polypeptide CRM197 conjugate coupled to Psoralea corylifolia polysaccharide, and most preferably a polypeptide-CRM197 conjugate coupled to Psoralea corylifolia polysaccharide; (c) CLEC conjugated to a polypeptide-carrier protein conjugate, preferably a polypeptide-KLH or polypeptide CRM197 conjugate coupled to Psoralea corylifolia polysaccharide, and most preferably a polypeptide-CRM197 conjugate coupled to Psoralea corylifolia polysaccharide; CLECs that bind to individual B and T cell epitopes of self-proteins (cancer) or pathogens (infectious diseases). CLECs that bind to known disease-specific T cell epitopes instead of promiscuous MHC/HLA-specific T cell epitopes, preferably CLECs coupled to Psoralea corylifolia polysaccharide; (d) CLECs that independently couple 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. (Here, "independent" means that the polypeptide chain does not exist as a fusion protein, a tandem repeat polypeptide or a peptide-protein conjugate, but exists as an independent entity; i.e., an independent polypeptide containing a B cell antigenic determinant and an independent polypeptide containing a T cell antigenic determinant); (e) CLECs independently coupled to T cell antigenic determinants representing linear MHC I and MHC II antigenic determinants or contained in proteins such as carrier proteins or target proteins ("independent", has the same meaning as (d)), for example, for the treatment of tumor diseases or autoimmune diseases.

In view of these favorable properties of the conjugates of the present invention, the conjugates and vaccines according to the present invention can be specifically used as active anti-Aβ, anti-Tau and/or anti-α-synuclein vaccines 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), AD with restricted Lewy bodies in the amygdala (AD/ALB), dementia in Down syndrome, Pick's disease, progressive supranuclear palsy (PSP), corticobasal degeneration, frontotemporal dementia and parkinsonism related to chromosome 17 (FTDP-17), and argyrophilic granulopathy.

Therefore, the conjugates according to the present invention can be used specifically for the prevention or treatment of diseases in humans, mammals or birds, for example, and in particular for the treatment and prevention of human diseases. Therefore, one aspect of the present invention is the use of the conjugates according to the present invention as a medical indication in the medical field. The present invention relates to the use of the conjugates according to the present invention for the treatment or prevention of diseases. Therefore, the present invention also relates to the use of the conjugates according to the present invention in the manufacture of a medicament for the prevention or treatment of diseases, preferably for the prevention or treatment of infectious diseases, chronic diseases, allergic reactions or autoimmune diseases. Therefore, the present invention also relates to a method for the prevention or treatment of diseases, preferably for the prevention or treatment of infectious diseases, chronic diseases, allergic reactions or autoimmune diseases, wherein an effective amount of the conjugates according to the present invention is administered to a patient in need thereof.

According to another aspect, the novel glycoconjugates according to the present invention can be used to prevent infectious diseases; preferably by providing (1,6) coupled monosaccharide moieties with non-β-(1,6) coupled monosaccharide moieties. Predominantly linear beta-(1,6)-glucan with a ratio of monosaccharide moieties of at least 1:1 as an antigen (eventually coupled to a carrier protein) for the prevention or treatment of direct or indirect diseases caused by fungi, especially Candida albicans Uses causing illness are excluded. Such diseases are, for example, microbial infections caused, for example, by Haemophilus influenzae type b (Hib), Streptococcus pneumoniae, Streptococcus meningitidis and Salmonella typhi or other infectious agents.

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

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

According to a preferred embodiment of the 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 including a needle adapter; ampoule , needle-free injection system, preferably a jet injector; patch, transdermal patch, microstructured transdermal system, microneedle array patch (MAP) (preferably solid MAP (S-MAP), coated MAP ( C-MAP) or dissolved MAP (D-MAP)); electrophoresis systems, ion electrophoresis systems, laser-based systems, especially erbium YAG laser systems; or gene gun systems. Conjugates according to the invention are not limited to any form of manufacture, storage or delivery. Therefore, all traditional and typical forms are suitable for use in the present invention. Preferably, the composition according to the invention may comprise the conjugate or vaccine of the invention in the form of a solution or suspension, a deep-frozen solution or suspension, a lyophilisate, a powder or a granule. The present invention is further illustrated by the following examples and drawings, but is not limited thereto.

Examples : Materials and Methods 1) Oxidation of CLEC/ dextran backbone

In order to form vaccine conjugates, polysaccharides, especially CLEC/β-glucan, need to be chemically modified to generate reactive groups that can be used to link proteins/peptides. Two common methods for polysaccharide activation are periodate oxidation at the ortho-hydroxyl group and cyanation of the hydroxyl group. Other methods of activating polysaccharides are possible and well known in the art. The example shown in this Examples section uses mild periodate oxidation.

Depending on their solubility, CLEC and β-glucans (such as mannan, lichenan, phyllan or β-glucan from barley) are oxidized using periodate oxidation in aqueous solution or DMSO.

Based on molar ratios (periodate: sugar subunit; 100% = 1 mole of periodate/moles of sugar monomer) of 1:5 (i.e. 20% oxidation) to 2,6:1 iodate solution to predetermine the degree of oxidation (260% oxidation degree).

Briefly, sodium periodate was added at a molar ratio of 1:5 to 2.6:1 (periodate:sugar subunit, corresponding to 20% and 260% oxidation degrees) to open the furanose ring of β-glucan between vicinal diols, leaving two aldehyde groups as substrates for subsequent coupling reactions. 10% (v/v) 2-propanol was added as a free radical scavenger. The reaction was incubated at room temperature for 4 hours in the dark on an orbital shaker (1000 rpm). Subsequently, the oxidized dextran was dialyzed three times against water using a Slide-A-Lyzer™ (ThermoScientific) or Pur-A-Lyzer™ (Sigma Aldrich) cassette with a cutoff of 20 kDa to remove sodium (periodate) and low molecular weight dextran impurities. The dialyzed dextran can be directly used for peptide conjugation reaction or stored at -20°C or freeze-dried and stored at 4°C for further use. 2) Conjugation of WISIT vaccine 2a. Formation by hydrazone

The peptide contains a hydrazine group at the N or C terminus for aldehyde coupling. Where the direction of coupling is intended to be via 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, such as succinic acid. Alternatively, intact proteins (eg CRM197) have been used for dextran coupling.

Typical examples of this type of peptide: N-terminal coupling of the peptide: H ₂ N-NH-CO-CH ₂ -CH ₂ -CO-polypeptide-COOH; C-terminal coupling: NH _2- polypeptide-NH-NH ₂ .

For coupling, an activated dextran solution (i.e., oxidized acanthus polysaccharide) is mixed with a dissolved chelazine-modified peptide or intact protein (e.g., CRM197) in a coupling buffer (either depending on the isoelectric point of the peptide). Stir in sodium acetate buffer pH 5.4 or DMEDA at neutral pH (6.8). The free hydrazine group in the peptide reacts with the aldehyde group to form a hydrazone bond, forming the final conjugate. For proteins, coupling to activated dextran is based on the reaction of the amine group of the lysine residue with an active aldehyde on the dextran moiety in the presence of sodium cyanoborohydride.

The conjugate is then reduced by the addition of sodium borohydride in borate buffer (pH 8.5). This step reduces the hydrazine bond to a stable diamine and converts unreacted aldehyde groups in the sugar backbone to primary alcohols. The carbohydrate concentration in the conjugate is estimated using the anthrone method, and the peptide concentration is estimated by UV spectroscopy or by amino acid analysis. 2b. Coupling using a heterobifunctional linker

The second conjugation technology applied relies on heterobifunctional linkers (e.g.: BMPH (N-β-maleyl iminopropionic acid hydrazide), MPBH (4-[4-N-maleyl dihydrazide)). acylimidophenyl]butyric acid hydrazine), EMCH (N-[ε-maleyl iminocaproic acid) hydrazine) or KMUH (N-[κ-maleyl iminocaproic acid) hydrazine Undecanoic acid] hydrazine) short maleic imine and hydrazine cross-linking agent, used to combine sulfhydryl (cysteine) and carbonyl (aldehyde) groups.

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

For coupling, an activated dextran solution (i.e., oxidized phylloxanthin) was reacted with BMPH overnight (in a ratio of 1:1 ratio (w/w) to a 2:1 ratio of BMPH:phenylalanine polysaccharide) followed by PBS Dialysis 3 times. The BMPH-activated dextran is then mixed with the solubilized Cys-modified polypeptide in a coupling buffer (eg, phosphate buffered saline, PBS). The maleimide 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 reactive aldehyde, produces a stable conjugate. The carbohydrate concentration in the conjugate was estimated using the anthrone method and analyzed by amino acid analysis or using Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid), DTNB). Mann analysis to determine peptide concentration. DTNB reacts with sulfhydryl groups to produce colored products, which provides a reliable method for measuring reduced cysteine and other free sulfhydryl groups in solutions through spectrophotometry (λmax=412 nm; ε=14,150/M·cm). 2c) Peptide KLH/CRM binding

By using the heterobifunctional cross-linker GMBS or SMCC (Thermo Fisher), the peptide (containing N- or C-terminal Cys residues, see above) is combined with the carrier CRM-197 (e.g. EcoCRM, Fina Biosolutions) or KLH (Sigma Aldrich) coupling. Briefly, CRM-197/KLH was mixed with excess GMBS or SMCC (according to the manufacturer's protocol) at room temperature for activation, then excess GMBS was removed by centrifugation on a desalting column, and excess peptide was added to Activated carrier was used for coupling (buffer: 200mM sodium phosphate (pH=6.8)), followed by dialysis three times with PBS. Coupling efficacy/peptide content was assessed by the Ellman assay (Ellman's reagent: 5,5'-dithio-bis-(2-nitrobenzoic acid)) for quantification of free thiol groups in solution. The peptide CRM-197/KLH conjugate was further formulated with Alum (Alhydrogel® Adjuvant 2%) and administered subcutaneously to the animals. When comparing the CRM-197/KLH vaccine with other vaccines according to the invention, each mouse was injected with the same amount of binding polypeptide. 2d) Formation of new glucose conjugates using peptides, KLH/CRM197 and dextran

The polypeptide-KLH and polypeptide-CRM197 conjugates produced as described in 2c) were also coupled to activated dextran at different ratios of polypeptide-KLH and polypeptide-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 the formation of the polypeptide conjugate, the Pep-KLH or Pep-CRM conjugate was reduced using dithiothreitol (DTT). The reduced carrier conjugate was coupled to activated dextran in the presence of an excess of the heterobifunctional linker BMPH. The cis-butylenediimide group of BMPH forms a stable thioether bond with the hydroxyl residue of the reduced KLH or CRM197 conjugate, while the aldehyde group in the glycan is coupled with the hydrazide group of BMPH. After 2 hours at room temperature, the generated hydrazone is reduced to a stable diamine by overnight reaction with sodium cyanoborohydride. Subsequently, the new sugar conjugate is dialyzed three times with PBS or water using a Slide-A-Lyzer™ (ThermoScientific) or Pur-A-Lyzer™ (Sigma Aldrich) cassette to remove low molecular weight impurities (see also: Example 23). 3) In vitro biological activity assay of CLEC conjugates

The in vitro bioactivity of mannosan and glucan conjugates was analyzed by ELISA using soluble mouse Fc-dectin-1a receptor (InvivoGen) or ConA as described by Korotchenko et al. (2020). Briefly, ELISA plates were coated with a reference glucan (CLR-agonist, CLEC), such as pyralidin, lichenin, or mannosan, and reacted with fluorescently labeled ConA (for mannosan) or soluble mouse Fc-dectin-1a receptor (for pyralidin and other β-D-glucans), which was detected by HRP-labeled secondary antibodies. Oxidized carbohydrates and glucose complexes were tested in competitive ELISAs (increasing concentrations of CLEC or conjugates were added to the soluble receptors used in the assay to reduce receptor binding to coated CLEC) to demonstrate functionality. _IC50 values were used to determine biological activity (i.e., binding efficacy to soluble receptors compared to non-oxidized, uncoupled ligand). 4) Activation assays using bone marrow-derived dendritic cells

Bone marrow-derived dendritic cells (BMDC) were collected from mouse femurs and tibias and cultured with 20 ng/mL mouse GM-CSF (Immunotools) as described by Korotchenko et al. (2020), and were subjected to subtle Change. By performing FACS analysis on CD11c ⁺ MHCII ⁺ CD11b ^int GM-CSF-derived DC (GM-DC), the effect of various conjugates and positive control (=LPS) on the expression of CD80 and MHCII was evaluated. 5) Determination of hydrodynamic radius

The hydrodynamic radius of the conjugates was analyzed by dynamic light scattering (DLS). Briefly, samples (i.e., conjugates) were centrifuged at 10,000<i>g for 15 min (Merck Millipore, Ultrafree-MC-VV Durapore PVDF). All sample wells were sealed with silicone oil to prevent evaporation, and data were collected sequentially for approximately 24 hours. All measurements were performed at 25°C using a WYATT DynaPro PlateReader-II1536 well plate (1536W SensoPlate, Greiner Bio-One). Samples were measured in triplicate. All measurements were filtered against a baseline value of 1.00±0.005, so only curves returning values between 0.995 and 1.005 were considered for further analysis (e.g. cumulative radius and regularization analysis). Sample analysis was performed according to https://www.wyatt.com/library/application-notes/by-technique/dls.html and DYNAMICS User Guide (M1406Rev.C, version 7.6.0), Technical Notes TN2004 and TN2005 (both at: www.wyatt.com). 6) Animal experiments

Female BALB/c mice, n = 5 mice per group, treated with different CLEC conjugates (id, im, sc), peptide-CRM-197/KLH conjugate (id) or peptide-CRM adsorbed to Alum Immunization was performed with -197/KLH conjugate (sc) and respective control groups (e.g., unconjugated CLEC, mixture of CLEC and peptides, etc.). Animals were vaccinated three times at biweekly intervals, and blood samples were collected the day before each vaccination and periodically two weeks after the last administration, unless otherwise stated. 7) Quantification of vaccine-induced antibodies in mouse plasma using ELISA

Whole blood was collected from mice using heparin as an anticoagulant, and plasma was obtained by centrifugation. The plasma samples were stored at -80°C. To detect anti-target-specific antibodies, ELISA plates (Nunc Maxisorb) are coated with peptide-BSA conjugates or recombinant proteins/fragments (typically at a concentration of 1 µg/ml) using 50mM sodium carbonate buffer and left overnight at 4°C. All anti-peptide ELISAs used in the examples provided use Pep-BSA conjugates (e.g., SeqID3 (Sequence: DQPVLPD), whose C-terminus is used for coupling to maleimide-activated BSA; nomenclature: Pep1c (DQPVLPD-C, SeqID3) was used as a bait for the anti-Pep1-specific response elicited by a conjugate vaccine containing Pep1b (SeqID2; DQPVLPD-(NH- _NH2 )) and Pep1c). ELISA plates were blocked using 1% bovine serum albumin (BSA), and plasma samples were serially diluted in the plates. Biotinylated anti-mouse IgG (Southern Biotech) was used for detection of target-specific antibodies, followed by streptavidin-POD (Roche) and TMB for color development. EC ₅₀ values were calculated through nonlinear regression analysis (four-parameter logistic fitting function) using GraphPad Prism software (Graph Pad Prism www.graphpad.com/scientific-software/prism/). target protein antibody Alpha synuclein recombinant (Anaspec) Anti-alpha synuclein 115-121 AB (LB509) (Biolegend) Alpha synuclein monomer (Abcam) Anti-alpha synuclein 115-121 AB (LB509) (Biolegend) Alpha synuclein fibers (Abcam) Anti-alpha synuclein 115-121 AB (LB509) (Biolegend) Amyloid beta 1-40 (Biolegend) Anti-amyloid beta 1-16 AB (6E10) (Biolegend) Amyloid beta 1-42 (Biolegend) Anti-amyloid beta 1-16 AB (6E10) (Biolegend) [Pyr3] Amyloid beta 3-40 (Anaspec) [Pyr3] Amyloid beta 3-42 (Anaspec) Tau 441 recombinant type (Anaspec) PD1 recombinant type (Abcam) Erb2/Her2 recombinant type (R&D Systems) Bet v1 recombinant type Extracellular membrane proximal domain (EMPD) recombinant Biozol KLH (Sigma) Anti-KLH AB (Sigma) CRM197 (FinaBiosolution) Anti-diphtheria AB (Abcam) 8) Evaluate the binding preference of aSyn- specific antibodies by inhibition ELISA

ELISA plates (Nunc Maxisorb) were coated with aSyn monomers (Abcam) or aSyn fibers (Abcam) and blocked with 1% bovine serum albumin (BSA). Control antibodies and plasma samples were incubated with serially diluted aSyn monomers or aSyn fibers in low-binding ELISA plates. Next, the pre-incubated antibody/plasma samples were added to the monomer/fiber coated plates and binding was detected using biotinylated anti-mouse IgG (Southern Biotech), followed by color development using 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 was used as an estimate of the selectivity of the Ab for the antigen under study. The _logIC50 values were calculated by nonlinear regression analysis (four-parameter logical fitting function) using GraphPad Prism software (Graph Pad Prism www.graphpad.com/scientific-software/prism/). 9) Quantification of aSyn aggregation

Protein aggregation assays were performed in an automated format in a GENIOS microplate reader (Tecan, Austria) with a reaction volume of 0.1 ml in black flat-bottom 96-well plates using continuous orbital shaking. Kinetics were monitored by top readings of fluorescence intensity every 20 min using 450 nm excitation and 505 nm emission filters. Fibril formation in the absence and presence of antibodies (antibody/protein molar ratio varied from 6× ^10-5 to 3× ^10-3 ) was initiated by shaking a 0.3 mg/ml (20.8 μM) aSyn solution in 10 mM HEPES buffer (pH 7.5), 100 mM NaCl, 5 μM ThT and 25 μg/ml heparin sulfate at 37°C in a plate reader (Tecan, Austria).

In addition, protofibril formation in the absence and presence of antibodies was also triggered by the presence of preformed protofibrils. Briefly, aSyn preformed protofibrils (1 µM) were aggregated in 100 µl PBS in the presence of activated aSyn monomers (10 µM) and 10 µM ThT for 0-24 h.

For data analysis, the mean value of the negative control samples, i.e. the background fluorescence of ThT, is calculated and removed from each sample at a given time point, e.g., in Microsoft Excel. For comparison of different conditions/inhibitors in aggregation analysis, each sample is normalized to the fluorescence reading measured at the start of the assay and set to 1. (t0=1).

To evaluate the kinetic curves, the Michaelis Menten kinetic model was used: GraphPad Prism software was used to calculate the Km (substrate concentration that produces half-maximal velocity) and Vmax (maximum velocity) values for each condition, and then the kinetic analysis of the enzyme was performed (Michaelis -Menten).

To compare different conditions/inhibitors in the aggregation analysis, the slope of the exponential growth phase of the ThT kinetics was calculated by linear regression using GraphPad Prism software . 10) Determination of the affinity of a single antigen-binding segment to the antigen and the affinity of an antibody molecule to the antigen

To determine the affinity of the antibody molecule to the antigen, a variation of the standard ELISA assay was used in which replicate wells containing antibodies bound to different antigens of each example were exposed to increasing concentrations of ionized thiocyanate ions. Resistance to thiocyanate dissolution was used as a measure of the affinity of the antibody molecule to the antigen, and the index representing 50% effective antibody binding (affinity index) was used to compare different sera. Briefly, plasma was diluted 1/500 in PBS and then dispensed onto coated and sealed ELISA plates (Nunc Maxisorb). After incubation for 1 hour, sodium thiocyanate (NaSCN, SIGMA; in PBS) was added to the sample at a concentration of 0.25 to 3 M. ELISA plates were incubated at room temperature for 15 minutes, then washed and detected using streptavidin-POD (Roche) and TMB and subsequent color development. The absorbance readings in the presence of increasing concentrations of NaSCN were subsequently converted to the appropriate percentage of total bound antibody, assuming that the absorbance readings in the absence of NaSCN represented effective total binding of the specific antibody (100% binding). The data were fit to a graph of (% bound) versus (log) concentration of NaSCN and the affinity index, representing the concentration of NaSCN required to reduce the initial optical density by 50%, was analyzed by linear regression. Data were excluded if the correlation coefficient of the linear fit was less than 0.88.

In order to measure the _kD value (binding affinity) to aSyn fiber, a displacement ELISA that can easily measure the _kD value of a complex formed by an antibody and its competing ligand was used. Briefly, equal concentrations of antibody were incubated with increasing concentrations of free aSyn fibers before measuring free antibody titers on disks with immobilized aSyn fibers. Relative binding of antibodies was expressed as a percentage of the maximum binding observed in the assay for each sample. Competition reactions with aSyn fiber (5 µg/ml) were defined to represent 0% binding (non-specific binding). Reactions with no competition were It is considered to represent 100% (maximum) binding in the displacement curve. Competition binding curves were analyzed based on a single point model using computer-assisted curve fitting software from GraphPad. 11) Induction of synucleinopathies in mice

To induce synucleinopathy, nine-week-old male C57BL/6 mice were stereotactically injected with preformed polymorphic protofibrils (PFFs, preformed sonicated τ-polymorphic aSyn protofibrils 1B) at the level of the right substantia nigra. PFFs were prepared and validated as described in Sci. Adv. 2020, 6, eabc4364, doi:10.1126/sciadv.abc4364; DOI: 10.1126/sciadv.abc4364. Briefly, each animal received a unilateral injection of 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]. The needle remained in place for 5 minutes and was then slowly removed from the brain.

Starting on the same day of vaccination, animals received three id vaccinations, once every two weeks (ie, weeks 0, 2, and 4), with CLEC-based vaccines (n=5) or unconjugated CLEC (n=10) ) served as the control group, followed by booster vaccination at week 10. After the end of the study (day 126), cerebrospinal fluid (CSF) was collected by cistern puncture, and the brains were carefully removed and fixed in paraformaldehyde (PFA; 4%). Coronal serial sections of the entire brain (6.72 mm from the frontal cerebral cortex to the striatum to the medulla oblongata - bregma) were collected and processed at 50 µm intervals using a cryostat and processed for immunohistochemistry. 12) Immunohistochemistry (IHC)

IHC staining for phospho-S129 aSyn (pS129aSyn) on coronal serial sections was performed as described previously [Sci. Adv. 2020, 6, eabc4364, doi:10.1126/sciadv.abc4364; DOI: 10.1126/sciadv.abc4364]. Monoclonal rabbit anti-pS129aSyn antibody EP1536Y (ab51253, Abcam) was used, followed by incubation with labeled multimeric HRP anti-rabbit (DakoEnVision+TM kit, K4011). Visualization of pS129aSyn staining was performed with DakoDAB (K3468), and sections were counterstained with Nissl stain. The actual number of pS129aSyn aggregates in each structure (cerebral cortex, striatum, thalamus, substantia nigra, and brainstem) and the total number of pS129aSyn aggregates were assessed by whole-section acquisition using Panoramic Scan II (3DHISTECH, Hungary) and further processing using the specially developed QuPath algorithm. Example 1 : In vitro determination of the bioactivity of CLEC conjugates

PAMPs (e.g., CLECs) are recognized by PRRs present in APCs. Binding of CLECs to their cognate PRRs (e.g., dectin-1 for β-glucan) is required to control various levels of adaptive immunity, for example, by inducing downstream carbohydrate-specific signaling and cell activation, cell maturation, and cell migration to draining lymph nodes or by cross-talk with other PRRs. Therefore, in order to provide the novel vaccine platform technology proposed in this application, it is critical that the CLECs used retain their PRR binding ability, which demonstrates the biological activity of the selected CLECs and CLEC-based conjugates.

Along these lines and to ensure that 1) the structure of the CLECs was not destroyed during mild periodate oxidation and 2) the polysaccharides remained bioactive after conjugation, binding to dectin-1 was assessed by ELISA. First, several different CLECs were oxidized by mild periodate oxidation to produce the active carbohydrate backbone of the proposed vaccine. These CLECs included: mannosaccharide, pyrifos polysaccharide (20 kDa), lichen polysaccharide (245 kDa), barley β-glucan (229 kDa), oat β-glucan (295 kDa), and oat β-glucan (391 kDa). Subsequently, different B cell epitope peptides (SeqID2, SeqID10, SeqID16) and SeqID7 were used as helper T epitope peptides for hydrazone coupling and vaccine conjugates were generated. All of these epitope peptides contained a C-terminal hydrazide linker for coupling. In addition, a peptide-A. pyrifos conjugate generated by coupling SeqID10 with the heterobifunctional linker BMPH was also used.

The bioactivity of non-oxidized and oxidized CLEC and the novel CLEC-based conjugates was then assessed using a competitive ELISA system based on competitive binding to soluble murine Fc-dectin-1a receptor (InvivoGen) or ConA as described by Korotchenko et al. (2020) .

The different CLECs tested showed different efficacy in binding to PRR. The binding efficacy of dectin-1 ligands Shigu polysaccharide, lichenin, barley β-glucan, oat β-glucan and dectin-1 was evaluated in a series of ELISA experiments. Subsequent experiments showed that the medium molecular weight (20 kDa) linear β-(1,6)-linked β-D-glucan polysaccharide was comparable to the larger, higher molecular weight linear β-(1,3)β Compared with -(1,4)-β-D glucan lichenan (approximately 245kDa), it shows significantly higher binding efficacy to dectin-1 (approximately 3 times) (see Figure 1).

This difference was even more pronounced when Pseudomonas aeruginosa was compared to other linear β-(1,3)β-(1,4)-β-D glucans from oats and barley (barley β-glucan (229 kDa), oat β-glucan: 265 and 391 kd), which showed only limited binding efficacy compared to Pseudomonas aeruginosa (e.g., barley β-glucan (229 kDa) was approximately 30 times lower).

Mild periodate oxidation of selected CLEC results in reduced binding to dectin-1. Oxidation of mannan polysaccharide reduces its binding capacity to the lectin ConA to an extent similar to the reduction described for oxidized dectin-1 binding following iodate oxidation. Likewise, oxidation of dextran resulted in a similar ratio of reduction in PRR binding (see Figure 1A).

Importantly, conjugate formation also resulted in a reduction in the PRR binding capacity of peptide-CLEC conjugates compared to unconjugated CLEC, such as mannan-containing conjugates as well as the different Achyris polysaccharides, lichenin or barley and oat polysaccharides tested. -β-glucan conjugates (see Figure 1B).

Experiments have shown that despite the small size and absence of β-(1,3) glycosidic linkages in the polysaccharide of Schizophora polysaccharides (note: glucan-containing β-(1,3) is described as the best ligand for dectin-1 ), but linear β-(1,6)-linked β-D-glucan polysaccharide can exert the highest binding effect, regardless of oxidation or binding. For example, conjugates containing the polysaccharide retained approximately 3 times higher binding capacity than lichenin-based constructs.

Regarding IC50 values, the binding results in Figure 1 show the binding of various constructs to soluble murine Fc-dectin-1a receptor. The IC50 values obtained were (Figure 1): - Oat β-glucan 265: 860µg/ml - Oat β-glucan 391: 820µg/ml - Barley β-glucan 229: 145µg/ml - Lichenin (Figure 1E): 13µg/ml - Lichenin 200% conjugate (Figure 1E): 27µg/ml (i.e., about half of the unconjugated lichenin) - β-glucan 229 binds at least 30 times more strongly than 145µg/ml) - Pyricularia polysaccharide conjugate (Figure 1D): 11, 14 and 15µg/ml (i.e., about half of the unconjugated yricularia polysaccharide) - Psoralea corylifolia BMPH conjugate (Figure 1F): 80µg/ml (peptide coupled to the heterobifunctional linker of Psoralea corylifolia).

Figures 1A and 1B further demonstrate that peptide conjugation via hydrazone formation or via a heterobifunctional linker is equally applicable to WISIT conjugates, as both types of conjugates retain high dectin-1 binding efficacy. Example 2 : Assay of DC activation after in vitro exposure to Psoralea corylifolia polysaccharide

An important function of the proposed vaccine is its ability to activate DCs upon PRR binding and uptake. To demonstrate that the CLEC-based conjugates not only bind to PRRs but also exert biological functions in their target cells (i.e., DCs), DC activation experiments were performed.

First, mouse bone marrow cells were cultured with mGM-CSF to generate BMDCs according to published protocols, and these GM-CSF DCs were then exposed to the peptide-glucan conjugate PSeqID2+SeqID7+Pseudomonas aeruginosa or an equivalent amount of oxidized but unconjugated saccharide. In each case, the conjugate/saccharide was titrated from 500µg to 62.5µg/mL each. For comparison, the strong activator LPS was used as a control group with a starting concentration of 2ng/ml. Importantly, the Pseudomonas aeruginosa preparation used for oxidation and conjugate formation also contained a small amount of LPS, so an equivalent amount of LPS was used to normalize this effect. DCs were then assessed for the expression of DC activation and maturation markers using FACS analysis (including CD80 and MHCII). Results:

GM-CSFDC stimulated in vitro with SeqId2-SeqID7-Pseudomonas aeruginosa conjugates showed a significant increase in the expression of CD80 and MHCII (see Figure 2). The levels were significantly higher than the effects observed with equivalent amounts of LPS included in the conjugate preparation. In contrast, an equivalent amount of oxidized but unconjugated sugars caused a slight decrease in CD80 expression, as expected from the levels of LPS in the home-preparation, and a significant decrease in the induction of MHCII compared to the Pseudomonas aeruginosa conjugate.

Taken together, upregulation of MHC-II indicates DC activation. In addition, CD80 was up-regulated beyond what would be expected for the same amount of LPS, strongly suggesting that the amygdala polysaccharide conjugate contributes significantly to DC maturation and activation (beyond what could be explained by exposure to LPS alone). Therefore, Examples 1 and 2 clearly demonstrate the biological activity of the Shi fungus polysaccharide vaccine. Example 3 : Determination of particle size by DLS

Separate experiments analyzing the particle size/hydrodynamic radius of different dextran conjugates have been performed.

For the DLS analysis, different peptide-dextran and peptide-carrier-dextran conjugates were analyzed and compared with unconjugated Shi Fuchsia polysaccharide respectively. All analyzes were performed in triplicate using WYATT DynaPro PlateReader-II. The results obtained indicate a particle size distribution with a maximum in the low nm spectrum for all tested conjugates. Tested conjugates: B cell epitope T cell epitope CLEC SeqID2 SeqID7 Shi fungus polysaccharide (80%) SeqID3 CRM197 Shi fungus polysaccharide (80%) na na Unoxidized polysaccharide result :

The current analysis shows that the peptide-SeqID2+SeqID7+SeqID7+SeqID2 polysaccharide used in this assay has an average major particle hydrodynamic radius (HDR) of approximately 5 nm. A smaller second peak detected at approximately 60 nm indicates the presence of very small amounts of aggregates in the formulation (see Figure 3A). However, most conjugate formulations appear to exist in monomeric form. This ubiquity of monomeric rather than cross-linked or aggregated conjugates is also explained by the fact that the monomeric Lycoris polysaccharide (approximately 20 kDa) can be detected at approximately 5 nm (as shown in the control sample, see also Figure 3C) This fact supports the universality of monomeric Acanthus polysaccharide conjugates (assuming that the HDR of monomeric Acanthus polysaccharide is about 5 nm). As shown by cumulative radius analysis over 24 hours, the HDR of the conjugate is also very stable and does not aggregate again, which supports the universality of the monomeric conjugate.

To characterize vaccines based on peptide-carrier-dextran conjugates, an additional SeqID6+CRM197 conjugate conjugated to Psoralea corylifolia was analyzed in this case. Again, DLS analysis showed an average HDR of 11 nm, with a second smaller peak at approximately 75 nm again indicating the presence of a small amount of aggregates (see Figure 3B). Since the size of CRM197 is approximately 60 kDa, the slight increase of 11 nm most likely reflects an increase in the molecular weight of the resulting conjugate. No significant aggregation or cross-linking of the CRM conjugate was detected, and the 24-hour cumulative radius analysis also showed that the HDR of the conjugate was stable and aggregation did not occur. Again, DLS analysis of this alternative type of CLEC-based vaccine supports the prevalence of monomeric conjugates.

The control sample (i.e., non-oxidized Auricularia polysaccharide) showed a larger HDR, averaging about 600 nm, and had two smaller peaks located at 5 nm and 46 nm respectively (see Figure 3C). The HDR of the fungus polysaccharide monomer is about 5 nm, which is in good agreement with the assumed molecular weight of 20kD. Larger aggregates can be easily detected, and most of the glucan exists in the form of large, high molecular weight particles. Importantly, cumulative radius analysis over 24 hours also showed that compared with the conjugated Acanthus polysaccharides, unconjugated Acanthus polysaccharides tend to aggregate strongly over time, causing the widespread formation of large particles, which is consistent with various literatures. The reports are consistent.

FIG3 depicts examples of these two conjugates and unoxidized Pyricularia polysaccharide as a control.

Compared to well-known examples in this technology (e.g. Wang et al., 2019; Jin et al., 2018), the results obtained in this example further demonstrate the unique characteristics of CLEC-based conjugates to date, namely showing The resulting small (i.e., 5-11 nm), predominantly monomeric glycosyl nanoparticles have an HDR much less than 150 nm, which is generally considered the preferred size for immunotherapeutic active conjugate vaccines. This is primarily due to the PRR binding and activation properties of larger particles, including whole glucan particles. Larger particles (>150 nm to 2-4 µm) are known to interact more efficiently with their receptors and initiate DC signaling, activation, maturation, and migration to draining lymph nodes, whereas smaller, even soluble, PRR-ligands are thought to bind to their receptors but prevent subsequent DC activation (Goodridge et al., 2011). However, these data, together with those described in Examples 1, 2 and 3 and other examples provided below, demonstrate for the first time small and soluble peptidylglucose-novel conjugates based on monomeric β-glucan, e.g. linear β (1,6)-β-D glucan fungus polysaccharide, as a skeleton, can effectively bind to PRR (dectin-1), activate the corresponding APC (such as GM-CSFDC) and show very high biological activity, and its skin The immunogenicity in a specific manner also significantly exceeds that of classic conjugate vaccines. Example 4 : In vivo comparison of different CLEC vaccines

CLEC-based vaccines that bind to their DC receptors (e.g., dectin-1 or ConA) were tested for their ability to induce a strong and specific immune response after repeated administration in n=5 Balb/c mice/group. Typical experiments were performed using 5µg of neat peptide content of B cell epitope peptide per dose.

Three different CLECs were compared in the first set of experiments. In this experiment, the α-synuclein-derived peptide SeqID2 or the amyloid β42 (Aβ42)-derived peptide SeqID10 and the hybrid helper T cell epitope SeqID7 were combined with oxidized auricularia polysaccharide through a C-terminal hydrazine linker. Coupled with beta-glucan (20% oxidation degree), mannan (20% oxidation degree) or barley β-glucan (229kDa, 20% oxidation degree). Vaccines used: B cell epitope T cell epitope CLEC SeqID2 SeqID7 Shi fungus polysaccharide (20%) SeqID2 SeqID7 Mannan (20% SeqID2 SeqID7 Barley β-glucan (229kDa, 20%) SeqID2 SeqID7 Shi fungus polysaccharide (80%) SeqID2 SeqID7 Lichenin (245kDa, 200% SeqID10 SeqID7 Shi fungus polysaccharide (80%) SeqID10 SeqID7 Lichenin (245kDa, 200%

Animals (female Balb/c mice) were vaccinated 3 times at 2-week intervals (Route: id), and mouse plasma collected 2 weeks after the third immunization was subsequently analyzed for the injected peptide (i.e., SeqID2, respectively). and SeqID10) immune response. result:

As shown in Figure 4A , all three CLEC vaccines (SeqID2+SeqID7+mannan, SeqID2+SeqID7+Shitu polysaccharide (linear β(1,6)β-glucan) and barley SeqID2+SeqID7+β-glucan (229kDa ) were able to induce a detectable immune response. Interestingly, immunization with a barley high molecular weight β-glucan-based vaccine induced only a very low anti-peptide response (OD _max/2 titer approximately 1/100 ). In contrast, the conjugates based on Schizophora polysaccharides induced significantly higher responses with an average titer of approximately 1/11000. Compared to the conjugates based on Schizophora polysaccharides, the mannan polysaccharide-based conjugates showed Approximately 7-fold reduction in immunogenicity since the average titer after immunization in this experiment reached approximately 1/1500.

Figure 4B shows the results of a second set of experiments comparing two different variants using the synuclein-derived peptide SeqID2 or the amyloid beta 42 (Aβ42)-derived peptide SeqID10 as a B-cell epitope and the T-cell epitope SeqID7. Immunogenicity of dextran-based conjugates. The first variant once again used Shigu polysaccharide as CLEC for binding, and the second variant system was modified by using linear β-(1,3)β-(1,4)-β-D glucan-lichenin (ca245kDa). ) produced. As shown in Figure 4B, both variants can induce high-titer immune responses against the injected peptides (i.e., SeqID2/3 (SeqID3=SeqID2 is suitable for BSA coupling) and SeqID10/11 (SeqID11=SeqID10 is suitable for BSA coupling)). However, the peptide-lichenin conjugates showed significantly lower immunogenicity than the peptide-lichenin conjugates in these experiments (4-8 times higher anti-peptide potency at the 5 μg dose), which is also consistent with Examples The lower dectin-1 binding capacity shown in 1 is consistent. This indicates that the binding efficacy of dectin-1 in vitro can be directly related to the in vivo immunogenicity and biological activity of the vaccine. This led to the identification of the fungus polysaccharide or its fragments (i.e. linear β(1,6)-β-D glucan) as the most potent glucan variant proposed in this application. The vaccine is also functional on different peptides, demonstrating the platform potential of this vaccine type. Example 5 : In vivo comparison of peptide Shigu polysaccharide conjugates and unconjugated peptide vaccines

In order to evaluate whether the conjugation of CLEC with peptide immunogens is necessary to induce the excellent immunogenicity of the vaccine according to the invention, a set of experiments was performed in which two conjugates (SeqID2+SeqID7+SeqID7+SeqID2+SeqID7+Mannan polysaccharide) were compared to a vaccine containing a mixed (not combined) formulation of all components (i.e., SeqID2 and SeqID7 plus non-oxidized auricularia polysaccharide or mannan polysaccharide, respectively). Similarly, n = 5 female Balb/c mice were immunized intradermally three times at biweekly intervals, and mouse plasma collected two weeks after the third immunization was analyzed subsequently for the injected peptide (i.e., SeqID3 ) immune response. Vaccines used: B cell epitope T cell epitope CLEC combine SeqID2 SeqID7 Shi fungus polysaccharide (20%) yes SeqID2) SeqID7 Mannan (20%) yes SeqID2 SeqID7 Shi fungus polysaccharide (not oxidized) unbound; mixed SeqID2 SeqID7 Mannan (not oxidized) unbound; mixed result:

Figure 5 shows a comparison of the anti-peptide (SeqID3) specific immune responses detected after three immunizations. In this experiment, the SeqID2+SeqID7+Pseudomonas aeruginosa conjugate (20% oxidized) was able to induce a 4-fold higher immune response than the mixture of unconjugated peptide SeqID2, SeqID7, and non-oxidized Pseudomonas aeruginosa (i.e., 1/12000 vs. 1/3000). Similarly, the SeqID2+SeqID7+Mannan polysaccharide conjugate (20% oxidized) was also more effective in inducing a peptide-specific immune response, while the mixture of these components was weaker (1/7000 vs. 1/4000; 1.75-fold increase). These data indicate that the peptide immunogen needs to be conjugated to activated CLECs to induce a strong and sustained immune response in vivo. Example 6 : In vivo comparison of SeqID5+SeqID7+ Psoralea corylifolia and SeqID2+ or Seq-7+ Psoralea corylifolia conjugates

To evaluate whether CLEC-based vaccines require both B-cell and T-cell epitopes to induce sustained anti-B-cell epitope-specific immune responses in vivo, a set of experiments was conducted to compare three conjugates: SeqID5+SeqID7+Pseudomonas aeruginosa, SeqID5+Pseudomonas aeruginosa, and SeqID7+Pseudomonas aeruginosa. n=5 female Balb/c mice were immunized intradermally three times at biweekly intervals, and subsequent immune responses to the injected peptide (i.e., SeqID6) were analyzed using mouse plasma collected two weeks after the third immunization. B cell antigen determinant T cell antigen determinant CLEC SeqID5 SeqID7 Pyricularia auricula polysaccharide (80%) SeqID5 na Pyricularia auricula polysaccharide (80%) na SeqID7 Pyricularia auricula polysaccharide (80%) result:

As shown in FIG6 , in this experiment, SeqID5+SeqID7+Auricularia auriculariae polysaccharide conjugate (80% oxidized) was able to induce a highly specific immune response against the injected peptide portion (i.e., α-synuclein-derived peptide SeqID6), with an average titer of 1/36,000. Peptide-Pseudo-Pseudo-Auricularia polysaccharide conjugates containing either SeqID5 or SeqID7 alone coupled to Pseudo-Auricularia polysaccharide by hydrazone coupling reaction were immunized three times at intervals of once every two weeks (route: i.d.) and induced a 12-fold lower immune response in the case of SeqID5-Pseudo-Auricularia polysaccharide (1/3000) or no SeqID6-specific immune response (for SeqID7-Pseudo-Auricularia polysaccharide conjugates, the titer was <1/100, which is below the detection limit).

These data demonstrate that conjugation of peptide immunogens to activated CLEC is required to induce strong and sustainable immune responses in vivo. However, it was also shown that in the absence of a T cell epitope (e.g., SeqID5 alone), the binding of acanthus polysaccharides to a single short B cell epitope can induce T cell independent B cell responses in vivo , although the potency is significantly lower than reported CLEC conjugates containing T cell and B cell epitopes. Example 7 : In vivo analysis of anti-Agaricus polysaccharide / dextran immune response after peptide - Acacia polysaccharide immunization

The analysis of anti-CLEC antibodies is important for the novelty and efficacy of the CLEC vaccine proposed according to the present invention on two levels:

1) β-glucan is the main component of the cell walls of various fungi, lichens and plants, giving the cell walls typical strength to resist intracellular osmotic pressure. Therefore, β-glucan is also considered to be a classic microbial pathogen-associated molecular pattern (PAMP) and a major target of high-titer circulating natural antibodies in healthy human subjects. PAMPs are common and relatively immutable molecular structures shared by many pathogens and are powerful activators of the immune system. (Chiani et al. Vaccine 27 (2009) 513-519, Noss et al. Int Arch Allergy Immunol 2012;157:98-108, Dong et al. J Immunol 2014; 192:1302-1312, Ishibashi et al. FEMS Immunology and Medical Microbiology 44 (2005) 99-109, Harada et al. Biol Pharm Bull. 2003 Aug;26(8):1225-8). IgG against β-(1,3)- and -β-(1,6)-glucan can be found in normal human serum, and β-(1,6)-glucan appears to be more sensitive than β-(1 -3) The variant is a more potent antibody. In addition, the β-(1-6)-β-glucan moiety has been identified as one of the typical microbial PAMPs, which serves as a focal point for recognition and surveillance of immune malignancies as well as defense against microbial invasion. The preferred glucan backbone for CLEC conjugates according to the present invention is the polysaccharide, which is composed of linear β-(1-6)-β-glucan moieties, and several research groups have reported anti- Immune responses can be detected in the plasma of human subjects who have not been immunized with Schizophora polysaccharide. Therefore, it is crucial to investigate the potential of CLEC-based vaccines to activate immune reactivity against Schizophora polysaccharides. Anti-β-glucan antibodies can specifically interact with peptide-Fructus polysaccharides in vivo and cause rapid elimination by forming antigen-antibody complexes, thereby precluding the induction of an effective immune response. Alternatively, the induction/enhancement of anti-Agaricus polysaccharide antibody responses following immunization may also promote immunogenicity, as potential cross-presentation of anti-Agaricus polysaccharide-specific IgG antibodies with CLEC conjugates and uptake of APCs may also enhance the efficacy of the administered vaccine. effect.

Prior to this, no formal studies have investigated the presence of anti-A. pyrenoidosa antibodies in untreated mice. However, Ishibashi et al. and Harada et al. were able to demonstrate the presence of β-glucan IgG against soluble sclerosant glucan/β-glucan (i.e., 1,3/1,6-β-glucan) in the serum of untreated DBA/2 mice.

2) As previously described (e.g., Torosantucci et al., Bromuro et al., Donadei et al., Liao et al.), CLEC-protein conjugates, such as CRM197-coupled laminarin, curdlan or synthetic β(1,3)-β-D-glucan, can act as potent immunogens, inducing not only high anti-CRM197 titers, but also high anti-glucan titers and protecting against fungal infection. Therefore, previous attempts to use such conjugates have been focused on using CLEC as a true disease/fungal infection specific immunogen, rather than using it as a carrier and immunologically inert backbone as proposed in the present application.

Along these lines, an extensive analysis of plasma samples from Balb/c mice (n=5/group) immunized with the first and peptide-CLEC conjugates was performed for the presence of anti-A. pyrenoidosa antibodies before and after repeated immunization. B cell antigen determinant T cell antigen determinant CLEC Combine SeqID2 SeqID7 Pyricularia auricula polysaccharide (20%) yes SeqID2 SeqID7 Mannopolysaccharide (20%) yes SeqID2 SeqID7 Pyricularia auricula polysaccharide (unoxidized) Unbound; Mixed SeqID2 SeqID7 Mannopolysaccharide (unoxidized) Unbound; Mixed result:

Therefore, samples from animals vaccinated with peptide-SeqID2+SeqID7+SeqID (20%) and peptide-mannan (SeqID2+SeqID7+mannan (20%)) were analyzed (all vaccines: 4µgaSyn targeted peptide/dose). For control purposes, animals receiving administration of a vaccine consisting of non-conjugated peptides and non-oxidized CLEC (i.e., SeqID2+SeqID7+non-oxidized mannan polysaccharide, SeqID2+SeqID7+non-oxidized mannan polysaccharide) were also used. Figure As shown in 7A, the Balb/c animals analyzed showed a pre-existing low-level immune response against Shigu polysaccharide/β(1,6)-β-D glucan. The two CLEC vaccines tested (SeqID2+ SeqID7 + Shigu polysaccharide (20%) and SeqID2 + SeqID7 + mannan polysaccharide (20%)) failed to induce strong de novo immune responses against the dextran backbone in vivo. In contrast, repeated administration of unbound polysaccharide present in the control group , non-oxidized agaric polysaccharides (containing a mixture of all three components) led to a strong anti-glucan immune response by increasing antibody levels against agaric polysaccharides 18.5-fold (compared to pre-immunization plasma) Induction. Conjugates or mixtures containing mannan polysaccharides were unable to induce anti-Sea polysaccharide titers, indicating the specificity of the anti-glucan response detected. Kinetic analysis of anti-Sea mannan antibody titers showed that they increased over time The titers increased steadily over time, with a strong increase after the third immunization in animals vaccinated with unconjugated and non-oxidized Shifu polysaccharide (see Figure 7B). The titers using gradually increasing amounts of natural Shifu polysaccharide Competitive ELISA also demonstrated the specificity of the antibody responses detectable in groups containing mixed components (Fig. 7C).

Taken together, these analyzes demonstrate that, despite the presence of low-level autoreactivity against Shigella polysaccharide (IgG) in untreated Balb/c mice, this does not occur after immunization with various CLEC conjugates. Induces/only induces a very low vaccination-dependent increase in immune reactivity against Schizophora polysaccharide. Therefore, CLEC used as peptide-CLEC conjugates according to the invention are indeed immunologically inert using the novel vaccine design according to the invention. This is in strong contrast to previously published results and thus constitutes a surprising and inventive new feature of the carbohydrate backbone according to the present invention (for example, β-glucan or mannan polysaccharide, especially the polysaccharide backbone).

Furthermore, pre-existing anti-WISIT polysaccharide responses do not appear to preclude an immune response to the peptide component of the WISIT vaccine, as the injected peptide responses in both experiments showed high anti-peptide titers. Example 8 : Immunogenicity analysis of dextran conjugates with N- or C -terminally coupled peptide immunogens

To evaluate whether the orientation of the linker used for conjugation would interfere with the immunogenicity of the vaccine, four different candidate vaccines were produced: In this experiment, α-synuclein-derived peptides SeqID1/2 and SeqID4/5 were conjugated to oxidized Pseudomonas aeruginosa (80%) via an N- or C-terminal hydrazide linker. In addition, each of the four vaccines carried a promiscuous helper T cell epitope SeqID7, conjugated to the CLEC backbone via a C-terminal hydrazide linker. Vaccines used: B cell antigen determinant T cell antigen determinant CLEC SeqI1 SeqID7 Pyricularia auricula polysaccharide (80%) SeqID2 SeqID7 Pyricularia auricula polysaccharide (80%) SeqID4 SeqID7 Pyricularia auricula polysaccharide (80%) SeqID5 SeqID7 Pyricularia auricula polysaccharide (80%)

Animals (female Balb/c mice) were vaccinated three times at biweekly intervals (Route: id), and mouse plasma collected two weeks after the third immunization was subsequently tested against the injected peptides (i.e., SeqID3 and SeqID6) and the immune response against the target protein (i.e., recombinant α-synuclein) were analyzed. result:

As shown in Figure 8, all four CLEC vaccines using N- or C-terminally coupled B cell epitopes were able to induce strong and highly specific immune responses against the injected peptide portion (Figure 8A) and the target protein (α-synuclein, Figure 8B). Interestingly, the coupling direction had different effects on immunogenicity. For example, the N-terminally coupled SeqID1+SeqID7+CLEC vaccine induced a 7-fold lower anti-injected peptide response and a 10-fold lower immune response against recombinant aSyn compared to the C-terminally coupled SeqID2+SeqID7+CLEC. In contrast, the C-terminally coupled SeqID5+SeqID7+CLEC vaccine induced approximately 4-fold lower injected peptide responses compared to the N-terminally coupled SeqID4+SeqID7+CLEC vaccine, but the same anti-aSyn response. Therefore, the direction of coupling can be varied depending on the specific characteristics of the peptide without affecting the development of a high titer immune response.

However, as shown in Figure 8, by changing the coupling direction of the immunogenic peptide, the specificity of the subsequent response against the target protein can be significantly increased and can therefore be used to generate new and unprecedented immune responses: For example: SeqID1 vaccination resulted in a 4.5-fold higher response against the peptide compared to the protein of interest and a 3.3-fold higher anti-peptide response compared to the protein induced by the SeqID2 vaccine.

In contrast, the SeqID4 vaccine induced a 1.7-fold higher response to the peptide compared to the protein, while the SeqID5 vaccine reversed this ratio, eliciting a 2.5-fold higher protein-specific response than could be detected with the injected peptide.

Taken together, these data clearly demonstrate that vaccines using either coupling orientation are biologically active and suitable for this administration. It also demonstrates that coupling orientation can be used to select particularly optimal and unprecedented active vaccines based on the peptide and target to be addressed. Example 9 : Analysis of the immunogenicity of CLEC conjugates using different helper T cell epitopes

In this example, the immunogenicity of a CLEC-based vaccine containing a non-native pan-DR epitope (PADRE containing an artificial cathepsin L cleavage site, SeqID7) was compared to other well-known helper T cell epitopes. For this purpose, several promiscuous epitopes were selected that were either engineered with a novel, artificially included cathepsin L cleavage site for efficient intracellular/lysosomal release following receptor-mediated uptake in APCs/DCs or remained unchanged. The epitopes selected included: Peptides sequence Antigenic determinant Cathepsin L cleavage site SeqID7 AKFVAAWTLKAAANRRA-(NH-NH ₂ ) PADRE + SeqID22 AKFVAAWTLKAAA-(NH-NH ₂ ) PADRE - SeqID23 KAAAVKAAFWTAL-NRRA-(NH-NH ₂ ) Artificial + SeqID24 DSETADNLEKTVAALSILPGHGC-(NH-NH ₂ ) diphtheria - SeqID25 DSETADNLEKTVAALSILPGHGCNRRA-(NH-NH ₂ ) diphtheria + SeqID26 ISITEIKGVIVHRIETILF-(NH-NH ₂ ) Measles virus fusion protein - SeqID27 ISITEIKGVIVHRIETILFNRRA-(NH-NH ₂ ) Measles virus fusion protein + SeqID28 ISQAVHAAHAEINEAGR-(NH-NH ₂ ) Chicken OVA (323-339) - SeqID29 ISQAVHAAHAEINEAGRNRRA-(NH-NH ₂ ) Chicken OVA (323-339) +

In order to evaluate whether peptide vaccines carrying these helper T cell epitope peptides can produce high immune responses after repeated immunizations and induce better immune responses than traditional conjugate vaccines, this experiment tested 10 different Vaccine candidates:

In this experiment, aSyn derived peptide SeqID2 was used as peptide-CLEC vaccine (i.e. SeqID2, conjugated to different helper T cell epitopes, coupled to oxidized Pseudomonas polysaccharide (80%;) via a C-terminal hydrazide linker) or peptide conjugates were generated using SeqID3 containing a C-terminal cysteine for coupling to GMBS-activated KLH. Vaccines used: vaccine B cell antigen determinant T cell antigen determinant / vector CLEC Adjuvant Way 1 SeqID2 SeqID7 Pyricularia auricula polysaccharide (80%) na id 2 SeqID2 SeqID22 Pyricularia auricula polysaccharide (80%) na id 3 SeqID2 SeqID23 Pyricularia auricula polysaccharide (80%) na id 4 SeqID2 SeqID24 Pyricularia auricula polysaccharide (80%) na id 5 SeqID2 SeqID25 Pyricularia auricula polysaccharide (80%) na id 6 SeqID2 SeqID26 Pyricularia auricula polysaccharide (80%) na id 7 SeqID2 SeqID27 Pyricularia auricula polysaccharide (80%) na id 8 SeqID2 SeqID28 Pyricularia auricula polysaccharide (80%) na id 9 SeqID2 SeqID29 Pyricularia auricula polysaccharide (80%) na id 10 SeqID3 KLH na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated three times at biweekly intervals (all vaccines: 5 μg of Syn targeting peptide/dose; route: id for CLEC-based vaccines, sc for KLH-based vaccines (with Alhydrogel as adjuvant)), and immune responses to the injected peptide (i.e., SeqID3) and to the target protein (i.e., recombinant human α-synuclein) were subsequently analyzed using mouse plasma collected two weeks after the third immunization. Results:

As shown in Figure 9, all nine CLEC vaccines and KLH conjugates using different helper T cell epitopes were able to induce responses to the injected peptide moiety (SeqID3, Figure 9A) and the target protein (recombinant α-synuclein, Figure 9B) strong and specific immune response.

All helper T epitopes induced anti-peptide titers similar to or superior to the known SeqID3+KLH conjugate. For example, vaccine 1 (comprising SeqID2 and SeqID7 coupled to Psoralea corylifolia) induced a 60% higher response than the KLH control, while vaccine 8 (comprising SeqID28, a well-known helper T epitope specifically suitable for Balb/c animals, SeqID2, and Psoralea corylifolia) induced a 5.5-fold higher response than the control. Even the promiscuous helper T antigenic determinant SeqID24 (derived from diphtheria toxin, a weak helper T antigenic determinant of Balb/c animals disclosed in WO2019/21355A1) was able to induce a sustainable immune response, although it was weaker than the KLH control group.

Similarly, all helper T epitopes induced anti-protein titers that were similar to or superior to traditional SeqID3-KLH conjugates. Importantly, for example, vaccine 1 (containing SeqID2 and SeqID7 coupled to Shigu polysaccharide) induced a 2.5-fold higher response than the KLH control group, whereas vaccine 8 (containing SeqID28, a well-known helper T epitope, specifically Suitable for application in Balb/c animals, SeqID2 and Shigu polysaccharide) can induce a response 3 times higher than that of the control group, again supporting the fact that the CLEC-based vaccine according to the present invention can induce an excellent anti-target response.

This example also demonstrates that the introduction of an additional cathepsin L cleavage site into a known helper T cell epitope leads to a more effective induction of an immune response compared to both the known vaccine and a CLEC-based vaccine without this artificial sequence.

For example, SeqID25, a modified variant of the weak helper T epitope SeqID24 that contains a cleavage site, induced a 7.5-fold higher anti-peptide response and a higher 3.6 times the anti-protein response. In addition, this change also resulted in a 40% increase in anti-protein titer compared to the KLH control. SeqID27 is a modified variant of the cathepsin L cleavage site of SeqID26 (an epitope derived from measles virus fusion protein, disclosed in WO2019/21355A1). Compared with the SeqID26-CLEC vaccine, it can also significantly increase the titer and anti- The peptide response increased by 1.8-fold, and the anti-protein titer increased by 3.2-fold (ie, vaccine 7 vs. vaccine 6). Compared with the KLH control group, vaccine 7 also induced a 2.2-fold higher anti-peptide response and a 1.6-fold higher anti-protein response. The SeqID7-based CLEC vaccine also induced higher anti-protein titers (20% increase) compared with unmodified variants (e.g., SeqID22), and compared with the KLH control group, these two peptides resulted in approximately Twice the anti-SeqID2 peptide and anti-aSyn titers.

Addition of a cathepsin cleavage site results in the formation of peptide variants with an additional N (e.g. a C-terminus that is released upon cleavage. For example: SeqID22, PADRE, is released as AKFVAAWTLKAAA, whereas for SeqID7 the modified PADRE is released as AKFVAAWTLKAAA-N). This N may also negatively influence further processing and MHCII presentation, thereby reducing the efficacy of the corresponding peptide. This phenomenon can be seen in the case of the very strong OVA-derived epitopes SeqID28 and SeqID29. The unmodified peptide induces a very high immune response while the modified variant pep17 induces a 75% reduction in anti-peptide titers and a 98% reduction in anti-protein titers compared to the unmodified variant. Example 10 : Immunogenicity analysis of CLEC conjugates using carrier protein as helper T cell antigen determinant : KLH

In this example, the immunogenicity of a CLEC-based conjugate vaccine containing the well-known carrier protein KLH was compared to conventional KLH vaccines. For this purpose, two aSyn-derived epitopes (SeqID3 and SeqID6) were selected for coupling with GMBS-activated KLH in this experiment. Subsequently, the Pep-KLH conjugate was coupled to the active aldehyde of the oxidized auricularia polysaccharide using a BPMH cross-linker to form a CLEC-based conjugate vaccine, in which KLH serves as a source of helper T cell epitopes to induce sustainable immune response. Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way SeqID3 htK Shi fungus polysaccharide (140%) na ID SeqID3 htK na na ID SeqID3 htK na Alhydrogel sc SeqID6 htK Shi fungus polysaccharide (80%) na ID SeqID6 htK na na ID SeqID6 htK na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (all vaccines: 20 µg aSyn targeting peptide/dose; route: CLEC-based vaccines and unadjuvanted KLH-based vaccines with id; KLH-based vaccines adjuvanted with Alhydrogel (sc) and subsequently used murine plasma collected two weeks after the third immunization to target the injected peptides (i.e., SeqID3 and SeqID6) as well as the target protein (i.e., recombinant human α-synuclein). ) were analyzed. result:

As shown in Figure 10A, all six vaccines using KLH as the source of the helper T epitope were able to induce strong and specific responses to the injected peptide portions (SeqID3 and SeqID6) and the target protein: recombinant alpha synuclein. immune response.

CLEC modification of KLH conjugates elicited highly superior immune responses using both peptides, SeqID3 and SeqID6, respectively. SeqID3+KLH+Pyrrolidone was able to induce a 2.3-fold higher anti-peptide response than SeqID3+KLH adjuvanted with Alhydrogel, and a 14-fold higher response than SeqID3+KLH administered intradermally without adjuvant. Similarly, anti-protein titers were also increased by 8.5-fold (compared to SeqID3+KLH adjuvanted with Alhydrogel) and 17-fold compared to the unadjuvanted material. The immune response effect of SeqID6+KLH+Psoralea corylifolia polysaccharide was also 2 (in terms of injected peptide) to 4.6 times (in terms of α-synaptobin) higher than that of SeqID6+KLH with adjuvant, while the immunogenicity was 8.7 (in terms of injected peptide) and 11 times (in terms of α-synaptobin) higher than that of SeqID6+KLH without adjuvant.

In addition to the general increase in immunogenicity of the CLEC-modified vaccines, the experimental results also showed that the CLEC modification according to the present invention resulted in a significant increase in the relative amount of induced antibodies bound to the target molecule (i.e., protein), thereby significantly increasing the subsequent immune response specific to the target. Thus, for the response induced by SeqID3+KLH+Pseudomonas aeruginosa, the relative amount of antibodies detecting α-synaptophysin (i.e., the ratio of total anti-injected peptide titer to anti-α-synaptophysin specific titer) was 3.7 times higher than that of the adjuvanted SeqID3+KLH, and in the case of SeqID6+KLH+Pseudomonas aeruginosa, it was 2.2 times higher than that of the adjuvanted conjugate.

In a second set of experiments, the induction of the same vaccines used (all vaccines: 5 µg aSyn targeting peptide/dose; route: CLEC-based vaccine with id; KLH-based vaccine with Alhydrogel as adjuvant with sc) was compared Ability to react with vector-specific antibodies. As expected, the traditional SeqID3+ and SeqID6+KLH-based vaccines were able to induce high anti-KLH titers (SeqID3+KLH: 1/2100 and SeqID6+KLH: 1/7700), while the CLEC-based SeqID3+KLH+ Shi fungus polysaccharide and The SeqID6+KLH+ Fungi polysaccharide vaccine is basically unable to induce sustained anti-carrier antibodies, and the titer obtained is close to the detection limit. SeqID3+KLH+ Fungus polysaccharide is 1/150, while SeqID6+KLH+ Fungus polysaccharide is less than 1/100. This resulted in a novel but undescribed strategy for optimizing peptide conjugate vaccines to increase target-specific potency while reducing unwanted anti-vector responses. Example 11 : Immunogenicity analysis of CLEC conjugates using carrier proteins as helper T cell epitopes : CRM197

In this example, the immunogenicity of a CLEC-based conjugate vaccine containing the well-known carrier protein CRM197 was compared to the conventional CRM197 vaccine. To this end, the α-synuclein-derived epitope SeqID6 was coupled to maleimide-activated CRM197. Subsequently, the SeqID6+CRM197 conjugate was coupled to the activated Schizophora polysaccharide using the heterobifunctional linker BPMH to form A CLEC-based conjugate vaccine in which CRM197 serves as a source of helper T cell epitopes to induce a sustainable immune response. Alternatively, SeqID5-(NH-NH2; SeqID5) and CRM197 were independently coupled to activated auricularia polysaccharide. This step is accomplished by reacting the hydrazine at the C-terminus of SeqID5 and the lysin present in CRM197 with the active aldehyde on the activated auricularia polysaccharide. Vaccines used: B cell epitope T cell epitope / carrier CLEC CLEC coupling Adjuvant way SeqID6 CRM197 Shi fungus polysaccharide (80%) in conjugate na ID SeqID5 CRM197 Shi fungus polysaccharide (80%) Independence na ID SeqID6 CRM197 na na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated three times at biweekly intervals (all vaccines: 20 µg α-synuclein targeting peptide/dose; route: id for CLEC-based vaccines, sc for CRM197-based vaccines adjuvanted with Alhydrogel) and subsequent immune responses to the injected peptide (i.e., SeqID6) and target protein (i.e., recombinant human α-synuclein and α-synuclein fibers) were analyzed using mouse plasma collected two weeks after the third immunization. Results:

As shown in Figure 11A, all 3 vaccines using CRM197 as the source of the helper T epitope were able to induce strong and specific immune responses against the injected peptide moiety (SeqID6) and the target protein: recombinant alpha synuclein. .

Likewise, CLEC modification of the CRM197 conjugate elicited an excellent immune response. SeqID6+CRM197+Shitu polysaccharide can induce 28 times higher anti-peptide response than SeqID6+CRM197 with Alhydrogel as adjuvant. Similarly, anti-protein titers increased 15-fold against recombinant alpha-synuclein (compared to SeqID6+CRM197 adjuvanted with Alhydrogel) and 11-fold against aggregated forms of aSyn (aSyn fibers). The vaccine generated by independently coupling SeqID5 and CRM197 to Shigu polysaccharide also induced a 1.7-fold higher injection peptide titer than the conventional SeqID6+CRM197 using Alhydrogel as an adjuvant, and the reactivity to recombinant aSyn also increased by 6.6 times, the anti-silk reaction increased by 4.25 times.

Comparison of anti-carrier-specific antibody responses showed that the traditional SeqID6+CRM197-based vaccine was able to induce high anti-CRM197 titers (1/6600), while the CLEC-based SeqID6+CRM197+Shitu polysaccharide vaccine was basically unable to induce sustained anti-carrier antibody. The obtained titer is close to the detection limit, and the titer of SeqID6+CRM197+Shitu polysaccharide is less than 1/100.

Therefore, this experiment shows that the CLEC modification of conventional peptide-protein conjugates significantly impairs the development of anti-carrier responses and results in a substantial enhancement of the target specificity of subsequent immune responses. For optimization, carrier proteins such as KLH and CRM197 are currently used as Basic advanced technology conjugate vaccines offer unprecedented new strategies.

Independent conjugation of CRM197 and SeqID6 to Psoralea corylifolia polysaccharide resulted in a sustained response to B cell epitopes present on CRM197, although the detection rate was lower than that of conventional non-CLEC modified conjugates (about 1/400 of the potency). This indicates that the CLEC backbone according to the present invention is also suitable for providing B cell epitopes from CLEC-coupled immunogenic proteins for use as vaccines. Example 12 : Selective analysis of immune responses elicited by CLEC -based vaccines in vivo

Aggregation of the presynaptic protein aSyn is believed to be the primary pathological culprit in synucleinopathies such as Parkinson's disease, and monomeric, non-aggregated aSyn has important neuronal functions. Therefore, it is believed to be critical for the treatment of synucleinopathies, for example by active or passive immunotherapy, to reduce/remove aggregated aSyn without affecting the available pool of non-aggregated molecules.

In order to further characterize the immune response induced by a CLEC-based vaccine containing aSyn targeting peptides SeqID2 and SeqID3 and SeqID5 and SeqID6 compared with conventional peptide carrier vaccines (i.e., SeqID3+KLH and SeqID6+CRM197), a set of The experiments analyzed the selectivity of the subsequent immune response to two different forms of the presynaptic protein aSyn: non-aggregated, predominantly monomeric aSyn, and aggregated aSyn fibers. Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way SeqID2 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID3 htK Shi fungus polysaccharide (80%) na ID SeqID3 htK na Alhydrogel sc SeqID5 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID6 CRM197 Shi fungus polysaccharide (80%) na ID SeqID6 CRM197 na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (all vaccines: 20 µg aSyn targeting peptide/dose; route: id for CLEC-based vaccines, sc for adjuvanted with Alhydrogel KLH and CRM197-based vaccines), subsequent immune responses against the target proteins (i.e., recombinant human alpha-synuclein and aSyn fibers) were analyzed using mouse plasma collected two weeks after the third immunization. ASyn-specific inhibition ELISA was performed on plasma samples, and IC50 values were determined. result:

Briefly, compared with traditional peptide conjugate vaccines (i.e., SeqID3+KLH and SeqID6+CRM, see Figure 12), all CLEC-based conjugates used in this experiment showed excellent immunogenicity and α-mutant Synuclein aggregation-specific target selectivity.

Conventional peptide conjugate vaccines are known to induce antibody responses with slightly increased selectivity for aSyn aggregates (i.e., fibrils) compared to monomeric/recombinant aSyn. Compared with recombinant aSyn, the selectivity of SeqID3+KLH with Alhydrogel as adjuvant for aSyn aggregates increased 9-fold. Compared with recombinant aSyn, which is mainly monomeric, SeqID6+CRM197 adjuvanted with Alhydrogel induced a less selective immune response, with 3.5-fold selective binding against aggregates.

In contrast, antibodies induced by CLEC-based peptide conjugate vaccines were characterized by several-fold increased selectivity compared to KLH or CRM197 conjugate vaccines. Plasma induced by SeqID2+SeqID7+Pseudomonas aeruginosa and SeqID5+SeqID7+Pseudomonas aeruginosa showed approximately 97-fold (i.e. 14-fold higher than the control vaccine SeqID3+KLH, Alhydrogel) and 50-fold (i.e. 14-fold higher than the control vaccine SeqID6+CRM, Alhydrogel) higher aggregation selectivity, respectively. SeqID3+KLH+Pseudomonas aeruginosa and SeqID6+CRM197+Pseudomonas aeruginosa showed 40-fold (i.e. 5-fold higher than SeqID3+KLH) and 50-fold (i.e. 14-fold higher than SeqID6+CRM) selectivity for aSyn aggregates, respectively.

Therefore, the experiments show that CLEC modification of peptide conjugates and peptide-protein conjugates greatly enhances the target specificity of the subsequent immune response, providing an unprecedented new strategy for optimizing current advanced technology conjugate vaccines. Example 13 : Analysis of the affinity of antibody molecules and antigens and the affinity of single antigen-binding segments to antigens in immune responses induced by CLEC - based vaccines

In order to further characterize the immune response induced by CLEC-based vaccines containing aSyn targeting peptides SeqID2, SeqID3, SeqID5 and SeqID6 compared with conventional peptide carrier vaccines (i.e., SeqID3+KLH and SeqID6+CRM197), a set of The experiment analyzes the affinity of the entire antibody molecule and a single antigen-binding segment to aSyn. Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way SeqID2 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID3 htK Shi fungus polysaccharide (80%) na ID SeqID3 htK na Alhydrogel sc SeqID5 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID6 CRM197 Shi fungus polysaccharide (80%) na ID SeqID6 CRM197 na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (all vaccines: 20 µg aSyn targeting peptide/dose; route: i.d. for CLEC-based vaccines, s.c. for adjuvanted with Alhydrogel KLH and CRM197-based vaccines), and subsequent immune responses against the target proteins (i.e., recombinant human aSyn and aSyn fibers) were analyzed using mouse plasma collected two weeks after each immunization. To determine the affinity of the induced antibody molecule ensemble for recombinant aSyn, a variation of the standard ELISA assay was used in which replicate wells containing antigen-bound antibodies were exposed to increasing concentrations of chaotropic thiocyanate ions. Resistance to thiocyanate elution is used as a measure of the overall affinity of the antibody molecule, and an index representing 50% of effective antibody binding (the overall affinity index of the antibody molecule) is used to compare plasma samples (between treatment groups and time points between).

In addition, the kD value of the antibody to aSyn fiber (the affinity of the single antigen-binding segment of the antibody to aSyn fiber) was also determined by aSyn competitive ELISA 2 weeks after the last immunization. result:

As shown in Figure 13, when comparing immune samples obtained two weeks after the second immunization (T2) or two weeks after the third immunization (affinity maturation of the antibody molecule ensemble (AM), the IC of the T2 and T3 samples were compared ₅₀ value: 1.1)), the conventional SeqID3+KLH conjugate (with Alhydrogel as adjuvant) shows only limited affinity maturation for binding to aSyn. In contrast, CLEC-based vaccines, such as SeqID2+SeqID7+SeqID7, induce strong maturation of the anti-aSyn response, as shown by an affinity index (AI) of 2.2, which is comparable to the affinity of the T3 sample for α-synuclein. Strongly increased correlation. Samples obtained from animals vaccinated with SeqID3+KLH+Fructus polysaccharide also showed significantly higher affinity and slightly increased maturity compared to SeqID3+KLH alone.

Similarly, the overall affinity of antibody molecules induced by SeqID5+SeqID7+Psoralea corylifolia and SeqID6+CRM197+Psoralea corylifolia vaccines for the immune response induced by aSyn protein was also significantly increased compared to the SeqID6+CRM197 baseline vaccine (analyzed at T3; i.e., 3-3.8-fold higher ionization salt levels were required to reduce binding), and the affinity maturation of the single antigen binding segment was also increased when comparing T2 and T3 values, respectively. When comparing T2 and T3, SeqID6+CRM197 did not induce an increase in the overall affinity of antibody molecules for aSyn, while the two CLEC-based vaccines induced a strong increase in specific binding to aSyn.

Quantitative experiments of aSyn fiber k _D values of immune responses elicited by CLEC-based vaccines and conventional baseline vaccines showed that the overall affinity of antibodies induced by CLEC-based vaccines for aSyn was significantly increased (see Figure 14). SeqID2+SeqID7+Shit fungus polysaccharide and SeqID3+KLH+Shit fungus polysaccharide conjugates showed 6-9 times higher affinity than the baseline vaccine SeqID3+KLH with Alhydrogel as adjuvant (i.e., Kd: 110nM and 160nM, with _kD of 1mM compared to). SeqID5+SeqID7+Shit fungus polysaccharide and SeqID6+CRM+Shit fungus polysaccharide conjugates showed 12-15 times higher Kd values than the baseline control group SeqID6+CRM197 with Alhydrogel as adjuvant (i.e. Kd: 50nM and 60nM, with a k of 750nM _D compared to).

Thus, this experiment demonstrates that CLEC modification of peptide conjugates and peptide-protein conjugates results in a significant enhancement of the target specificity and affinity of the subsequent immune response, providing an unprecedented new strategy to optimize current state-of-the-art conjugate vaccines. Example 14 : In vitro functional analysis of immune responses elicited by CLEC -based vaccines

In order to analyze whether the aSyn-specific antibodies elicited by the CLEC-based vaccine (containing aSyn targeting peptides SeqID2/3 and SeqID5/6) have biological activity, this example conducted a set of experiments to analyze the ability of the antibodies to inhibit aSyn aggregation in vitro. Vaccine used: B cell antigen determinant T cell antigen determinant / vector CLEC Adjuvant Way SeqId2 SeqID7 Pyricularia auricula polysaccharide (80%) na id SeqID3 KLH na Alhydrogel sc SeqID5 SeqID7 Pyricularia auricula polysaccharide (80%) na id SeqID6 CRM197 Pyricularia auricula polysaccharide (80%) na id SeqID6 CRM197 na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (all vaccines: 20 µg aSyn targeting peptide/dose; route: id for CLEC-based vaccines, sc for adjuvanted with Alhydrogel of vaccines based on KLH and CRM197). The in vitro aggregation inhibition capabilities of mouse plasma samples collected two weeks after each immunization and their corresponding control samples (for example: non-aSyn-binding antibodies or pre-immune plasma obtained before immunization) were analyzed. result:

As shown in Figure 15A, control antibodies or plasma extracted from animals before immunization had no significant effect on aSyn aggregation kinetics, confirming the specificity of the assay. Antibodies induced by the known SeqID3+KLH conjugate (with Alhydrogel as adjuvant) can significantly reduce aSyn aggregation, as shown by a 40% decrease in slope values (aSyn monomer only: 100%; KLH: 60%). Antibodies induced by the SeqID2 + SeqID7 + Shigu polysaccharide vaccine strongly inhibited aSyn aggregation, as shown by an 85% decrease in slope values (aSyn monomer only: 100%; CLEC: 15%), indicating that compared with antibodies induced by classic vaccines, their Inhibitory capacity is significantly higher.

Vaccine-induced antibodies based on SeqID5-SeqID7-SeqFructus polysaccharide and SeqID6+CRM+SeqFructus polysaccharide showed 86-92% inhibition of aggregate formation starting from recombinant aSyn (low-content aggregates), with preformed The formation of aggregates (=real aggregates) starting from fibrils has an inhibitory effect of 67-82%, and the inhibitory effect of antibodies induced by the benchmark vaccine SeqID6+CRM using Alhydrogel as an adjuvant on the formation of the above two aggregates is respectively 68% and 57% (see Figure 15B). Example 15 : Analysis of the impact of immune pathways on immune responses elicited by CLEC -based vaccines

This example performed a series of immunizations to compare id administration with alternative routes, including subcutaneous (sc) and intramuscular (im). Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way SeqID2 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID2 SeqID7 Shi fungus polysaccharide (80%) na sc SeqID2 SeqID7 Shi fungus polysaccharide (80%) na im

Animals (female Balb/c mice) were vaccinated three times at biweekly intervals (all vaccines: 1µg, 5µg and 20µg aSyn targeting peptide/dose), and immune responses to the subsequently injected peptides and target proteins (i.e., recombinant human alpha-synuclein and aSyn filaments) were analyzed using mouse plasma collected two weeks after the third immunization. Results:

Tables 1 and 2 and Figure 16 show that the SeqID2 + SeqID7 + Shia polysaccharide vaccine administered by intramuscular or subcutaneous route can induce a hyperimmune response against the injected peptide (Figure 16A) and an anti-aSyn response (Figure 16B). At all doses tested, the maximum titers achieved were significantly lower than those after id administration. sc administration showed a similar dose-response behavior to id, whereas im showed no significant difference between 5 and 20 µg, suggesting that saturation is reached at these doses/administration volumes. Similar results were obtained for the reactivity of monomeric and aggregated aSyn respectively. These results demonstrate that, unlike other pathways/tissues, the CLEC scaffold presented in the present invention is highly selective for application to skin. 1µg 5µg 20µg ID 16.000 83.000 140.000 sc 1000 2.000 12.000 im 4.000 16.000 15.000 Table 1: Antibody responses induced by anti-SeqID2/3 after application of WISIT vaccine by different routes 1µg 5µg 20µg ID 2.000 5.000 10.000 sc 100 1.000 4.000 im 2.000 2.000 5.000 Table 2: Antibody responses induced by anti-aSyn after application of WISIT vaccine by different routes Example 16 : Analysis of B cell epitopes using post-translationally modified peptides : Aβ

To evaluate whether peptide vaccines carrying peptides characterized by post-translational modifications (e.g. including phosphorylation, acetylation or pyroglutamic acid modification of amino acids) can generate high immune responses and induce superior immune responses after repeated immunizations. Based on the known immune response of conjugated vaccines, this experiment tested 2 different candidate vaccines:

In this experiment, an amyloid beta (Aβ) 40/42-derived peptide carrying an N-terminal pyroglutamine amino acid as an example of post-translational modification was used as a peptide-CLEC vaccine (i.e., SeqID33, coupled to the promiscuous T-cell epitope SeqID7 via a C-terminal hydrazide linker to oxidized Pseudomonas aeruginosa (80%;)), or SeqID32 containing a C-terminal cysteine was used to generate a known peptide conjugate for coupling to GMBS-activated KLH. Vaccines used: B cell antigen determinant T cell antigen determinant / vector CLEC Adjuvant Way SeqID33 SeqID7 Pyricularia auricula polysaccharide (80%) na id SeqID32 KLH na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (route: CLEC-based vaccine with id, KLH-based vaccine (with Alhydrogel as adjuvant) with sc), using the third immunization Rat plasma collected two weeks after vaccination was analyzed for subsequent immune responses to the injected peptides (i.e., SeqID32/33) and to the proteins of interest (i.e., recombinant Aβ(pE)3-40 and Aβ(pE)3-42). result:

As shown in Figure 17, both vaccines were able to induce strong and specific immune responses against the injected peptide moiety and target proteins (Aβ1-40/42, Aβ(pE)3-40 and Aβ(pE)3-42) . However, compared to SeqID32+KLH adjuvanted with Alhydrogel, the vaccine based on SeqID33+SeqID7+CLEC showed a 6-fold higher immune response against the injected peptide moiety. Most importantly, the vaccine was against the target protein/peptide AβpE3-42. The immune response was also 3.7 times higher, and the immune response to the Aβ variant AβpE3-40 was 1.6 times higher. Surprisingly, both vaccines tested also induced responses against Aβ1-42, showing immunogenicity from pyroglutamic acid (pE)-modified truncated Aβ forms (i.e., AβpE3-42 and AβpE3-40). Unexpected extension to intact, unmodified amyloidogenic and pathological molecules (i.e., Aβ1-40/42) expands the potential therapeutic activity of this class of vaccines. Likewise, the SeqID33+SeqID7+CLEC-based vaccine showed several times higher (3-fold) immune response against this unmodified form of Aβ compared to SeqID32+KLH with Alhydrogel as adjuvant, demonstrating that the CLEC-based vaccine excellent immunogenicity.

Furthermore, avidity analysis using anti-thiocyanate elution (NaSCN) showed that the antibody induced by SeqID33+SeqID7+CLEC had a 2.6-fold higher affinity for AβpE3-42 compared with the antibody induced by SeqID32+KLH.

Therefore, CLEC-based vaccines are well suited to use post-translationally modified peptides as immunogens (whether they constitute autoantigens (e.g., SeqID32/33) or foreign target structures), and when administered as CLEC-based vaccines, such antigens determine The gene can induce excellent immune responses, thereby endowing CLEC-based vaccines with higher immune responses and higher target-specific responses compared with traditional vaccines.

Furthermore, this example provides clear evidence that CLEC-based vaccines using target protein epitopes present in amyloidosis (including Alzheimer's disease, Lewy body dementia or Down syndrome) induce surprisingly more specific immune responses than prior art vaccines. Example 17 : Analysis of B cell epitopes of intracellular proteins and self-antigens : Tau

To assess whether peptide vaccines carrying peptides derived from intracellular proteins that are subject to extensive modifications (e.g., hyperphosphorylation, truncation, and aggregation), regardless of whether they constitute autoantigens (e.g., SeqID32/33) or foreign target structures, can After repeated immunization, a high immune response was produced and the immune response was better than that of conventional conjugate vaccines. This experiment was tested with 2 different candidate vaccines:

In this experiment, a Tau-derived peptide was used as a peptide-CLEC vaccine (i.e., SeqID35) with the hybrid T-cell epitope SeqID7 via a C-terminal hydrazine linker with oxidized auricularia polysaccharide (80%; ) coupling), or use SeqID36 containing a C-terminal cysteine to generate conventional peptide conjugates for coupling to GMBS-activated KLH. Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way SeqID36 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID35 htK na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (route: CLEC-based vaccine for id, KLH-based vaccine for sc with Alhydrogel as adjuvant), after the third immunization Two weeks of mouse plasma were analyzed for immune responses to the injected peptide (i.e., SeqID35/36) and to the target protein (i.e., recombinant human Tau441). SeqID35/36 is a well-known and effective Tau epitope, spanning aa294-305 in human 4Rtau441, and was selected as a functionally effective epitope in EP2758433. result:

As shown in Figure 18, both vaccines were able to induce strong and specific immune responses against the injected peptide moiety and the target protein Tau441. However, compared to SeqID35+KLH with Alhydrogel as adjuvant (the better vaccine conjugate against Tau441 according to EP2758433), the vaccine based on SeqID36+SeqID7+CLEC showed a 2.3-fold higher immune response against the injected peptide moiety, most importantly Interestingly, the immune response against the target protein Tau441 was also 3.3 times higher.

In addition, avidity analysis using anti-thiocyanate elution (NaSCN) showed that the affinity of SeqID36+SeqID7+CLEC-inducing antibodies to SeqID35 was 2.3 times higher than that of SeqID35+KLH-inducing antibodies.

Therefore, CLEC-based vaccines are well suited to use antigenic determinants against intracellular proteins as immunogens, thereby conferring higher immune responses and higher target-specific responses as compared to conventional vaccines.

Furthermore, this example provides clear evidence that CLEC-based vaccines using target protein epitopes present in tauopathy and Alzheimer's disease are inducing surprisingly superior and more specific immune responses against self-epitopes present in tau compared to current state-of-the-art tau-targeted vaccines. Tauopathy is a neurodegenerative disease characterized by abnormal tau protein deposits in the brain. The spectrum of tauopathies extends beyond the traditionally discussed disease forms such as Pick's disease, progressive supranuclear palsy, corticobasal degeneration, and argyrophilic granulopathy to include glomerular neurological tauopathies, primary age-related tauopathies including neurofibrillary entanglement dementia, chronic traumatic encephalopathy (CTE), and aging-related tauo-astrocytopathy. Example 18 : B -cell epitope analysis of secreted proteins, autoantigens, and conformational epitopes : IL23

To evaluate whether peptide vaccines carrying peptides derived from secreted proteins, whether they constitute self-antigens (e.g., SeqID37 to SeqID42) or foreign target structures, can generate high immune responses after repeated immunizations and induce immune responses superior to those of traditional conjugate vaccines, six different candidate vaccines were tested:

In this experiment, three different IL23-derived peptides were used as peptide-CLEC vaccines (i.e., SeqID38, SeqID40, SeqID42 in combination with the pan-T cell epitope SeqID7, coupled to oxidized Pseudomonas aeruginosa (80%;) via a C-terminal hydrazide linker) or as known peptide conjugates, generated using SeqID37, SeqID39 and SeqID41 containing a C-terminal cysteine, for coupling to GMBS-activated KLH. Vaccines used: B cell antigen determinant T cell antigen determinant / vector CLEC Adjuvant Way SeqID38 SeqID7 Pyricularia polysaccharide (80%) na id SeqID37 KLH na Alhydrogel sc SeqID40 SeqID7 Pyricularia polysaccharide (80%) na id SeqID39 KLH na Alhydrogel sc SeqID42 SeqID7 Pyricularia polysaccharide (80%) na id SeqID41 KLH na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated three times at biweekly intervals (route: id for CLEC-based vaccines, sc for KLH-based vaccines with Alhydrogel as adjuvant) and subsequent immune responses to the injected peptides (i.e., SeqID37, SeqID39, and SeqID41) and to the target protein (i.e., recombinant human IL23) were analyzed using mouse plasma collected two weeks after the third immunization. Results:

As shown in Figure 19, all six vaccines were able to induce strong and specific immune responses against the injected peptide moieties and the target protein: IL23 (p12/p40).

However, compared with SeqID37+KLH adjuvanted with Alhydrogel, the vaccine based on SeqID38+SeqID7+CLEC showed a 2-fold higher immune response against the injected peptide moiety and, most importantly, a higher immune response against the target protein IL23. 3 times. SeqID37/SeqID38 represent conformational epitopes on the D1 domain of the p40 subunit of IL-12 and IL-23, which reflect specific binding to IL-12/IL-23p40 and neutralization of human IL-12 and IL- 23 epitopes of the biologically active fully human monoclonal antibody Ustekinumab (Luo et al., J Mol Biol 2010 Oct 8; 402(5)): 797-812.).

In contrast, Alhydrogel-adjuvanted SeqID39+KLH and SeqID40+SeqID7+CLEC-based vaccines induced similar responses against the injected peptide moiety and the target protein IL23. Interestingly, SeqID39/SeqID40 is a peptide spanning the linear epitope aa38-46 in the p40 subunit of IL12 and IL23 respectively (Guane et al., 2009).

Compared to SeqID38 and SeqID40, which showed comparable or very superior anti-target responses using the CLEC backbone, SeqID41/42 did not show the same characteristics, which supports the fact that the selected peptide immunogens are suitable for the surprising effects provided in these examples. SeqID41/42 are peptides spanning the linear epitope aa144-154 in the p19 subunit of IL23, respectively. SeqID41+KLH adjuvanted with Alhydrogel induced 15-fold higher responses against the injected peptide portion and 8-fold higher responses against the target protein IL23 compared to the SeqID42+SeqID7+CLEC vaccine used in this experiment.

In summary, CLEC-based vaccines are well suited to use epitopes targeting secreted proteins (including signaling molecules or interleukins/chemokines) as immunogens, which can confer higher immune responses and higher immunity than conventional vaccines. target-specific response.

Furthermore, this example provides clear evidence that CLEC-based vaccines are suitable for the use of conformational epitopes and that conformational epitopes can induce better immune responses when administered as CLEC-based vaccines.

This example also provides evidence that CLEC-based immunogens using IL12/IL23 epitopes surprisingly induce more specific immune responses than prior art vaccines directed against these self-epitopes. Therefore, such vaccines may be useful in treating IL12/IL23-related autoimmune inflammatory diseases, including: psoriasis, chronic inflammatory bowel disease, rheumatoid arthritis. Example 19 : B cell epitope analysis of self-epitopes present in transmembrane proteins : Extracellular membrane proximal domain (EMPD) of membrane-bound IgE

In this experiment, peptides derived from human EMPD were used. SeqID43/SeqID44 constitutes an epitope disclosed in WO2017/005851A1 and is used as a peptide-CLEC vaccine (i.e.: SeqID44 is combined with the pan-T cell epitope SeqID7, via a C-terminal hydrazine linker and oxidized stone Auritic polysaccharides (80%; coupling) or as known peptide conjugates were generated using SeqID43 containing a C-terminal cysteine for coupling to GMBS-activated KLH. Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way SeqID44 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID43 htK na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (route: CLEC-based vaccine with id, KLH-based vaccine with Alhydrogel as adjuvant with sc), using the third immunization Rat plasma collected two weeks later was subjected to subsequent immunoreactions against the injected peptide (i.e. SeqID43) and against the 41 amino acid fragment of the target protein region EMPD (revealed in WO2017/005851A1 as a suitable surrogate for protein recognition). analyzed. result:

As shown in Figure 20, both vaccines were able to induce strong and specific immune responses against the injected peptide portion and EMPD protein fragment.

However, compared with SeqID43+KLH adjuvanted with Alhydrogel, the vaccine based on SeqID44+SeqID7+CLEC showed an approximately 60% increase in immune response against the injected peptide portion and, most importantly, a 30% increase in immune response against the target protein fragment.

Furthermore, avidity analysis using anti-thiocyanate elution (NaSCN) showed that the SeqID44+SeqID7+CLEC-induced antibody had a 3.8-fold higher affinity for the EMPD peptide compared to the SeqID43+KLH-induced antibody.

In summary, CLEC-based vaccines are well suited to use epitopes targeting transmembrane proteins, including the extracellular membrane proximal domain (EMPD) of membrane-bound IgE, which can confer higher immune responses than conventional vaccines and Higher target-specific response.

Therefore, it is apparent that the CLEC-based vaccine according to the present invention can be preferably used for active anti-EMPD vaccination for the treatment and prevention of IgE-related diseases. IgE-related diseases include allergic diseases such as seasonal, food, pollen, mold spores, poisonous plants, drugs, insect, scorpion or spider venom, latex or dust allergy, pet allergy, allergic bronchial asthma, non-allergic asthma, Churg-Strauss syndrome, allergic rhinitis and conjunctivitis, atopic dermatitis, nasal polyposis, Kimura's disease, adhesive contact dermatitis, antibacterial agents, fragrances, hair dyes, metals, rubber components, topical medications, rosin, wax, polishes, cement and leather, chronic sinusitis, atopic eczema, Ig E-related autoimmune diseases ("autoallergies"), chronic (idiopathic) and autoimmune urticaria, choleretic 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, eosinophilic-related diseases, such as eosinophilic asthma, eosinophilic gastroenteritis, eosinophilic otitis media, and eosinophilic esophagitis (see Holgate World Allergy Organization Journal 2014, 7:17, US8,741,294B2, Usatine Am Fam Physician. 2010 Aug 1;82(3):249-55.). In addition, the vaccine according to the present invention can be used to treat lymphoma or prevent the allergic side effects of antacid therapy, especially for gastric or duodenal ulcers or reflux. For the present invention, the term "IgE-related disease" includes or is used synonymously with the term "IgE-dependent disease" or "IgE-mediated disease". Example 20 : B cell epitope analysis of mimetic epitopes and conformational epitopes of allergens : Betv1

To evaluate whether peptide vaccines carrying peptides derived from foreign proteins/allergens can generate high immune responses after repeated immunizations and induce immune responses superior to traditional conjugate vaccines, 2 different vaccine candidates were tested:

The present experiments used peptides SeqID45/SeqID46 derived from the well-known major birch ( Betula verrucosa ) pollen antigen Bet v 1. SeqID45/SeqID46 constitutes a mimetic epitope of the native sequence of Bet v 1 (Immunol Lett. 2009 Jan 29; 122(1): 68-75.). In addition, the authors showed that antibodies induced by this mimetic epitope can bind to two different regions in Bet v 1, namely amino acids 9-22 and 104-113. Therefore, the mimetic epitope SeqID45/SeqID46 is also an example of a conformational epitope.

SeqID45/SeqID46 are used as peptide-CLEC vaccines (i.e., SeqID46 in combination with the pan-T cell epitope SeqID7, coupled to oxidized auricularia polysaccharide (80%;) via a C-terminal hydrazine linker) or as a habit Peptide conjugates, generated using SeqID45 containing a C-terminal cysteine, were used for coupling to GMBS-activated KLH. Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way SeqID46 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID45 htK na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (route: CLEC-based vaccine with id, KLH-based vaccine with Alhydrogel as adjuvant with sc), using the third immunization Rat plasma collected two weeks later was analyzed for subsequent immune responses to the injected peptide (i.e., SeqID45) and to the target protein (i.e., recombinant Betv1). result:

As shown in Figure 21, both vaccines were able to induce strong and specific immune responses against the injected peptide moiety and the target protein: Betv1.

However, compared with SeqID45+KLH adjuvanted with Alhydrogel, the vaccine based on SeqID46+SeqID7+CLEC showed a 3.3-fold higher immune response against the injected peptide moiety and, most importantly, a 2-fold higher immune response against the target protein Betv1. times.

In addition, affinity analysis using anti-thiocyanate elution (NaSCN) showed that the affinity of SeqID46+SeqID7+CLEC induced antibodies to recombinant BetvI was 1.9-fold higher than that of SeqID45+KLH induced antibodies.

In conclusion, CLEC-based vaccines are well suited to use allergen epitopes including Betv1, conferring higher immune responses and higher target-specific responses compared with conventional vaccines. Furthermore, this example provides clear evidence that CLEC-based vaccines are suitable for the use of mimetic and conformational epitopes and that such mimetic and conformational epitopes when administered as a CLEC-based vaccine can induce excellent immune responses. Therefore, such vaccines can be used to treat allergic diseases such as hay fever, seasonal, food, pollen, mold spores, poisonous plants, drugs, insect, scorpion or spider venom, latex or dust allergy, pet allergies, allergic bronchial asthma , allergic rhinitis and conjunctivitis, atopic dermatitis, adhesive contact dermatitis, antibacterial agents, fragrances, hair dyes, metals, rubber ingredients, topical drugs, rosin, wax, polishes, cement and leather, chronic sinusitis, Atopic eczema, autoimmune diseases in which IgE plays a role ("autoallergy"), chronic (idiopathic) and autoimmune urticaria, allergic reactions, especially idiopathic and exercise-induced allergic reactions. Example 21 : Analysis of B cell epitopes present in different forms of cancer / tumor diseases ( i.e. oncogenes ) : Her2

To evaluate whether peptide vaccines carrying peptides derived from cancer-associated antigens/oncogenes can generate high immune responses after repeated immunizations and induce immune responses superior to traditional conjugate vaccines, 2 different candidates were tested in this experiment vaccine:

In this experiment, peptides SeqID47/SeqID48 derived from Her2, a well-known member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family, were used.

SeqID47/SeqID48 constitute the epitope of the native sequence of the human Her2 extracellular domain: aa positions 610-623. The epitope SeqID47/SeqID48 has been revealed as a powerful antigen by Wagner et al. (2007) and Tobias et al. (2017), which are respectively present in conventional conjugate vaccines, such as peptide-tetanus toxoid and peptide-CRM197 conjugates. .

SeqID47/SeqID48 were used as peptide-CLEC vaccines (i.e., SeqID48 combined with the pan-T cell epitope SeqID7, coupled to oxidized Pseudomonas aeruginosa (80%; ) via a C-terminal hydrazide linker) or as known peptide conjugates, generated using SeqID47 containing a C-terminal cysteine (part of the native sequence), for coupling to maleimide-activated CRM197. Vaccines used: B cell antigen determinant T cell antigen determinant / vector CLEC Adjuvant Way SeqID48 SeqID7 Pyricularia auricula polysaccharide (80%) na id SeqID47 CRM197 na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (route: CLEC-based vaccine for id, CRM197-based vaccine for sc with Alhydrogel as adjuvant), using the third immunization Rat plasma collected two weeks later was analyzed for subsequent immune responses to the injected peptide (i.e., SeqID47) and to the target protein (i.e., recombinant human Her2). result:

As shown in Figure 22, both vaccines were able to induce strong and specific immune responses against the injected peptide moiety and the target protein: Her2.

However, compared to SeqID47+CRM197 with Alhydrogel as adjuvant, the vaccine based on SeqID48+SeqID7+CLEC showed a 23% higher immune response against the injected peptide part and, most importantly, a 30% higher immune response against the target protein Her2.

In conclusion, CLEC-based vaccines are very suitable for use as cancer vaccines, conferring higher immune responses and higher target-specific responses compared to conventional vaccines (such as CRM197-based conjugate vaccines). Therefore, it is clear that such vaccines can be used to treat neoplastic diseases. Example 22 : Analysis of B cell epitopes present in different forms of neoplastic disease / cancer ( i.e. oncogenes ) : PD1

To evaluate whether peptide vaccines carrying peptides from cancer-related antigens/oncogenes can generate high immune responses after repeated immunizations and induce immune responses superior to those of traditional conjugate vaccines, different candidate vaccines targeting programmed cell death protein 1 (PD1) were tested:

PD-1 is an immune checkpoint that prevents autoimmunity by two mechanisms: 1) It promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes. 2) It reduces apoptosis of regulatory T cells (anti-inflammatory, suppressive T cells). Downregulating the activity of immune checkpoints such as PD1 signaling (by blocking PD1 activation) is a recently developed strategy to activate the immune system to attack tumors, which is currently used to treat certain types of cancer. Suitable B cell epitopes and prototype vaccines targeting human PD1 have been revealed.

In this experiment, peptide SeqID49/SeqID50, derived from human PD1, aa position 92-110, has been used as a B cell epitope. This epitope has been revealed by Kaumaya et al. (ONCOIMMUNOLOGY 2020, VOL. 9, NO. 1, e1818437) as a potent antigen in a fusion peptide-based vaccine, in which the PD1 epitope is linked to measles virus fusion peptide (MVF) amino acids (aa288-302) via a tetraamino acid residue (GPSL) and emulsified in the adjuvant Montanide ISA 720VG to induce antibodies that block PD-1 signaling.

SeqID49/SeqID50 were either used as peptide-CLEC vaccines (i.e., SeqID50 combined with the pan-T cell epitope SeqID7 coupled to oxidized Pseudomonas aeruginosa (80%; ) via a C-terminal hydrazide linker) or as known peptide conjugates using SeqID49 containing a C-terminal cysteine (part of the native sequence) coupled to maleimide-activated KLH. Vaccines used: B cell antigen determinant T cell antigen determinant / vector CLEC Adjuvant Way SeqID50 SeqID7 Pyricularia auricula polysaccharide (80%) na id SeqID49 KLH na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated 3 times every two weeks (route: CLEC-based vaccine with id, KLH-based vaccine with Alhydrogel as adjuvant with sc), after the third immunization Two weeks of mouse plasma were analyzed for subsequent immune responses to the injected peptide (i.e., SeqID49) and to the protein of interest (i.e., recombinant human PD1). result:

As shown in Figure 23A, both vaccines were able to induce strong and specific immune responses against the injected peptide portion and the target protein: PD1.

However, compared to SeqID49+KLH with Alhydrogel as adjuvant, the vaccine based on SeqID50+SeqID7+CLEC showed similar high immune responses against the injected peptide part, but most importantly, also 2-fold higher immune responses against the target protein PD1, indicating a different development of antibodies that specifically detect the selected target protein compared to the conventional vaccine.

In addition, affinity analysis using anti-thiocyanate elution (NaSCN) showed (Figure 23B) that the antibodies induced by SeqID50+SeqID7+CLEC had a 4.5-fold higher affinity for SeqID49 compared to the antibodies induced by SeqID49+KLH.

In summary, CLEC-based vaccines are very suitable for use as cancer vaccines, especially by targeting immune checkpoints, such as the PD-PDL1/2 system or CTLA4, to confer higher immune responses, especially when compared with conventional vaccines (e.g., KLH-based vaccines). Conjugate vaccines also have higher target-specific responses than conjugate vaccines. It is therefore also evident that such vaccines could be used to treat neoplastic diseases. Example 23 : Immunogenicity Analysis of CLEC Conjugates Using Carrier Proteins as Helper T Cell Epitopes : Different Conjugate /CLEC Ratios

In this example, the immunogenicity of CLEC-based conjugate vaccines containing the well-known carrier protein CRM197 and using different peptide-CRM/CLEC ratios was compared. To this end, the aSyn-derived epitope SeqID6 was coupled to cis-butylenediamide-activated CRM197. Subsequently, the SeqID6+CRM197 conjugate was coupled to activated Psoralea corylifolia polysaccharide via the heterobifunctional linker BPMH at different w/w ratios to form CLEC-based conjugate vaccines, in which CRM197 served as a source of helper T cell epitopes to induce a sustained immune response. Vaccines used: B cell antigen determinant T cell antigen determinant / vector CLEC Ratio (w/w) Adjuvant Way SeqID6 CRM197 Pyricularia auricula polysaccharide (80%) 1/1 na id SeqID6 CRM197 Pyricularia auricula polysaccharide (80%) 1/2.5 na id SeqID6 CRM197 Pyricularia auricula polysaccharide (80%) 1/5 na id SeqID6 CRM197 Pyricularia auricula polysaccharide (80%) 1/10 na id SeqID6 CRM197 Pyricularia auricula polysaccharide (80%) 1/20 na id

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (all vaccines: 5 μg aSyn targeting peptide/dose; route: CLEC-based vaccine id) and immune responses against the injected peptide (i.e., SeqID6), against the target protein (i.e., recombinant human α-synuclein), and against aSyn fibers were analyzed using mouse plasma collected 2 weeks after the third immunization. Results:

As shown in FIG. 24 , all five vaccines using CRM197 as a source of helper T antigenic determinants were able to induce strong and specific immune responses against the injected peptide portion (SeqID6) and the target protein: recombinant α-synuclein.

The CLEC modification of the CRM197 conjugate induced a highly efficient immune response, and all w/w conjugate/CLEC ratios were tested in this example. SeqID6-CRM197-Pyrrolidone (w/w 1/10) provided the highest anti-aSyn specific immune response compared to the other variants tested. Therefore, SeqID6+CRM197 conjugates with medium/high conjugate/CLEC ratios were particularly suitable for inducing the best immune response (e.g., 1/5, 1/10, and 1/20).

Therefore, this experiment demonstrates that CLEC modification of known peptide-protein conjugates causes subsequent immune responses with strong target specificity, thus providing an unprecedented new strategy to optimize the construction of carrier proteins (such as KLH, CRM197 or other proteins), currently advanced technology conjugate vaccines. Example 24 : Immunogenicity analysis of CLEC conjugates and peptide conjugates using carrier proteins as helper T cell epitopes - aSyn N- terminus (aa1-10)

In this example, it was assessed whether the CLEC-based conjugate vaccine according to the invention could induce a better immune response against aSyn aggregates compared to the corresponding peptide conjugate using a state-of-the-art carrier protein as source of T cell antigenic determinants.

Therefore, a set of experiments was initiated comparing two conjugates containing an epitope suggested to be suitable as aSyn targeting epitope. The experiments allowed to demonstrate the selectivity of the immune response elicited against the injected peptide and aSyn protein, and subsequently against two different forms of the presynaptic protein aSyn: non-aggregated, mainly monomeric aSyn and aggregated aSyn filaments.

For example, Weihofen et al. (Neurobiology of Disease 124 (2019) 276-288, aa1-10 as an epitope of Cinpanemab) and WO2016/062720 (aa1-8 as an epitope in VLP-based immunotherapy) showed that derived from The N-terminal aSyn sequence at position aa1-10 serves as a potentially suitable epitope for aSyn-targeted immunotherapy. In order to evaluate whether CLEC modification indeed results in a better immune response, a CLEC-based vaccine containing aSyn sequence aa1-8 (SeqID12+SeqID7+Fructus polysaccharide) was compared with the corresponding conventional peptide-KLH vaccine (using Alum as adjuvant). SeqID13+KLH) were compared. Vaccines used: B cell epitope T cell epitope/ carrier CLEC Adjuvant way SeqID12 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID13 htK na Alum ID

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (all vaccines: 5 μgaSyn targeting peptide/dose; route: CLEC-based vaccine with id, KLH-based vaccine with Alhydrogel as adjuvant The subsequent immune response against the injected peptide and target protein was determined sc) and by ELISA and EC50 values. Additionally, to assess the selectivity of the immune response, aSyn-specific inhibition ELISA was performed on plasma samples and expressed as percent maximal binding. result:

Compared to the known peptide-conjugate vaccine (i.e., SeqID13+KLH, see Figure 25A), the targeted CLEC-based conjugate vaccine used in this experiment (SeqID12+SeqID7+Pseudomonas aeruginosa) exhibited superior immunogenicity against aSyn protein. As a comparison group, the CLEC-based vaccine induced a 1.8-fold increase in anti-aSyn titers, accompanied by a 3-fold increase in the ratio of anti-peptide to anti-protein responses. This strongly supports the teaching of the present invention that CLEC modification elicits a superior immune response compared to similar known vaccines.

Furthermore, the known peptide KLH-coupled vaccine induced a greatly increased (about 10-fold) antibody response that was selective for aSyn monomers compared to aggregates (i.e., filaments, see Figure 25B). In contrast to this finding and very surprisingly, the CLEC-based conjugates elicited a completely different selectivity: the SeqID12+SeqID7+Pseudomonas polysaccharide-induced antibodies showed a significant increase in selectivity for aSyn aggregates compared to recombinant aSyn, by about 10-fold, completely changing the profile of antibody induction (see Figure 25B).

Thus, the present experiments demonstrate that known peptide vaccines using aSyn aa1-8 are poorly suited to generate effective and selective immune responses in vivo, indicating that this antigenic determinant is not suitable for aggregate-selective immunotherapy. Importantly, the results also demonstrate that CLEC modification of the peptide conjugates results in a greatly enhanced target specificity of the subsequent immune response and a change in selectivity for aggregates, thus providing an unprecedented conjugate vaccine targeting aSyn. Example 25 : Immunogenicity Analysis of CLEC Conjugates and Peptide Conjugates Using Carrier Proteins as Helper T Cell Epitopes - aSyn aa100-108

Here, a set of experiments was performed to compare two conjugates, both containing an epitope suggested to be suitable as an aSyn targeting epitope, by analyzing the selectivity of the immune response elicited against the injected peptide and aSyn protein and the subsequent immune response elicited against two different forms of the presynaptic protein aSyn: non-aggregated, mainly monomeric aSyn and aggregated aSyn filaments.

For example, WO2011/020133 and WO2016/062720 suggest aSyn sequences derived from positions aa 100-108/109 (as native sequences or simulated epitopes, i.e. 100-108) as potentially suitable antigens for aSyn targeted immunotherapy Determining base. In order to evaluate whether CLEC modification would indeed produce a better immune response using this epitope region, a CLEC-based vaccine containing aSyn aa 100-108 (SeqID16+SeqID7+Shitia polysaccharide) was compared with the corresponding conventional peptide-KLH vaccine. (SeqID17+KLH using Alum as adjuvant) was compared. Vaccines used: B cell epitope T cell epitope/ carrier CLEC Adjuvant way SeqID16 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID17 htK na Alum ID

Animals (female Balb/c mice) were vaccinated 3 times every two weeks (all vaccines: 5 μg aSyn targeting peptide/dose; route: id for CLEC-based vaccines, sc for KLH-based vaccines with Alhydrogel as adjuvant) and the subsequent immune responses against the injected peptides and target proteins were determined by ELISA and EC50 values. In addition, to assess the selectivity of the immune response, aSyn-specific inhibition ELISA was performed on plasma samples and expressed as percentage of maximum binding. Results:

The CLEC-based aSyn-targeted conjugate vaccine (SeqID16+SeqID7+Pseudomonas aeruginosa) used in this experiment demonstrated an overall very low anti-aSyn protein response, which was also lower than the known peptide-conjugate vaccine (i.e., SeqID17+KLH, see Figure 26A). Compared to the CLEC-based vaccine, the known vaccine induced an immune response characterized by a 2.1-fold increase in anti-aSyn titer, but at the same time a 2-fold decrease in anti-peptide/anti-protein titer. The latter finding supports the teaching of the present invention that CLEC modifications elicit better anti-target protein responses, even at a lower overall immunogenicity than similar known vaccines.

Furthermore, both vaccines, namely the conventional peptide conjugate and the CLEC-based vaccine, are not well suited for inducing aggregation-selective immune responses (see Figure 26B). Therefore, the experiments provided show that CLEC-based peptide vaccines targeting the aa100-108 region and conventional peptide vaccines are less suitable for generating effective and selective immune responses in vivo, indicating that this epitope may be used in accordance with the present invention. Not the best choice for aggregate-selective immunotherapy. Example 26 : Immunogenicity analysis of CLEC conjugates and peptide conjugates using carrier proteins as helper T cell epitopes - aSyn aa91-100

In this example, a set of experiments was initiated to compare two conjugates, both of which contained an antigenic determinant that was considered suitable as an aSyn targeting antigenic determinant by analyzing the immune response elicited against injected peptides and aSyn protein.

For example, US 2014/0377271 A1 showed that the antigenic determinant aa91-99 acts as an autoantigen in PD patients and should therefore constitute a potentially suitable antigenic determinant for aSyn targeted immunotherapy. In order to assess whether CLEC modification indeed elicits a superior immune response using this antigenic determinant, a CLEC-based vaccine containing aSyn aa91-100 (SeqID14+SeqID7+Pyricularia auriculariae) was compared with the corresponding known peptide-KLH vaccine (SeqID15+KLH with Alum as adjuvant). Vaccines used: B cell antigen determinant T cell antigen determinant/ vector CLEC Adjuvant Way SeqID14 SeqID7 Pyricularia auricula polysaccharide (80%) na id SeqID15 KLH na Alum id

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (all vaccines: 5 μg aSyn targeting peptide/dose; route: id for CLEC-based vaccines, sc for KLH-based vaccines with Alhydrogel as adjuvant) and the subsequent immune responses against the injected peptides and the target protein aSyn were determined by ELISA and EC50 values.

Surprisingly, both vaccines induced relevant anti-peptide titers, but failed to induce detectable anti-aSyn protein titers (see Figure 27). Thus, the experiments provided show that CLEC-based and traditional peptide vaccines targeting the aa 91-100 region are less suitable for generating effective and selective immune responses in vivo, suggesting that this epitope may not be the best choice for polymerized selective immunotherapy according to the present invention. Example 27 : Immunogenicity Analysis of CLEC Conjugates and Peptide Conjugates Using Carrier Protein as Helper T Cell Epitope - aSyn C- terminal Region aa131-140

In this example, a set of experiments was initiated to compare two conjugates, both of which contained an epitope that was deemed suitable by analyzing the immune response elicited against the injected peptide and the aSyn protein. As aSyn targets epitopes. In addition, the selectivity of the subsequent immune response elicited against two different forms of the presynaptic protein aSyn was evaluated.

For example, US 2015/0232524 and WO 2016/062720 proposed C-terminal aSyn sequences from positions aa-126-140 and 131-140 as potential suitable antigenic determinants for aSyn targeted immunotherapy. To evaluate whether CLEC modification would indeed result in a better immune response, a CLEC-based vaccine containing aSyn aa131-140 (SeqID20+SeqID7+Pyricularia auriculariae) was compared with the corresponding known peptide-KLH vaccine (SeqID21+KLH with Alum as adjuvant). Vaccines used: B cell antigen determinant T cell antigen determinant/ vector CLEC Adjuvant Way SeqID20 SeqID7 Pyricularia auricula polysaccharide (80%) na id SeqID21 KLH na Alum id

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (all vaccines: 5 μgaSyn targeting peptide/dose; route: CLEC-based vaccine with id, KLH-based vaccine with Alhydrogel as adjuvant The subsequent immune response against the injected peptide and target protein was determined sc) and by ELISA and EC50 values. Additionally, to assess the selectivity of the immune response, aSyn-specific inhibition ELISA was performed on plasma samples and expressed as percent maximal binding. result:

Compared to the known peptide-conjugate vaccine (i.e., SeqID21+KLH, see Figure 28A), the CLEC-based aSyn-targeted conjugate vaccine (SeqID20+SeqID7+Pseudomonas aeruginosa) used in this experiment demonstrated an overall lower anti-aSyn protein response. Compared to the CLEC-based vaccine, the immune response induced by the known vaccine was characterized by a 1.8-fold increase in anti-aSyn titer, but a 45% decrease in the ratio of anti-peptide/anti-protein titer. The latter finding supports the teaching of the present invention that CLEC modification elicits a better anti-target protein response, even at a lower overall immunogenicity than a similar known vaccine.

Furthermore, known peptide conjugates are less suitable for inducing polymerization-selective immune responses (see Figure 28B). In contrast, antibodies generated by CLEC-based vaccines were approximately 10-fold more selective for monomeric aSyn at the expense of aggregated aSyn (see Figure 28B). Therefore, the experiments presented show that CLEC-based and conventional peptide vaccines targeting the aa131-140 region are less suitable to generate an effective and selective immune response to aggregated aSyn in vivo, indicating that this antigen determines the may not be the best choice for aggregation-selective immunotherapy. Example 28 : Immunogenicity analysis of CLEC conjugates and peptide conjugates using carrier proteins as helper T cell epitopes - aSyn C- terminal region aa103-135

In this example, it was assessed whether the CLEC-based conjugate vaccine according to the present invention could induce a better immune response against aSyn compared to the corresponding peptide conjugate using a state-of-the-art carrier protein as the source of T cell antigenic determinants.

Therefore, a set of experiments was initiated to compare several binders derived from epitope regions suggested to be suitable as aSyn targeting epitopes. These experiments allowed to demonstrate the selectivity of the immune response elicited against the injected peptide and aSyn protein, and subsequently against two different forms of the presynaptic protein aSyn: non-aggregated, mainly monomeric aSyn and aggregated aSyn filaments.

Several studies have shown that the C-terminal aSyn sequence originating from the position aa103-135 is a potential suitable epitope for aSyn targeted immunotherapy, either as a source of self-epitopes or peptides containing its original sequence or its mimicking epitopes. In order to assess whether CLEC modification would indeed lead to a better immune response using this region in aSyn, several CLEC-based vaccines (using peptides within the region 107-126) were compared with the corresponding known peptide-CRM vaccine (using Alum as adjuvant). Vaccines used: B cell antigen determinant T cell antigen determinant / vector CLEC Adjuvant Way SeqID56 SeqID7 Pyricularia polysaccharide (80%) na id SeqID58 CRM na Alum sc SeqID53 SeqID7 Pyricularia auricula polysaccharide (80%) na id SeqID55 CRM na Alum sc SeqID51 SeqID7 Pyricularia auricula polysaccharide (80%) na id SeqID52 CRM na Alum sc SeqID65 SeqID7 Pyricularia auricula polysaccharide (80%) na id SeqID66 CRM na Alum sc SeqID67 SeqID7 Pyricularia auricula polysaccharide (80%) na id SeqID68 CRM na Alum sc SeqID69 SeqID7 Pyricularia auricula polysaccharide (80%) na id SeqID70 CRM na Alum sc SeqID71 SeqID7 Pyricularia auricula polysaccharide (80%) na id SeqID72 CRM na Alum sc

Animals (female Balb/c mice) were vaccinated 3 times every two weeks (all vaccines: 5 μg aSyn targeting peptide/dose; route: id for CLEC-based vaccines, sc for CRM-based vaccines with Alum as adjuvant) and the subsequent immune responses against the injected peptides and target proteins were determined by ELISA and EC50 values. In addition, to assess the selectivity of the immune response, aSyn-specific inhibition ELISA was performed on plasma samples and expressed as percentage of maximum binding. Results: vaccine Peptide titer (x1000) aSyn fiber potency (x1000) SeqDI56+SeqID7+Pyricularia polysaccharide 0,1 0,1 SeqID58+CRM+Alum 9,781 0,1 SeqDI53+SeqID7+Pyricularia auricula 0,1 0,1 SeqID55+CRM+Alum 17,647 0,8 SeqDI51+SeqID7+Pyricularia auricula 19,694 1,687 SeqID52+CRM+Alum 13,115 1,594 SeqDI65+SeqID7+Pyricularia polysaccharide 9,04 3,256 SeqID66+CRM+Alum 7,984 1,108 SeqDI67+SeqID7+Pyricularia auricula polysaccharide 12,016 7,335 SeqID68+CRM+Alum 18,126 1,864 SeqDI69+SeqID7+Pyricularia auricula 28,125 3,523 SeqID70+CRM+Alum 37,88 1,054 SeqDI71+SeqID7+Pyricularia auricula polysaccharide 7,783 5,788 SeqID72+CRM+Alum 14,603 2,429 Table 3: Immune responses induced by vaccines covering aa107-126

Both CLEC-based and CRM-based vaccines containing 5- and 6-mer peptides were less suitable for inducing high anti-aSyn silk titers in this experiment. Compared with conventional peptide conjugate vaccines, the CLEC-based conjugate vaccines (7- to 12-mer peptides) targeting the C-terminus of aSyn used in this experiment (see Table 3 and Figures 29A, 30A, and 31A) all showed strong efficacy against aSyn. Excellent immunogenicity of filaments (see Table 3, up to 4-fold increase). This strongly supports the teaching of the present invention that CLEC modifications elicit superior immune responses compared to similar conventional vaccines using epitopes derived from 103-135, especially 107-126.

Selectivity analysis of polymerized aSyn further supports this teaching. As shown in Figures 29B and 30B, a CLEC vaccine containing an epitope derived from sequence aa115-126 was unexpectedly effective in eliciting a highly aggregated selective immune response. As shown in Figure 29B, the CLEC-based vaccine SeqID51+SeqID7+Shitu polysaccharide contains an 8-mer aSyn targeting epitope, and the induced antibodies are 10 times more selective for aSyn aggregates, while the corresponding conventional vaccine (SeqID52+CRM+Alum) cannot induce aggregation-selective antibodies. Similarly, the CLEC-based vaccine SeqID67+SeqID7+Shitu polysaccharide, which contains a 10-mer aSyn targeting epitope, induced approximately 10 times higher selectivity of aSyn aggregates compared with monomers, and the corresponding The antibodies elicited by the conventional vaccine (SeqID68+CRM+Alum) are more selective for monomers, about 3 times more selective than aggregates (see Figure 30B).

Selectivity analysis of vaccines containing the epitope aa107-114 (SeqID73+SeqID7+Fructus polysaccharide and SeqID74+CRM+Alum, see Figure 31B) surprisingly showed that despite the presence of high resistance elicited by the CLEC-based vaccine aSyn titer (i.e., superior immunogenicity), however neither the CLEC vaccine nor the conventional vaccine was able to induce aggregation-selective antibodies, suggesting that only the highly selected peptide sequence in aa103-135 is suitable as a specific target for aggregation aSyn's immunotherapy.

As shown above (see Figures 26-28), it can also be noted that the epitopes derived from aa91-100, aa100-108 and aa131-140 are all less suitable as potential immunotherapeutic regions for specifically targeting aggregated aSyn. Example 29: In vitro functional analysis of immune responses elicited by CLEC -based vaccines

In order to analyze whether aSyn-specific antibodies elicited by a CLEC-based vaccine (containing aSyn-targeting peptide from the epitope region aa103-135) are biologically active, a set of experiments was performed to analyze the inhibition of aSyn in vitro by the antibodies. The ability to gather. Vaccines used: B cell epitope T cell epitope/ carrier CLEC Adjuvant way SeqID67 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID68 CRM na Alhydrogel sc SeqID71 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID72 CRM197 na Alhydrogel ID SeqID73 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID74 CRM197 na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (all vaccines: 20 µg aSyn targeting peptide/dose; route: id for CLEC-based vaccines, sc for adjuvanted with Alhydrogel of vaccines based on CRM197). The in vitro aggregation inhibition ability was analyzed through mouse plasma samples collected two weeks after each immunization and respective control samples (e.g., aSyn-conjugated antibody LB509, epitope aa115-122, or pre-immune plasma obtained before immunization). result:

As shown in Figure 32D, plasma obtained from animals before immunization had no significant effect on the aggregation kinetics of aSyn, confirming the specificity of the assay.

Antibodies induced by the SeqID67+SeqID7+Skeleton polysaccharide vaccine (containing a 10-mer aSyn-derived peptide) strongly inhibited aSyn aggregation, as demonstrated by a 40% reduction in aggregation over time in this assay, whereas the corresponding CRM conjugate vaccine showed only minimal effect, indicating that it has a higher inhibitory capacity compared with classical vaccine-induced antibodies (Figure 32A). Similar results were obtained by analyzing antibodies induced by SeqID71+SeqID7+ Shifu polysaccharide (containing 12-mer aSyn-derived peptide), which reduced aggregation more strongly (70-80% inhibition) and exceeded that of the conventional CRM vaccine (SeqID72+ The inhibitory capacity of antibodies induced by CRM+Alhydrogel is 2 to 2.5 times. In comparison, Figure 32C shows that neither the CLEC-based vaccine constructed based on the epitope aa107-114 (containing an 8-mer aSyn-derived peptide) nor the antibodies induced by conventional vaccines inhibited aSyn aggregation.

As shown in Figure 32D, aSyn-specific antibody LB509 was unable to inhibit aSyn aggregation. In contrast, a slight increase in aggregation was detectable in this analysis.

This is a very surprising effect in view of the teachings of the present invention (see Example 14 and Figure 15 for analysis of antigenic determinants derived from the aSyn sequence aa115-126, in particular aa115-121), as well as Table 1 and Figures 29-32 describing vaccines containing antigenic determinants 115-126), as the single strain LB509 (antigenic determinant aa:115-122) is known to bind to different forms of aSyn (Jakes et al. Neurosci. Lett. 1999 Jul 2;269(1):13-6) and has the same antigenic determinant as the biologically effective vaccine according to the present invention (see Figure 32D). Therefore, the superior biological effects obtained using the CLEC-based vaccine are indeed surprising and suggest that the highly selected peptide sequence contained in aa115-126 is superior as an immunotherapeutic agent that specifically targets aggregated aSyn in the aa103-135 region. Example 30 : Determination of in vitro biological activity of peptide -CRM197-CLEC conjugates against murine dectin -1 receptor

In a series of ELISA experiments, the binding efficacy of murine dectin-1 was evaluated for conjugates containing the dectin-1 ligands pyrrolidone, lichenin and laminarin. The bioactivity of the peptide + CRM197 + CLEC conjugate was indicated by its PRR binding capacity. Along these lines and to ensure that the structure of CLEC (pyrrolidone, lichenin, laminarin) remained bioactive after conjugation, binding to mouse dectin-1 was assessed. The bioactivity of unoxidized and oxidized Psoralen, lichenin and laminarin, as well as the CRM conjugate vaccine and the novel conjugate based on peptide+CRM197+CLEC, was then assessed using a competitive ELISA system based on competitive binding of soluble murine Fc-dectin-1a (InvivoGen) as described by Korotchenko et al. (2020) .

Subsequent experiments showed that the medium molecular weight (20 kDa) linear β-(1,6)-linked β-D-glucan, Pyrrolizan, and the β(1-6)-linked linear β(1-3)-glucan, Laminaria japonica, exhibited approximately 10 times higher efficiency in binding to mouse dectin-1 than the larger high molecular weight linear β-(1,3) β-(1,4)-β-D-glucan, lichenan (approximately 245 kDa) (see Figure 33).

As shown in Figure 33A, the dectin-1 ligand Schistocaryon polysaccharide, oxidized Schizophyllum polysaccharide, SeqID6+CRM conjugate (CRM conjugate 1) and SeqID6+CRM+ Schistocaryon polysaccharide conjugate (CRM-Sequencer polysaccharide) were evaluated by ELISA analysis. Binding effect of otic polysaccharide conjugate 1) on mouse dectin-1. Subsequent experiments showed that the peptide+CRM197+Fructus polysaccharide conjugate showed similar binding efficacy to murine dectin-1 as the oxidized Fructus polysaccharide. In contrast, traditional CRM conjugate 1 showed no specific mouse dectin-1 binding ability. Five new CRM-Fructus polysaccharide conjugates (SeqID52/66/68/70/72) also showed high binding efficacy to murine dectin-1 (Figure 33B). Subsequent experiments showed that the peptide-CRM197-Fructus polysaccharide interacts with different B cell epitopes, ranging from a 7-mer B cell epitope (SeqID6+CRM+Fructus polysaccharide; Figure 33A) to a 12-mer B cell epitope The base (SeqID71+CRM+Fructus polysaccharide; Figure 33B) showed similar mouse dectin-1 binding efficacy to oxidized Fructus polysaccharide. As shown in Figure 33C, whether oxidized or combined, linear β-(1,3) β-(1,4)-β-D glucan-lichen with high molecular weight (approximately 22-245 kDa) is more abundant than linear-based β-(1,3)-lichenin. The construct of β-(1,6)-linked β-D-glucan polysaccharide exhibits lower binding efficacy. For example, auricularia polysaccharides containing CRM197 peptide conjugates retained approximately 10-fold higher binding capacity than lichenin-based constructs. Laminarin, a linear β(1-3)-glucan with β(1-6)-linkages, also showed high binding efficacy to murine dectin-1 (Figure 33D). Subsequent experiments showed that regardless of oxidation or conjugation, the peptide + CRM197 + laminarin conjugate showed similar murine dectin-1 binding efficacy to the fungus polysaccharide-based construct.

The experiment showed that the peptide + CRM197 + CLEC conjugate exhibited biological activity against dendritic cells by binding to dectin-1 in the mouse system. Example 31 : Determination of the in vitro biological activity of the peptide -CRM197-CLEC conjugate against the human dectin -1 receptor

The binding efficacy of the dectin-1 ligands Shiguan, lichenin and laminarin to human dectin-1 was evaluated in a series of ELISA experiments. The biological activity of the peptide+CRM197+CLEC conjugate is expressed by its PRR binding ability. Along these lines and in order to ensure that the structure of CLEC (leafia polysaccharide, lichenin, laminarin) still maintains biological activity after coupling, through competitive binding based on soluble human Fc-dectin-1a receptor (InvivoGen) Competitive ELISA system to assess binding to human dectin-1. result:

As shown in FIG. 34 , the binding efficacy of SeqID6+CRM conjugates conjugated with lichenin (Lich conjugate), Psoralen polysaccharide (Pus conjugate) or laminarin polysaccharide (Lam conjugate) to human dectin-1 was assessed by ELISA analysis.

Subsequent experiments showed that the peptide + CRM197 + Psoralea corylifolia polysaccharide vaccine had a significantly higher binding potency to human dectin-1 than the vaccine combined with lichen polysaccharide (about 30 times) (see Figure 34). In contrast, the peptide + CRM197 + laminarin polysaccharide vaccine showed weak binding to human dectin-1. Example 32 : In vivo comparison of different vaccines based on peptide + CRM197 + Psoralea corylifolia polysaccharide

The novel CRM197-Auricularia polysaccharide vaccine has different B cell epitopes ranging from 8-mer to 11-mer that are able to bind to their DC receptors (e.g. dectin-1) and is tested for its ability to induce a strong and specific immune response after repeated administration to n=5 Balb/c mice/group. Typical experiments were performed using 5µg of the B cell epitope peptides per dose.

In this experiment, aSyn-derived peptides SeqID52+CRM197 and SeqID66/68/70+CRM conjugates were coupled to oxidized auricularia polysaccharides. Animals (female Balb/c mice) were vaccinated with β-glucan-modified or unmodified peptide-CRM conjugates 3 times at biweekly intervals (Route: id), using two days after the third immunization. Murine plasma collected weekly was analyzed for subsequent immune responses to the injected peptides (i.e., SeqID52/66/68/70, respectively) and to aggregated aSyn fibers. Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way SeqID52 CRM Shi fungus polysaccharide (80%) na ID SeqID66 CRM Shi fungus polysaccharide (80%) na ID SeqID68 CRM Shi fungus polysaccharide (80%) na ID SeqID70 CRM Shi fungus polysaccharide (80%) na ID SeqID52 CRM na Alhydrogel sc SeqID66 CRM na Alhydrogel sc SeqID68 CRM na Alhydrogel sc SeqID70 CRM na Alhydrogel sc result:

As shown in Figure 35A, compared with the unmodified peptide CRM-based vaccine with Alhydrogel as adjuvant, all four CRM-Agaricus polysaccharide-based vaccines (SeqID52/66/68/70/72) were able to Significantly enhanced responses were produced against injected peptide moieties (eg: SeqID52/66/68/70) and against aggregated aSyn fibers.

Compared with the unmodified peptide-CRM-based conjugate vaccine, the conjugate based on peptide+CRM+Fructus polysaccharide can induce a 2-5 times increase in potency relative to the respective peptides (the highest potency is 1/190.000 ) and increase the potency against aSyn fiber by 3-13 times (the highest potency is 1/29.000). Example 33 : Selective analysis of immune responses elicited in vivo by vaccines based on peptide +CRM+ Fructus polysaccharides

Compared with the conventional peptide + CRM197 vaccine, in order to further characterize the immune response triggered by the peptide + CRM197 + Shigu polysaccharide vaccine containing different B cell epitopes, a set of experiments was performed in this example to analyze the aSyn fibers against aggregated Selectivity of the subsequent immune response elicited.

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (all vaccines: 5 μg aSyn targeting peptide/dose; route: 4 peptide+CRM197+CLEC-based vaccines (SeqID52/SeqID66/68 /70-CRM197-Shifu polysaccharide) with id; sc for 4 peptide + CRM197-based vaccines with Alhydrogel as adjuvant (SeqID52/SeqID66/68/70-CRM197)) and for two days after the third immunization The subsequent immune response to target proteins, namely recombinant human alpha-synuclein and aSyn fibers, was analyzed in mouse plasma collected weekly. ASyn-specific inhibition ELISA was performed on plasma samples, and IC50 values were determined. Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way SeqID52 CRM Shi fungus polysaccharide (80%) na ID SeqID66 CRM Shi fungus polysaccharide (80%) na ID SeqID68 CRM Shi fungus polysaccharide (80%) na ID SeqID70 CRM Shi fungus polysaccharide (80%) na ID SeqID52 CRM na Alhydrogel sc SeqID66 CRM na Alhydrogel sc SeqID68 CRM na Alhydrogel sc SeqID70 CRM na Alhydrogel sc result:

Briefly, compared to conventional peptide-CRM197 conjugate vaccines, all CLEC-based conjugates used in this experiment demonstrated excellent aSyn aggregate-specific target selectivity by targeting aSyn fibers with significantly higher as determined by low IC50 values (see Figure 36).

All 4 known peptide-CRM197 conjugate vaccines tested in this experiment induced antibodies that showed very weak selectivity for aSyn fibers, indicated by very high IC50 values of 400-1700 ng/ml. display.

In contrast, all antibodies induced by the novel peptide+CRM197+Fructus polysaccharide-based conjugate vaccine were characterized by significantly lower IC50 values for aSyn fibers, ranging from 3.5-15 ng/ml.

Therefore, the experiments show that regardless of the epitope used, CLEC modification of the CRM197 conjugate causes a greatly enhanced target specificity of the subsequent immune response, providing an unprecedented new strategy to optimize the current advanced technology conjugate vaccine. Example 34 : Affinity analysis of immune responses elicited by vaccines based on peptide +CRM197+ Fructus polysaccharides

In order to further characterize the immune response elicited by peptide-CRM197-Auricularia auriculariae polysaccharide-based vaccines containing different B cell epitopes compared to known peptide-CRM197 vaccines, a set of experiments was conducted in this example to analyze the overall avidity of antibodies elicited against aSyn fibers.

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (all vaccines: 5 μg aSyn targeting peptide/dose; route: CLEC-based vaccine with id (SeqID52/66/68/70+CRM197+ fungus polysaccharide); sc for a CRM197-based vaccine adjuvanted with Alhydrogel (SeqID52/66/68/70-CRM197), subsequent analysis of target proteins using mouse plasma collected two weeks after each immunization (i.e., aSyn fibers) immune response. For induction of antibodies against aSyn fibers, a variation of the standard ELISA assay was used in which replicate wells containing antibodies bound to the antigen were exposed to increasing concentrations of chaotropic thiocyanate ions. Resistance to thiocyanate elution is used as a measure of the overall affinity of the antibody molecule, and an index indicating 50% effective antibody binding (the overall antibody molecule affinity index) is used to compare plasma samples. Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way SeqID52 CRM197 Shi fungus polysaccharide (80%) na ID SeqID66 CRM197 Shi fungus polysaccharide (80%) na ID SeqID68 CRM197 Shi fungus polysaccharide (80%) na ID SeqID70 CRM197 Shi fungus polysaccharide (80%) na ID SeqID52 CRM197 na Alhydrogel sc SeqID66 CRM197 na Alhydrogel sc SeqID68 CRM197 na Alhydrogel sc SeqID70 CRM197 na Alhydrogel sc result:

As shown in Figure 37, the antibodies induced by all tested conventional peptide-CRM197 conjugates (with Alhydrogel as adjuvant) showed only limited binding strength to aSyn fibers, as indicated by very low affinity indices ranging from 0.25 to 0.85. Proven. In contrast, the antibodies induced by all novel peptide+CRM197+Fructus polysaccharide-based vaccines showed significantly higher binding strengths to aSyn fibers, with AI ranging from 0.5 to 2.2.

Thus, the experiments show that CLEC modification of peptide-CRM197 conjugates leads to a strong enhancement of target-specific immune responses (titer), and that, regardless of the antigenic determinant used, the target specificity and affinity of the induced antibody response are strongly enhanced, providing an unprecedented new strategy to optimize the most advanced protein conjugate vaccines, including CRM197. Example 35 : In vivo comparison of different peptides + CRM197 + CLEC - based vaccines

In this example, aSyn-derived peptide SeqID6+CRM197 conjugates coupled to Shiguan, lichenin, or laminarin were tested to determine their potent and specific induction after repeated administration in n=5 Balb/c mice/group The ability of the immune response. A typical experiment was performed using B cell epitope peptides at a net peptide content of 5 µg per dose, with animals (female Balb/c mice) vaccinated 3 times at biweekly intervals (Route: id), followed by a third dose Mouse plasma collected two weeks after immunization was analyzed for immune responses to the injected peptide (i.e., SeqID6) and aggregated aSyn fibers. Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way SeqID6 CRM197 Shi fungus polysaccharide (80%) na ID SeqID6 CRM197 Lichenin (200%) na ID SeqID6 CRM197 Laminaria polysaccharide (200%) na ID SeqID6 CRM197 na Alhydrogel sc result:

The vaccines tested induced significant immune responses to injected peptides such as SeqID6 and to aSyn fibers that aggregated after repeated immunizations in mice.

Compared to conventional peptide-CRM based vaccines and to peptide-CRM based vaccines conjugated to laminarin or lichenin, peptide+CRM+Psoralen based conjugates induced high titers against the respective peptides and aSyn fibers (see FIG. 38 ).

Specifically, SeqID+CRM197+Lynaridin induced a 1.6-fold higher potency against the injected peptide SeqID6 compared to SeqID6+CRM197+lichenin, and a 12-fold higher potency compared to SeqID6+CRM197+laminarin. Compared with SeqID6+CRM197+laminarin, SeqID6+CRM197+lichenin induced a 7.5-fold higher titer.

Similarly, the potency induced by SeqID+CRM197+lichenin against aSyn aggregates (filaments) was 3.1 times higher than that of SeqID6+CRM197+lichenin, 7.6 times higher than that of SeqID6+CRM197+laminarin, and 7.6 times higher than that of SeqID6+CRM197+laminarin. The non-CLEC modified SeqID6+CRM197 with Alhydrogel as an adjuvant was 6 times higher. Compared with SeqID6+CRM197+laminarin, SeqID6+CRM197+lichenin can induce a 2.4-fold higher titer, and compared with the non-CLEC-modified SeqID6+CRM197 using Alum as an adjuvant, it can induce a 2-fold higher titer.

CLEC modification of peptide-CRM197 conjugates provides an unprecedented new strategy to optimize currently the most advanced protein conjugate vaccines, including CRM197. Example 36 : Determination of in vitro biological activity of peptide -CLEC- conjugates on murine and human dectin-1 receptors

In a series of ELISA experiments, the binding efficacy of the dectin-1 ligands Shiguan, lichenin and laminarin on murine and human dectin-1 was evaluated. The biological activity of the peptide-CLEC conjugate is represented by its PRR binding ability. Along these lines and in order to ensure that the structure of CLEC (leafia polysaccharide, lichenin, laminarin) remains biologically active after coupling, through competition based on soluble murine and human Fc-dectin-1a receptors (InvivoGen) A competitive ELISA system assessing binding to murine and human dectin-1. result:

As shown in FIG. 39 , the binding efficacy of SeqID5+SeqID7+CLEC conjugates conjugated to lichenan (Lich conjugate), Psoralen polysaccharide (Pus conjugate) or laminarin polysaccharide (Lam conjugate) to mouse and human dectin-1 was evaluated by ELISA analysis.

Subsequent experiments showed that the peptide-Auricularia polysaccharide vaccine had significantly higher binding efficacy against mouse and human dectin-1 than the vaccine combined with lichen polysaccharide (see Figure 39A+B). In contrast, the peptide-laminarin polysaccharide vaccine showed very high binding efficacy against mouse dectin-1 (Figure 39A), but only weak binding efficacy against human dectin-1 (Figure 39B). Example 37: In vivo comparison of different peptide -CLEC- based vaccines

CLEC-based vaccines that bind murine and/or human dectin-1 were tested for their ability to induce a strong and specific immune response following repeated administration in n=5 Balb/c mice/group. Typical experiments were performed using 5µg of neat peptide per dose of B cell epitope peptide.

In this experiment, the alpha-synuclein-derived peptide SeqID5 and the promiscuous helper T cell epitope SeqID7 were coupled to oxidized Psoralen, Lichenin or Laminaria polysaccharide via a C-terminal hydrazide linker. B cell antigen determinant T cell antigen determinant CLEC SeqID5 SeqID7 Pyricularia auricula SeqID5 SeqID7 Lichen polysaccharide SeqID5 SeqID7 Kelp Polysaccharide

Animals (female Balb/c mice) were vaccinated 3 times at 2-week intervals (dose: 5µg and 20µg; route: id), followed by analysis of mouse plasma collected 2 weeks after the third immunization for injection Subsequent immune response to the peptide (i.e., SeqID6). result:

As shown in Figure 40A (dose: 5µg SeqID5 peptide equivalent) and B (dose: 20µg SeqID5 peptide equivalent), all three CLEC vaccines (SeqID5+SeqID7+Pseudomonas aeruginosa, SeqID5+SeqID7+Lichenin, and SeqID5+SeqID7+Laminaria polysaccharide) were able to induce detectable immune responses in a dose-dependent manner. Interestingly, immunization with the laminarin-based vaccine induced only very low anti-peptide and anti-aSyn responses, regardless of the dose used. In contrast, the Pseudomonas aeruginosa-based conjugate induced significantly higher anti-peptide and anti-aSyn responses. The lichenin-based conjugate showed lower immunogenicity compared to the Psoralea corylifolia-based conjugate, but induced higher titers than the laminarin-based conjugate in this experiment.

This indicates that the in vitro binding efficacy to dectin-1, especially to humans, can be directly related to the in vivo immunogenicity and biological activity of the vaccine. This conclusion leads to the identification of Psoralea corylifolia polysaccharide or its fragment (i.e., linear β(1,6)-β-D glucan) as the most effective glucan variant proposed in this application. Example 38 : In vitro biological activity assay of CLEC -modified oligosaccharide / polysaccharide + CRM197 and oligosaccharide / polysaccharide + TT- sugar conjugates

The biological activity of the oligo/polysaccharide + CRM197 + Shia polysaccharide and oligo/polysaccharide + TT + Shia polysaccharide conjugates is expressed by their PRR binding ability. In this example, two commercially available conjugates that could be coupled to or left unchanged were analyzed: i) conjugation with N. meningitidis oligosaccharides (A, C, W135 and Y) containing CRM197 Biological vaccine Menveo® and ii) Haemophilus influenza type b virus capsular polysaccharide (polyribose phosphate, PRP) tetanus toxoid (TT) conjugate ActHIB®.

To ensure that the structure of Pseudomonas aeruginosa remained bioactive after conjugation to Menveo® and ActHIB®, binding to dectin-1 was assessed using a competitive ELISA system based on competitive binding to soluble murine Fc-dectin-1a receptor (InvivoGen) as described in Korotchenko et al. (2020). The bioactivity of unmodified and Pseudomonas aeruginosa-modified CRM197 and TT conjugate vaccines was then assessed and compared to relevant control groups. Results:

In ELISA experiments, the binding efficacy of the oxidized dectin-1 ligand Pseudomonas aeruginosa, the Pseudomonas aeruginosa modified or unmodified Haemophilus influenzae type b capsular polysaccharide (polyribosyl-ribitol-phosphate, PRP) tetanus toxoid (TT) conjugate ActHIB®, and the β-glucan modified or unmodified meningococcal oligosaccharide (A, C, W135 and Y) conjugate vaccine Menveo® containing CRM197 was evaluated with dectin-1. Subsequent experiments (Figure 41) showed that CRM-Pseudomonas aeruginosa and TT-Pseudomonas aeruginosa conjugates showed similar binding efficacy to dectin-1 as oxidized Pseudomonas aeruginosa. In contrast, traditional non-modified CRM- and TT conjugates showed no specific dectin-1 binding.

The experiment showed that the oligosaccharide/polysaccharide-CRM197/TT-Auricularia auricularia polysaccharide conjugate exhibited biological activity against dendritic cells by binding to dectin-1. Example 39 : In vivo comparison of vaccines based on different oligosaccharides / polysaccharides + CRM197 + Auricularia auricularia polysaccharide and vaccines based on oligosaccharides / polysaccharides + TT + Auricularia auricularia polysaccharide

ActHIB®, a conjugate of Haemophilus influenzae type B capsular polysaccharide (polyribosyl-ribitol-phosphate, PRP) and tetanus toxoid (TT) and Menveo®, a conjugate vaccine containing CRM197 of meningococcal oligosaccharides (A, C, W135 and Y), were coupled to Pseudomonas oxidans and tested for their ability to induce robust and specific immune responses after repeated administration in n=5 Balb/c mice/group.

In this experiment, animals (female Balb/c mice) were inoculated three times at biweekly intervals with β-glucan-modified (route: id) or unmodified conjugates (route im), using the Rat plasma collected two weeks after three immunizations was analyzed for subsequent immune responses to ActHIB® and Menveo®. Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way Neisseria meningitidis (A, C, W135, Y) CRM197 Shi fungus polysaccharide (80%) na ID Neisseria meningitidis (A, C, W135, Y) CRM197 na na im Haemophilus influenzae (b) PRP TT Shi fungus polysaccharide (80%) na ID Haemophilus influenzae (b) PRP TT na na im result:

As shown in Figure 42, all vaccines tested were able to induce significant immune responses against the immunoconjugates after repeated immunization in mice.

Animals treated with CLEC-modified Menveo® and ActHIB® showed 2.4-fold and 1.4-fold higher anti-conjugate responses than unmodified vaccines, indicating improved immunogenicity of oligosaccharide/polysaccharide carrier vaccines. These results also indicate that CLEC modification of existing clinically proven oligosaccharide/polysaccharide carrier vaccines according to the present invention can improve the immunogenicity of these vaccines.

Furthermore, the examples provided demonstrate that peptide- and oligo/polysaccharide-CRM/TT-β-glucan vaccines are functional in vivo and are suitable as novel vaccine compositions for the treatment of infectious diseases according to the present invention. Example 40 : B cell epitope analysis of secreted proteins, autoantigens and conformational epitopes : IL31

To evaluate whether peptide vaccines carrying peptides derived from secreted proteins, whether they constitute autoantigens (e.g., SeqID132 to SeqID147) or foreign target structures, can generate high immune responses after repeated immunizations and induce superior immune responses than traditional conjugates Immune response to the vaccine, 8 different vaccine candidates were tested:

In this experiment, 8 different IL31-derived peptides were used as peptide-CLEC vaccines (i.e.: SeqID132; SeqID 134; SeqID 136; SeqID 138; SeqID 140; SeqID 142; SeqID 144; and SeqID146) with pan-T cell antigenic determination based on SeqID7 conjugation via a C-terminal hydrazine linker coupled to oxidized Schizophora polysaccharide (80%; material containing a C-terminal cysteine for coupling to GMBS-activated CRM197. Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way SeqID132 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID133 CRM197 na Alhydrogel sc SeqID134 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID135 CRM197 na Alhydrogel sc SeqID136 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID137 CRM197 na Alhydrogel sc SeqID138 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID139 CRM197 na Alhydrogel sc SeqID140 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID141 CRM197 na Alhydrogel sc SeqID142 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID143 CRM197 na Alhydrogel sc SeqID144 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID145 CRM197 na Alhydrogel sc SeqID146 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID147 CRM197 na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (route: CLEC-based vaccine with id, CRM-based vaccine with Alhydrogel as adjuvant with sc), using the third immunization Rat plasma collected two weeks later was analyzed for subsequent immune responses to the injected peptides (i.e., SeqID133, SeqID135, SeqID137, SeqID139, SeqID141, SeqID143, SeqID145, and SeqID147) and to the protein of interest (i.e., recombinant human IL31). result:

As shown in FIG. 43 , the vaccine was able to induce a strong and specific immune response against the injected peptide portion ( FIG. 43A ) as well as the target protein: human IL31 ( FIG. 43B ).

However, compared to the CRM197 peptide conjugate vaccine adjuvanted with Alhydrogel, the peptide-CLEC-based vaccine of this example showed similar or significantly higher immune responses against the injected peptide portion and, most importantly, also against the full-length target - human IL31.

Furthermore, analysis of affinity using thiocyanate elution resistance (NaSCN) showed that peptide-CLEC-induced antibodies had significantly higher affinity for full-length human IL31 compared to peptide-CRM197-induced antibodies (see Figure 43C; Example: Comparison of antibodies induced by SeqID132+SeqID7+Shitia polysaccharide and SeqID133+CRM Alum).

In conclusion, the CLEC-based vaccines tested are well suited to use antigenic determinants against secreted proteins, including signaling molecules or cellular/tendinogens, especially human IL31, as immunogens, which have high immune responses and high target-specific responses compared to conventional vaccines.

This example also provides results showing that CLEC-based immunogens using human IL31 epitopes surprisingly induced immune responses with higher avidity compared to prior art vaccines directed against these self-epitopes.

Therefore, it is clear that the CLEC-based vaccine according to the present invention can be preferably used for active anti-IL31 immunization. Example 41 : Immunogenicity Analysis of CLEC Conjugates Targeting IL31 Using Carrier Proteins as Helper T Cell Epitopes : CRM197

In this example, the immunogenicity of a CLEC-based conjugate vaccine containing the well-known carrier protein CRM197 was compared with the known CRM197 vaccine. To this end, human IL31-derived epitopes SeqID133, SeqID135, SeqID137, SeqID139, SeqID141, SeqID143, SeqID145, SeqID147, SeqID149 and SeqID151 were coupled to cis-butylene imide-activated CRM197. Subsequently, the CRM197 conjugate was coupled to activated Psoralea corylifolia polysaccharide using the heterobifunctional linker BPMH to form a CLEC-based conjugate vaccine, in which CRM197 served as a source of helper T cell antigenic determinants to induce a sustainable immune response. B cell antigen determinant T cell antigen determinant / vector CLEC Adjuvant Way SeqID133 CRM197 Pyricularia auricula polysaccharide (80%) na id SeqID133 CRM197 na Alhydrogel sc SeqID135 CRM197 Pyricularia auricula polysaccharide (80%) na id SeqID135 CRM197 na Alhydrogel sc SeqID137 CRM197 Pyricularia auricula polysaccharide (80%) na id SeqID137 CRM197 na Alhydrogel sc SeqID139 CRM197 Pyricularia auricula polysaccharide (80%) na id SeqID139 CRM197 na Alhydrogel sc SeqID141 CRM197 Pyricularia auricula polysaccharide (80%) na id SeqID141 CRM197 na Alhydrogel sc SeqID143 CRM197 Pyricularia polysaccharide (80%) na id SeqID143 CRM197 na Alhydrogel sc SeqID145 CRM197 Pyricularia polysaccharide (80%) na id SeqID145 CRM197 na Alhydrogel sc SeqID147 CRM197 Pyricularia polysaccharide (80%) na id SeqID147 CRM197 na Alhydrogel sc SeqID149 CRM197 Pyricularia auricula polysaccharide (80%) na id SeqID149 CRM197 na Alhydrogel sc SeqID151 CRM197 Pyricularia auricula polysaccharide (80%) na id SeqID151 CRM197 na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (all vaccines: 5 μg IL31 targeting peptide/dose; route: CLEC-based vaccine with id, CRM197-based vaccine with Alhydrogel as adjuvant sc) and used murine plasma collected two weeks after the third immunization against the injected peptides (i.e., SeqID133, SeqID135, SeqID137, SeqID139, SeqID141, SeqID143, SeqID145, SeqID147, SeqID149, and SeqID151) and against full-length IL31 The subsequent immune responses generated were analyzed. result:

The IL31-peptide + CRM197 vaccine induced a strong and specific immune response against the injected peptide portion (Figure 44A) and the target protein: human IL31 (Figure 44B).

CLEC modification of the CRM197 conjugate targeting IL31 elicited similar or significantly higher immune responses against the immunizing peptide compared to the non-CLEC modified Alhydrogel adjuvanted CRM197-based known vaccine. Importantly, target-specific anti-full-length IL31 titers elicited by the non-CLEC modified Alhydrogel adjuvanted CRM197-based known vaccine were similar (SeqID141+CRM and SeqID147+CRM) or 2-9-fold lower than those of the CLEC-modified vaccine.

In addition, affinity analysis using thiocyanate elution resistance (NaSCN) showed that the antibody induced by IL31-peptide+CRM197+CLEC had significantly higher affinity for full-length human IL31 than the antibody induced by IL31-peptide+CRM197 (see Figure 44C, example: comparison of antibodies induced by SeqID133+CRM+Pseudomonas aeruginosa and SeqID133+CRMAlum).

Thus, this experiment demonstrates that CLEC modification of known peptide-protein conjugates results in a greatly enhanced target specificity of the subsequent immune response, thereby providing an unprecedented new strategy to optimize current state-of-the-art conjugate vaccines constructed on carrier proteins such as KLH, CRM197 or other proteins.

This example also provides results showing that CLEC-based immunogens using human IL31 epitopes unexpectedly induced immune responses with higher potency and affinity compared to state-of-the-art IL31 vaccines.

Therefore, it is clear that the CLEC-based vaccine according to the present invention is preferably used for active anti-IL31 immunization. Example 42 : Inhibition of IL31 signaling by anti -IL31 antibodies induced by WISIT vaccine

In order to study the inhibition of natural IL-31 signaling by WISIT vaccine-induced antibodies and conventional CRM197 vaccine-induced antibodies, human adenocarcinoma alveolar basal epithelial cells-A549 cells (ATCC, Virginia, USA) were used in this experiment. Cells were treated with different vaccine-induced antibodies (1000 ng/ml) and human IL-31 was added. The vaccine-induced antibodies used were from animals that were repeatedly immunized as described in Examples 40 and 41. All samples were administered at an anti-IL31 antibody concentration of 1000 ng/ml. In this assay, controls include IL31-blocking antibody (immunogen against E. coli -derived recombinant human IL-31Ser24-Thr164, accession #Q6EBC2 at 1000 ng/ml) used as a positive control and Negative control murine plasma without inhibitory antibodies.

After incubation for 20 minutes, cells were lysed and STAT3 phosphorylation analyzed using PathScan Phospho-Stat3 (Tyr705) Sandwich ELISA Kit (Cell Signaling Technologies, Danvers, MA, USA). result:

Antibodies induced by known peptide + carrier, peptide + CLEC, and peptide + carrier + CLEC vaccines can specifically inhibit IL31 signaling using this cell-based in vitro assay (Figure 45 and Figure 46), proving that it Able to modify the effects produced by IL31 activity (i.e., demonstrate biological activity and therapeutic potential).

The CLEC modification of IL31-targeted vaccines (two types, including peptide conjugates and peptide-CRM conjugates) unexpectedly resulted in improved performance compared with prior art and non-CLEC-modified conventional CRM197-based vaccines with Alhydrogel as adjuvant. Similar or significantly higher suppressive capacity of the immune response.

Figure 45 summarizes the composition of the IL31-peptide + SeqID7 + Shi fungus polysaccharide conjugate (IL31 peptide: SeqID132, SeqID134, SeqID136, SeqID138, SeqID140, SeqID142, SeqID144, SeqID146) and the conventional IL31-peptide + CRM conjugate (IL31 peptide: SeqID133 , SeqID135, SeqID137, SeqID139, SeqID141, SeqID143, SeqID145, SeqID147). Analysis of the inhibitory ability of antibodies induced.

Figure 46 summarizes the conventional IL31 conjugates composed of IL31-peptide + CRM + Shigu polysaccharide (IL31 peptide: SeqID133, SeqID135, SeqID137, SeqID139, SeqID141, SeqID143, SeqID145, SeqID147, SeqID149, SeqID151) and Alum as adjuvant. - Analysis of the inhibitory ability of antibodies induced by peptide + CRM conjugates (IL31 peptides: SeqID133, SeqID135, SeqID137, SeqID139, SeqID141, SeqID143, SeqID145, SeqID147, SeqID149, SeqID151).

Therefore, it is clear that the CLEC-based vaccines according to the present invention are preferably used for active anti-IL31 immunization. Therefore, such vaccines can be used to treat and prevent IL31-related diseases and autoimmune inflammatory diseases. Analysis of the inhibitory ability of vaccine-induced antibodies also showed that immunogenic peptides SeqID132/133, SeqID134/135, SeqID138/139, SeqID146/147, SeqID148/149 and SeqID150/151 can induce more effective antibodies (both in inhibiting IL31 activity and compared with known vaccine-induced antibodies), and are therefore very suitable for treating and preventing the aforementioned diseases, while SeqID136/137, SeqID140/141, SeqID142/143 and SeqID144/145 are less suitable. Example 43 : B cell epitope analysis of secreted proteins, autoantigens and conformational epitopes : CGRP

To evaluate whether peptide vaccines carrying peptides from secreted proteins (regardless of whether they constitute self-antigens or foreign target structures) can generate high immune responses after repeated immunizations and induce immune responses superior to those of learned conjugate vaccines, different candidate vaccines were tested:

In this experiment, different CGRP (calcitonin gene related peptide) derived peptides were used as peptide+CLEC vaccines (i.e.: SeqID152, SeqID154, SeqID156, SeqID158, SeqID160 and SeqID162) in combination with the pan-T cell epitope SeqID7, by The C-terminal hydrazine linker was conjugated to oxidized Schizophora polysaccharide (80%; C-terminal cysteine of activated CRM197. Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way SeqID152 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID153 CRM197 na Alhydrogel sc SeqID154 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID155 CRM197 na Alhydrogel sc SeqID156 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID157 CRM197 na Alhydrogel sc SeqID158 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID159 CRM197 na Alhydrogel sc SeqID160 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID161 CRM197 na Alhydrogel sc SeqID162 SeqID7 Shi fungus polysaccharide (80%) na ID SeqID163 CRM197 na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated three times at biweekly intervals (route: id for CLEC-based vaccines, sc for CRM-based vaccines with Alhydrogel as adjuvant), and subsequent immune responses against the injected peptides (i.e., SeqID153, SeqID155, SeqID157, SeqID159, SeqID161, and SeqID163) and against the target protein (i.e., recombinant human CGRP) were analyzed using mouse plasma collected two weeks after the third immunization. Results:

The vaccines tested were able to induce strong and specific immune responses against the injected peptide moiety and the target protein: human CGRP (Figure 47).

Compared to the CRM197 peptide conjugate vaccine adjuvanted with Alhydrogel, the peptide-CLEC based vaccine of this example showed similar or significantly higher immune responses against the injected peptide portion ( FIG. 47A ) and, most importantly, also against the full-length target - human CGRP ( FIG. 47B ).

In addition, affinity analysis using thiocyanate washout resistance (NaSCN) showed that the peptide + CLEC-induced antibody had a significantly higher affinity for full-length human CGRP than the peptide + CRM197-induced antibody ( Figure 47C ).

In conclusion, the CLEC-based vaccines tested are well suited to use antigenic determinants against secreted proteins, including signaling molecules or cellular/tendering factors, especially human CGPR, as immunogens, with high immune responses and high target-specific responses compared to conventional vaccines.

This example also provides results showing that CLEC-based immunogens using human CGPR epitopes surprisingly induced immune responses with higher affinity compared to prior art vaccines targeting these self-epitope.

Therefore, it is clear that the CLEC-based vaccine according to the present invention can be preferably used for active anti-CGRP immunization. Therefore, such vaccines can be used to treat CGRP-related diseases, including episodic and chronic migraines and cluster headaches, hyperalgesia, and hyperalgesia in dysfunctional pain states such as rheumatoid arthritis, osteoarthritis, visceral Pain hypersensitivity syndrome, fibromyalgia, inflammatory bowel disease, neuropathic pain, chronic inflammatory pain and headaches. Example 44 : Immunogenicity Analysis of Targeted CGRPCLEC Conjugates Using Carrier Proteins as Helper T Cell Epitopes : CRM197

In this example, the immunogenicity of a CLEC-based conjugate vaccine containing the well-known carrier protein CRM197 was compared to a conventional peptide + CRM197 vaccine. To this end, the human CGRP-derived epitopes SeqID153, SeqID155, SeqID157, SeqID159, SeqID161 and SeqID163 were coupled to maleimide-activated CRM197. Subsequently, the heterobifunctional linker BPMH was used to couple the CRM197 conjugate with activated Shigu polysaccharide to form a CLEC-based conjugate vaccine, in which CRM197 served as a source of helper T cell epitopes to induce a sustainable immune response. Vaccines used: B cell epitope T cell epitope / carrier CLEC Adjuvant way SeqID153 CRM197 Shi fungus polysaccharide (80%) na ID SeqID153 CRM197 na Alhydrogel sc SeqID155 CRM197 Shi fungus polysaccharide (80%) na ID SeqID155 CRM197 na Alhydrogel sc SeqID157 CRM197 Shi fungus polysaccharide (80%) na ID SeqID157 CRM197 na Alhydrogel sc SeqID159 CRM197 Shi fungus polysaccharide (80%) na ID SeqID159 CRM197 na Alhydrogel sc SeqID161 CRM197 Shi fungus polysaccharide (80%) na ID SeqID161 CRM197 na Alhydrogel sc SeqID163 CRM197 Shi fungus polysaccharide (80%) na ID SeqID163 CRM197 na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated 3 times at biweekly intervals (all vaccines: 5 µg CGRP targeting peptide/dose; route: CLEC-based vaccine with ID, CRM197-based vaccine with Alhydrogel as adjuvant sc), and subsequent immune responses to the injected peptides (i.e., SeqID153, SeqID155, SeqID157, SeqID159, SeqID161, and SeqID163) and to full-length CGRP were performed using murine plasma collected two weeks after the third immunization. analyze. result:

The CRM197-based vaccine induced a strong and specific immune response against the injected peptide moiety and the target protein: human CGRP (Figure 48).

Compared with the non-CLEC-modified known CRM197-based vaccine with Alhydrogel as adjuvant, the CLEC-modified CRM197 conjugate targeting CGRP induced similar or higher immune responses against the anti-immunizing peptide ( FIG. 48A ) and anti-full-length CGRP ( FIG. 48B ).

In addition, affinity analysis using thiocyanate washout resistance (NaSCN) showed that the CGRP-peptide+CRM197+CLEC-induced antibody had significantly higher affinity for full-length human CGRP than the CGRP-peptide+CRM197-induced antibody ( FIG. 48C ).

Thus, the experiments demonstrate that CLEC modification of known peptide-protein conjugates results in high target specificity of the subsequent immune response, providing an unprecedented new strategy to optimize current state-of-the-art conjugate vaccines constructed on carrier proteins such as KLH, CRM197 or other proteins.

This example also provides results showing that CLEC-based immunogens using human CGPR epitopes unexpectedly induced immune responses with higher potency and affinity compared to state-of-the-art CGRP vaccines.

Therefore, it is apparent that the CLEC-based vaccines according to the present invention are preferably used for active anti-CGRP immunization. Such vaccines can therefore be used to treat CGRP-related diseases, including sporadic and chronic migraine and cluster headaches, allergic pain, allergic pain in dysfunctional pain states, such as rheumatoid arthritis, osteoarthritis, visceral pain hypersensitivity syndrome, fibromyalgia, inflammatory bowel disease, neuropathic pain, chronic inflammatory pain and headache. Example 45: In vivo functional analysis of immune responses induced by CLEC -based vaccines

To determine whether aSyn-specific antibodies elicited by CLEC-based vaccines can inhibit aSyn fibril formation in vivo, a proof-of-concept experiment was initiated using an established synucleinopathy vaccination model [Sci. Adv. 2020, 6, eabc4364, doi:10.1126/sciadv.abc4364; DOI: 10.1126/sciadv.abc4364]. Vaccine used: B cell antigen determinant T cell antigen determinant / vector CLEC Adjuvant Way SeqID5 SeqID7 Pyricularia auricula polysaccharide (80%) na id na na Pyricularia auricula na id

In this model, C57BL/6 mice are stereotactically injected with α-syn preformed fibrils (PFF) at the level of the right substantia nigra, subsequently causing extensive synucleinopathy characterized by phosphosynuclein immunopositive species. Lewy neurites and intracytoplasmic aggregates along anatomical junctions. Animals were immunized four times at 0, 2, 4 and 10 weeks, using SeqID5+SeqID7+Shitia polysaccharide vaccine or uncoupled CLEC as a control group, and the first immunization was started on the day of PFF vaccination. 126 days after PFF injection, the animals were sacrificed and the brains were analyzed for the presence of phosphorylated S129aSyn-positive aggregates in selected brain regions, including the cerebral cortex, striatum, thalamus, substantia nigra, and brainstem. result:

Plasma and CSF obtained at sacrifice were used to analyze subsequent immune responses. High antibody titers against the injected peptides were detected in the plasma of animals treated with the SeqID5+SeqID7+Psoralea corylifolia vaccine. In contrast, no signal above background was detected in the control group treated with CLEC alone (Figure 49). Analysis of anti-peptide titers in CSF also showed high levels of SeqID5+SeqID7+Psoralea corylifolia vaccine-induced antibodies, while no signal above background was detected for animals treated with the vehicle (Figure 49). Immunohistochemistry of brain sections showed the presence of large numbers of phosphorylated S129aSyn-positive aggregates in all analyzed areas of the vehicle-treated group, indicating a strong dissemination of aSyn pathology. In contrast, synuclein pathology was significantly reduced in mice vaccinated with SeqID5+SeqID7+Pseudomonas aeruginosa vaccine (Figure 49). Of note, there was a strong and significant correlation between the magnitude of the antibody response and the level of synuclein pathology in vaccine recipients (Figure 49). Example 46 : Immunogenicity Analysis of Peptide +CRM+CLEC Conjugates

In this example, the vector-specific immunogenicity of a CLEC-based conjugate vaccine was compared with that of a conventional vector vaccine.

To this end, α-synuclein derived epitopes SeqID6 or IL31 derived epitopes SeqID133, SeqID135 and SeqID137 were coupled to cis-butylenediamide activated CRM197. Subsequently, the peptide-CRM197 conjugate was coupled to activated Pseudomonas aeruginosa polysaccharide using the heterobifunctional linker BPMH to form a CLEC-based conjugate vaccine, in which CRM197 served as a source of helper T cell epitopes to induce a sustained immune response. Vaccines used: B cell antigen determinant T cell antigen determinant / vector CLEC Adjuvant Way SeqID6 CRM197 Pyricularia auricula polysaccharide (80%) na id SeqID6 CRM197 na Alhydrogel sc SeqID133 CRM197 Pyricularia auricula polysaccharide (80%) na id SeqID133 CRM197 na Alhydrogel sc SeqID135 CRM197 Pyricularia auricula polysaccharide (80%) na id SeqID135 CRM197 na Alhydrogel sc SeqID137 CRM197 Pyricularia auricula polysaccharide (80%) na id SeqID137 CRM197 na Alhydrogel sc

Animals (female Balb/c mice) were vaccinated three times at biweekly intervals, and immune responses against the carrier protein CRM197 were analyzed using mouse plasma collected two weeks after the third immunization. Vaccine doses containing SeqID6: 20 µg and 100 µg alpha-synuclein targeting peptide/dose; route: id for CLEC-based vaccine, sc for CRM197-based vaccine with Alhydrogel as adjuvant (Figure 50A). Dose for vaccines containing SeqID133, SeqID135 and SeqID137: 5 µg IL31 targeting peptide/dose; route: id for CLEC-based vaccines and sc for CRM197-based vaccines with Alhydrogel as adjuvant. result:

Comparison of anti-vector-specific antibody responses demonstrated that conventional SeqID6+CRM197-based vaccines were able to induce high anti-CRM197 titers in a dose-dependent manner. In comparison, the CLEC-based SeqID6+CRM197+Fructus auricularia polysaccharide vaccine used induced significantly reduced anti-CRM responses after repeated immunizations with both 20 µg and 100 µg doses (degree of reduction: 4.5-5-fold; Figure 50A).

Similarly, non-CLEC-modified SeqID133-, SeqID135-, and SeqID137+CRM197-based vaccines used at 5 µg IL31-targeting peptide/dose induced 3.7- to 5.8-fold higher anti-CRM197 titers than the CLEC-modified peptide-CRM conjugates, respectively ( FIG. 50B ).

Therefore, experiments show that covalent CLEC modification of conventional peptide-protein conjugates significantly impairs the development of anti-carrier responses, providing an unprecedented new strategy to optimize the construction of carrier proteins (such as KLH, CRM197 or other proteins) The current advanced technology conjugate vaccine. Example 47 : In vivo analysis of the immune response against Shigu polysaccharide / glucan following immunization

As discussed in Example 7, analysis of anti-CLEC antibodies induced by CLEC-based immunogens is important for the novelty and efficacy of the CLEC-vaccines proposed according to the present invention on two levels.

Along these lines, before immunization and after repeated immunization, the results of the first immunization, peptide+CLEC immunization and peptide+CRM+CLEC conjugate immunization of Balb/c mice (n=5/group) Plasma samples were extensively analyzed for anti-S. Vaccines used: B cell epitope T cell epitope CLEC Adjuvant Ratio (w/w) na na Shi fungus polysaccharide na na SeqID6 CRM197 Shi fungus polysaccharide (80%) na 1/1 SeqID6 CRM197 Shi fungus polysaccharide (80%) na 1/2,5 SeqID6 CRM197 Shi fungus polysaccharide (80%) na 1/5 SeqID6 CRM197 Shi fungus polysaccharide (80%) na 1/10 SeqID6 CRM197 Shi fungus polysaccharide (80%) na 1/20 SeqID6 CRM197 Lichenin (200%) na SeqID6 CRM197 Laminaria polysaccharide (200%) na SeqID132 SeqID7 Shi fungus polysaccharide (80%) na SeqID133 CRM197 Shi fungus polysaccharide (80%) na SeqID133 CRM197 na Alhydrogel SeqID134 SeqID7 Shi fungus polysaccharide (80%) na SeqID135 CRM197 Shi fungus polysaccharide (80%) na SeqID135 CRM197 na Alhydrogel SeqID136 SeqID7 Shi fungus polysaccharide (80%) na SeqID137 CRM197 Shi fungus polysaccharide (80%) na SeqID137 CRM197 na Alhydrogel result:

In this example 4 different types of samples are analyzed:

Figure 51A shows the anti-Pseudomonas polysaccharide immune reactivity of samples obtained from animals that underwent repeated immunizations with SeqID6+CRM+Pseudomonas polysaccharide, SeqID6+CRM+Lichen polysaccharide, or SeqID6+CRM+Laminaria polysaccharide (all vaccines: 20 μg aSyn targeting peptide/dose). Figure 51B shows the anti-Pseudomonas polysaccharide immune reactivity of samples from animals that underwent repeated immunizations with SeqID6+CRM+Pseudomonas polysaccharide using vaccines containing different w/w peptide+CRM conjugate/CLEC ratios (i.e., conjugate/CLEC ratios of 1/1, 1/2, 5, 1/5, 1/10, and 1/20) (all vaccines: 5 μg aSyn targeting peptide/dose). Figure 51C shows the anti-Pseudomonas polysaccharide immune reactivity of samples obtained from animals that underwent repeated vaccinations with SeqID133+CRM+Pseudomonas polysaccharide, SeqID135+CRM+Pseudomonas polysaccharide, or SeqID137+CRM+Pseudomonas polysaccharide (all vaccines: 5 μg IL31 targeting peptide/dose). Figure 51D shows the anti-Pseudomonas polysaccharide immune reactivity of samples obtained from animals that underwent repeated vaccinations with SeqID132+SeqID7+Pseudomonas polysaccharide, SeqID134+SeqID7+Pseudomonas polysaccharide, or SeqID136+SeqID7+Pseudomonas polysaccharide (all vaccines: 5 μg IL31 targeting peptide/dose).

For control purposes, samples from animals before immunization and from animals treated with non-oxidized CLEC were used in this experiment. In addition, samples from animals administered with vaccines consisting of non-CLEC modified peptide + CRM conjugates (SeqID133 + CRM, SeqID135 + CRM or SeqID137 + CRM with Alum as adjuvant) were also included in the analysis.

As shown in Figure 51, the Balb/c animals analyzed showed a pre-existing low-level immune response against glucan/Pyropolysaccharide/[beta](1,6)-[beta]-D glucan.

All CLEC vaccines tested (peptide+CLEC and peptide+CRM+CLEC conjugates) failed to significantly increase pre-existing anti-glucan responses or to induce de novo high immune responses against the glucan backbone in vivo (all samples tested: <2x pre-immune levels; mean: 0.8+/-0.5-fold change).

In contrast, repeated administration of unconjugated, unoxidized Psoralea corylifolia polysaccharide present in the control group induced a strong anti-glucan immune response by increasing the antibody levels against Psoralea corylifolia polysaccharide by >5-fold (compared to pre-immune plasma). Non-CLEC modified peptide + CRM conjugates and conjugates containing lichenin and laminarin failed to induce anti-Psoralea corylifolia polysaccharide titers above pre-immune levels, indicating the specificity of the anti-glucan response detected.

Taken together, these analyzes demonstrate that despite low levels of pre-existing autoreactivity against Shigella polysaccharide (IgG) in untreated Balb/c mice, after immunization with various CLEC conjugates , no or only very low vaccination-related changes in immune reactivity against Shia polysaccharide could be detected. This shows that administration of the novel vaccine design according to the invention significantly reduces dextran immunogenicity. This is in strong contrast to previously published results and thus constitutes an unexpected and inventive new feature of the carbohydrate backbone according to the invention (eg, β-glucan, especially the polysaccharide backbone).

Furthermore, pre-existing anti-A. pyrifos polysaccharide responses do not appear to preclude immune responses to the peptide component of the WISIT vaccine, as all experimental responses to injected peptides showed high anti-peptide titers. Example 48 : In vivo comparison of the effect of dextran conjugation on the immunogenicity of peptide + vector vaccines

To assess whether conjugation of CLEC to the peptide + carrier immunogen is necessary to induce superior immunogenicity of the vaccine according to the present invention, this example initiated a set of experiments to compare three vaccine formulations: a peptide + carrier conjugate covalently modified with β-glucan, a vaccine formulation comprising a mixture of the peptide + carrier conjugate and uncoupled β-glucan, and an unmodified peptide + carrier vaccine without Alum adjuvant.

Similarly, n=5 female Balb/c mice were immunized three times at two-week intervals, and subsequent immune responses to the injected peptide and aSyn fibers (i.e., SeqID6) were analyzed using mouse plasma collected two weeks after the third immunization. Vaccines used: B cell antigen determinant Carrier CLEC CLEC Combined SeqID6 CRM197 Pyricularia auricula polysaccharide (80%) yes SeqID6 CRM197 Pyricularia auricula polysaccharide (unoxidized) No; mixed only SeqID6 CRM197 na, unadjuvanted no result:

Figure 52 shows a comparison of the anti-peptide (SeqID6) and anti-aSyn monomer specific immune responses detected after three immunizations. Compared with the mixture of SeqID6+CRM197 and unoxidized Psoralea corylifolia polysaccharide, the conjugate of SeqID6+CRM197+Psoralea corylifolia polysaccharide can induce an immune response against the injected peptide that is about 10 times higher (Figure 52A) and a 4-fold higher anti-aSyn titer (Figure 52B). Compared with SeqID6+CRM197 (without adjuvant), the conjugate of SeqID6+CRM197+Psoralea corylifolia polysaccharide can also induce an immune response against the injected peptide that is about 10 times higher. Interestingly, the mixture of SeqID6+CRM197 and unoxidized Psoralea corylifolia polysaccharide did not induce an immune response significantly different from that of SeqID6+CRM197.

These data demonstrate the need to combine peptide carrier immunogens with activated CLEC in accordance with the present invention to induce better immune responses in vivo.

The B cell antigenic determinant sequence disclosed in the examples is as follows: SeqID sequence Paternal protein SeqID1 (H2N-NH-CO-CH2-CH2-CO)-DQPVLPD alpha-synuclein SeqID2 DQPVLPD-(NH-NH2) alpha-synuclein SeqID3 C-DQPVLPD alpha-synuclein SeqID4 (H2N-NH-CO-CH2-CH2-CO)-DMPVDPD alpha-synuclein SeqID5 DMPVDPD-(NH-NH2) alpha-synuclein SeqID6 C-DMPVDPD alpha-synuclein SeqID10 DAEFRH-(NH-NH2) Aß SeqID11 DAEFRH-C Aß SeqID12 MDVFMKGL-(NH-NH2) alpha-synuclein SeqID13 MDVFMKGL-C alpha-synuclein SeqID14 ATGFVKKDQL-(NH-NH2) alpha-synuclein SeqID15 ATGFVKKDQL-C alpha-synuclein SeqID16 LGKNEEGAP-(NH-NH2) alpha-synuclein SeqID20 EGYQDYEPEA-(NH-NH2) alpha-synuclein SeqID21 EGYQDYEPEA-C alpha-synuclein SeqID32 p(E)FRHDS-C Aß SeqID33 p(E)FRHDS-(NH-NH2) Aß SeqID35 KDNIKHVPGGGS-C Tau SeqID36 KDNIKHVPGGGS-(NH-NH2) Tau SeqID37 WDPGMLQSELIQKEFGDY-C IL12/23 SeqID38 WDPGMLQSELIQKEFGDY-(NH-NH2) IL12/23 SeqID39 TPDAPGETV-C IL12/23 SeqID40 TPDAPGETV-(NH-NH2) IL12/23 SeqID41 TQQIPSLSPSQ-C IL12/23 SeqID42 TQQIPSLSPSQ-(NH-NH2) IL12/23 SeqID43 QQQGLPRAAGG-C EMPD SeqID44 QQQGLPRAAGG-(NH-NH2) EMPD SeqID45 QQFLSVRAL-C Bet v1 SeqID46 QQFLSVRAL-(NH-NH2) Bet v2 SeqID47 YMPIWKFPDEEGAC Her2 SeqID48 YMPIWKFPDEEGA C -(NH-NH2) Her2 SeqID49 GAISLAPKAQIKESLRAEL-C PD1 SeqID50 GAISLAPKAQIKESLRAEL-(NH-NH2) PD1 SeqID51 DMPVDPDN-NH-NH2 alpha-synuclein SeqID52 CDMPVDPDN alpha-synuclein SeqID53 DMPVDP-(NH-NH2) alpha-synuclein SeqID55 C-DMPVDP alpha-synuclein SeqID56 DMPVD-(NH-NH2) alpha-synuclein SeqID58 C-DMPVD alpha-synuclein SeqID65 DMPVDPDNE-(NH-NH2) alpha-synuclein SeqID66 DMPVDPDNE-C alpha-synuclein SeqID67 DMPVDPDNEA-(NH-NH2) alpha-synuclein SeqID68 DMPVDPDNEA-C alpha-synuclein SeqID69 DMPVDPDNEAY-(NH-NH2) alpha-synuclein SeqID70 DMPVDPDNEAY-C alpha-synuclein SeqID71 DMPVDPDNEAYE-(NH-NH2) alpha-synuclein SeqID72 DMPVDPDNEAYE-C alpha-synuclein SeqID73 APQEGILE-(NH-NH2) alpha-synuclein SeqID74 APQEGILE-C alpha-synuclein SeqID132 SKMLLKDVEEEKG-NHNH2 IL31 SeqID133 SKMLLKDVEEEKG-C IL31 SeqID134 EELQSLSK-NHNH2 IL31 SeqID135 EELQSLSK-C; IL31 SeqID136 KGVLVS-NHNH2 IL31 SeqID137 KGVLVS-C IL31 SeqID138 SVIDEIIEHLDKLI-NHNH2 IL31 SeqID139 SVIDEIIEHLDKLI-C IL31 SeqID140 SPAIRAYLKTIRQLDNKSVI-NHNH2 IL31 SeqID141 SPAIRAYLKTIRQLDNKSVI-C IL31 SeqID142 HERKRFILT-NHNH2 IL31 SeqID143 HERKRFILT-C IL31 SeqID144 HESKRFILT-NHNH2 IL31 SeqID145 HESKRFILT-C IL31 SeqID146 SVPTDTHERKRF-NHNH2 IL31 SeqID147 SVPTDTHERKRF-C IL31 SeqID148 SVPTDTHESKRF-NHNH2 IL31 SeqID149 SVPTDTHESKRF-C IL31 SeqID150 KRFILTISQQFS-NHNH2 IL31 SeqID151 KRFILTISQQFS-C IL31 SeqID152 RLAGLLSR-NHNH2 CGRP SeqID153 RLAGLLSR-C CGRP SeqID154 RLAGLLSRSGGVVKN-NHNH2 CGRP SeqID155 RLAGLLSRSGGVVKN-C CGRP SeqID156 RSGGVVKN-NHNH2 CGRP SeqID157 RSGGVVKN-C CGRP SeqID158 NNFVPTNVGSKAF-NHNH2 CGRP SeqID159 NNFVPTNVGSKAF-C CGRP SeqID160 VPTNVGSKAF-NHNH2 CGRP SeqID161 VPTNVGSKAF-C CGRP SeqID162 NVGSKAF-NHNH2 CGRP SeqID163 NVGSKAF-C CGRP

Based on the general disclosure of the present invention and these examples, the following preferred embodiments of the present invention are disclosed: 1. A conjugate consisting of or containing the following: at least one β-glucan or mannan polysaccharide; and at least one β-glucan or mannan polysaccharide; A B cell or T cell epitope polypeptide, wherein β-glucan or mannan polysaccharide is covalently combined with the B cell and/or T cell epitope polypeptide to form β-glucan or mannan polysaccharide with B cells and/or Or conjugates of T cell epitope polypeptides. 2. The conjugate according to embodiment 1, wherein the β-glucan is a predominantly linear β-(1,6)-glucan with a β-(1,6) coupled monosaccharide moiety and a non-β-glucan. The ratio of -(1,6) coupled monosaccharide moieties is at least 1:1, preferably at least 2:1, more preferably at least 5:1, especially at least 10:1. 3. The conjugate according to Embodiment 1 or 2, wherein the β-glucan is dectin-1 combined with β-glucan, preferably agaric polysaccharide, lichenin, laminarin, cardranan, β-glucan. Polyglycan peptide (BGP), schizophyllan, scleroglucan, whole glucan granules (WGP), zymosan or mushroom polysaccharide, more preferably Shitu polysaccharide, laminarin, lichenin, mushroom polysaccharide, schizophyllan Or scleroglucan, especially Shigu polysaccharide; and/or wherein the β-glucan is a strong dectin-1 binding β-glucan, preferably measured by competitive ELISA, with less than 10 mg/ ml, preferably with an IC50 value of less than 1 mg/ml, even more preferably with an IC50 value of less than 500 µg/ml, especially with an IC50 value of less than 200 µg/ml, binding to soluble murine Fc - beta-glucan of the dectin-1a receptor; and/or wherein the conjugate has an IC50 value of less than 1 mg/ml, preferably less than 500 µg/ml, as determined by competitive ELISA Binding to the soluble murine Fc-dectin-1a receptor with an IC50 value of less than 200 µg/ml, especially with an IC50 value of less than 100 µg/ml; and/or - beta-glucan Sugar with an IC50 value below 10 mg/ml, preferably below 1 mg/ml, even better with an IC50 value below 500 µg/ml, especially below 200 µg/ml binds to soluble human Fc-dectin-1a receptor with an IC50 value; and/or - wherein the conjugate binds with an IC50 value of less than 1 mg/ml, preferably less than 500 µg/ml, as determined by competitive ELISA Binds to soluble human Fc-dectin-1a receptor with an IC50 value of ml, even better with an IC50 value of less than 200 µg/ml, especially with an IC50 value of less than 100 µg/ml. 4. The conjugate according to any one of embodiments 1 to 3, wherein the polypeptides comprise at least one B cell epitope and at least one T cell epitope, preferably one covalently linked to β-glucan. B cell epitope+CRM197 conjugate, especially peptide+CRM197+linear β-(1,6)-glucan or peptide+CRM197+linear Shi fungus polysaccharide conjugate. 5. The conjugate according to any one of embodiments 1 to 4, wherein the ratio of β-glucan to B cell and/or T cell epitope polypeptide in the conjugate, especially the ratio of Shi fungus polysaccharide to peptide is 10 :1(w/w) to 0.1:1(w/w), preferably 8:1(w/w) to 2:1(w/w), especially within the range of 4:1(w/w) , the restriction is that if the conjugate contains a carrier protein, the preferred ratio of β-glucan to B cell epitope-carrier polypeptide is 50:1 (w/w) to 0.1:1 (w/w), Especially 10:1 to 0.1:1. 6. The conjugate according to any one of embodiments 1 to 5, wherein the B cell epitope and the pan-specific/hybrid T cell epitope independently bind to the β-glucan. 7. The conjugate according to any one of embodiments 1 to 6, wherein the length of the B cell epitope polypeptide is 5 to 20 amino acid residues, preferably 6 to 19 amino acid residues, Especially 7 to 15 amino acid residues; and/or the length of the T cell epitope polypeptide is 8 to 30 amino acid residues, preferably 13 to 29 amino acid residues, especially Is 13 to 28 amino acid residues, wherein the B cell epitope and/or the T cell epitope are preferably connected to β-glucan and/or carrier protein through a linker, more preferably through a half A cystine residue or a linker comprising a cysteine or glycine residue; or a linker produced by: hydrazine-mediated coupling, via a heterobifunctional linker such as N-β-cis Butenediimidopropionic acid hydrazide (BMPH), 4-[4-N-maleimidophenyl]butyric acid hydrazine (MPBH), N-[ε-maleic acid hydrazide Coupling of acylimidocaproic acid) hydrazine (EMCH) or N-[κ-maleimidodecanoic acid] hydrazine (KMUH)), imidazole-mediated coupling, reductive amination, carbon Diimine coupling; -NH- _NH linker, -NRRA, NRRA-C or NRRA-NH- _NH linker; peptide linkers such as dimers, trimers, tetramers (or longer Polymers) peptide groups, such as CG or CG; or cleavage sites, such as cathepsin cleavage sites; or combinations thereof, especially by cysteine or NRRA-NH- _NH2 linkers; wherein the T cell antigen determines The base is preferably a polypeptide comprising the amino acid sequence AKFVAAWTLKAAA, optionally connected to a linker, such as a cysteine residue or a linker comprising a cysteine residue, NRRA, NRRA-C or NRRA-NH- _NH linker; or a variant of the amino acid sequence AKFVAAWTLKAAA; wherein such variants include the amino acid sequence AKFVAAWTLKAA; wherein the first residue alanine is modified by an amino acid sequence such as glycine, valine, isoleucine, and leucine. Variants of amino acids in which aliphatic amino acid residues are substituted; variants in which the third amino acid residue, phenylalanine, is substituted by L-cyclohexylphenylalanine; wherein the thirteenth amino acid residue, alanine, is substituted by an aliphatic amine group Variants with substitutions of acid residues (such as glycine, valine, isoleucine and leucine); variants containing aminocaproic acid, preferably coupled to the C-terminus of the amino acid sequence AKFVAAWTLKAA Variants comprising aminocaproic acid; variants having the amino acid sequence AX ₁ FVAAX ₂ TLX ₃ AX ₄ A, wherein X ₁ is selected from the group consisting of W, F, Y, H, D, E, N, Q, I and K; X ₂ is selected from the group consisting of F, N, Y and W; X ₃ is selected from the group consisting of H and K, and X ₄ is selected from the group consisting of A, D and E. The restriction is that the oligopeptide sequence is not AKFVAAWTLKAAA; in particular, the T cell epitope is selected from the group consisting of AKFVAAWTLKAAANRRA-(NH-NH ₂ ), AKFVAAWTLKAAAN-C, AKFVAAWTLKAAA-C, AKFVAAWTLKAAANRRA-C, aKXVAAWTLKAAaZC, aKXVAAWTLKAAaZCNRRA, aKXVAAWTLKAAa, aKXVAAW TLKAAaNRRA,aA (X)AAAKTAAAAa, aA(X)AAATLKAAa, aA(X)VAAATLKAAa, aA(X)IAAATLKAAa, aK(X)VAAWTLKAAa and aKFVAAWTLKAAa, where X is L-cyclohexylalanine, Z is aminocaproic acid, and a is an aliphatic amino acid residue selected from the group consisting of alanine, glycine, valine, isoleucine and leucine; and/or an alpha synapse in which the T cell epitope is selected from the following group Nucleoprotein peptides: GKTKEGVLYVGSKTK (aa31-45), KTKEGVLYVGSKTKE (aa32-46), EQVTNVGGAVVTGVT (aa61-75), VTGVTAVAQKTVEGAGNIAAATGFVK (aa71-86), DPDNEAYEMPSE (aa116-130), DNEAYEMPSEEGYQ (aa 121-135) and EMPSEEGYQDYEPEA (aa126- 140). 8. The conjugate according to any one of embodiments 1 to 7, wherein the conjugate further comprises a carrier protein, preferably a non-toxic cross-reactive material (CRM) of diphtheria toxin, especially CRM197, KLH, diphtheria toxoid (DT) ), tetanus toxoid (TT), Haemophilus influenzae protein D (HipD) and meningococcal serogroup B outer membrane protein complex (OMPC), recombinant non-toxic form of Pseudomonas aeruginosa exotoxin A ( r EPA ), flagellin, E. 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 peptides Dendrimers, for example, multiple antigenic peptides (MAP), especially wherein the ratio of carrier protein to β-glucan or mannan 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; the preferred restriction condition is that if the conjugate contains a carrier protein, the conjugate contains at least one other independently bound T cell or B cell epitope polypeptide, wherein preferably, the conjugate is composed of or includes the following: (a) β-glucan (b) at least B cell or T cell epitope polypeptide, and (c) carrier protein , wherein the three components (a), (b) and (c) are in the order (a)-(b)-(c), (a)-(c)-(b) or (b)-(a) -(c), especially covalently bonded to each other in the order (a)-(c)-(b); and preferably all such components (a), (b) and (c) are bonded via a linker . 9. The conjugate according to any one of embodiments 1 to 8, wherein the polypeptide is or includes a B cell or T cell epitope polypeptide, wherein the polypeptide is preferably or includes a B cell and T cell epitope, especially wherein the epitope polypeptide is selected from the following group: Tau polypeptide, preferably 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, Tau260-264, Tau 267-273, Tau294-305, 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, Tau 418-426, Tau 420-426; including mimics of the above-mentioned Tau-derived polypeptides that mimic epitopes, and amino acid substitutions containing simulated phosphorylated amino acids (including substitution of phosphorylated S by D and substitution by E Phosphorylated T) peptides include Tau176-186, Tau200-207, Tau210-218, Tau213-221, Tau225-234, Tau379-408, Tau389-408, Tau391-408, Tau393-402, Tau393-406, and Tau418 respectively. -426, Tau420-426; Tau379-408 with pSp396 and pS404, dual phosphorylated peptides Tau195-213 [pS202/pT205], Tau207-220 [pT212/pS214] and Tau224-238 [pT231], fused to a 7-mer (Tau418-426) or 11-mer (Tau417-427) N-terminal YGG linker, Tau position sequence 195-213 SGYSSPGSPGTPGSRSRTP 207-220 GRSRSRTPSLPTPPT 224-238 KKVAVVRTPPKSPSS 393-408 VYKpSPVVSGDTpSPRHL 379-408[P-Ser396,404] RENAKAKTDHGAEIVYKpSPVVSGDTpSPRHL Tau260-264[P-Ser262] ikB 294-305 KDNIKHVPGGGS* 251-280 PDLKNVKSKIGSTENLKHQPGGGKVQIINK 256-285 VKSKIGSTENLKHQPGGGKVQIINKKLDLS 256-285-pS262 VKSKIGpSTENLKHQPGGGKVQIINKKLDLS 259-288 KIGSTENLKHQPGGGKVQIINKKLDLSNVQ 275-304 VQIINKKLDLSNVQSKCGSKDNIKHVPGGG 201-230-pT217 GSPTPGSRSRTPSLPpTPPTREPKKVAVVR 379-408-pS396pS404 RENAKAKTDHGAEIVYKpSPVVSGDTpSPRHL 181-210-pS202pT205 TPPSSGEPPKSGDRSGYSSPGpSPGpTPGSRS 300-317 VPGGGSVQIVYKPVDLSK 267-273 KHQPGGG 298-304 KHVPGGG 329-335 HHVPGGG 361-367 THVPGGG 200-207(pS202/pT205) PGpSPGpTPG 200-207(pS202/pT205) phosphate mimetics PGDPGEPG 200-207(pS202/pT205) phosphate mimetics SPGDPGEPG 210-218 (pT212/pS214) SRpTPpSLPTP 210-218 (pT212/pS214) Phosphomimetic SREPDLPTP 210-218 (pT212/pS214) Phosphomimetic SREPDLP 213-221 (pT217) PSLPpTPPTR 213-221 (pS214/pT217) PpSLPpTPPTR 213-221 (pT217) Phosphomimetic PSLPEPPTR 213-221 (pS214/pT217) Phosphomimetic PDLPEPPTR 393-402 (pSer396) VYKpSPVVSGD 393-402 (pSer396) Phosphomimetic VYKDPVVSG 393-406 (pSer396, pSer404) VYKpSPVVSGDTpSPR 393-406 (pSer396, pSer404) Phosphomimetics VYKDPVVSGDTDPR 176-186 (pT181) PPAPKpTPPSSG 176-186 (pT181) Phosphomimetic PPAPKEPPSSG 225-234 (pT231) KVAVVRpTPPK 225-234 (pT231) Phosphomimetic KVAVVREPPKS Tau379-408[P-Ser396,404] RENAKAKTDHGAEIVYKpSPVVSGDTpSPRHL Tau379 - 408[P-Ser396,404] phosphate mimetics RENAKAKTDHGAEIVYKDPVVSGDTDPRHL 389-408 GAEIVYKpSPVVSGDTpSPRHL 391-408 EIVYKpSPVVSGDTpSPRHL 389-408 Phosphate Mimic GAEIVYKDPVVSGDTDPRHL 391-408 Phosphate mimetic EIVYKDPVVSGDTDPRHL 418-426 DMVDpSPQLA 418-426 Phosphate Mimics DMVDDPQLA 420-426 VDpSPQLA 420-426 Phosphate Mimic VDDPQLA 362-366 HVPGG (LDNIT HVPGG GNKKIE) 235-246 SPSSAKSRLQTA ; IL12/23 polypeptide, preferably FYEKLLGSDIFTGE, FYEKLLGSDIFTGEEPSLLPDSP, VAQLHASLLGLSQLLQP, GEPSLLPDSPVAQLHASLLGLSQLLQP, PEGHHWETQQIPSLSPSQP, PSLLPDSP, LPDSPVA, FYEKLLGSDIFTGEEPSLLPDSPVAQLHASLLGLSQLLQP, LLPDSP, LLGSDIFTGEPS LLPDSPVAQLHASLLG, FYEKLLGSDIFTGEEPSLLPDSPVAQLHASLLG, QPEGHHW, LPDSPVGQLHASLLGLSQLLQ and QCQQLSQKLCTLAWSAHPLV; GHMDLREEGDEETT, LLPDSPVGQLHASLLGLSQ and LLRFKILRSLQAFVAVAARV; IL12/23 p40 sub-unit aa136-145, aa136-143, aa 136-151, aa137-146, aa144-154, aa144-155; QPEGHHWETQQIPSLS, GHHWETQQIPSLSPSQPWQRL, QPEGHHWETQ, TQQIPSLSPSQ, QPEGHHWETQQIPSLSPSQ, QPEGHHWETQQIPSLSPS; Native human IL12/23p40-aa15-66, aa38-46 , aa53-71, aa119-130, aa160-177, aa236-253, aa274-285, aa315-330; LLLHKKEDGIWSTDILKDQKEPKNKTFLRCE and KSSRGSSDPQG; aa38-46, aa53-71, aa119-130, aa of mouse IL12/23 160-177 , aa236-253, aa274-285, aa315-330; IgE polypeptides, preferably SVNPGLAGGSAQSQRAPDRVL, HSGQQQGLPRAAGGSVPHPR; AVSVNPGLAGGSAQSQRAPDRVLCHSGQQGLPRAAGGSVP, QQQGLPRAAGG, QQLGLPRAAGG, QQQGLPRAAEG, QQLGLPRA AEG, QQQGLPRAAG, QQLGLPRAAG, QQQGLPRAAE, QQLGLPRAAE, HSGQQQGLPRAAGG, HSGQQLGLPRAAGG, HSGQQQGLPRAAEG, HSGQQLGLPRAAEG, QSQRAPDRVLCHSG , GSAQSQRAPDRVL and WPGPPELDV; Her2 polypeptides, preferably LHCPALVTYNTDTFESMPNPEGRYTFGASCV, ACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEK and CPLHNQEVTAEDGTQRCEK; KLLSLIKGVIVHRLEGVE; Her2 sequence aa266-296, aa563-598, aa585-598 , aa597-626 and aa613-626; AVLDNGDPLNNTTPVTGA, LKGGVLIQRNPQLC, YNTDTFESMPNPEGRYTFGAS, PESFDGDPASNTAPLQPEQLQ, PHQALLHTANRPEDE, CRVLQGLPREYVNARHC, YMPIWKFPDEEGAC; RHSPESFDGDPASNTAPLQPYMPIWKFPDEEGAC; C-KLYWADGEFT-C, C-VDYHYEGTIT-C, C-VDYHYEGAIT-C; RLVPVGLERGTVDWV, TRWQKGLALGSGDMA, QVSHWVSGLAEGSFG, LSHTSGRVEGSVSLL, LDSTSLAGGPYEAIE, HVVMNWMREEFVEEF, SWASGMAVGSVSFEE. QVSHWVSGLAEGSFG and LSHTSGRVEGSVSLL; RLSLTEI LKGGVLIQRNPQLC, VLIQRNPQLCYQDTILWKDI, YQDTILWKDIFHKNNQLALT, FHKNNQLALTLIDTNRSRAC, LIDTNRSRACHPCSMPCKGS, HPCSMPCKGSRCWGESSEDC, RCWGESSEDCQSLTRTVCAG, QSLTRTVCAGGCARCKGPLP, GCARCKGPLPTDCCHEQCAA, TDCCHEQCAAGCTGPKHSDC, GCTGPKHSDCLACLHFNHSG, LACLHFNHSGICELHCPALV, ICELHCPALVTYNTDTFESM, TYNTDTFESMPNPEGRYTFG, PNPEGRYTFGASCVTACPYN, GASCVTACPYNYLSTDVGS, PYNYLSTDVGSCTLVCPLHNQE, TLVCPLHNQEVTAED PD1, PDL1 and CTLA-4 polypeptides are preferably GAISLAPKAQIKESLRAEL, PGWFLDSPDRPWNPP, FLDSPDRPWNPPTFS, SPDRPWNPPTFSPA, ISLHPKAKIEESPGA and FMTYWHLLNAFTVTVPKDL, especially GAISLAPKAQIKESLRAEL; Aβ polypeptides are preferably native human Aβ1-40 and/or Aβ1-42 or those having Aβ1-42 sequences. aa1-6, aa1-7, aa1-8, aa1-9, aa1-10, aa1-11, aa1-12, aa1-13, aa1-14, aa1-15, aa1-21, aa2-7, aa2- 8. aa2-9, aa2-10, aa3-8, aa3-9, aa3-10, aa pE3-8, aa pE3-9, aa 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 , polypeptide fragments of aa30-42, aa37-42; NYSLDKIIVDYNLQSKITLP, LINSTKIYSYFPSVISKVNQ, LEYIPEITLPVIAALSIAES; cyclized Aβ1-14; DKELRI, DKELRID, DKELRIDS, DKELRIDSG, DKELRIDSGY, SWEFRT, SWEFRTD, SWEFRTDS, SWEFRTDSG, SWEFRTDSGY ,TLHEFRH,TLHEFKH,THTDFRH, THTDFKH, AEFKHD, AEFKHG, SEFRHD, SEFRHG, SEFKHD, SEFKHG, ILFRHG, ILFRHD, ILFKHG, ILFKHD, IRWDTP, IRYDAPL, IRYDMAG; IL31 polypeptide, preferably native human IL31 (Genbank: AAS86448.1), native canine IL31 (Genbank :BAH97742.1), native feline IL31 (UNIPROT: A0A2I2UKP7), native equine IL31 (UNIPROT F7AHG9), or at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to any of the foregoing Any peptide sequence, IL31 protein-derived polypeptides are selected from the mimetics of the above-mentioned IL31-derived polypeptides, including mimetic epitopes and peptides containing amino acid substitutions, for human IL31: sequences aa98-145, aa87-150, Peptides derived from aa105-113, aa85-115, aa84-114, aa86-117, aa87-116; or fragments and peptides thereof SDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQNYTL; DVQKIVEELQSLSKMLLKDV, EELQSLSK and DVQK, LDNKSVIDEIIEHLDKLIFQDA; and DEIIEH, TD THECKRFILTISQQFSECMDALKS,TDTHESKRF,TDTHERKRF HESKRF,HERKRF, HECKRF; KLIFQDAPETNI, FQDAPETNISVP, APETNISVPTDT, TNISVPTDTHEC, SVPTDTHESKRF, TDTHECKRFILT, TDTHESKRFILT, TDTHERKRFILT, HECKRFILTISQ, HESKRFILTISQ, HERKRFILTISQ, KRFILTISQQFS, ILTISQQFSECM, ILTISQQFSESM, ILTISQQFSERM, ISQQFSECMDLA, ISQQFSESMDLA, ISQQFSERMDLA, QFSECMDALKS, QFSESMDLALKS, QFSERMDLALKS, SKMLLKDVEEEKG, EELQSLSK, KGVLVS, SPAIRAYLKTIRQLDNKSVIDEI For Canine IL31: 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 other Peptides and peptides composed of fragments: SDVRKIILELQPLSRGLLEDYQKKETGV, DVRKIILELQPLSRGLLEDY ELQPLSR LSDKNIIDKIIEQLDKLKFQHE, LSDKNIIDKIIEQLDKLKFQ, KLKFQHE, LSDKNI, LDKL, LSDKN, ADTFECKSFILTILQQFSACLESVFKS and ADNFERKNF For feline IL31: feline IL-31 sequence aa124-135 and peptides SDVRKIILELRPMSKGLLQDYVSKEIGL and DVRKIILELRPMSKGLLQDY, LSDKNTIDKIIEQLDKLKFQRE, ADNFERKNFILAVLQQFSACLEHVLQS and ADNFERKNF For equine IL31: aa118-129 and peptides of equine IL-31 sequence: LQPKEIQAIIVELQNLSKKLLDDY, EIQAIIVELQNLSKKLLDDY, SLNNDKSLYIIEQLDKLNFQ and TDNFERKRFILTILRWFSNCLEHRAQ CGRP polypeptide, preferably: native human CGRP α (ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF ); calcitonin isoform α-CGRP preproprotein aa83 -119, accession number NP_001365879.1 or aa82-228 of native human CGRP β (ACNTATCVTHRLAGLSRSGGMVKSNFVPTNVGSKAF); aa82-118 of calcitonin gene-related peptide 2 precursor, accession number NP_000719.1 or its precursor molecule (NP_001365879.1 and NP_000719.1), preferably selected from the sequence of aa8-35, aa11-37, aa1-20 or fragments thereof and the sequence ACDTATCVTH; ACDTATCVTHRLAGL; ACDTATCVTHRLAGLLSR; ACDTATCVTHRLAGLLSRSG; ACDTATCVTHRLAGLLSRSGGVVKN; VVKN; RSGGVVKN; RLAGLLSRSGGVVKNNFVPT; RLAGLLSRSGGVVKNNFVPTNVG ; RLAGLLSRSGGVVKNNFVPTNVGSK; RLAGLLSRSGGVVKNNFVPTNVGSKAF; LLSRSGGVVKNNFVPTNVGSKAF; RSGGVVKNNFVPTNVGSKAF; GGVVKNNFVPTNVGSKAF; VVKNNFVPTNVGSKAF; NNFVPTNVGSKAF; VPTNVGSKAF; NVGSKAF; GSKAF allergen epitope polypeptide , preferably: polypeptides derived from native allergens, polypeptides derived from allergen proteins, the polypeptides are selected from Mimetics of the above-mentioned allergen-derived polypeptides include simulated epitopes, restricted peptides, peptides containing amino acid substitutions and conformational epitopes, see Table A and Table B. Preferably selected from: Allergen source allergens UNIPROT common alder Aln g 1 P38948 Alternaria alternata Alt a 1 P79085 Alt a 3 P78983 Alt a 4 Q00002 Alt a 5 P42037 Alt a 6 Q9HDT3 Alt a 7 P42058 Alt a 8 P0C0Y4 Alt a 10 P42041 Alt a 12 P49148 Alt a 13 Q6R4B4 Ragweed Amb a 1 P27761 Amb a 1.05 P27762 Amb a 3 P00304 Amb a 5 P02878 Amb a 6 O04004 Amb a 7 No complete sequence available Amb a 8 Q2KN24 Amb a 9 Q2KN27 Amb a 10 Q2KN25 European honey bee Api m 1 P00630 Api m 2 Q08169 Api m 3 Q4TUB9 Api m 4 01501 Api m 5 B2D0J4 Api m 6 Q27SJ8 Api m 7 Q8MQS8 Api m 8 B2D0J5 Api m 9 C9WMM5 Api m 10 Q1HHN7 Api m 11 B3GM11 Api m 12 Q868N5 celery Api g 1 P49372 Api g 2 E6Y8S8 Api g 3 P92919 Api g 4 Q9XF37 Api g 5 P81943 peanut Ara h 1 P43238 Ara h 2 Q6PSU2-1 Ara h 3 O82580 Ara h 3.02 Q9SQH7 Ara h 5 Q9SQI9 Ara h 6 Q647G9 Ara h 7 Q9SQH1 Ara h 8 Q6VT83 weeping birch Bet v 1 P15494 Bet v 2 P25816 Bet v 3 P43187 Bet v 4 Q39419 Bet v 6 O65002 Bet v 7 P81531 domestic dog Can f 1 O18873 Can f 2 O18874 Can f 3 P49822 Can f 4 D7PBH4 Can f 5 P09582 Can f 6 H2B3G5 Can f 7 Q28895 Can f 8 F1PHB6 European hornbeam car b 1 P38949 European chestnut Cas s 1 B7TWE3 Cas s 5 Q42428 Cas s 8 Unknown sequence Mycosporium Cla h 2 No complete sequence available i h 5 P42039 i h 6 P42040 i h 7 P42059 i h 8 P0C0Y5 ci h 9 B7ZK61 CI h 10 P40108 CI h 12 P50344 filbert Cor a 1 Q08407 Cor a 2 Q9AXH5 Cor a 6 A0A0U1VZC8 Cor a 8 Q9ATH2 Cor a 9 Q8W1C2 Cor a 10 Q9FSY7 Cor a 11 Q8S4P9 Japanese cedar Cry j 1 P18632 Cry j 2 P43212 European carp Cyp c 1 Q8UUS3 wild carrot Dau c 1 O04298 Dau c 4 Q8SAE6 European house dust mite Der p 1 P08176 (variant) Der p 2 P49278 Der p 3 P39675 Der p 4 Q9Y197 Der p 5 P14004 Der p 6 P49277 Der p 7 P49273 Der p 8 P46419 Der p 9 Q7Z163 Der p 10 O18416 Der p 11 Q6Y2F9 Der p 14 Q8N0N0 Der p 20 B2ZSY4 Der p 21 Q2L7C5 Der p39 XP_027203190.1 european beech Fags 1 B7TWE6 domestic cat Fel d 1 P30438 (chain 1), P30440 (chain 2) Fel d 2 P49064 Fel d 3 Q8WNR9 Fel d 4 Q5VFH6 Fel d 5 No complete sequence available Fel d 6 No complete sequence available Fel d 7 E5D2Z5 Fel d 8 F6K0R4 rubber tree Hev b 1 P15252 Hev b 2 P52407 Hev b 3 O82803 Hev b 4 Q6T4P0 Hev b 5 Q39967 Hev b 6 P02877 Hev b 7.01 O04008 Hev b 7.02 O65811 Hev b 8 O65812 Hev b 9 Q9LEJ0 Hev b 10 P35017 Hev b 11 Q949H3 Hev b 12 Q8RYA8 Hev b 13 Q7Y1X1 American cedar Jun a 1 P81294 Jun a 2 Q9FY19 Jun a 3 P81295 apple Mal d 1 P43211 Mal d 2 Q9FSG7 Mald 3.0101w Q5J026 Mal d 4 Q9XF42 white oak Que a 1 B6RQS1 timothy grass PhIp 1 Q40967 PhI p 2 P43214 PhI p 4 Q5ZQK5 PhI p 5 Q40960 PhI p 6 P43215 PhI p 7 O82040 PhI p 11 Q8H6L7 PhI p 12 P35079 PhI p 13 Q9XG86 Polistes annularis Pol a 1 Q9U6W0 Pol a 5 Q05109 paper wasp Pol d 1 Q6Q252 Pol d 4 Q7Z269 Pol d 5 Q68KJ8 Guinea Paper Wasp (Polistes exclamans) Pol e 1 No complete sequence available Pol e 4 No complete sequence available Pol e 5 Q68KJ9 Northern paper wasp (Polistes fuscatus) Pol f 5 P35780 Polistes gallicus Pol g 1 P83542 Pol g 5 P83377 Polistes metricus Pol m 5 P35780 and/or selected from: allergens UNIPROT Epitope / Mock epitope Amb a 1 P27761 GMLATVAFNTFTDNVDQR, AFNKFTDNVDQR, MPRCRFGF, WRTQNDVLENG TFTDNVDQRMPRCRH RVVELMDWTVLH, GSAMTWGMLAAE, SYNIIATGIHPV, TMVATGLMPVLI, QDFDDIL, CLFSQGNRC, VANLKVGV, NPGGLSAAPAGS, RHASTLLGRHG, EAAESMWRVASG, QNRLNSNGNNGG SQ, DDDLQHQFDDQD Amb a 3 P00304 CDIKDPIRLEPGGPD, EVWREEAYHACDIKD, GKVYLVGGPELGGWK, LGGWKLQSDPRAYAL, NFTTGEDSVAEVWRE, PGGPDRFTLLTPGSH, PGGPDRFTLLTPGSH, QFKTTDVLWFNFTTG, RAYALWSARQQFKTT, TPGSHFICTKDQKFV Alt a1 P79085 MISTSRK, QKRNTIT, KISEFYGRKP, VATATLPNYC, YSCGENSFMD, YYNSLGFNIK Ara h 1 P43238 REREREEDWRQPREDWRRPS, RTRGRQPGDYDDDRRQPRRE, DDDRRQPRREEGGRWGPAGP, TTNQRSPPGERTRGRQPGDY, QPREDWRRPSHQQPRKIRPE, GREGEQEWGTPGSHVREETS, PVNTPGQFEDFFPASSRDQS, AGGEQEERGQRRWSTRSSEN, REGEPDLSNNFGKLFEVKPD, NASSELHLLGFGINAENNHR, IDQ IEKQAKDLAFPGSGEQV, LAFPGSGEQVEKLIKNQKES AKSSPYQKKT, QEPDDLKQKA, LEYDPRLUYD, GERTRGRQPG, PGDYDDDRRQ, PRREEGGRWG, REREEDWRQP, EDWRRPSHQQ, QPKKIRPEGR, TPGQFEDFFP, SYLQEFSRNT, FNAEFNEIRR, EQEERGQRRW, DITNPINL RE , NNFGKLFEVK, GTGNLELVAV, RRYTARLKEG, ELHLLGFGIN, HRIFLAGDKD, IDOIEKOAKD, KDLAFPGSGE, KESHFVSARP, PEKESPEKED Ara h 2 Q6PSU2-1 ARQQWELQGDRRCQSQLERA, ERDPYSPSQDPYSPSPYDRR, RRCQSQLERANLRPCEQHLM, RDPYSSPYDRRGAGSSQHQ, GRDPYSPSQDPYSPSQDPDR, PYSPSQDPDRRDPYSPSPYD, QKIQRDEDSYERDPYSPSQD, HASARQQWEL, QWELQGDRRC, DRRCQSQLER, LRPCEQHLM Q, KIQRDEDSYE, YERDPYSPSQ, SQDPYSPSPY, DRLQGRQQEQ, KRELRNLPQQ, QRCDLDVESG, HASARQQWEL, ERDPYSPSQDPYSPS, RRCQSQLER, CDLEVESGGRDRY, ERDPYSPSQDPYSPS, NLRPCEQHLMQKIQRD, PQRCDLE, RQQEQQFKRELRNLPQQ, SDRLQGRQQ, RRCQSQLERANLRPCEQHLMQKIQRDEDSYGRDPYSPSQDPY Ara h 3 O82580 IETWNPNNQEFECAG, GNIFSGFTPEFLEQA, VTVRGGLRILSPDRK, DEDEYEYDEEDRG, EDEYEYDEEDRRRGRGSRGR, NIFSGFTPEFLEQAFQVDDR, ESEEEGAIVTVRGGLRILSP, SGFTPEFLEQAFQVDDRQIV, EEGAIVTVRGGLRILSPDRK, TYEEPAQQGRRYQSQRPPRR, RRADEEEEYDEDEYDEED , NHEQEFLRYQQQSRQSRRRS, QEFLRYQQQSRQSRRRSLPY, QEEREFSPRGQHSRRERAGQ Ara h 6 Q647G9 CDELNEMENTQR, CEALQQIMENQCD, CNFRAPQRCDLDV, KPCEQHIMQRI, YDSYDIR, KRELRMLPQQ, MRRERGRGGDSSSS Bet v 1 P15494 FILDG aa 32-37, aa 42-50, aa138 -153. CQQFLSVRALC, QQFLSVRALC, CQQFLSVRAL Cry j 1 P18632 DALTLRTATNIW, DGDALTLRTATN, DGRGAQVYIGNG, NATPQLTKNAGV, NGGPCVFIKRVS, NGNATPQLTKNA, NSDDDPVNPAPG, VENGNATPQLTK, NAGVLTCSLSK Cry j 2 P43212 KWVNGREI, GQCKWVNGREICNDRDRPTA, GRENSRAEVSYVHVNGAKFI, PGNKKFVVNNLFFNGPCQPH, SHIIYENVEMINSENPILINQFYCT, YCTSASACQNQRSAVQIQDV, TYKNIRGTSATAAAIQLKCS, AEVSYVHVNGAK Der p 1 P08176 (variant) KGIPNTKAP, DMFQIGKYG, GIREVWPAG, SSMGAYWGG, KGTTGVRNT, CQIYPPNANKIREALAQ, GYSNAQGVDYWI, NQSLDLAEQELVDCASQHGC, VRNSWDTNWGDNGY, EQSYPCWLSGTPSTP, TPLCDYAAARVGACG, KEQLPTSYPPERAGW, NCLSSDEPLHIRWCQ, EALSEEEWPRYTS HP, SCDATQRAQGRCS, SCDSSQKKQGRCS, SCDESRRRQGRCS, KGIPNTKAP, DMFQIGKYG, GIREVWPAG, SSMGAYWGG, KGTTGVRNT, CKGIPNTKAP, CDMFQIGKYG, CGIREVWPAG, CSSMGAYWGG, CKGTTGVRNT, CKGIPNTKAPC, CDMFQIGKYGC, CGIREVWPAGC, CSSMGAYWGGC, KGTTGVRNTC, KGIPNTKAPC, DMFQIGKYGC, GIREVWPAGC, SSMGAYWGGC, KGTTGVRNTC, Der p 2 P49278 FVVEYTKKW, SWWNLPQIG, KGITTKWMA, AGISYTKTW, DQVDVKDCANHEIKK, VPGIDPNACHYMKCK, APKSENVVVTVKVMGDNGVLACAIATHAKIRD, CHGSEPCIIHRGKPFQLEAVFEANQNSKTAK, DQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGK, EANQNSKTAKIE IKASIEGLEVDVPGIDPNAC, EVDVPGIDPNACHYMKCPLVKGQQYDIKYTWIVPKIAPKSEN, FVVEYTKKW, SWWNLPQIG, KGITTKWMA, AGISYTKTW, FVVEYTKKWC, SWWNLPQIGC, KGITTKWMAC, AGISYTKTWC, CFVVEYTKKWC, CSWWNLPQIGC, CKGITTKWMAC, CAGI SYTKTWC, CFVVEYTKKW, CSWWNLPQIG, CKGITTKWMA, CAGISYTKTW Der p39 XP_027203190.1 QEELREAFRMY, TSALREILRAL, NDELDEMIAEI Fel d 1 P30438 (chain 1), P30440 (chain 2) KALPVVLENARILKNCVDAKMTEEDKE, KRDVDLFLTGTPDEYVEQVAQYKALPV, DAKMTEEDKENALS, EICPAVKRDVDLFLTG, EPERTAMKKIQDCY, FAVANGNELLLDLS, KKIQDCYVENGLIS, LLDKIYTSPLC, VAQYKALPVVLENA, VKMAETCPIFYDVF, VQNTVEDLKLNTLGR, EICPAVKRDVD LFLTGTPDEYVEQVAQYK, SSKDCMGEAVQNTVEDLKLNTLGR, VKMAITCPIFYDVFFAVANGNELLLDLSLTKVNA, CPAVKRDVDLFLT, EQVAQYKALPVVLENA KALPVVLENARILNCV RILKNCVDAKMTEEDKE KENALSLLDKIYTSPL TAMKKIQDCY VENGLI SRVLDGLVMTTISSSK, ENLAYTKALLTG; NNISVDGLLTS; VIRNELLLTYA; VINDLLLVLA; GDFYPL; NDFYPE; LDLNP, FAVANGNELL; LKLNTLGREICPAVKRGVDL; YVEQV; ENARILKNCVDAKM; aa21-31, aa21-47 PhIp 1 Q40967 VRYTTEGGTKTEAEDVIPEGWKADTSYESK, APYHFDLSGHAFGAM, HITDDNEEPIAPYHFDLSGHA, NEEPIAPYHFDLSGHAFG, CFEIKCTKPEACSGEPVVV, CTKPEACSGEPVVVHITDDNEEPIAPYHFDLSGH, DNEEPIAPYHF, EPIAPYHFDLSGH, EQKLRSAGELELQFRRVKC, GEPVVVHITDDNEEPIA PYHFDLSGHAFGAMAKKG, GYKDVDKPPFSGMTGCGNTPIFKSGRGCGSCFEIKCTKPEACS, HITDDNEEPIAPYHF, HITDDNEEPIAPYHFDLSGHAFGA, HVEKGSNPNYLALLVKYVNGDGDV VAV, KCTKPEACSGEPVVVHITDDNEEPIAPYHFDLS, KPPFSGMTGCGNT, KTEAEDVIPEGWKADTSYESK, LTGPFTVRYTTEGGTKTEAEDVIPEGWKADTSYESK, PIAPYHFD, PIAPYHFDLSGHAFG PhI p 2 P43214 VPKVTFTVEKGSNEKHLAVLVKYEGDTMAEVEL, VEKGSNEKHLAVLVKYEGDTMAEVELREHGSD, REHGSDEWVAMTKGEGGVWTFDSEEPLQGPFN, FRFLTEKGMKNVFDDVVPEKYTIGATYAPEE, CFRFLTEKGMKNVFDDVVPEKYTIGATYAPEE, FRFLTEKGMKNVFDDVVPEKYTIGATYAPEEC PhIp5 Q40960 AEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDK, ATTEEQKLIEKINAGFKAALAAAAGVQPADKYR, FVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSK, ADLGYGPATPAAPAAGYTPATPAAPAEAAPAGK, AYKLAYKTAEGATTPEAKYDAYVATLSEALRI, EAAFNDAIKASTGGAYESYKFIPALEAAVK, TVATAPEV KYTVFETALKKAITAMSEAQKAAK, SRLGRSSAWV, THWQLGERPD, PSTPGERVRH, RGGPDDLTAL, PFWVRGTTW, PSTPGSRQNM, PSTPGDNPLV, KFVVNGRWID, KFLVNGRWID, RLTENTEPLL, FTWGGLRDKS, ERAGAMERAN, RSVSKEEPGM, KLGKFGAARV, VQDLMKSSGV, KLGKF GAARV, CKLGKFGAARVC, CKLGKFGAARV, KLGKFGAARVC, PhI p 6 P43215 GKATTEEQKLIEDVNASFRAAMATTANVPPAD, YKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYN, DAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKY, AADHAAPEDKYEAFVLHFSEALRIIAGTPEVHA Human PCSK9 polypeptide, preferably native human PCSK9 or a polypeptide comprising amino acid residues aa150 to 170, aa153-162, aa205 to 225, aa211-223, aa368-382 or composed thereof, which has an amino acid sequence ( Registration number: Q8NBP7): And/or PCSK9 protein-derived polypeptides, which are selected from mimics of the above-mentioned polypeptides, including simulated epitopes and peptides containing amino acid substitutions, and/or PCSK9 derived sequences NVPEEDGTRFHRQASK, NVPEEDGTRFHRQASKC, PEEDGTRFHRQASK, CPEEDGTRFHRQASK, PEEDGTRFHRQASKC, AEEDGTRFHRQASK, TEEDGTRFHRQASK , PQEDGTRFHRQASK, PEEDGTRFHRRASK, PEEDGTRFHRKASK, PEEDGTRFHRQASR, PEEDGTRFHRTASK, SIPWNLERITPPR, PEEDGTRFHRQASK, PEEDGTRFHRQA, EEDGTRFHRQASK, EEDGTRFHRQAS, SIPWNLERITP, SIPWNLERITPC, SIPWNLERIT, SIPWNLERITC, LRPRGQPNQC, SRHLAQASQ, SRHLAQASQC, SRSGKRRGER, SRSGKRRGERC, IIGASSDCSTCFVSQ, IIGASSDSSTSFVSQ, IIGASSDSSTSFVSQC, CIGASSDSSTSFVSC, IGASSDSSTSFVSC, CDGTRFHRQASKC , DGTRFHRQASKC, CDGTRFHRQASK, AGRDAGVAKGAC, RDAGVAKC, RDAGVAK, SRHLAQASQLEQC; SRHLAQASQLEQ, GDYEELVLALRC; GDLLELALKLP, EEDSSVFAQC, EEDSSVFAQ, NVPEEDGTRFHRQASKC, NVPEEDGTRFHRQASK, CKSAQRHFRTGDEEPVN, KSAQRHFRTGDEEPVN, alpha synapse Nucleoprotein polypeptides, preferably native α-synuclein or polypeptides composed of the following amino acid residues of the amino acid sequence of native human α-synuclein: 1 to 5, 1 to 8, 1 to 10, 60 to 100, 70 to 140, 85 to 99, 91 to 100, 100 to 108, 102 to 108, 102 to 109, 103 to 129, 103 to 135, 107 to 130, 109 to 126, 110 to 130 , 111 to 121, 111 to 135, 115 to 121, 115 to 122, 115 to 123, 115 to 124, 115 to 125, 115 to 126, 118 to 126, 121 to 127, 121 to 140 or 126 to 135, the The amino acid sequence is: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA (human aSyn (1-140 aa): UNIPROT accession number P3 7840), preferably comprising or consisting of the following amino acid residues Polypeptides: 1 to 8, 91 to 100, 100 to 108, 103 to 135, 107 to 130, 110 to 130, 115 to 121, 115 to 122, 115 to 123, 115 to 124, 115 to 125, 115 to 126 , 118 to 126, 121 to 127 or 121 to 140; or a simulated epitope selected from the group consisting of: DQPVLPD, DQPVLPDN, DQPVLPDNE, DQPVLPDNEA, DQPVLPDNEAY, DQPVLPDNEAYE, DSPVLPDG, DHPVHPDS, DTPVLPDS, DAPVTPDT, DAPVRPDS and YDRPVQPDR. 10. The conjugate of any one of embodiments 1 to 8, wherein the conjugate includes a T cell epitope and does not contain a B cell epitope, wherein the conjugate preferably contains more than one T cell epitope. , especially containing two, three, four or five T cell epitopes. 11. The combination according to any one of embodiments 1 to 10, which is used for preventing or treating diseases in humans, mammals or birds, preferably for preventing or treating infectious diseases, chronic diseases, especially in humans. Allergic or autoimmune diseases; preferably, use in the prevention or treatment of diseases caused directly or indirectly by fungi, especially Candida albicans, is excluded. 12. The conjugate according to any one of embodiments 1 to 11 for active anti-Tau vaccination against: synucleinopathies, Pick disease, progressive supranuclear palsy (PSP) ), corticobasal degeneration, frontotemporal dementia associated with chromosome 17 (FTDP-17) and argyrophilic granulopathy; and/or active immunotherapy for IL12/IL23 related diseases and autoimmune inflammatory diseases, Such diseases are especially selected from the group consisting of: psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, diabetes (preferably type 1 diabetes), atherosclerosis, inflammatory bowel disease (IBD) /Crohn's disease (M. Crohn), multiple sclerosis, Behcet's disease, ankylosing spondylitis, Vogt-Koyanagi-Harada disease, chronic granulomatous disease, hidradenitis suppurativa, anti-neutropenia ANCA-associated vasculitis, neurodegenerative disease (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; and/or as an active anti-EMPD vaccine for the treatment and prevention of IgE-related diseases , preferably allergic diseases such as seasonal, food, pollen, mold spores, poisonous plants, agents/medicines, insect, scorpion or spider venom, latex or dust allergies; pet allergies; allergic bronchial asthma; non-allergic asthma ;Chager-Strauss syndrome; allergic rhinitis and conjunctivitis; atopic dermatitis; nasal polyps; Kimura's disease; reactions to adhesives, antimicrobials, fragrances, hair dyes, metals, rubber ingredients, topical Contact dermatitis with agents, rosins, waxes, polishes, cement and leather; chronic sinusitis; atopic eczema; autoimmune diseases in which IgE plays a role ("autoallergy"); chronic (idiopathic) chronic or autoimmune urticaria; choline-induced urticaria; mastocytosis, especially cutaneous mastocytosis; allergic bronchopulmonary zoomycosis; chronic or recurrent idiopathic angioedema; intermittent Qualitative cystitis; anaphylaxis, especially idiopathic and exercise-induced anaphylaxis; immunotherapy, eosinophil-related diseases (such as eosinophilic asthma, eosinophilic gastroenteritis, eosinophilic otitis media and eosinophilic esophagitis); or for the treatment of lymphoma or prevention of sensitizing side effects of antacid therapy, especially for gastric or duodenal ulcers or reflux; and/or for active anti-human epidermal growth factor receptor 2 (anti- Her2) vaccination to treat and prevent Her2-positive neoplastic diseases; and/or for individualized neoantigen-specific therapy, preferably NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-C1, MAGE- In the case of C2, MAGE-C3, survivin, gp100, tyrosinase, CT7, WT1, PSA, PSCA, PSMA, STEAP1, PAP, MUC1, 5 T4, KRAS or Her2; and/or for the control of cancer microorganisms Environmental active anti-immune checkpoint vaccination for the treatment and prevention of neoplastic diseases and for the treatment and prevention of T cell dysfunction in cancer/neoplastic diseases (e.g., avoidance of CD8 T cell infiltration into cancer tissue depletion) and chronic degenerative diseases Diseases, including diseases with reduced T cell activity, such as Parkinson's disease; and/or for familial and sporadic AD, familial and sporadic Aβ cerebral amyloid angiopathy, hereditary cerebral hemorrhage with amyloidosis (HCHWA), dementia with Lewy bodies and Down syndrome, retinal ganglion cell degeneration in glaucoma, inclusion body myositis/myopathy; and/or as an active vaccine for the treatment and prevention of synaptic nuclei Proteinopathy, preferably Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), Parkinson's disease dementia (PDD), neuroaxonal dystrophy, amygdala damage Lewy body-limited Alzheimer's disease (AD/ALB); and/or its use as an active vaccine containing an antigen or neoantigen selected from the group consisting of: NY-ESO-1, MAGE-A1, MAGE-A3 , MAGE-C1, MAGE-C2, MAGE-C3, Survivin, gp100, tyrosinase, CT7, WT1, PSA, PSCA, PSMA, STEAP1, PAP, MUC1, 5 T4 and KRAS; and/or for the treatment of or Prevention of IL31-related diseases associated with IL31 protein-derived polypeptides, such as fragments of IL31 protein, preferably pruritus-causing allergic diseases, pruritus-causing inflammatory diseases, and pruritus-causing inflammatory diseases in mammals (including humans, dogs, cats, and horses) Autoimmune diseases; atopic dermatitis, prurigo nodularis, psoriasis, cutaneous T-cell lymphoma (CTCL) and other itching conditions, 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 conditions including lichen planus, cutaneous amyloidosis, stasis dermatitis, scleroderma, pruritus associated with wound healing, and non-pruritic conditions such as Allergic asthma, allergic rhinitis, inflammatory bowel disease (IBD), osteoporosis, follicular lymphoma, Hodgkin lymphoma and chronic myelogenous leukemia; especially anti-IL31 therapy and anti-IL4 and/or anti-IL13 peptide vaccine combination; and/or for the treatment or prevention of CGRP-related diseases associated with CGRP-derived polypeptides, such as fragments of CGRP, preferably intermittent and chronic migraine and cluster headache, hyperalgesia, and dysfunction Hyperalgesia in painful conditions such as rheumatoid arthritis, osteoarthritis, visceral pain hypersensitivity syndrome, fibromyalgia, inflammatory bowel disease, neuropathic pain, chronic inflammatory pain and headache; and/or use In specific allergen immunotherapy (AIT) for the treatment of IgE-mediated type I allergic diseases. Such illnesses include (but are not limited to) hay fever, seasonal, food, pollen, mold spores, poisonous plants, agents/medicines, insect, scorpion or spider venom, latex or dust allergies; pet allergies; allergic bronchial asthma; allergies Rhinitis and conjunctivitis; atopic dermatitis; contact dermatitis to adhesives, antimicrobials, fragrances, hair dyes, metals, rubber ingredients, topical agents, rosin, waxes, polishes, cement and leather; Chronic sinusitis; atopic eczema; autoimmune diseases in which IgE plays a role ("autoallergy"); chronic (idiopathic) and autoimmune urticaria; systemic allergic reactions, especially idiopathic and exercise-induced anaphylaxis; and/or used to improve the target-specific immune response of existing vaccines, especially anti-infectious vaccines, while inducing no or only very limited CLEC-specific or carrier protein-specific antibodies. reaction, these vaccines are preferably selected from the group of vaccines: PedvaxHIB®, ActHIB®, Hiberix®, Recombivax HB®, PREHEVBRIO®, Engerix-B, HEPLISAV-B®, Gardasil®, Gardasil 9®, Cervarix®, Menveo®, Menactra®, MenQuadfi®, Prevnar-13®, Prevnar 20®, Pneumovax-23®, Vaxneuvance®, Typhim V®, Typhim VI®, Typherix®, combined with non-toxic recombinant Pseudomonas aeruginosa exotoxin A Vi polysaccharide, Typbar-TCV®, Shingrix®; and/or for the prevention of infectious diseases, such as microbial infections or viral infections, preferably caused by: Haemophilus influenzae type B (Hib), Streptococcus pneumoniae, meningitis Diplococcus and Salmonella typhi or other infectious agents, including those causing hepatitis A or B, human papillomavirus infection, influenza, typhoid, measles, mumps and rubella. In addition, there are meningococci group B, cytomegalovirus (CMV), respiratory syncytial virus (RSV), Clostridium difficile, extraintestinal pathogenic Escherichia coli (Expec), Klebsiella pneumoniae, Shigella spp. Staphylococcus aureus, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae, coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2), Ebola virus , Borrelia burgdorferi, HIV and other infections; and/or used to treat or prevent proprotein convertase subtilisin kexin type 9 related diseases, which include (but are not limited to) hyperlipidemia, hypercholesterolemia disease, atherosclerosis, increased serum levels of low-density lipoprotein cholesterol (LDL-C) and cardiovascular events, stroke or various forms of cancer, and/or for inducing target-specific immune responses without inducing or only Induces a very limited CLEC-specific or carrier protein-specific antibody response; and/or is used to induce a target-specific immune response while inducing no or only a very limited CLEC-specific or carrier protein-specific antibody response; and/or Used in diseases associated with reduced or dysfunctional Treg populations to enhance the weakened/reduced Treg number and activity, and thereby reduce the autoimmune response of disease-specific T effector cells and suppress the patient's autoimmune response, where the use is suitable As a Treg epitope or with Treg inducers such as rapamycin, low-dose IL-2, TNF receptor 2 (TNFR2) agonists, anti-CD20 antibodies (e.g., rituximab), prednisolone , T cell epitopes of a combination of isoprinosine, glatiramer acetate or sodium butyrate; and/or treatment for enhancing or maintaining T cell numbers, especially T effector cell numbers and T cell functions in PD patients , which preferably includes a combination of checkpoint inhibitors or vaccines that use anti-immune checkpoint inhibitor epitopes to induce an anti-immune checkpoint inhibitor immune response, to enhance or maintain the number of T cells, especially the T effect, in PD patients cell number and T cell function, among which PD patients are preferably selected from those with an overall decrease in CD3+ cells, especially those with an overall decrease in CD3+CD4+ cells typical of PD patients in all disease stages; preferably, those in H+Y1-4 stages, It is better for patients with H+Y stage 1-3, and the best is H+Y stage 2-3. 13. The conjugate according to any one of embodiments 1 to 12, wherein β-glucan or mannan is used as a C-type lectin (CLEC) polysaccharide adjuvant, preferably for enhancing the response to a given T cell T cell response to an epitope polypeptide, wherein the T cell epitope is preferably a linear T cell epitope, especially the T cell epitope is a polypeptide, and the polypeptide includes or consists of the following amino acid sequence: SeqID7 , 8, 22-29, 87-131, GKTKEGVLYVGSKTK, KTKEGVLYVGSKTKE, EQVTNVGGAVVTGVT, VTGVTAVAQKTVEGAGNIAAATGFVK, MPVDPDNEAYEMPSE), DNEAYEMPSEEGYQD, EMPSEEGYQDYEPEA or combinations thereof. 14. The conjugate according to any one of embodiments 1 to 13 for increasing affinity maturation against a specific polypeptide antigen or for inducing an increased immune response relative to a human autologous antigen. 15. The conjugate according to any one of embodiments 1 to 14, further comprising a carrier protein containing a T cell epitope for reducing or eliminating the response and/or enhancement of B cells to CLEC and/or the carrier protein The reaction of T cells to the T cell epitope of a carrier protein, wherein the carrier protein is preferably a non-toxic cross-reactive material (CRM) of diphtheria toxin, especially CRM197, KLH, diphtheria toxoid (DT), tetanus toxoid (TT) ), Haemophilus influenzae protein D (HipD) and meningococcal serogroup B outer membrane protein complex (OMPC), recombinant non-toxic form of Pseudomonas aeruginosa exotoxin A ( rEPA ), flagellin, Escherichia coli Heat-labile enterotoxin (LT), cholera toxin (CT), mutant toxins (e.g. LTK63 and LTR72), virus-like particles, albumin-binding proteins, bovine serum albumin, ovalbumin, synthetic peptide dendrimers, e.g. Multiple antigen peptide (MAP), 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, especially T cell epitopes in vaccines containing linear T cell epitopes with enhanced efficacy, for example by adding lysosomes at the N or C terminus Enhanced by a protease cleavage site, such as a cathepsin L-like cleavage site or a cathepsin S-like cleavage site, wherein the cathepsin L-like cleavage site is preferably defined by the following common sequence: X _n -X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X _{6 -X 7} -X _{8 Xn} : 3-27 amino acids from immunogenic peptide X ₁ : Any amino acid X ₂ : Any amino acid Amino acid X ₄ : N/D/A/ _Q /S/R/G/L; preferably N/D, more preferably N Preferably F or R, _more preferably R X ₆ : F/R/A/K/V/S/Y; Preferably F or R, more preferably R /P/F, preferably AX ₈ : cysteine or linker, such as NHNH ₂ , where the best sequence is _{X n -X 1} Preferably defined by the following common sequence: X _n -X ₁ -X ₂ -X ₃ -X ₄ -X _{5 -X 6} -X ₇ -X ₈ X ₁ : any amino acid X ₂ : any amino acid X ₃ : any amino acid, preferably V, L, I, F, W, Y, H, more preferably V X ₄ : any amino acid, Preferably V, L, I, F, W, Y, H, more preferably V X ₅ : K, R, E, D, Q, N, preferably K, R, more preferably R X ₆ : any amine Amino acid X ₇ : any _amino acid _, preferably AX ₈ : _preferably AX ₈ : cysteine or linker, such as NHNH ₂ , where the best sequence is 16. A method for producing a conjugate as any one of embodiments 1 to 15, wherein the β-glucan or mannan is activated by oxidation and wherein the activated β-glucan or mannan is combined with The B cell epitope polypeptide and/or the T cell epitope polypeptide are contacted, thereby obtaining a relationship between the β-glucan or mannan polysaccharide and the B cell epitope polypeptide and/or the T cell epitope polypeptide. conjugate. 17. The method according to embodiment 16, wherein the β-glucan or mannan is obtained by periodate oxidation, reductive amination or cyanation of the hydroxyl group at the ortho-position. 18. According to the method of embodiment 16 or 17, the β-glucan or mannan polysaccharide is oxidized to the following oxidation degree, which is defined as the degree of reactivity with Schiff’s fuchsin reagent, which is equivalent to 0.2-2.6 , preferably at a molar ratio of 0.6-1.4, especially at a molar ratio of 0.7-1, for the oxidation degree of an equal amount of the fungus polysaccharide oxidized with periodate. 19. The method according to any one of embodiments 16 to 18, wherein the conjugate is conjugated to the carbonyl (aldehyde) by hydrazone-based coupling, or by using a heterobifunctional maleimide and Hydrazine linker (for example: BMPH (N-β-maleyl imino propionic acid hydrazine, MPBH (4-[4-N-maleyl imino-phenyl] butyric acid hydrazine Hydrazine), EMCH (N-[ε-maleimidocaproic acid] hydrazine) or KMUH (N-[κ-maleimidodecanoic acid] hydrazine) couple the thiol group (For example: cysteine) is produced by combining with a carbonyl group (aldehyde). 20. A vaccine product designed to vaccinate an individual against a specific antigen, wherein the product contains a compound containing β-(1 , 6)-glucan or mannan as a C-type lectin (CLEC) polysaccharide adjuvant covalently coupled to a specific antigen. 21. The vaccine product according to embodiment 20, wherein the product comprises the method according to embodiments 1 to 16 Any one or a conjugate obtainable by/has been obtained by the method of any one of embodiments 16 to 19. 22. The vaccine product according to embodiment 20 or 21, wherein the antigen comprises at least one B cell epitope and at least one T cell epitope, preferably, wherein the antigen is a polypeptide comprising one or more B cell epitopes and T cell epitopes. 23. The vaccine product according to any one of embodiments 20 to 22, wherein The covalently coupled antigen and CLEC polysaccharide adjuvant are present in particles with a size of 1 to 5000 nm, preferably 1 to 200 nm, especially 2 to 160 nm, which size is dynamically determined in the form of hydrodynamic radius (HDR) Determined by light scattering (DLS). 24. The vaccine product according to any one of embodiments 20 to 23, wherein the covalently coupled antigen and CLEC polysaccharide adjuvant have a size of 1 to 50 nm, preferably 1 to 25 nm, In particular particles of 2 to 15 nm are present, the size being determined by DLS in HDR format. 25. The vaccine product according to any one of embodiments 20 to 24, wherein the covalently coupled antigen and the CLEC polysaccharide adjuvant are in size Particles of less than 100 nm, preferably less than 70 nm, especially less than 50 nm are present, the size being determined by DLS in HDR format. 26. A pharmaceutical composition comprising as in any one of embodiments 1 to 25 The defined conjugate or vaccine and a pharmaceutically acceptable carrier. 27. The pharmaceutical composition according to embodiment 26, wherein the pharmaceutically acceptable carrier is a buffer, preferably a phosphate or TRIS-based buffer 28. The pharmaceutical composition according to embodiment 26 or 27, which 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 including a needle adapter ; Ampoules, needle-free injection systems, preferably jet syringes; patches, transdermal patches, microstructured transdermal systems, microneedle array patches (MAP) (preferably solid MAP (S-MAP), coated MAP (C-MAP) or dissolved MAP (D-MAP)); electrophoresis systems, ion electrophoresis systems, laser-based systems, especially erbium YAG laser systems; or gene gun systems. 29. The pharmaceutical composition according to any one of embodiments 26 to 28, wherein the conjugate or vaccine is included in the form of a solution or suspension, a deep-frozen solution or suspension, a lyophilisate, a powder or a granule. 30. The use of a conjugate according to any one of embodiments 1 to 15, for the manufacture of a composition for preventing or treating diseases, preferably for preventing or treating infectious diseases, chronic diseases, allergic reactions or autoimmunity Elixir for disease. 31. A method for preventing or treating diseases, preferably for preventing or treating infectious diseases, chronic diseases, allergic reactions or autoimmune diseases, wherein an effective amount according to the embodiment is administered to a patient in need A combination of any of 1 to 15.

without

Figure 1 Display: CLEC Conjugate ConA and DC Receptor ( Right now dectin -1) of In vitro binding activity.

A) Shitu polysaccharide (Pus) was shown to have higher binding efficacy to dectin-1 when compared with lichen (Lich); and B) Compared with Shifu polysaccharide, from oat (Oat_BG265, Beta-glucans from oats_BG391) and barley (Barley_BG229) show limited binding efficacy; C) Different glucan types (i.e., Shigu polysaccharide, mannan polysaccharide and barley glucan (229kd)) in Dextran retains high or moderate receptor binding activity after oxidation, as assessed by competitive binding assays. "20% and 40% oxidized" indicates the oxidation state of the dextran portion used for conjugation. % inhibition represents the degree of inhibition of the binding of β-glucan or mannan to the disc by inhibiting the binding of soluble dectin-1 receptors (Shitia polysaccharide and barley_BG229) or ConA (mannan) in the presence of the specified concentration of test CLEC. . D) Schizophora polysaccharide conjugates and E) lichenin conjugates retain approximately 50% of the dectin-1 binding capacity compared to uncoupled β-glucan, as assessed by competitive binding assays. F) The fungus polysaccharide conjugate produced by the heterobifunctional linker maintains high dectin-1 binding efficacy. Data are presented in relative light units (RLU) for luminescence ELISA. Pus70 conjugates 1-3 refer to three different CLEC peptide conjugates (SeqID2, SeqID10 and SeqID16) respectively. Pus 70% and Lich 200% refer to the polysaccharide and lichen polysaccharide in their respective oxidation states. BMP HPus refers to activated agaricus polysaccharide. BMPH conjugate 2 refers to the CLEC-SeqID10 conjugate. Figure 2 shows: Flow cytometric analysis of dendritic cells activated by lipopolysaccharide (LPS) and different auricularia polysaccharide preparations.

Immature bone marrow-derived mouse dendritic cells (BMDCs) were generated in vitro using granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF-BMDCs were stimulated with LPS (equivalent amounts contained in oxidized Psoralea corylifolia and Psoralea corylifolia conjugate preparations), SeqID2+SeqID7+Psoralea corylifolia conjugate, or oxidized Psoralea corylifolia conjugate alone for 24 hours. The doses of Psoralea corylifolia conjugate and Psoralea corylifolia conjugate alone were increased starting from 62.5 µg/mL (up to 500 µg/mL). DCs were identified by CD11c/CD11b expression, and the surface expression of CD80 and class II major histocompatibility complex (MHC) according to A) and C) SeqID2+SeqID7+ Pseudomonas aeruginosa conjugates or B) and D) oxidized Pseudomonas aeruginosa only was measured by flow cytometry. The expression of activation markers in DCs treated with Pseudomonas aeruginosa preparations (=measured) and treated with an equal amount of LPS (=expected) was analyzed by CytExpert software. Figure 3 shows: Particle size of CLEC conjugates was determined by dynamic light scattering (DLS) .

Particle size is determined by DLS by measuring random changes in the intensity of light scattered from a suspension or solution. Shown are the regularized analysis and the corresponding results within 24 hours of A) SeqID5+SeqID7+Shit fungi polysaccharide (80% oxidation state) conjugate, B) SeqID6+CRM+Shit fungus polysaccharide conjugate, and C) unmodified Shi fungus polysaccharide. Cumulative radius analysis. Figure 4 shows: Comparison of immunogenicity of vaccines based on different CLECs .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations with an interval of 2 weeks. Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. Samples were collected 2 weeks after the third administration and analyzed for A) anti-peptide responses (SeqID3) to vaccines based on mannoses, barley and pyriferan (SeqID2+SeqID7+CLEC) and B) anti-peptide responses (SeqID3 and SeqID11) to vaccines based on pyriferan and lichenin (SeqID2+SeqID7+CLEC and SeqID10+SeqID7+CLEC). Figure 5 shows: Comparative analysis of the immunogenicity of pyriferan conjugates and vaccines consisting of unconjugated peptides and CLEC .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations, administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to understand the dynamics of the subsequent immune response. Samples were taken 2 weeks after the 3rd dose and analyzed for anti-peptide responses (SeqID3). The vaccines used are: SeqID2+SeqID7+CLEC or a mixture of unconjugated SeqID2, SeqID7 and CLEC. Figure 6 shows: Comparative analysis of the immunogenicity of the Shi fungus polysaccharide conjugates containing B cell and T cell epitopes and the conjugates containing only the respective B cell or T cell epitopes.

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations with an interval of 2 weeks. Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. The vaccines used were: SeqID5+SeqID7+CLEC or SeqID5+CLEC, and SeqID7+CLEC. Samples were taken 2 weeks after the third administration and analyzed for anti-peptide responses (SeqID6). Figure 7 shows: Comparative analysis of anti-Pseudomonas aeruginosa antibody responses in mice after repeated immunization with peptide - Pseudomonas aeruginosa conjugates or vaccines containing the corresponding unconjugated components.

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations, administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. Pre-plasma and t1-t3 represent immune responses detectable before (pre-plasma) or after the first (t1), second (t2), or third (t3) immunization. Samples were taken 2 weeks after the third administration and analyzed for anti-Psoralen responses. A) Analysis of anti-Psoralen responses elicited 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. FIG8 shows a comparative analysis of immune responses elicited by CLEC-based vaccines using different peptide coupling orientations .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations with an interval of 2 weeks between administrations. Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. Four different CLEC-based candidate prototype vaccines were tested (two different peptides coupled to Psoralea corylifolia polysaccharide via their C-terminus or N-terminus). Samples were taken 2 weeks after the third administration and analyzed for A) anti-peptide and B) anti-aSyn protein responses. The vaccines used were: SeqID1/2/4/5+SeqID7+CLEC. Figure 9 shows: Comparative analysis of the immunogenicity of CLEC -based vaccines using different promiscuous helper T cell epitopes .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations, administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. The immune responses elicited by 9 different CLEC-based vaccines (vaccines 1-9) containing the same B cell epitope and different helper T epitopes (i.e., SeqID7, SeqID22-29) were evaluated with the corresponding peptide-KLH conjugate (vaccine 10). Samples were taken 2 weeks after the third administration and analyzed for A) anti-peptide and B) anti-aSyn protein responses. FIG. 10 shows a comparative analysis of the target protein and carrier protein- specific immunogenicity induced by CLEC -based vaccines and known peptide - protein conjugate vaccines using the carrier protein KLH as a source of helper T cell antigenic determinants .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal or subcutaneous (sc) vaccinations administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to understand the dynamics of the subsequent immune response. Compared to that induced by conventional peptide-KLH conjugates (i.e. SeqID3+KLH and SeqID6+KLH) co-administered with Alum/Alhydrogel (sc, i.e. subcutaneous injection) or without additional adjuvant (id, i.e. intradermal injection) Response, the immune response elicited by two peptide-protein conjugate vaccines using KLH as the source of the helper T epitope and combined with CLEC modifications (SeqID3+KLH+Shitia polysaccharide and SeqID6+KLH+Shit fungus polysaccharide, respectively) was evaluated. . Samples were taken 2 weeks after the 3rd administration and analyzed by ELISA for A) anti-peptide and anti-aSyn protein responses and B) anti-KLH responses. Figure 11 shows: Comparative analysis of target protein and carrier protein specific immunogenicity induced by CLEC -based vaccines and conventional peptide - protein conjugate vaccines using carrier protein CRM197 as a source of helper T cell epitopes .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal or subcutaneous vaccinations, administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to understand the dynamics of the subsequent immune response. This study used 2 different CRM-based vaccine types. SeqID6+CRM+Pus represents the peptide-CRM conjugate that is subsequently coupled to the fungus polysaccharide, while SeqID5+CRM+Pus represents the conjugate in which the peptide component and the carrier molecule have been coupled to CLEC respectively. The immune responses induced by both types were evaluated relative to respective known peptide-CRM conjugates (i.e., SeqID6+CRM, adjuvanted with Alum/Alhydrogel and administered subcutaneously). Samples were taken 2 weeks after the 3rd administration and analyzed by ELISA for A) anti-peptide and anti-aSyn protein responses and B) anti-CRM responses. Figure 12 shows: Comparative analysis of the selectivity of immune responses elicited by CLEC -based vaccines against two different aSyn forms in vivo .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal or subcutaneous vaccinations with 2-week intervals. CLEC-based vaccines (SeqID2+SeqID7+Pus and SeqID5+SeqID7+Pus; intradermal) and CLEC-based alternative vaccines (SeqID3+KLH+Pus and SeqID6+CRM+Pus; intradermal) were evaluated relative to known peptide component vaccines (SeqID3+KLH+Alum and SeqID6+CRM+Alum, subcutaneous). Samples were taken 2 weeks after the third administration and analyzed for aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn for inhibition; dashed line: filamentous aSyn for inhibition. FIG. 13 shows a comparative analysis of the affinity of antibody molecules to antigens in immune responses induced by CLEC -based vaccines .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal or subcutaneous vaccinations, with an interval of 2 weeks between administrations. CLEC-based vaccines (SeqID2+SeqID7+Pus and SeqID5+SeqID7+Pus, intradermally administered) and CLEC-based alternative vaccines (SeqID3+KLH+Pus and SeqID6+CRM+Pus, intradermally administered) were evaluated relative to known peptide component vaccines (SeqID3+KLH+Alum and SeqID6+CRM+Alum, subcutaneously administered). Samples were taken 2 weeks after the second immunization (T2) or 2 weeks after the third immunization (T3), and antibody affinity to aSyn was assessed by ELISA-based affinity analysis. FIG. 14 shows a comparative analysis of the affinity of single antigen binding segments to antigens in immune responses elicited by CLEC -based vaccines .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal or subcutaneous vaccinations, administered 2 weeks apart. Evaluate CLEC-based vaccines (SeqID2+SeqID7+Pus and SeqID5+SeqID7+Pus, administered intradermally) relative to conventional peptide-component vaccines (SeqID3+KLH+Alum and SeqID6+CRM+Alum, administered subcutaneously) and CLEC-based alternatives Vaccines (SeqID3+KLH+Pus and SeqID6+CRM+Pus, administered intradermally). Samples were taken 2 weeks after the 3rd administration, and the antibody equilibrium dissociation constant (Kd) to aSyn was assessed by aSyn shift ELISA analysis. Figure 15 shows: Comparative in vitro functional analysis of immune responses elicited by CLEC -based vaccines .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal and subcutaneous vaccinations with an interval of 2 weeks. Samples were taken 2 weeks after the third administration and the modulation of aSyn aggregation in the presence of aSyn-specific antibodies was assessed by ThT fluorescence analysis. A ) aSyn aggregation in CLEC vaccine-induced antibodies (SeqID2+SeqID7+Pus; intradermal administration), antibodies induced by the known peptide component (SeqID3+KLH+Alum, subcutaneous administration) or mouse plasma for 0-72 hours. B) Aggregation of aSyn or aSyn with preformed protofibrils in the presence of CLEC vaccine-induced antibodies (SeqID5+SeqID7+Pus and SeqID6+CRM+Pus, intradermally administered), known peptide component-induced antibodies (SeqID6+CRM+Alum, subcutaneously administered), or mouse plasma for 0-92 hours. Kinetic curves were calculated by normalizing to 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 the ThT kinetics. Figure 16 shows: Comparative analysis of the effects of the immunization route on the immune response elicited by CLEC -based vaccines.

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations at 2-week intervals. Blood samples were collected at baseline and after each vaccination to understand the dynamics of the subsequent immune response. Two alternative routes including subcutaneous (sc) and intramuscular (im) administration were compared with intradermal (id) administration of CLEC-based vaccines. Three doses of CLEC-based vaccine (SeqID2+SeqID7+Pus) were administered per route. Samples were taken 2 weeks after the 3rd dose and analyzed for A) anti-peptide and B) anti-aSyn protein responses. Figure 17 shows a comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes characterized by post-translationally modified peptide Aβ .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations, administered 2 weeks apart (id and sc). Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. CLEC-based vaccines (SeqID33+SeqID7+Pus, id) were evaluated against traditional peptide conjugate-based vaccines (SeqID32+KLH+Alum, sc). Samples were taken 2 weeks after the third administration and analyzed for A) anti-peptide, anti-AβpE3-40, anti-AβpE3-42, and anti-Aβ1-42 responses. B) Antibody affinity to AβpE3-42 was evaluated by ELISA-based affinity assay. Figure 18 shows: Comparative analysis of immune responses elicited by CLEC vaccines containing B cell antigenic determinants derived from intracellular proteins and self-antigens : Tau .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations administered 2 weeks apart (id and sc). Blood samples were collected at baseline and after each vaccination to understand the dynamics of the subsequent immune response. The CLEC-based vaccine (SeqID36+SeqID7+Pus, id) was evaluated against a traditional peptide-component-based vaccine (SeqID35+KLH+Alum, sc). Samples were taken 2 weeks after the 3rd dose and analyzed for A) anti-peptide and anti-recombinant Tau441 protein responses. B) The affinity of the antibodies for SeqID35 was assessed by ELISA-based affinity assay. Figure 19 shows: Comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes, autoantigens and conformational epitopes derived from the secreted protein : IL23 .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations, administered 2 weeks apart (id and sc). Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. Three CLEC-based vaccines (SeqID38/SeqID40/SeqID42 all conjugated to SeqID7 and Psoralea corylifolia, id) were evaluated against a traditional peptide conjugate-based vaccine (SeqID37/SeqID39/SeqID41 conjugated to KLH and Alhydrogel (Alum), sc). Samples were taken 2 weeks after the third administration and analyzed for anti-peptide and anti-IL23 protein responses. Figure 20 shows: Comparative analysis of immune responses elicited by CLEC vaccines containing B cell antigenic determinants derived from self antigenic determinants present in a transmembrane protein : the extracellular membrane proximal domain (EMPD) of membrane-bound IgE .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations administered 2 weeks apart (id and sc). Blood samples were collected at baseline and after each vaccination to understand the dynamics of the subsequent immune response. The CLEC-based vaccine (SeqID44+SeqID7+Pus, id) was evaluated against a traditional peptide component-based vaccine (SeqID43+KLH+Alum, sc). Samples were taken 2 weeks after the 3rd administration and analyzed for A) anti-injection peptide and anti-EMPD peptide responses. B) The affinity of the antibodies for the EMPD peptide was assessed by an ELISA-based affinity assay. Figure 21 shows: Comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes derived from allergens, mimic epitopes and conformational epitopes : Betv1 .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations with an interval of 2 weeks between administrations (id and sc). Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. CLEC-based vaccines (SeqID46+SeqID7+Pus, id) were evaluated against traditional peptide-based vaccines (SeqID45+KLH+Alum, sc). Samples were taken 2 weeks after the third administration and analyzed for A) anti-peptide and anti-Bet v 1 protein responses. B) Antibody affinity for Bet v 1 was assessed by ELISA-based affinity assay. Figure 22 shows: Comparative analysis of immune responses elicited by CLEC vaccines containing B cell antigenic determinants present in different forms of cancer / tumor diseases ( i.e., oncogenes ) : Her2 .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations, with an interval of 2 weeks between administrations (id and sc). Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. CLEC-based vaccines (SeqID48+SeqID7+Pus, id) were evaluated relative to traditional peptide-based vaccines (SeqID47+KLH+Alum, sc). Samples were taken 2 weeks after the third administration and analyzed for anti-peptide and anti-Her2 protein responses. Figure 23 shows: Comparative analysis of immune responses elicited by CLEC vaccines containing B cell antigenic determinants present in different forms of neoplastic diseases / cancers ( i.e. , oncogenes ) : PD1 .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations with an interval of 2 weeks between administrations (id and sc). Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. CLEC-based vaccines (SeqID50+SeqID7+Pus, id) were evaluated against traditional peptide-based vaccines (SeqID49+KLH+Alum, sc). Samples were collected 2 weeks after the third administration and analyzed for A) anti-peptide and anti-PD1 protein responses. B) The affinity of antibodies to SeqID49 was evaluated by ELISA-based affinity assay. Figure 24 shows: Comparative analysis of target protein-specific immunogenicity induced by CLEC- based peptide -CRM197 conjugate vaccines using different peptide -CRM197/CLEC ratios .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations, administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to understand the dynamics of the subsequent immune response. This study used 5 different peptide-CRM-based vaccines with different peptide-CRM/fungal polysaccharide ratios (w/w). All 5 groups were immunized with SeqID6+CRM+Pus conjugate. 1:1, 1:2.5, 1:5, 1:10 and 1:20 represent peptide-CRM conjugate/CLEC w/w ratios of 1/1, 1/2.5, 1/5, 1/10 and 1/ A combination of 20. Induced immune responses were assessed using samples collected 2 weeks after the 3rd administration, and anti-aSyn protein responses were analyzed by ELISA. Potency determination is based on calculation of ODmax/2. Figure 25 shows: Comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes from aSyn (aal-8) .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations with an interval of 2 weeks between administrations (id and sc). Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. CLEC-based vaccines (SeqID12+SeqID7+Psoralea corylifolia, id) were evaluated against traditional peptide component conjugate-based vaccines (SeqID13 conjugated with KLH and Alhydrogel (Alum), sc). Samples were taken 2 weeks after the third administration 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. Figure 26 shows: Comparative analysis of immune responses elicited by CLEC vaccines containing B cell antigenic determinants from aSyn (aa100-108) .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations administered 2 weeks apart (id and sc). Blood samples were collected at baseline and after each vaccination to understand the dynamics of the subsequent immune response. The CLEC-based vaccine (SeqID16 + SeqID7 and Shigu polysaccharide, id) was evaluated against a conventional vaccine based on a conjugate of peptide components (SeqID17 combined with KLH and Alhydrogel (Alum), sc). Samples were taken 2 weeks after the 3rd dose and analyzed for A) anti-peptide and anti-aSyn protein responses and B) aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: filamentous aSyn used for inhibition. Figure 27 shows: Comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes from aSyn (aa91-97) .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations with an interval of 2 weeks between administrations (id and sc). Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. CLEC-based vaccines (SeqID14+SeqID7 and Psoralea corylifolia, id) were evaluated against known peptide component conjugate-based vaccines (SeqID15 conjugated with KLH and Alhydrogel (Alum), sc). Samples were taken 2 weeks after the third administration and analyzed for anti-peptide and anti-aSyn protein responses. Figure 28 shows: Comparative analysis of immune responses elicited by CLEC vaccines containing B cell antigenic determinants from aSyn (aa130-140) .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations with an interval of 2 weeks between administrations (id and sc). Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. CLEC-based vaccines (SeqID20+SeqID7 and Psoralea corylifolia, id) were evaluated against a known peptide component conjugate-based vaccine (SeqID21 conjugated with KLH and Alhydrogel (Alum), sc). Samples were taken 2 weeks after the third administration 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. Figure 29 shows: Comparative analysis of immune responses elicited by CLEC vaccines containing B cell antigenic determinants from aSyn (aa115-122) .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations administered 2 weeks apart (id and sc). Blood samples were collected at baseline and after each vaccination to understand the dynamics of the subsequent immune response. The CLEC-based vaccine (SeqID51 + SeqID7 and Shigu polysaccharide, id) was evaluated against a conventional vaccine based on a conjugate of peptide components (SeqID52 combined with CRM and Alhydrogel (Alum), sc). Samples were taken 2 weeks after the 3rd dose and analyzed for A) anti-peptide and anti-aSyn silk responses and B) aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: filamentous aSyn used for inhibition. Figure 30 shows: Comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes from aSyn (aa115-124) .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations administered 2 weeks apart (id and sc). Blood samples were collected at baseline and after each vaccination to understand the dynamics of the subsequent immune response. The CLEC-based vaccine (SeqID67 + SeqID7 and Shigu polysaccharide, id) was evaluated against a traditional peptide conjugate-based vaccine (SeqID68 combined with CRM and Alhydrogel (Alum), sc). Samples were taken 2 weeks after the 3rd dose and analyzed for A) anti-peptide and anti-aSyn silk responses and B) aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: filamentous aSyn used for inhibition. Figure 31 shows: Comparative analysis of immune responses elicited by CLEC vaccines containing B cell epitopes from aSyn (aa107-113) .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations administered 2 weeks apart (id and sc). Blood samples were collected at baseline and after each vaccination to understand the dynamics of the subsequent immune response. The CLEC-based vaccine (SeqID73 + SeqID7 and Shigu polysaccharide, id) was evaluated against a traditional peptide conjugate-based vaccine (SeqID74 combined with CRM and Alhydrogel (Alum), sc). Samples were taken 2 weeks after the 3rd dose and analyzed for A) anti-peptide and anti-aSyn silk responses and B) aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: filamentous aSyn used for inhibition. Figure 32 shows: Comparative in vitro functional analysis of immune responses elicited by CLEC -based vaccines .

Female BALB/c mice aged 8-12 weeks received a total of 3 vaccinations administered 2 weeks apart (id and sc). Blood samples were collected at baseline and after each vaccination to understand the dynamics of the subsequent immune response. Samples were collected 2 weeks after the 3rd administration, and ThT kinetic measurements were assessed in the presence of AC, CLEC vaccine-induced antibodies (SeqID67/71/73+SeqID7 and Shigu polysaccharide, id), or known peptide components ( i.e., the fibrillar part of aSyn)-induced antibodies (SeqID68/72/74 combined with CRM and Alhydrogel (Alum), sc), or D) aSyn-specific monoclonal antibody LB09 or untreated mouse plasma. Figure 33 shows the binding activity of CRM197-CLEC conjugate to murine DC receptor ( ie, dectin -1) in vitro .

Comparative analysis of dectin-1 binding capacity determined by ELISA is shown. A) Pus refers to unmodified Pseudomonas polysaccharide, and pus oxi refers to activated Pseudomonas polysaccharide. CRM-pus conjugate 1 refers to SeqID6+CRM197+Pseudomonas polysaccharide conjugate, and CRM conjugate 1 refers to CRM197+SeqID6 conjugate without β-glucan modification. Negative control group refers to samples without inhibitor. B) SeqID52/66/68/70/72 refer to CRM197-Pseudomonas polysaccharide conjugates with the indicated B cell epitopes; C) Lich oxi refers to activated lich oxi and CRM-Lich conjugate 1 refers to SeqID6+CRM197+lich oxi conjugate. D) Lam oxi refers to activated laminarin, and CRM-Lam conjugate 1 refers to SeqID6+CRM197+laminarin conjugate. Figure 34 shows the binding activity of CRM197-CLEC conjugate to human DC receptor ( ie, dectin-1) in vitro .

Shows comparative analysis of dectin-1 binding capacity determined by ELISA. Lich conjugate refers to SeqID6+CRM197+lichenin conjugate, Pus conjugate refers to SeqID6+CRM197+Pyricularia polysaccharide conjugate, and Lam conjugate refers to SeqID6+CRM197+laminan conjugate. Negative control group refers to samples without inhibitor. Figure 35 shows: Comparison of immunogenicity of different CRM -Pyricularia polysaccharide-based vaccines .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations, with an interval of 2 weeks between administrations. Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. Samples were taken 2 weeks after the third administration and analyzed for A) anti-peptide response B) anti-aggregated aSyn fiber response. Figure 36 shows: Comparative analysis of the selectivity of the immune response elicited in vivo by vaccines based on peptides + CRM + Pseudomonas aeruginosa versus aSyn fibers .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal or subcutaneous vaccinations with an interval of 2 weeks. CRM-Pseudomonas aeruginosa based vaccines were evaluated relative to the known CRM vaccine. Samples were taken 2 weeks after the third administration and aSyn selectivity analysis (inhibition ELISA) was performed. The IC50 values of antibodies inhibited with increasing doses of aSyn fibers are shown. Figure 37 shows: Avidity of antibody molecules induced by peptide + CRM197 + Pseudomonas aeruginosa vaccine to antigen .

The stability and affinity index of aSyn-antibody complexes induced by peptide+CRM197+Pseudomonas aeruginosa or peptide+CRM197 vaccines after challenge with different concentrations of the isocyanate sodium thiocyanate (NaSCN) are shown. Figure 38 shows: Comparison of immunogenicity of different CLEC- based vaccines .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations, administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to understand subsequent immune response dynamics. Anti-SeqID6 peptide responses (A) and anti-aSyn fiber responses (B) induced by peptide+vector+dextran-based vaccines or non-CLEC-modified vaccines adjuvanted with Alum were sampled and analyzed 2 weeks after the 3rd administration. ); Dosage: 20 µg peptide equivalent/injection; Schizophyllum polysaccharide means SeqID6+CRM+ Schizophyllum polysaccharide, lichenin means SeqID6+CRM+lichenin, laminarin means SeqID6+CRM+laminarin, and sc+Alum means non-CLEC modified with Alum Vaccine SeqID6+CRM as adjuvant. Figure 39 shows the in vitro binding activity of peptide -CLEC- conjugates to murine (A) and human (B) DC receptors ( ie, dectin-1) .

Comparative analysis of dectin-1 binding capacity measured by ELISA is shown. Lich conjugate refers to SeqID5+SeqID7+lichenin conjugate, Pus conjugate refers to SeqID5+SeqID7+pyracantha conjugate, Lam conjugate refers to SeqID5+SeqID7+laminan conjugate. Negative control group refers to samples without inhibitor. Figure 40 shows: Comparison of immunogenicity of different CLEC- based vaccines .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations, administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to understand the dynamics of the subsequent immune response. Samples were taken 2 weeks after the 3rd dose and analyzed for anti-peptide responses (SeqID6, expressed as peptide) and peptide-dextran-based vaccine-induced anti-aSyn responses (expressed as protein) (i.e.: SeqID5+SeqID7+CLEC, dose ) Figure 41 shows the in vitro DC receptor ( ie, dectin -1) binding activity of the glycoconjugate - Fructus polysaccharide conjugate .

Both CLEC-modified vaccines, oligosaccharide + CRM197 + Psoralea corylifolia polysaccharide conjugate and polysaccharide + TT + Psoralea corylifolia polysaccharide conjugate, maintained high dectin-1 binding efficacy. Figure 41 shows a comparative analysis of dectin-1 binding capacity measured by ELISA. Act-Pus refers to the tetanus toxoid (TT) conjugate ActHIB® of Haemophilus influenzae type b capsular polysaccharide (polyribosyl-ribitol-phosphate, PRP) modified with Pseudomonas aeruginosa polysaccharide, Act refers to ActHIB® conjugate vaccine without β-glucan modification, Men refers to CRM197 conjugate vaccine Menveo® containing meningococcal oligosaccharides (A, C, W135, Y) without β-glucan modification, Men-Pus refers to Menveo® vaccine modified with Pseudomonas aeruginosa polysaccharide, and pus oxi refers to the active Pseudomonas aeruginosa polysaccharide used for modification. Figure 42 shows the comparison of immunogenicity of different CLEC -based saccharide conjugate vaccines .

Female BALB/c mice aged 8-12 weeks received a total of 3 id/im vaccinations with an interval of 2 weeks. Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. Samples were taken 2 weeks after the third administration and analyzed for anti-vaccine responses induced by oligo/polysaccharide carrier-dextran or non-dextran-modified conjugate vaccines. A) Shows the response induced by: Menveo® conjugated with Psoralea corylifolia (Menveo®+Psoralea corylifolia): N. meningitidis (A, C, W135, Y) + CRM197 + Psoralea corylifolia (80%), or unmodified Menveo®: N. meningitidis (A, C, W135, Y) + CRM197, (dose: 5µg); B) Shows the response induced by: ActHIB® conjugated with Psoralea corylifolia (ActHIB®+Psoralea corylifolia): H. influenzae (b) PRP + TT + Psoralea corylifolia (80%), or unmodified ActHIB®H: H. influenzae (b) PRP + TT (dose: 2µg). FIG. 43 shows: Comparative analysis of the immunogenicity of CLEC -based vaccines using different IL31 peptide epitopes .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations, with an interval of 2 weeks between administrations. Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. Eight different CLEC-based vaccines (SeqID132+SeqID7+Pseudomonas aeruginosa; SeqID134+SeqID7+Pseudomonas aeruginosa; SeqID136+SeqID7+Pseudomonas aeruginosa; SeqID138+SeqID7+Pseudomonas aeruginosa; SeqID140+SeqID7+Pseudomonas aeruginosa; SeqID142+SeqID7+Pseudomonas aeruginosa; SeqID144+SeqID7+Pseudomonas aeruginosa; SeqID146+SeqID 7+eqIDS147) relative 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 taken 2 weeks after the third administration and analyzed for A) anti-peptide and B) anti-IL31 protein responses. C) shows the affinity of antibodies induced by SeqID132+SeqID7+Pseudomonas aeruginosa or SeqID133+CRM vaccines determined by challenging with different concentrations of the isocyanate sodium thiocyanate (NaSCN). Figure 44 shows: Comparative immunogenicity analysis of CLEC -based vaccines using different IL31 peptide epitopes .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations, administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. The 10 different CLEC-based vaccines were evaluated relative to the respective unmodified peptide-CRM197 conjugates adjuvanted with alum (i.e., SeqID133+CRM197; SeqID135+CRM197; SeqID137+CRM197; SeqID139+CRM197; SeqID141+CRM197; SeqID143+CRM197; SeqID145+CRM197; SeqID147+CRM197; SeqID149+CRM197; and SeqID151+CRM197). (SeqID133+CRM197+Pyricularia auricularia polysaccharide; SeqID135+CRM197+Pyricularia auricularia polysaccharide; SeqID137+CRM197+Pyricularia auricularia polysaccharide; SeqID139+CRM197+Pyricularia auricularia polysaccharide; SeqID141+CRM197+Pyricularia auricularia polysaccharide; SeqID143+CRM197+Pyricularia auricularia polysaccharide; SeqID145+CRM197+Pyricularia auricularia polysaccharide; SeqID147+CRM197+Pyricularia auricularia polysaccharide; SeqID149+CRM197+Pyricularia auricularia polysaccharide; and SeqID151+CRM197+Pyricularia auricularia polysaccharide). Samples were taken 2 weeks after the third administration and analyzed for A) anti-peptide and B) anti-IL31 protein responses; C) The affinity of antibodies induced by SeqID133+CRM197+Pyricularia auriculariae or SeqID133+CRM vaccines was determined by challenging with different concentrations of the isocyanate (NaSCN) . Figure 45 shows: Inhibition of IL31 signaling by anti -IL31 antibodies induced by IL31 peptide -CLEC vaccine.

The inhibitory effects of vaccine-induced antibodies on human IL-31 signaling were assessed in human A549 cells (ATCC, Virginia, USA). The vaccine-induced antibodies used were obtained from animals immunized with IL31-peptide + SeqID7 + Psoralea corylifolia polysaccharide conjugate (CLEC; IL31 peptides: SeqID132, SeqID134, SeqID136, SeqID138, SeqID140, SeqID142, SeqID144, SeqID146) and the known IL31-peptide + CRM conjugate with Alum as adjuvant (CRM-Alum; IL31 peptides: SeqID133, SeqID135, SeqID137, SeqID139, SeqID141, SeqID143, SeqID145, SeqID147). Positive control group: commercial anti-IL31 blocking antibody; without inhibitor: IL31 stimulation only, bg: background without IL31 stimulation. FIG46 shows the inhibition of IL31 signaling by anti - IL31 antibodies induced by IL31 peptide - vector -CLEC vaccine.

The inhibitory effect of vaccine-induced antibodies on human IL-31 signaling was evaluated in human A549 cells (ATCC, VA, USA). The vaccine-induced antibodies used were derived from the use of IL31-peptide+CRM197+Crimsonose polysaccharide conjugate (CRM-CLEC; IL31 peptide: SeqID133, SeqID135, SeqID137, SeqID139, SeqID141, SeqID143, SeqID145, SeqID147, SeqID149, SeqID 151) and Animals were repeatedly immunized with Alum as adjuvant with conventional IL31-peptide + CRM conjugate (CRM-Alum; IL31 peptide: SeqID133, SeqID135, SeqID137, SeqID139, SeqID141, SeqID143, SeqID145, SeqID147, SeqID149, SeqID 151). Positive control group: commercially available anti-IL31 blocking antibody; no inhibitor: only IL31 stimulation, bg: no background of IL31 stimulation. Figure 47 shows: Comparative immunogenicity analysis of CLEC -based vaccines using different CGRP peptide epitopes .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations, administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to understand the dynamics of the subsequent immune response. Relative to the respective Alum-adjuvanted peptide-CRM197 conjugates (i.e., SeqID153+CRM197; SeqID155+CRM197; SeqID157+CRM197; SeqID159+CRM197; SeqID161+CRM197; and SeqID163+CRM197), six different Caused by CLEC-based vaccines (SeqID152+SeqID7+Shit fungus polysaccharide; SeqID154+SeqID7+Shit fungus polysaccharide; SeqID156+SeqID7+Shit fungus polysaccharide; SeqID158+SeqID7+Shit fungus polysaccharide; SeqID160+SeqID7+Shit fungus polysaccharide; and SeqID162+SeqID7+Shit fungi polysaccharide). immune response. Samples were taken 2 weeks after the 3rd dose and analyzed for A) anti-peptide and B) anti-CGRP protein responses; C) Shown by SeqID152+SeqID7+ determined by challenge with varying concentrations of the chaotrope sodium thiocyanate (NaSCN) Affinity of antibodies induced by Shigu polysaccharide or SeqID153+CRM vaccine. Figure 48 shows: Comparative immunogenicity analysis of CLEC -based vaccines using different CGRP peptide epitopes .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations, administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to understand subsequent immune response dynamics. Six different CLEC-based vaccines were evaluated (SeqID153+CRM197+Shit fungus polysaccharide; SeqID155+CRM197+Shit fungus polysaccharide; SeqID157+CRM197+Shit fungus polysaccharide; SeqID159+CRM197+Shit fungus polysaccharide; SeqID161+CRM197+Shit fungus polysaccharide; and SeqID163+CRM197+ Fungi polysaccharide) versus the corresponding unmodified peptide+CRM197 conjugates with Alum as adjuvant (i.e., SeqID153+CRM197; SeqID155+CRM197; SeqID157+CRM197; SeqID159+CRM197; SeqID161+CRM197; and SeqID163+CRM197). Samples were taken 2 weeks after the 3rd dose and analyzed for A) anti-peptide and B) anti-CGRP protein responses. C) shows the avidity of antibodies induced by SeqID153+CRM197+Shitu polysaccharide or SeqID153+CRM vaccine, determined by challenge with different concentrations of the chaotropic agent sodium thiocyanate (NaSCN). Figure 49 shows: Antibodies induced by SeqID5+SeqID7+ Shitu polysaccharide vaccine inhibit aSyn aggregation in the in vivo PFF model .

Beginning on the day of PFF vaccination, C57BL/6 mice with recombinant aSynPFF stereotactically injected into the right substantia nigra were immunized for four times using SeqID5+SeqID7+Shitia polysaccharide vaccine (vaccine) or unconjugated CLEC (vector) as a control group. Second-rate. Plasma was collected after the third immunization. Brain, plasma and CSF were harvested 126 days after PFF inoculation. Plasma titers (A) of peptide-specific antibodies used for immunization were collected two weeks after the third immunization on day 126. (B) Comparison of cerebrospinal fluid and plasma titers of vaccine B cell peptide-specific antibodies at day 126. (C) Analysis of phospho-S129 aSyn-positive aggregates in all brain regions of mice vaccinated with SeqID5+SeqID7+SeqID7+vaccinated with CLEC. (D) Correlation between antibody response and synucleinopathy levels in vaccine recipients (r=-0.9391; CI (95%) -0.9961 to -0.3318, p=0.0179, R2=0.882). (EH) Representative pSer129aSyn staining at the level of (E,F) substantia nigra and (G,H) striatum in the injected cerebral hemisphere. (E,G) Mice treated with vehicle and (F,H) mice treated with vaccine and then injected with PFF. Error bars represent mean ± SEM from n = 5-9 animals per group. Statistical differences were assessed by unpaired t-test; **p<0.01;*p<0.05. Figure 50 shows: carrier-specific immunogenicity analysis of peptide +CLEC and peptide +CRM+CLEC conjugates.

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal/subcutaneous vaccinations, administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. The immune responses elicited by four different CLEC-based vaccines (CRM-Psoralea corylifolia; i.e., SeqID6+CRM197+Psoralea corylifolia; SeqID133+CRM197+Psoralea corylifolia; SeqID135+CRM197+Psoralea corylifolia; and SeqID137+CRM197+Psoralea corylifolia) were evaluated relative to respective peptide-CRM197 conjugates adjuvanted with Alum (CRM-Alum; i.e., SeqID6+CRM197; SeqID133+CRM197+Psoralea corylifolia; SeqID135+CRM197+Psoralea corylifolia; and SeqID137+CRM197+Psoralea corylifolia). Samples were taken 2 weeks after the third administration and analyzed for A) SeqID6+CRM197+Psoralea corylifolia polysaccharide-induced in vivo anti-CRM response and B) SeqID133+CRM197+Psoralea corylifolia polysaccharide; SeqID135+CRM197+Psoralea corylifolia polysaccharide; and SeqID137+CRM197+Psoralea corylifolia polysaccharide-induced in vivo anti-CRM response. Figure 51 shows: CLEC- specific immunogenicity analysis of peptide +CLEC and peptide +CRM+CLEC conjugates .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations, administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. The immune responses elicited by 14 different CLEC-based vaccines were evaluated. Samples were taken 2 weeks after the third application and analyzed for in vivo anti-Pseudomonas polysaccharide responses; A) Samples: SeqID6+CRM197+Pseudomonas polysaccharide, SeqID6+CRM197+Lichen polysaccharide; SeqID6+CRM197+Laminaria polysaccharide ; B) Samples SeqID6+CRM197+Pseudomonas polysaccharide; Pseudomonas polysaccharide coupled at the specified conjugate/Pseudomonas polysaccharide ratio (w/w); C) Samples: SeqID133+CRM197+Pseudomonas polysaccharide; SeqID135+CRM197+Pseudomonas polysaccharide; and SeqID137+CRM197+Pseudomonas polysaccharide; D) SeqID132+SeqID7+Pyricularia polysaccharide; SeqID134+SeqID7+Pyricularia polysaccharide; and SeqID136+SeqID7+Pyricularia polysaccharide; Pre-serum: samples obtained before immunization; Positive control group: samples from animals immunized with only non-oxidized Pyricularia polysaccharide. Figure 52 shows: immunogenicity analysis of peptide - carrier - dextran conjugates and vaccines composed of peptide - carrier conjugates and unconjugated dextran .

Female BALB/c mice aged 8-12 weeks received a total of 3 intradermal vaccinations, with an interval of 2 weeks between administrations. Blood samples were collected at baseline and after each vaccination to understand the kinetics of the subsequent immune response. Samples were taken 2 weeks after the third administration and analyzed for anti-SeqID6 peptide (A) and anti-aSyn monomer (B) responses. Vaccines used: SeqID6+CRM197+Pseudomonas aeruginosa, SeqID6+CRM197 mixed with unoxidized Pseudomonas aeruginosa, and non-CLEC-modified, unadjuvanted SeqID6+CRM197.

TW202409077A_112124769_SEQL.xml

Claims (15)

A conjugate comprising β-glucan and a B cell and/or T cell epitope polypeptide, wherein the β-glucan is covalently bound to the B cell and/or T cell epitope polypeptide to form the The conjugate of β-glucan and the B cell and/or T cell epitope polypeptide, the β-glucan is mainly linear β-(1,6)-glucan, and its (1,6 ) The ratio of 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, especially at least 10:1 . The conjugate of claim 1, wherein the β-glucan is pustulan. The conjugate of claim 1 or 2, wherein the polypeptides comprise at least one B cell antigenic determinant and at least one T cell antigenic determinant. The conjugate of 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). The conjugate of any one of claims 1 to 4, wherein the B cell antigen determinant and the pan-specific/promiscuous T cell antigen determinant are independently bound to the β-glucan. The conjugate of 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, especially 7 to 15 amino acid residues. A conjugate according to any one of claims 1 to 6, wherein the length of the T cell antigen determinant polypeptide is 8 to 30 amino acid residues, preferably 13 to 29 amino acid residues, and especially 13 to 28 amino acid residues. The conjugate of any one of claims 1 to 7, wherein the conjugate further comprises a carrier protein, preferably a non-toxic cross-reactive material (CRM) selected from diphtheria toxin, especially CRM197, KLH, diphtheria toxoid (DT) ), tetanus toxoid (TT), Haemophilus influenzae protein D (HipD), meningococcal serogroup B outer membrane protein complex (OMPC), Pseudomonas aeruginosa exotoxin Recombinant non-toxic form of A ( r EPA), flagellin, E. 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, for example, multiple antigenic peptides (MAP), with the proviso that if the conjugate includes a carrier protein, the conjugate includes at least one other independently bound T cell or B cell epitope base polypeptide, especially wherein the ratio of carrier protein to β-glucan in the conjugate is 1/0.1 to 1/50, preferably 1/0.1 to 1/40, more preferably 1/0.1 to 1/20 , especially 1/0.1 to 1/10. A conjugate according to any one of claims 1 to 8, wherein the polypeptide is or comprises a B cell or T cell antigenic determinant polypeptide, wherein the polypeptide is preferably a B cell and T cell antigenic determinant or comprises a B cell and T cell antigenic determinant. The conjugate of any one of claims 1 to 8, wherein the conjugate contains a T cell epitope and does not contain a B cell epitope, wherein the conjugate preferably contains more than one T cell epitope, especially Is composed of two, three, four or five T cell epitopes. The conjugate according to any one of claims 1 to 10, which is used for preventing or treating a disease, preferably for preventing or treating an infectious disease, a chronic disease, an allergy or an autoimmune disease. For example, the conjugate of any one of claims 1 to 11 is used 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), neuroaxonal dystrophy, Alzheimer's disease Alzheimer's disease (AD), AD with Lewy bodies restricted by the amygdala (AD/ALB), dementia in Down syndrome, Pick disease, progressive supranuclear palsy (PSP) ), corticobasal degeneration, chromosome 17-related frontotemporal dementia and Parkinson's disease (FTDP-17), and argyrophilic granulopathy. A method for producing a conjugate as in any one of claims 1 to 12, wherein the β-glucan is activated by oxidation and wherein the activated β-glucan and the B cell epitope polypeptide and /or the T cell epitope polypeptide comes into contact, thereby obtaining a conjugate of the β-glucan and the B cell epitope polypeptide and/or the T cell epitope polypeptide. The method of claim 13, wherein the β-glucan is obtained by periodate oxidation, reductive amination or cyanation of the hydroxyl group at the ortho-position. The method of claim 13 or 14, wherein the β-glucan is oxidized to a degree of oxidation, which is defined as a reactivity with Schiff's fuchsin-reagent, which is equivalent to the degree of oxidation of an equivalent amount of Pyrrolidone polysaccharide oxidized with periodate at a molar ratio of 0.2-2.6, preferably 0.6-1.4, and especially 0.7-1.

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